WO2007022346A2 - Cellular function underlying bone micro-structure characteristic of type 2 diabetes - Google Patents

Abstract

The present invention relates to methods of characterizing and modifying bone quality and pathogenesis in patients suffering from bone pathologies, including those associated with metabolix disorders such as Type 2 diabetes. These methods may involve increasing bone density and/or thickness, as necessary in the patient, and reducing bone tissue degradation as, for instance, due to type 2 diabetes. The methods of the present invention may applied to cortical and/or cancellous bone.

Description

CELLULAR FUNCTION UNDERLYING BONE MICRO-STRUCTURE CHARACTERISTIC OF TYPE 2 DIABETES

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Patent Application Serial No. 60/708,987 filed August 15, 2005, the disclosure of which is incorporated by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made in collaboration with inventors at the Veterans Administration. The U.S. government has certain rights to the invention.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER

PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK. [0003] APPLICABLE

FIELD OF THE INVENTION

[0004] The present invention relates to methods of characterizing and/or modifying bone quality in subjects and patients suffering from bone pathology, including bone pathologies associated with metabolic disorders, such as Type 2 diabetes. These methods may involve increasing bone density and/or altering micro-structural parameters such as thickness, as necessary in the patient, and reducing bone tissue degradation due to the pathology. The methods of the present invention may be applied to cortical and/or cancellous bone.

BACKGROUND OF THE INVENTION

[0005] Insulin-independent diabetes mellitus, so-called Type 2 diabetes, affects bone quality. However, clinical and animal study results are inconclusive in terms of either increase or decrease of bone mineral density (BMD). Recent in vitro experiments show the importance of wingless proteins (Wnts) in differentiation of adult mesenchymal cells (MSCs) into osteoblasts, which are responsible for bone formation.

[0006] Pathologies that impede normal remodeling merit attention because of the clinical need to restore normality and because of the information that the pathological process may provide about the normal process. Diabetes, one of the pathologies that slows the bone healing process, merits particular attention. Diabetes affects a significant percentage of the U.S. population, and its complications are severe. In fact, almost 20% of the adult population was affected by diabetes in 2002 with approximately 1.3 million people presenting new cases every year afterwards. Women with Type 2 diabetes have a 1.70-fold higher risk of hip fracture than women without diabetes. The longer period of time associated with fracture healing in diabetic patients has side effects. Also, oral diabetes medications are associated with higher incidence of fractures.

[0007] While decreased BMD is an established complication of type 1 diabetes (Krakauer et al., 1995; van Daele PL et al., 1995), reports on Type 2 diabetes show conflicting results about its relation with BMD. Studies on BMD at the lumbar spine and the femoral neck report unaltered (Barrett-Connor et al., 1992; Bartos et al., 2001; Christensen et al., 1999; Isaia et al,, 1999; Miedany et al., 1999; Sert et al., 2003; Sosa et al., 1996; Tuominen et al., 1999; Wakasugi et al., 1993), increased (Akin et al., 2003; Barrett-Connor et al., 1992; Christensen et al., 1999; Isaia et al., 1999; Miedany et al., 1999; Sahin et al., 2001; Sert et al., 2003; van Daele et al., 1995) or rarely decreased (Gregorio et al., 1994, Sert et al., 2003; Wakasugi et al., 1993) BMD results. Moreover, BMD at the distal radius has been reported as unaltered (Barrett-Connor et al., 1992; Christensen et al., 1999), increased (Barrett-Connor et al., 1992), or decreased (Krakauer et al., 1995; Levin et al., 1976). These various results may indicate that positive or negative effects on BMD may depend on the bone site of Type 2 diabetic patients investigated. Further, Type 2 diabetes may alter the cortical and cancellous bone ratio in different measure as a function of site.

[0008] The studies mentioned above measured BMD as the only bone quality parameter. However, it now appears that additional parameters need to be investigated and that higher resolution imaging should be employed. In the present invention, the micro-structure in a Type 2 diabetes model is evaluated. Unlike many other Type 2 diabetes models, the Goto- Kakizaki (GK) rat, bred from a Wistar rat (Goto et al., 1976), is non-obese and shows similar metabolic, hormonal and vascular disorders to the human diabetes disease. Its characteristics include fasting hyperglycemia, impaired secretion of insulin in response to glucose, both in vivo and in isolated pancreatic cells, and hepatic and peripheral insulin resistance. Bone research on the GK rat is currently limited to evaluation of contents of bone gamma- carboxyglutamic acid-containing protein (Takeshita, 1993), osteocalcin (Ostenson et al., 1997) and macro-structural observations (Ahmed et al., 2003).

[0009] Type 2 diabetes delays bone repair in both human and animal models in the absence of obesity (Levin et al., 1976; Ishida et al., 1985; Ostenson et al., 1997) through diminished formation of new bone (He et. al., 2004). While slow bone formation can be the consequence of increased apoptosis of osteoblasts (Weinstein et al., 1998; Hock et al., 2001), the impact of diabetes Type 2 on osteoblast precursors is unknown. Cellular events that may result in fewer osteoblasts on bone surfaces of diabetic compared to the normal counterparts (Leidig- Bruckner and Ziegler, 2001) need to be analyzed, hi particular, Wnt signaling via β-catenin requires attention because of compelling data from the diabetes literature. Specifically, glycogen synthase (GS) is the rate-limiting enzyme that catalyzes the last step of the glycogen synthetic pathway (Frame et al., 2001; Ni et al., 2003; Doble and Woodgett, 2003). Activity of GS is regulated by glycogen synthase kinase 3 (GSK-3). GSK-3 phosphorylates GS, thereby inhibiting its activity and glycogen production (Figure 1). One of insulin's effects is to inhibit GSK-3 and allow glycogen synthesis to proceed. In canonical Wnt signaling pathway, GSK-3 also phosphorylates β-catenin. As a result, β-catenin is targeted for degradation by the proteosome. Upon binding of certain Wnts to cellular receptors, GSK-3 activity is inhibited, β-catenin levels accumulate and the protein translocates to the nucleus. GSK-3 activity is elevated in human skeletal muscle and fat cells with Type 2 diabetes (Nikouliaa, 2000), providing a possible mechanism for insulin resistance (Pearce, 2004). Altered GSK-3 activity has frank implications for Wnt/β-catenin signaling in diabetic cells. The altered GSK-3 activity may affect the cells' ability to respond to the canonical Wnt signal. Alternatively, the balance between canonical and non-canonical signaling may be shifted. Further, GSK-3 is present in MSCs (Etheridge et al., 2004) and the effects of Type 2 diabetes on MSCs' differentiation into osteoblasts is unknown.

[0010] In the present invention, the roles of Wnt3a and other Wnts on the proliferation and differentiation on cells from the bone marrow of GK rat are studied. Since plasma level of osteocalcin is well related to bone formation and turnover, the low plasma values in these animal models suggest that bone formation and turnover decrease in non-insulin-dependent diabetes mellitus. Information on the cellular events underlying bone formation and turnover and therefore bone repair remain unknown. Because adequate bone quality depends on adequate function of osteoblasts, proliferation of MSCs and differentiation of MSCs into pre- osteoblasts and osteoblasts merits attention. The present invention also focuses on some of the bone quality parameters that should be affected by Type 2 diabetes, regionally in long bones. It has been demonstrated that the osteoblastic function is decreased whereas the osteoclastic function was elevated in Type 2 diabetic patients (Okazaki R et al., 1997, 1999; Suzuki K et al., 1997, 2005). Whether diabetes' slow bone repair process depends on MSC altered proliferation, differentiation, or other factors involving altered vascularization, is not known. Whether the poor tissue quality of Type 2 diabetes is due to unsuitable function of MSCs versus osteoblasts or osteoclasts, is not known. The present invention, however, sheds light on these unknowns, and provides methods useful in the characterization, diagnosis and development of treatments for bone pathologies.

[0011] The system analysis approach in the present invention is broadly applicable to bone biology to investigate MSCs of humans, as well as in osteoblasts and osteoclasts: in particular, to find out whether Wnt/β-catenin targeting/trajectory varies with age, bone density, or strain levels and with the presence of pathologies that hinder bone repair. Because the invention points to the Wnt processes affected by diabetes Type 2, results of the invention are expected to help the formulation of a subsequent study of the effect of Wnt on human MSCs of individuals affected by diabetes Type 2 by age cohort. In fact, recent research (Akune et, al., 2004) indicates that aging MSCs may be more prone to differentiate towards the adipocytic lineage rather than the osteogenic lineage. The present invention helps to illuminate the cause of bone tissue degradation due to diabetes Type 2. Such knowledge is fundamental to therapy development. Indeed, the understanding of the differences between diabetic and normal bone may lead to development of interventions on GSK-3 focused on correcting the altered spatio-temporal targeting/trajectory of β-catenin so as to re-establish normality through the understanding of cytoskeleton components involved in normal and altered trajectories. In the long run, this invention is expected to lead to the development of methods to manipulate the local environment through use of biologic agents to improve bone repair. [0012] Accordingly, in a first aspect, this invention provides methods for determining the capacity for bone repair or formation in a mammal by characterizing the amount or intracellular localization of β-catenin in a multipotent mesenchymal cell (MSC) from the mammal. In preferred embodiments, the multipotent mesenchymal cell is isolated from bone marrow, or isolated from blood, and cultured. In some embodiments, the total amount of β- catenin in the mesenchymal cell is determined. In still other embodiments, the physical distribution of β-catenin in the mesenchymal cell is determined. In yet other embodiments, the relative localization of β-catenin in the nucleus and the cytoplasm, which may be expressed as a ratio, is to be compared in determining the capacity for bone repair or formation. In some embodiments, the β-catenin to be detected is fluorescently labeled (e.g., attached to a fluorescently labeled monoclonal antibody, fused or coupled to a fluorescent protein such as a green fluorescent protein) and the fluorescence of the label is detected to indicate the amount and/or location of the β-catenin. In preferred embodiments of the above, the mammal has osteoporosis or altered bone mineral density. The mammal may have Type 2 diabetes or is particularly a mammal with Type 2 diabetes and an altered bone mineral density associated with Type 2 diabetes. The mammal may have insulin resistance or is particularly a mammal with insulin resistance and an altered bone mineral density associated with insulin resistance. In some further embodiments, a modulator of insulin resistance, Wnt, or a protein kinase inhibitor is administered to the cells and the effect of the treatment on β- catenin intracellular amounts and/or intracellular location is determined in comparison to that of cells from the same population which were not contacted with the modulator. The response may be examined in cells contacted with Wnt or other modulators of bone repair and formation, and the response monitored over the course of the differentiation of the MSC to an osteoblast or other cell type (e.g., adipocyte).

[0013] In some embodiments of any of the above, the β-catenin is detected and its intracellular localization tracked using scanning microscopy including, but not limited to, multiphoton scanning microscopy. In other embodiments, the location of intracellular β- catenin molecules is tracked over time in a single cell using scanning microscopy including, but not limited to, multiphoton scanning microscopy. In preferred embodiments of the above, confocal microscopy is used. [0014] In embodiments of any of the above, the amount of β-catenin or nuclear localization of β-catenin is different from than that conducted and observed for a comparison population of the mammal and the mammal is thereby identified or diagnosed as having reduced ability to repair or form bone tissue with respect to the comparison population. In some embodiments, the mammal has Type 2 diabetes or insulin resistance and the comparison population does not have Type 2 diabetes or insulin resistance. In some further embodiments, a modulator of insulin resistance, Wnt, or a protein kinase inhibitor is administered to the cells and the effect of the treatment on β-catenin intracellular amounts and/or intracellular location is determined in comparison to that of cells from the same population which were not contacted with the modulator. The response to the modulator may then be compared to the response of the comparison population to the modulator.

[0015] In a second aspect, the invention provides a method of identifying a modulator of bone repair or formation by performing a first determination of the amount or intracellular distribution of β- catenin in a mammalian multipotent mesenchymal cell, osteoclast, or osteoblast; and contacting the cell with the agent and performing a second determination of the amount or the intracellular distribution of β-catenin in the cell; and comparing the amount or intracellular distribution of β-catenin determined in the first determination to that of the second determination, wherein changes in the amount or nuclear localization of β-catenin identifies the agent to be a modulator of bone repair or formation. In some embodiments, wherein contacting the agent alters the total amount or nuclear localization of β-catenin and the agent is identified as being useful in promoting bone repair, formation, osteroporosis, or pathological alterations of bone density and structure. In other embodiments, the first determination and second determination monitor the amount and/or intracellular localization of β-catenin over time and the amount and movement of the β-catenin is compared over time. In some embodiments, the modulator to be tested is a GSK3 inhibitor or a WNT modulator. In preferred embodiments, the mammalian multipotent mesenchymal cell is a human multipotent mesenchymal cell. In other embodiments of any of the above, the β-catenin is fluorescently labeled and the fluorescence signal is used to detect the β-catenin (e.g., fused to a fluorescent protein and the fluorescence of the fusion protein is monitored to determine the amount or localization of β-catenin in the cell). [0016] In yet other embodiments of any of the above, the multipotent mesenchymal cell is obtained from a subject having Type 2 diabetes, insulin resistance, osteoporosis, a bone fracture, a bone healing disorder, a bone pathology, or a metabolic disorder that affects bone tissue repair or formation.

[0017] In a third aspect, the invention provides methods of treating a subject having Type 2 diabetes, insulin resistance, osteoporosis, a bone fracture, a bone healing disorder, a bone pathology, or a metabolic disorder that affects bone tissue repair or formation, by administering an agent which is a modulator of GSK, Wnt, or β-catenin. In some embodiments, the invention provides methods of treating such conditions by administering an agent identified according to the second aspect of this invention. The agents are administered as pharmaceutical compositions and in therapeutically effective amounts.

[0018] hi a fourth aspect, the invention provides methods of characterizing mechanisms promoting bone repair or formation in a cell selected from the group consisting of a multipotent mesenchymal cell, an osteoblast, or an osteoclast, said method comprising tracking the amount, location or movement of fluorescently labeled B-catenin in the cell using scanning microscopy or multiphoton microscopy. In preferred embodiments, confocal microscopy is used, hi some embodiments, the β-catenin is fused to a fluorescent protein.

[0019] hi a fifth aspect, the invention provides the GK rat as an animal model for studying local effects of Type 2 diabetes and β-catenin, GSK, or Wnt modulators on bone tissue, hi some embodiments, the GK rat is used as an animal model for identifying such modulators. Preferably, calcification of MSC-derived osteoblasts can be used as the end measure.

[0020] In a sixth aspect, the invention provides a method of characterizing the pathogenesis of local tissue in bone from a mammal by assessing the histomorphometry of the local tissue in bone and evaluating β-catenin amounts and intracellular localization of MSC from the local tissue, hi some embodiments, an effect of Wnt, insulin modulators, or GSK modulators, or protein kinase inhibitors on β-catenin intracellular amounts or movement is determined, hi some embodiments, the effect is monitored in the local tissue of affected bone and of unaffected bone and the differences evaluated to characterize the pathogenesis of the local pathology, hi some embodiments, the bone is from the tibia. In other embodiments, the mammal has insulin resistance or Type 2 diabetes, hi preferred embodiments, the histomorphometry is performed on bone microstructures from a region and bone marrow from the region. The histomorphometry generally can be any or all of a histological examination of cartilage, bone and marrow, bone surface area, and osteoblast number, density, surface are and shape.

[0021] Li a seventh aspect, the invention provides a method of characterizing the MSC response to Wnt in a subject having a bone pathology by obtaining a first sample of MSC from a first mammal having a bone pathology and culturing the first sample of MSC in the presence of Wnt to obtain a first culture and staining the first culture with an osteogenic stain to determine the number of osteoblasts therein , wherein the number of osteoblasts in the first culture provides a first measure of the differentiation of MSC into osteoblasts; obtaining a second sample of MSC from a second mammal which is different from the first mammal in not having the bone pathology and culturing the second sample of MSC in the presence of Wnt to obtain a second culture and staining the second culture with an osteogenic stain to determine the number of osteoblasts therein , wherein the number of osteoblasts in the second culture provides a second measure of the differentiation of MSC into osteoblasts; and comparing the first and second measure to characterize the response of bone to Wnt signaling in the bone pathology. In some preferred embodiments, the first subject has Type 2 diabetes and the second subject does not. In still other embodiments, the Wnt is Wnt 3a, Wntl 1, or Wnt 5.

[0022] In other embodiments of any of the above aspects, the present invention further provides the steps of quantitatively describing the bone micro-structure of a bone pathology optionally in conjunction with assessing the β-catenin intracellular amounts or localization. The bone micro-structure effects can be correlated with the β-catenin measures. For instance, in one embodiment, the β-catenin effects and bone micro-structure of a subject having Type 2 diabetes is compared to those of a subject not having Type 2 diabetes. Type 2 diabetes affects the micro-structural properties of cortical and cancellous bone differently in relation to location. At specific sites, the micro-structure shows the characteristics of osteoporotic tissue. At other specific sited, the micro-structure shows increased density and or thickness. Accordingly, analysis of the localized MSC (MSC obtained from bone marrow proximal to the portion of bone displaying pathology; e.g. affected cancellous bone and cortical bone) β- catenin amounts and intracellular localization associated with a particular β-catenin response to Wnt, Wnt modulators, insulin modulators, GSK inhibitors, and β-catenin modulators can be used to characterized the pathogenesis of the diverse effects of Type 2 diabetes on MSC function in different bone regions.

[0023] An objective of the present invention is to describe quantitative differences in MSCs differentiation towards the osteoblastic lineage in a diabetic and a non-diabetic rat. MSCs have a higher propensity to differentiate into osteoblasts and produce a higher mineralized matrix in diabetic versus non-diabetic rat. Accordingly, the higher mineralized matrix is a particular microstructural analysis and target for drug screening and therapy.

[0024] Yet another objective of the present invention is to describe qualitative and quantitative differences in the translocation of β-catenin in MSCs stimulated by Wnt3a between diabetic and non-diabetic rat. Type 2 diabetes affects Wnt3/β-catenin negatively through specific pathological localization.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1. Wnt/β-catenin signaling pathway.

[0026] Figure 2. MicroCT of tibia for (a) GK and (b) Wistar rat in Figure 3.

[0027] Figure 3. MicroCT of tibia midshaft of 90 day old GK rat #1 (a) and 91 day old

Wistar rat #3 (b) suggests osteoporosis of GK rat.

[0028] Figure 4. Comparison of corresponding regions suggests osteoporosis in the GK rat.

[0029] Figure 5. Comparison of trabecular bone in corresponding regions between GK and Wistar rats. Differences appear to depend on the chosen region.

[0030] Figure 6. Alzarin red stain of mesenchymal cells. The dishes relative to two GK rats (GK2 and GK6) appear fuller in comparison to Wistar rats (WT5 and WTl) for each plate density. Precautions are taken to avoid formation of "ring" at the inner border of the dishes.

[0031] Figure 7. Previous models (Ascenzi M.-G. et al., 2003) of the lamellar type that appears (a) extinct and (b) bright on cross section of secondary osteons. Newly proposed models of extinct (c) and bright (d) lamella. Five layers in each image represent micro- structural layers in the osteon radial direction. The colors are indicative of collagen-apatite orientation relative to osteon axis: blue (longitudinal), red (oblique acute), green (transverse) and grey (oblique obtuse).

[0032] Figure 8. (a) Detail of osteon transverse section is modeled in (b) by software that reproduces osteocyte lacunar and canalicular network, (c) 3D view of model's detail above and below the indicated plane of focus.

[0033] Figure 9. Eight frames extracted from a 2D confocal movie of fluorescent beta- catenin in cells of sea urchin embryo (Weitzel et al., 2003). β-catenin fluorescence appears bright in the cell nuclei.

[0034] Figure 10. (A) Ellipses with marked axes drawn on the first frame shown in Figure 3. (B) Parametric equations of the ellipses shown in Figure 4 where a and b are the major and minor axes respectively, φ is the angle between the major axis and the x-axis, (xo, yo) are the coordinates of the center and θ is the parameter, 0 < θ < 2π. (C) Plot of ellipses specified by the equations in Figure 10(B).

[0035] Figure 11. Relative percent area (upper curve) and relative percent distance of centers (lower curve) of modeled fluorescent region with respect to modeled cell.

[0036] Figure 12. Time course of β-catenin stabilization by Wnt3a in L cells (A) or bovine articular chondrocytes (B). Whole cell lysates were prepared from cells stimulated with Wnt3a - or control L cell-conditioned media for the indicated intervals. Western blots (5000 cells/lane) were probed with anti-β-catenin antibody (Santa Cruz Biologicals).

[0037] Figure 13. Overall experimental design of the present invention.

DETAILED DESCRIPTION

[0038] The present invention relates to the investigation of cellular events underlying bone microstructure characteristic of bone pathologies such as those associated with Type 2 diabetes and to testing of a new qualitative and quantitative method for intervention.

[0039] The present invention also tests in a rat model the novel hypothesis that altered MSC differentiation and function is reflected in altered micro-structural properties of bone tissue. Further, the invention tests an innovative technique that tracks Wnt/5-catenin movement from cell membrane to nucleus in three dimensions as a function of time. This allows for characterization of MSC behavior and development of intervention therapies of cellular trafficing to modify bone quality for Type 2 diabetes.

DEFINITIONS

[0040] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag (1991); GRANT AND HACKH¹S CHEMICAL DICTIONARY, 5th Edition, (McGraw Hill, 1987); HAWLEY¹S CONDENSED CHEMICAL DICTIONARY (14th Edition, Lewis, Ed., John Wiley and Sons, 2001)and the CONCISE DICTIONARY OF BIOMEDICINE AND MOLECULAR BIOLOGY, 2nd Edition (CRC Press, 2002); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0041] It is noted here that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0042] Mesenchymal cells are part of the embryonic mesoderm, consisting of loosely packed, unspecialized cells set in a gelationous ground substance from which connective tissue, bone, cartilage, and the circulatory and lymphatic systems develop. When found in the bone marrow they are often called "stromal cells", and these cells can be extracted from the bone marrow and grown in a culture. Depending on the signal proteins in the environment, these mesenchymal cells may replicate or differentiate into fat cells, cartilage cells, or bone cells. Signal proteins dictate the fate of the cells. The option of renewal or differentiation into a variety of cell types leads to the name some researchers prefer for multipotent mesenchymal cells: mesenchymal stem cells.

[0043] Pathologies of bone include, but are not limited to, osteoporosis, Paget's disease of bone, osteogenesis imperfecta, and primary hyperparathyroidism, fibrous dysplasia (monostotic, polyostotic McCune- Albright syndrome), osteopetrosis. Metabolic diseases associated with bone pathology include Type 2 diabetes, familial hypophosphatemia, vitamin D-resistant rickets, vitamin D-dependent rickets type I, receptor defect rickets, vitamin D- dependent rickets type II, defective 25-hydroxylase, Fanconi syndrome, oncogenous syndrome, osteodystrophy, renal rickets, hypophosphatasia, metaphyseal dysplasia.

[0044] A "mammal" includes any mammal including primates, humans, rodents, mice, rats, rabbits, guinea pigs, and horses.

[0045] Wnt includes all proteins from this family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis or cellular differentiation. Wnt proteins bind to receptors of the Frizzled and LRP families which are found on the cell surface. Via several cytoplasmic relays, the signal is transduced to β- catenin, which thereupon enters the nucleus to form a complex with TCF to then activate transcription of Wnt target genes. Wnt proteins include, Wnt 1, 2, 2B/13, 4, 3, 3A, 5A and 5B, 6, 7A, 7B, 8A, 8B, 9A, 9B, 1OA, 1OB, 11, and 16. Wnt includes polypeptides, polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence or specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising a referenced amino acid sequence; immunogenic fragments respectively thereof, and conservatively modified variants respectively thereof. A Wnt polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal. The proteins for use in the invention include both naturally occurring or recombinant molecules. Particularly preferred Wnt proteins are mammalian (e.g., rat, mouse, human). Wnt3A, wingless-type MMTV integration site family, member 3A, see NCBI GeneBank Accession No. AB060284 for amino acid and cDNA sequence (incorporated herein by reference with regard to such sequence information); Wnt5A, wingless-type MMTV integration site family, member 5 A, see NCBI GeneBank Accession No. L20861 for amino acid and cDNA sequence (incorporated herein by reference). Wtn5B, wingless-type MMTV integration site family, member 5B, see NCBI GeneBank Accession No. AB060966 for amino acid and cDNA sequence (incorporated herein by reference with regard to such sequences). Wntl 1, wingless-type MMTV integration site family, member 11, see NCBI GeneBank Accession No. Y12692 for amino acid and cDNA sequence (incorporated herein by reference with regard to such sequence information).

[0046] β-catenin is a member of the armadillo family of proteins. These proteins have multiple copies of the so-called armadillo repeat domain which is specialized for protein- protein binding, β-catenin includes polypeptides, polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence or specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising a referenced amino acid sequence; immunogenic fragments respectively thereof, and conservatively modified variants respectively thereof. A β-catenin polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal. The proteins for use in the invention include both naturally occurring or recombinant molecules. A particularly preferred β-catenin is human β-catenin, see NCBI GeneBank Accession No. NPOO 1895 for amino acid and cDNA sequence (incorporated herein by reference with regard to such sequence information). Other preferred β-catenins are the rat β-catenin, see, NCBI GeneBank Accession No. NP445809 for amino acid and cDNA sequence (incorporated" herein by reference with regard to such sequence information) and mouse β-catenin see, NCBI GeneBank Accession No. NP031640 for amino acid and cDNA sequence (incorporated herein by reference with regard to such sequence information). In some embodiments, the β- catenin is labeled. In some embodiments of any of the aspects, the levels of β-catenin nucleic acids (e.g., mRNA, cDNA) are alternatively or additionally used in characterizing the amounts or turn-over of β-catenin in a cell at one or more time points. Methods of detecting nucleic acids in a cell preparation or in cells are well known to those of ordinary skill in the art.

[0047] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. Particularly preferred labeled are fluorescent proteins which may be fused to β-catenin to detect the β-catenin molecule. Methods of making such fusion proteins are well known to persons of ordinary skill in the art. Other labels are attached to antibodies which are capable of specifically binding β- catenin.

[0048] Compact or cortical bone consists of about 40% minerals, 40% collagen, and 20% fluids. The major internal spaces or discontinuities of compact bone include the vascular system, pits and cavities (lacunae), narrow channels (canaliculae), fine porosity, and spaces between the mineral phases.

[0049] Cancellous bone consists of trabeculae, i.e. osseous structures with either a sheet- like or a rod-like configuration. These structures interlace to form a lattice-like or spongy biological structure.. The cancellous bone porosity, can range from 30% to more than 90%, is mainly due to the wide vascular and bone marrow intrabecular spaces. As is seen in compact bone, levels of calcification vary from trabecula to trabecula and within trabeculae.

[0050] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity) .

[0051] An "agonist" refers to an agent that acts to stimulate, increase, activate, facilitate, enhance activation, sensitize or up regulate the activity or expression of the referenced protein. For instance, an agonist of Wnt or β-catenin stimulate, increase, activate, facilitate, enhance activation, sensitize or up regulate the activity or expression of the referenced protein to promote bone repair or formation.

[0052] An "antagonist" refers to an agent that partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity of the referenced protein. An antagonist of GSK may partially or totally block stimulation, decrease, prevent, delays activation, inactivate, desensitize, or down regulate the activity of GSK to promote bone repair or formation.

[0053] "Inhibitors," "activators," and "modulators" of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term "modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of a polypeptide or polynucleotide of the invention, e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, and then determining the functional effects. Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%. Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher. Accordingly, modulators include agonists, partial agonists and antagonists of the referenced endogenous agents themselves or their effects. Modulators of insulin resistance are well known in the art. Modulators of GSK activity (e.g., The results show that a serine/threonine kinase inhibitors, glycogen synthase kinase-3 protein kinase inhibitors, insulin modulators, insulin resistance modulators) are also well known in the art. Modulators include antisense nucleic acids of a protein of interest.

[0054] Where comparisons are made and a difference or altered measure is recited therebetween the difference can be, for instance, a biologically or pathogenically distinct difference or a statistically different difference at the 90% or 95% or 99% confidence level according to an appropriate statistical test. In some embodiments for the biologically or pathogenically distinct difference, the distance between compared values can be of the order of 80% or less, 60% or less, 40% or less, 20% or less, or 10% or less, in which the smaller of the two comparision values is expressed as a percent of the larger value.

[0055] The term "test compound" or "drug candidate" or "modulator" or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, RNAi, siRNA, antisense nucleic acids, oligonucleotide, etc. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

[0056] A "small organic molecule" refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 Daltons and less than about 2500 Daltons, preferably less than about 2000 Daltons, preferably between about 100 to about 1000 Daltons, more preferably between about 200 to about 500 Daltons.

[0057] "Determining the functional effect" or "measuring the response" and the like refer to assays increased or decreased or under the influence of an agent or candidate compound for use in methods according to the invention, e.g., measuring physical and chemical or phenotypic effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein; measuring inducible markers or transcriptional activation of the protein; measuring binding activity or binding assays, e.g. binding to antibodies; measuring changes in ligand binding affinity; e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.

[0058] Samples or assays for identifying the modulators according to the invention are conducted in the presence of the candidate modulator and then the results are compared to control samples without the modulator to examine for the desired activity or to determine the functional effect of the candidate inhibitor. A positive reference control which is an agent having the desired activity may be used. For instance, in the case of Wnt modulators, insulin modulators or insulin resistance modulators, the positive control agent may be the native peptide (e.g., Wnt or insulin) or a modulator of insulin resistance (e.g., pioglitazone). Control samples (untreated with modulators) are assigned a relative of 100%. Inhibition is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25 to 0%. Stimulation is achieved when the control activity value relative to the modulator is about65%, preferably 50%, more preferably 25 to 10% or less, or less than 5%. Suitable methods for identifying modulators for use according to the invention are well known in the art and are further exemplified in the Examples.

[0059] The modulators for use according to the invention can be used to treat bone pathologies, including those associated with type 2 diabetes. The terms "treating" or "treatment" of includes:

(1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a mammal that does not yet experience or display symptoms of the disease,

(2) inhibiting the disease, i. e. , arresting or reducing the development of the disease or its clinical symptoms, or.

(3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.

[0060] An "siRNA" or "RNAi" refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene (e.g., Wnt, β-catenin) when the siRNA expressed in the same cell as the gene or target gene. "siRNA" or "RNAi" thus refers to the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene of the Wnt/β-catenin signaling pathway (e.g., β-catenin, GSK) and forms a double stranded siRNA. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. [0061] The design and making of siRNA molecules and vectors are well known to those of ordinary skill in the art. For instance, an efficient process for designing a suitable siRNA is to start at the AUG start codon of the mRNA transcript (e.g., see, Figure 5) and scan for AA dinucleotide sequences (see, Elbashir et al. EMBO J 20: 6877-6888 (2001). Each AA and the 3' adjacent nucleotides are potential siRNA target sites. The length of the adjacent site sequence will determine the length of the siRNA. For instance, 19 adjacent sites would give a 21 Nucleotide long siRNA siRNAs with 3' overhanging UU dinucleotides are often the most effective. This approach is also compatible with using RNA pol III to transcribe hairpin siRNAs. RNA pol ITI terminates transcription at 4-6 nucleotide poly (T) tracts to create RNA molecules having a short poly(U) tail. However, siRNAs with other 3' terminal dinucleotide overhangs can also effectively induce RNAi and the sequence may be empirically selected. ror selectivity, target sequences with more than 16-17 contiguous base pairs of homology to other coding sequences can be avoided by conducting a BLAST search (see, www.ncbi.nlm.nih.gov/BLAST).

[0062] The design and making of siRNA molecules (those which are complementary to a nucleic acid sequence of, and can silence, Wnt or β-catenin expression) and vectors are well known to those of ordinary skill in the art. For instance, an efficient process for designing a suitable siRNA is to start at the AUG start codon of the mRNA transcript and scan for AA dinucleotide sequences (see, Elbashir et al. EMBO J 20: 6877-6888 (2001). Each AA and the 3' adjacent nucleotides are potential siRNA target sites. The length of the adjacent site sequence will determine the length of the siRNA. For instance, 19 adjacent sites would give a 21 Nucleotide long siRNA siRNAs with 3' overhanging UU dinucleotides are often the most effective. This approach is also compatible with using RNA pol III to transcribe hairpin siRNAs. RNA pol III terminates transcription at 4-6 nucleotide poly(T) tracts to create RNA molecules having a short poly(U) tail. However, siRNAs with other 3' terminal dinucleotide overhangs can also effectively induce RNAi and the sequence may be empirically selected. For selectivity, target sequences with more than 16-17 contiguous base pairs of homology to other coding sequences can be avoided by conducting a BLAST search (see, www.ncbi.nlm.nih.gov/BLAST).

[0063] The siRNA expression vectors to induce RNAi can have different design criteria. A vector can have inserted two inverted repeats separated by a short spacer sequence and ending with a string of T's which serve to terminate transcription. The expressed RNA transcript is predicted to fold into a short hairpin siRNA . The selection of siRNA target sequence, the length of the inverted repeats that encode the stem of a putative hairpin, the order of the inverted repeats, the length and composition of the spacer sequence that encodes the loop of the hairpin, and the presence or absence of 5'-overhangs, can vary. A preferred order of the siRNA expression cassette is sense strand, short spacer, and antisense strand. Hairp siRNAs with these various stem lengths (e.g., 15 to 30) can be suitable. The length of the loops linking sense and antisense strands of the hairpin siRNA lean have varying lengths (e.g., 3 to 9 nucleotides, or longer). The vectors may contain promoters and expression enhancers or other regulatory elements which are operably linked to the nucleotide sequence encoding the siRNA. These control elements may be designed to allow the clinician to turn oix or on me expression ot the gene by adding or controlling external factors to which the regulatory elements are responsive.

[0064] The siRNA can be administered directly or an siRNA expression vectors can be used to induce RNAi can have different design criteria. A vector can have inserted two inverted repeats separated by a short spacer sequence and ending with a string of T's which serve to terminate transcription. The expressed RNA transcript is predicted to fold into a short hairpin siRNA . The selection of siRNA target sequence, the length of the inverted repeats that encode the stem of a putative hairpin, the order of the inverted repeats, the length and composition of the spacer sequence that encodes the loop of the hairpin, and the presence or absence of 5'-overhangs, can vary. A preferred order of the siRNA expression cassette is sense strand, short spacer, and antisense strand. Hairp siRNAs with these various stem lengths (e.g., 15 to 30) can be suitable. The length of the loops linking sense and antisense strands of the hairpin siRNA lean have varying lengths (e.g., 3 to 9 nucleotides, or longer). The vectors may contain promoters and expression enhancers or other regulatory elements which are operably linked to the nucleotide sequence encoding the siRNA. The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. These control elements may be designed to allow the clinician to turn off or on the expression of the gene by adding or controlling external factors to which the regulatory elements are responsive.

[0065] Construction of suitable vectors containing the desired therapeutic gene coding and control sequences employs standard ligation and restriction techniques, which are well understood in the art (see Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.

[0066] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0067] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences; refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0068] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0069] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat' I. Acad. ScL USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)). [0070] A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et at, J. MoI. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix {see Henikoff & Henikoff, Proc. Natl. Acad. ScL USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0071] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0072] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0073] A particular nucleic acid sequence also implicitly encompasses "splice variants." Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. "Splice variants," as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. An example of potassium channel splice variants is discussed in Leicher, et al., J. Biol. Chem. 273(52):35095-35101 (1998). [0074] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0075] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0076] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0077] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified valiant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0078] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) {see, e.g., Creighton, Proteins (1984)).

[0079] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0080] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein such as a β-catenin-green fluorescent protein fusion protein). [0081] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10⁰C lower than the thermal melting point (T_n,) for the specific sequence at a defined ionic strength pH. The T_n, is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42⁰C, or, 5x SSC, 1% SDS, incubating at 65⁰C, with wash in 0.2x SSC, and 0.1% SDS at 65⁰C.

[0082] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37⁰C, and a wash in IX SSC at 45⁰C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al., John Wiley & Sons.

[0083] For PCR, a temperature of about 36⁰C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length. For high stringency PCR amplification, a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50⁰C to about 65°C, depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90⁰C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCi? Protocols, A Guide to Methods and Applications, Academic Press, Inc. N. Y.). [0084] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.

[0085] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0086] Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'₂, a dimer of Fab which itself is a light chain joined to V_H-C_HI by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'₂ dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region {see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries {see, e.g., McCafferty et al, Nature 348:552-554 (1990)).

[0087] For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used {see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity {see, e.g., Kuby, Immunology (3^rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies {see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al, Bio/Technology 10:779-783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al, Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens {see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens {see, e.g., WO 93/08829, Traunecker et al, EMBO J. 10:3655-3659 (1991); and Suresh et al, Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two co valently joined antibodies, or immunotoxins {see, e.g., U.S. Patent No. 4,676,980 , WO 91/00360; WO 92/200373; and EP 03089).

[0088] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers {see, e.g., Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0089] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0090] By "therapeutically effective dose or amount" herein is meant a dose that produces effects for which it is administered. The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques {see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)).

[0091] The term "pharmaceutically acceptable salts" or "pharmaceutically acceptable carrier" is meant to include salts of modulatory compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When modulatory compounds for used according to the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds for use according to the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for the present invention.

[0092] The neutral forms of the modulator compounds may be regenerated by contacting the salt with a base or acid and isolating the parent modulator compound in the conventional manner. The parent form of the modulator compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the puiposes of the present invention.

[0093] In addition to salt forms, the modulator for use according to the present invention can be in a prodrug form. Prodrugs are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0094] Certain modulator compounds for use according to the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain modulator compounds for use according to the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention. [0095] Where modulator compounds for use according to the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present

[0096] Pharmaceutical compositions of modulators for use according to the invention suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

[0097] The modulator of choice, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0098] Suitable formulations of modulators according to the invention for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the compound of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

[0099] Formulations of modulators suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0100] Injection solutions and suspensions of modulators for use according to the invention can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.

[0101] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can, if desired, also contain other compatible therapeutic agents.

[0102] Preferred pharmaceutical preparations deliver one or more modulators optionally in combination with one or more therapeutic agents in a sustained release formulation.

[0103] In therapeutic use for the treatment of bone pathologies, the modulators utilized in the pharmaceutical method of the invention are administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. For example, dosages can be empirically determined considering the type and stage of the pathology diagnosed in a particular patient. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of the modulator. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.

[0104] The pharmaceutical preparations for use according to the invention are typically delivered to a mammal, including humans and non-human mammals. Non-human mammals treated using the present methods include domesticated animals (i.e., canine, feline, murine, rodentia, and lagomorpha) and agricultural animals (bovine, equine, ovine, porcine).

Screening Methods

[0105] The present invention also provides methods of identifying modulators of the Wnt/β-catenin pathway wherein the modulators promote the repair and/or formation of bone.

[0106] Using the assays described herein, one can identify lead compounds that are suitable for further testing to identify those that are therapeutically effective modulating agents by screening a variety of compounds and mixtures of compounds for their ability to modulate (e.g., increase) the activity of β-catenin and other components of the Wnt/β-catenin pathway. Compounds of interest can be either synthetic or naturally occurring.

[0107] Screening assays can be carried out in vitro or in vivo. Typically, initial screening assays are carried out in vitro, and can be confirmed in vivo using cell based assays or animal models. For instance, proteins of the regenerating gene family are involved with cell proliferation. Also, the binding of a modulator to GSK, Wnt, or β-catenin molecule can be assessed and/or the effects of expression of the Wnt/β-catenin target genes measured.

[0108] Usually a compound that modulates the Wnt/β-catenin system is synthetic. The screening methods are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). [0109] The invention provides in vitro assays for identifying modulators which are readily adapted to a high throughput format. For each of the assay formats described, "no modulator" control reactions which do not include a modulator provide a background level of activity. With regard to some screening methods, in the high throughput assays of the invention, it is possible to screen up to several thousand different modulators in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many different plates per day; assay screens for up to about 6,000-20,000, and even up to about 100,000-1,000,000 different compounds is possible using the integrated systems of the invention. The steps of labeling, addition of reagents, fluid changes, and detection are compatible with full automation, for instance using programmable robotic systems or "integrated systems" commercially available, for example, through BioTX Automation, Conroe, TX; Qiagen, Valencia, CA; Beckman Coulter, Fullerton, CA; and Caliper Life Sciences, Hopkinton, MA.

[0110] Essentially any chemical compound can be tested as a potential modulator of the Wnt/ β-catenin signaling pathway. Most preferred are generally compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika- Biochemica Analytika (Buchs Switzerland), as well as providers of small organic molecule and peptide libraries ready for screening, including Chembridge Corp. (San Diego, CA), Discovery Partners International (San Diego, CA), Triad Therapeutics (San Diego, CA), Nanosyn (Menlo Park, CA), Affymax (Palo Alto, CA), ComGenex (South San Francisco, CA), and Tripos, Inc. (St. Louis, MO).

[0111] In one preferred embodiment, modulators are identified by screening a combinatorial library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical or peptide libraries" can be screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[0112] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

[0113] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with β-D-glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see, Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like). [0114] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem. Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433 A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford,

MA).

[0115] Methods and systems for modelling bone structure are disclosed in U.S. Patent Application Publication Nos. 20050131662, 20040062786 and 20020082779 which are each incorporated herein by reference in their entirety and in particular with respect to such modeling methods and procedures used therein. Further information on the multidirectional morphology structure and mechanics of bone including the mechanics of osteonic lamella are described in U.S. Patent Application Publication Nos. 20030216899 and 20020155162 which are each incorporated herein by reference in their entirety, particularly with reference to such descriptions and the analytical methods and procedures used therein.

DIAGNOSTIC METHODS

[0116] The present invention provides methods of diagnosing or providing prognosis of bone pathologies by determining the intracellular amounts and/or localization of β-catenin or β-catenin encoding nucleic acids (e.g., cDNA, mRNA) in MSCs at one or more time points, preferably after contacting the MSCs with a Wnt (e.g., Wnt3A, Wnt5A, Wntl 1), and optionally or alternatively by determining the micromorphometry of bone in a sample from a subject suspected of having or at risk of a bone pathology. Such subjects include, but are not limited to, those who have an age, gender, or metabolic condition associated with a bone pathology. Diagnosis preferably involves determining the level and location of β-catenin protein throughout the cytoplasm and nucleus. A preferred measures include the relative amounts of β-catenin in the nucleus and cytoplasm and the total amount pf β-catenin in the cell. Levels of β-catenin nucleic acids may also be used. The levels and localization and amount may be compared to a baseline or range. Typically, the baseline value is representative of levels of the levels and localization in a healthy subject not suffering from the bone pathology as optionally measured using a biological sample or other clinical methods or in a subject not having the condition placing the subject at an increased risk. Variation of levels or localization of the β-catenin protein or nucleic acids from the baseline range (either up or down) indicates that the patient has a bone pathology or is at risk of developing a bone pathology, depending on the marker used. In the case of β-catenin a reduced amount or localization of β-catenin or β-catenin nucleic acids would be consistent with a diagnosis of a bone pathology.

[0117] Antibody reagents can be used in assays to detect β-catenin in samples using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, "Principles and Practice of Immunoassay," 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach," Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used. See, e.g., Self et al, Curr. Opin. Biotechnol, 7:60-65 (1996). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction, with laser induced fluorescence. See, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, /. Chromatogr. B. Biomed. ScL, 699:463-80 (1997). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. See, e.g., Rongen et al, J. Immunol. Methods, 204:105-133 (1997). In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention. Nephelometry assays are commercially available from Beckrnan Coulter (Brea, CA; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et ah, J. Clin. Chem. Clin. Biochem., 27:261-276 (1989)).

[0118] Specific immunological binding of the antibody to nucleic acids can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with iodine- 125 (¹²⁵I) can be used. A chemiluminescence assay using a chemiluminescent antibody specific for the nucleic acid is suitable for sensitive, non-radioactive detection of protein levels. An antibody labeled with fluorochrome is also suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β- galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. An urease detection system can be used with a substrate such as urea- bromocresol purple (Sigma Immunochemicals; St. Louis, MO).

[0119] A signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of ¹²⁵I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.

[0061] The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

[0120] Alternatively, nucleic acid binding molecules such as probes, oligonucleotides, oligonucleotide arrays, and primers can be used in assays to detect differential RNA expression in patient samples, e.g., RT-PCR. In one embodiment, RT-PCR is used according to standard methods known in the art. In another embodiment, PCR assays such as Taqman^® assays available from, e.g., Applied Biosystems, can be used to detect nucleic acids and variants thereof. In other embodiments, qPCR and nucleic acid microarrays can be used to detect nucleic acids. Reagents that bind to selected biomarkers can be prepared according to methods known to those of skill in the art or purchased commercially.

[0121] Analysis of nucleic acids can be achieved using routine techniques such as Southern analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), or any other methods based on hybridization to a nucleic acid sequence that is complementary to a portion of the marker coding sequence (e.g., slot blot hybridization) are also within the scope of the present invention. Applicable PCR amplification techniques are described in, e.g., Ausubel et al and Innis et al, supra. General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization," BIOS Scientific Publishers, 1999. Amplification or hybridization of a plurality of nucleic acid sequences (e.g., genomic DNA, mRNA or cDNA) can also be performed from mRNA or cDNA sequences arranged in a microarray. Microarray methods are generally described in Hardiman, "Microarrays Methods and Applications: Nuts & Bolts," DNA Press, 2003; and Baldi et al, "DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling," Cambridge University Press, 2002.

[0122] Analysis of nucleic acid markers and their variants can be performed using techniques known in the art including, without limitation, microarrays, polymerase chain reaction (PCR)-based analysis, sequence analysis, and electrophoretic analysis. A non- limiting example of a PCR-based analysis includes a Taqman^® allelic discrimination assay available from Applied Biosystems. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al, Methods MoI Cell Biol, 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al, Nat. Biotechnol, 16:381-384 (1998)), and sequencing by hybridization. Chee et al, Science, 274:610-614 (1996); Drmanac et al, Science, 260:1649-1652 (1993); Drmanac et al, Nat. Biotechnol, 16:54-58 (1998). Non- limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Other methods for detecting nucleic acid variants include, e.g., the INVADER^® assay from Third Wave Technologies, Lie, restriction fragment length polymorphism (RFLP) analysis, allele-specific oligonucleotide hybridization, a heteroduplex mobility assay, single strand conformational polymorphism (SSCP) analysis, single- nucleotide primer extension (SNUPE) and pyrosequencing.

[0123] A detectable moiety can be used in the assays described herein. A wide variety of detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions. Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes {e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, and the like.

[0124] Useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different markers. Such formats include microarrays and certain capillary devices. See, e.g., Ng et al., J. Cell MoI. Med., 6:329-340 (2002); U.S. Pat. No. 6,019,944. In these embodiments, each discrete surface location may comprise antibodies to immobilize one or more markers for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more markers for detection.

nalysis can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate diagnosis or prognosis in a timely fashion.

[0126] Alternatively, the antibodies or nucleic acid probes of the invention can be applied to sections of patent biopsies immobilized on microscope slides. The resulting antibody staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art. COMPOSITIONS, KITS AND INTEGRATED SYSTEMS

[0127] The invention provides compositions, kits and integrated systems for practicing the assays described herein using reagents, nucleic acids, and/or antibodies specific for the polypeptides or nucleic acids specific for the polynucleotides of the invention.

[0128] Kits for carrying out the diagnostic assays of the invention typically include a probe that comprises an antibody or nucleic acid sequence that specifically binds to polypeptides or polynucleotides of the invention, and a label for detecting the presence of the probe. The kits may include several antibodies or polynucleotide sequences encoding polypeptides of the invention, e.g., a cocktail of antibodies that recognize β-catenin.

[0129] The present invention is next described by means of the following examples. The use of these and other examples anywhere in the specification is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified form. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the claims, along with the full scope of equivalents to which the claims are entitled.

EXAMPLES

Study Design And Procedures

[0130] Remove the soft tissue from the rat limbs of both diabetic and control rats. Conduct histomorphometry on long bone sections and microCT analysis at specific sites of long bones. Isolate and culture MSCs from both diabetic and control rats separately. Differentiate MSCs from diabetic control rats separately into osteoblasts. Employ confocal microscopy to track intracellular Wnt/β-catenin movement upon stimulation of MSCs with Wnt3a. Characterize pathways geometrically and quantify fluorescent data.

PRELIMINARY WORK

[0131] Bone Microstructure: The posterior limbs of 6 GK and 6 Wistar male rats, aged between 75 and 91 days old were employed. The posterior limbs were Faxitron X-rayed. Faxitron X-rays (obtained with slightly different parameters) of 90 day old GK rat (a) and 91 day old Wistar rat confirmed previous results (Ahmad et al., 2003). In particular, femur and tibia of GK rat are slightly shorter than Wistar rat.

[0132] MicroCT was employed on a small portion of the trabecular proximal tibia, at 4% of tibia total length, below the growth plate (Fig. 2 and Table 1). The trabeculae appear thicker, closer to each other, and more plate-like in the GK rat (Fig. 2a) in comparison to the Wistar rat (Fig. 2b).

Table I: MicroCT results on rat tibias

MicroCT on tibia cortical midshaft (Fig, 3 and Table 1) indicates slight osteoporosis in the GK rat.

[0133] Femurs and tibiae were then sectioned longitudinally with a small saw whose blade was water-cooled to prevent heating of material. Figure 4 shows the proximal (a, b) and distal

(c, d) femoral sections of 90 day old GK and of 91 day old Wistar rat.

[0134] Figure 5 shows tibia longitudinal proximal sections of the same rats whose femurs appear in Figure 4. The black rectangular region below the growth plate was compared between OK and Wistar by histomorphometry and microCT. The trabeculae within the red regions clearly show differences: the trabeculae of the GK rat appear more longitudinally oriented and closer to each other. The black larger rectangular region encloses compared region where cancellous bone appears osteoporotic in the GK rat.

[0135] Observation of micro-structure is indicative of regional effect of diabetes Type 2. MicroCT at various bone sites and histomorphometry on longitudinal sections are expected to confirm and deepen the meaning of these results. Because of the regional differences observed, MSCs should be separately isolated for femur, proximal tibia and tibia midshaft. Regional differences may be due simply to mechanical adaptation, or to a combination of mechanical adaptation and cellular function.

In Vitro Studies [0136] MSCs were isolated from the bone marrow of femur and tibia of 2 GK and 2 Wistar female rats, aged 84 and 61 days, respectively. MSCs were cultured following the protocol described above. Three different stains were tested to study the propensity of cells to differentiate towards osteobasts and calcify the matrix. Cells were plated in 35 turn dishes at various densities (5x 10³, 10⁴, 5x 10⁴ cells/dish) at passage 1 after the primary culture was grown in DBX for 7 days. None of the stained plates were quantified. Nevertheless, the cell predisposition for matrix calcification is apparently higher for the GK rat.

Alkaline phosphatase (ALP) - at 9 days

[0137] This stain detects alkaline phosphatase, a gene commonly used as a late marker for differentiation towards the osteoblastic lineage in vitro. Alkaline phosphatase expression precedes matrix calcification. In general earlier expression of ALP results in earlier detection of matrix calcification. Plates were stained 9 days after plating. ALP assays were run on unstained plates at 14 days to quantify relative levels of ALP. No significant differences were detected between GK and Wistar rat in this assay at 14 days. The timing is critical. ALP assays run at an earlier time (7 days) may indicate earlier expression of ALP in one strain, although the maximum level of expression may eventually be the same (at 14 days) for both cells types. Two plates for each of GK and Wistar cells were prepared at 5xlO³ concentration and one plate for each of GK and Wistar were prepared at 5x10⁴ concentration. In some instances the cells were also counterstained with Mayer's Hematoxylin to visualize the cell nucleus to assess the proportion of cells stained by alkaline phosphatase.

Alzarin Red - at 14 days

[0138] This stain detects calcium with a bright red color (Fig. 6) that can be extracted and quantitated spectrophotometrically. 14 day cultures were chosen as starting point to look at calcification. Three dishes of each of GK and Wistar cells were prepared.

Van Kossa - at 21 days.

[0139] This stain also detects calcium but produces a dark brown insoluble precipitate. 21 - day cultures were chosen as the end point of calcification. Two flasks of GK cells (with different density per flask) and Wistar cells were stained. Under regular light microscopy we see a increased number and size of clusters for the GK rat cells in comparison to the Wistar rat. [0140] These results are surprising. It is not believed that they are due to age difference between the rats. Furthermore, it is expected that a higher calcified matrix gives rise to a stiffer bone tissue more prone to fracture in patients. This would be in line with the so far unexplained higher risk of bone fracture in patients with Type 2 diabetes.

FURTHER RELATED RESEARCH Coπfocal Microscopy

[0141] The inventor has employed confocal microscopy in two recent studies of bone micro-specimens (Ascenzi M.-G. et al., 2003; Ascenzi M.-G. and Lomovtsev, 2005). The research was motivated by a long-standing question, first raised by Leeuwenhoek in the late 1600's about the structural make up of lamellae of human secondary osteons that he was able to see under a self-made microscope. Since then, many researchers have worked to answer this question. Since lamellae appear either extinct or bright in cross-section under circularly polarized light, the question was framed as understanding the structural difference between the two lamellar types. Over the centuries, scientists divided into a first group that believed that the lamellar difference in appearance under polarized light corresponded to a difference in collagen fibril orientation; and a second group believing that the difference was instead due to a difference in collagen fibril density.

[0142] The inventor tackled the issue by: (1) developing and implementing a demanding micro-dissection technique for single lamellar isolation; and (2) examining the isolated specimen microscopically under a novel direction, parallel to the original osteon radial direction and perpendicular to the collagen fibrils in the lamella.

[0143] Results: The present invention comprises a new "parabolic distribution" model of lamellar structure (Fig. 7). This work has established that human secondary osteons consist of two lamellar types that differ from each other by characteristic patterns of collagen orientation. For the first time, confocal microscopy was applied to the specimens, clearly revealing the collagen fibril orientation without superimposition of sub-structures. This confocal study was expanded to a larger number of specimens (Ascenzi M.-G. et al., 2005, incorporated herein by reference in its entirety and in particular with regard to such experimental procedures, methods and results) with the purpose of quantifying the collagen orientation and acquiring unique information on collagen fibrils' orientation distribution along the specimen thickness, that is, the original osteon's radial direction. Such results are now included in the present osteon modeling (see Data Management and Biological System Modeling).

[0144] The inventor was able to conduct a delicate and time-consuming lamellar isolation technique on the two lamellar types. The proposed research is expected to expand this confocal microscopy skill to confocal microscopy scanning of the kinematics process of the Wnt/β-catenin pathway.

Data Management and Biological System Modeling

[0145] Because bone quality assessment requires attention to bone tissue micro- and ultra- structure, the inventor has worked towards the realization of biological system modeling, and specifically of the virtual rendering of human bone structure that respects the naturally hierarchical bone structure and biological variation (Ascenzi A. et al., 1997; Ascenzi M.-G., 1999; Ascenzi M.-G. et al., 2004; Ascenzi M.-G. and Lomovtsev, 2005). First, the inventor has created large databases obtained from histomorphometric measurements on images of bone micro-structure and of specimens isolated along a variety of orientations. Second, the inventor devised software that chooses from the databases and either recreates geometry of existing micro-structures or builds geometry of virtual micro-structures along the experimentally observed statistical distributions (Fig. 8). Statistics used are the paired t-test and non-parametric test, if necessary. The micro-structures are then assigned the mechanical properties observed experimentally. The models show high adherence to functional behavior of micro-structures and are geared towards the simulation of macroscopic bone tissue that reflects the natural hierarchical organization of bone and respects the biological variation. This line of research is oriented towards the understanding of the micro-structural alterations due to pathology that affect bone quality and the virtual rendering of them. The present invention is expected to apply data management to the data to be collected on the confocal images of a cell under Wnt/β conditions and of biological system modeling to the β-catenin pathway to be modeled in the proposed research.

Data Collection and Analysis of Two-Dimensional Images of β-Catenin Kinetics

[0146] To show feasibility of proposed data collection and modeling of kinetic systems from biological imaging data, a time-lapse movie of β-catenin movements was analyzed. The dynamics of β-catenin expression during development have been studied by 4-D confocal laser-scanning microscopy of sea urchin embryos (Weitzel et al., 2004). Those investigators produced a two dimensional time-lapse movie from through-focus projections of fluorescently-tagged β-catenin in the embryo (Weitzel et al, 2004). In accordance with the present invention, 9 consecutive frames were extracted from the published confocal movie and magnified (2x) (Figure 3. One cell in each extracted frame was analyzed in terms of position and area of cell and of fluorescent β-catenin with the imaging system XaraXl (Xara Group Limited, UK)). Specifically, the cell membrane and boundary of each fluorescent area were fitted with ellipses drawn on each imported image, and an x,y-eoordinate reference system was fixed and applied to all 8 images (Figure 10A). For each ellipse, the coordinates of the center, the major and minor axes' measurements and the measurement of the angle between major axis and x-axis (θ) were recorded. The data were imported into a file of Maple (Waterloo Inc. software), a program for mathematical manipulations. An algorithm was written with Maple to produce equations that describe the shape of the ellipses (Figure 10B), verify the accuracy of data collection by plotting the equations and comparing them to the ellipses previously drawn on the confocal images (Figure 10C), and map the sequence of centers to measure the movement of the fluorescent region in respect of the cell from frame to frame.

[0147] Two sets of values were calculated for each of the 8 frames (represented on the horizontal axis of Figure 11): the percent distance between center of modeled fluorescent region and center of modeled cell; and the percent area of modeled fluorescent region with respect to cell (represented on the vertical axis of Figure 11). The values were interpolated (see the two curves through each of the two sets of data points in Figure 11) by means of four-degree splines (Piegl and Tiller, 1997) with respect to frame number. These preliminary data show how position and size of fluorescent regions can be mathematically tracked, quantified, and compared among frames of 2D time-lapse confocal movies. Moreover, animated cartoons that approximate the live image movements can be generated from the mathematical models. Analysis of the 3-D imaging is expected to include data collection in terms of x and y coordinates from a stack of images, rather than from one image, in terms of time, as done here. Further, the level of each image in the stack is expected to provide for a third coordinate. \

Wnt Signaling and Reagents

[0148] The following data are included to demonstrate feasibility of the experimental approaches with recombinant Wnt proteins. An important aspect of the present invention is the window of image acquisition. Experiments with mouse L cells and bovine chondrocytes showed that β-catenin levels stabilize within 3 hours of Wnt3a treatment (Fig. 12). That result is consistent with published work from other labs (Shibamoto et al., 1998).

[0149] A preliminary experiment is expected to determine the optimal 20 minute window for image acquisition in MSCs, i.e., when the shift from cytosolic to nuclear P-catenin is maximal.

Overall Experimental Design (Figure 13)

[0150] It is expected that 12 diabetic Goto-Kakizaki rats and 12 Wistar rats are needed. Rats, aged 6 months, may be purchased from Taconic Farms, Inc. (Germantown, New York). Isolate the MSC from the rats' bone marrow and culture MSC in the laboratory. Use Wnt- conditioned media (Wnt3a, Wnt 5a), as well as the GFP-β-catenin fusion construct.

[0151] One objective of the present invention is to describe quantitatively the bone micro- structure of diabetic rat in comparison to non-diabetic rat model.

[0152] Histomorphometry is conducted on digitally recorded images of longitudinal sections of femur and tibia. Images are enlarged to 4x and analyzed by XaraXl and Metamorph software. A grid is superimposed to each image. Density and thickness are measured within each square of the grid. The percent density within each site (for example for the proximal femur; femoral neck, Ward's triangle, great trochanter, and intermediate region between epiphysis and diaphysis) is computed by counting the number of grid vertices that fall on the trabeculae and then dividing such number by the total number of vertices and multiplying by 100 (Parfitt, 1983). Trabecular thickness is estimated by measuring the 2D thickness of each trabecula every 50μm on the grid edges (Odgaard, 2001). The measurement error is at most equal to ±10 μm and comparable to current studies (Link et al., 2002). Data is entered in Excel files.

[0153] MicroCT is conducted at a resolution of 20μm at chosen sites of femur and tibia. Results from microCT are compared to histomorphonnetry results.

[0154] Expected Results: Cortical and cancellous bone are expected to be affected differently by Type 2 diabetes in relation to location. The micro-structure is expected to show either the characteristics of osteoporotic tissue at specific locations or of increased density, thickness and orientation at other locations in the bone. Regional results are discussed in terms of force distribution on the region.

[0155] In the event that micro-structural differences are very small between the diabetic and the control rats, the analysis may be repeated on 8 month old rats. It is expected that later in life the phenotype becomes more apparent.

[0156] Another objective of the present invention is to describe quantitative differences in osteoblastic differentiation for diabetic and non-diabetic rat models in terms of the inherent capacity of MSCs to process a Wnt signal.

[0157] Each of the Goto-Kakizaki and Wistar rats is sacrificed as described in the ARC approved protocol. Bone marrow MSCs are harvested from rat femurs and tibiae separately under aseptic conditions (Lennon et al., 1995). The humeri are also dissected and stored (fixed in 2% paraformaldehyde) for histology, if needed. The ends of each of femur and tibia are cut off and an 18-gauge needle containing 1 ml DMEM containing 20% FBS, glutamine, and antibiotics inserted into the distal end. The marrow plug is flushed out and mechanically disrupted by passage through the needle. The marrow cell suspension is plated in T75 flasks for expansion as needed.

[0158] Cells are plated at a seeding density of 1 x 10 cells/cm in 60mm dishes for CFU-F and CFU-F/ ALP+ assays (primary culture), and in 100mm dishes for expansion in (secondary) culture.

[0159] Expansion (i.e., basal medium) consists of DMEM containing 10% FBS and antibiotics. MSCs in primary culture are passaged at 70-80% confluence and plated at a seeding density of 1 x 10⁴ cells/cm² in 100mm tissue culture dishes. A whole dish of GK rat and of Wistar rat cells are set aside for transfection (see above).

[0160] Additional experiments include determination of colony forming unit- fibroblast (cfu-f) and cfu-osteoblast (cfu-o) after expansion of the MSCs in either the presence or absence of dexamethasone. Dexamethasone induces cells to differentiate towards the osteoblastic lineage. This is expected to give a more accurate assessment of osteoblastic potential in the bone marrow from age-matched GK and Wistar rats by distinguishing between pre-osteoblastic precursors already present in the bone marrow (seen when expanded in absence of DEX) and stem cells that can become osteoblast precursors only when treated with DEX. Expansion in the absence of DEX is also expected to allow analysis of the multipotent character of the GK and Wistar cultures through differentiation to other lineages (Westendorf et al., 2004; Bennet et al., 2005; Kennek and Macdougald, 2005; Yates et al., 2005). Oil Red stain is used to compare the potential for adipocyte differentiation in the GK and Wistar cultures.

[0161] For osteogenic differentiation, basal medium containing osteogenic supplements (10 mM β-glycerophosphate, 100 nM dexamethasone, and 0.05 mM ascorbic acid 2-phosphate) are added 24 h after subculturing. Four experimental groups are used for both diabetic and control MSCs: Wnt3a conditioned media, Wnt5a conditioned media, control LiCl cell conditioned media, and a control with no conditioned media. Osteogenic assays (Alizarin red staining and quantitative alkaline phosphatase assay) are performed on days 12-21 (to be determined by the osteogenic response of non-diabetic controls). The diabetic osteogenic response is compared between groups, as well as compared to control rat MSCs. For CFU-F assays, non-adherent cells are washed off of primary cultures on day 3. Fresh basal medium is added and the cultures maintained for 5 additional days, or until colonies >2 mm in diameter are present. The cultures are fixed in ice-cold methanol and stained with Giemsa and the colonies are counted (Phinney et al., 1999). For CFU-F/ ALP+ assays, osteogenic medium is added to wash primary cultures on day 3. After 2 weeks in osteogenic media, the cultures are fixed and stained for alkaline phosphatase using commercially available reagents from Sigma (D'Ippolito et al., 1999) or Alzarin red staining for calcium. The proposed sample size (n=6 rats in each group) has 90% power to detect a 20% difference in the number of CFU-F (p<0.01).

[0162] Expected Results: Based on reports in humans, mice, and cats (Martin et al., 2002), it is anticipated that the frequency of MSC in control rat marrow will be ~1 per 0.5 million cells. Therefore, it is expected that -40 CFU-Fs will be found in a 60-ram dish. CFU-F/AlkP⁺ frequency may be reduced in diabetic rats. It is expected that there will be ample cells from both femurs in each rat to perform the assays with sufficient replicates. One may adjust the size of dishes used, or combine cells from femurs and tibiae (and use another bone for histology), if it is necessary to increase the initial cell number.

[0163] In the event that no differences are detected in either MSC response to Wnts or MSC differentiation into osteoblasts between the diabetic and the control rats, two explanations are possible: either the present methods do not detect the difference or osteoblastic differentiation is normal. To see how normal osteoblastic differentiation is, bone histology (Yates et al., 2002) and histomorphometry (Ascenzi M.-G. et al., 2004) are performed on the previously fixed rat humeri. The histological evaluation consists of a complete description of cartilage, bone and marrow; and use of histomorphometry to assess bone surface area and osteoblasts' number, surface area and shape.

[0164] Another objective of the present invention is to describe qualitative and quantitative differences in β-catenin trajectory of Wnt signaling in MSCs of diabetic and non-diabetic rat models. The experimentation methods include transiently transfecting MSCs with a GFP-β- catenin fusion protein (Kim et al., 2000) under confocal microscopy, observing the transiently transfected MSCs with confocal microscopy (see for instance Ascenzi M.-G. and Lomovtsev, 2005), and acquiring images.

[0165] Confocal Imaging: MSCs previously set aside are employed. Under normal conditions, β-catenin levels are low in rat MSC (Etheridge et al., 2004). Stimulation of MSCs with either Wnt3a or LiCl stabilizes β-catenin (for human cells: Boland et al., 2004; and Preliminary Work). Sodium chloride is used as a control that is not expected to stabilize β- catenin.

[0166] Determination of window for capture of confocal images: A series of preliminary experiments are performed with control rat MSCs to determine the optimal time period for image capture of β-catenin movement. A GFP-β-catenin expression construct is used (Kim et al., 2000). MSC is plated onto coverslips or glass slides. The cells are transfected with FuGENE lipofection reagent (Roche Molecular Biochemicals) on the following day. Image analysis is performed 48 h after addition of FuGENE/DNA complexes to the cultures. MSC transfected with the GFP-β-catenin expression construct is viewed by inverted fluorescence microscopy at time intervals. The cells are also viewed under the confocal microscope to determine the optimal sampling time for a low signal-to-noise ratio. Transfected MSC are stimulated with Wnt3a conditioned media or 5 raM lithium chloride. The cells are viewed under regular inverted and confocal microscopes to evaluate the accumulation of GFP-O- catenin in the cytosol and nucleus and determine when to collect images within a 20-minute time period. The estimated 20 min time period within a two hour window is estimated on the basis of published data and the experience of those of ordinary skill with mouse L cells and bovine chondrocytes (see above experiments and data). The optimal 20-rnin window for image acquisition is determined. The information gained from this experiment is used to determine the time frame for confocal image capture. [0167] Acquisition of confocal images of GFP-β-catenin movement: Images are acquired of transiently transfected MSC during a 20-min window in which β-catenin movements are maximal, as determined by the experiments described above. Confocal imaging is performed using real-time imaging of live cells (Gangalum et al., 2004; Ehrlich M. et al., 2004) and a Leica TCS-SP confocal microscope (Heidelberg, Germany). A 488/568/647 nm Kr/Ar multi-line laser and a IOOX objective with a Planapochromat water immersion lens is employed. The thickness of the plane of focus is estimated at 0.5 microns. Each cell is scanned every 0.5μm through its volume along so-called z-direction to avoid either missing or overlapping data. Each scan is expected to take <2 min to complete and to produce 10-18 images. The set of images of a scan is defined as a z-stack. Fluorescence detected by photomultipliers is converted by the microscope to pseudo-color for good visualization. Images are digitally memorized and processed using Adobe Photoshop 6.0.

[0168] Identification of z-stacks for data collection: Five z-stacks that are representative of β-catenin movements are selected for analysis. Data from the confocal images of five individual cells per each of the two groups obtained by stimulating separately either GK or Wistar MSC with Wnt3a are collected and analyzed.

[0169] Expected Results: Measurement differences in β-catenin localization are appreciated on the confocal images because the cell membrane are well differentiated from the nucleus with enough space between them (approximately 2.5 cm with the 10Ox objective). A 20-minute image acquisition time is anticipated on the basis on the above prelimiary data (as well as published reports) which show maximal accumulation of β-catenin in L cells between 1-3 h after stimulation.

[0170] If the MSCs transfect poorly, the electroporator Amaxa Nucleofector Device can be used as an alternative to put the vector into the cells. Because transiently transfected cells are used here, there may be a range of expression levels of GFP-β-catenin. Selection of cells for image acquisition includes an analysis of signal-to-noise in cells with different levels of brightness. Cells of similar size and shape are also selected for analysis to reduce potential artifacts. If needed, the cell population can be synchronized with a GQ/G_I block by serum deprivation, and the imaging can be performed within 2h after release, when β-catenin movement is maximal (Olmeda et al., 2003).

IMAGE DATA COLLECTION [0171] Confocal images are analyzed and geometric data (i.e., pathway and volume of fluorescence) is collected. The boundary of the cell membrane and nucleus is marked and the amount of fluorescence therein is measured. The following is repeated for each of the 5 imaged cells per each of the two groups obtained by stimulating separately either GK or Wistar MSC with Wnt3a.

[0172] Method Details: The images of each of the 5 selected z-stacks are examined for data collection. Both the XaraXl and the Metamorph imaging systems are calibrated at the magnification employed for confoeal microscopy. For each selected stack, each image is open with XaraX 1. One x,y reference system is chosen and fixed at the center of the cell nucleus of each image. For each cell analyzed, the adherence of the MSC renders the reference system consistent within each z-stack and from one z-stack to the next. To describe the geometry of the cell, tracing with XaraXl curves on the image along the cell membrane and the boundary of the nucleus and of the fluorescent regions is performed. Note that from this point on, the process is more complex than the one described above under Further Related Research. The β-catenin is expected to transfect through multiple paths. This is a very time consuming process. Metamorph selects points on each curve (Ascenzi A. et al., 1997) by means of the "Linescan" tool, which is set automatically to choose along the x,y coordinates every pixel on the curve. Detailed information is obtained about each curve because a pixel measures less than 0.25 microns with our calibration. Metamorph collects the data automatically on an Excel file. A z-coordinate will be added to the Excel file through a computation that involves the position of the image in the z-stack and the thickness of the plane of focus. Each Excel file is then be imported into a Microsoft Access database that allows for easy management of large data sets. The work conducted for the data collection of Preliminary Work indicates a couple of hours as the time necessary to complete the above task for each image. Data collection is performed at 10-18 images per z-stack, at 5 z-stacks per cell, for 5 cells. After data is collected from the images relative to the first cell, modeling preparation commences while the data is collected from the remaining 3 cells. The collected data is imported into Maple software for modeling preparation (Ascenzi M.-G. et al., 2004).

IMAGE DATA ANALYSIS

[0173] The trajectory of GFP/β-catenin is mathematically described by determining the movement of the fluorescent areas relative to each other within a reference system. The following are prepared: A. 3D virtual rendering of cell

[0174] An algorithm is written to automatically compute from the data points of all the images of any given stack with software Maple: (a) the equations of surfaces that approximate the cell membrane and (b) the goodness-to-fit of the surfaces to the selected points. The present inventor has developed the similar process for 2D curves (Ascenzi A. et al., 1997). Approximation is appropriate here because approximation conveys the idea of pattern or shape. Usually, approximation uses polynomials of relatively low degree for easy manageability (see for instance Piegl and Tiller, 1997). If the complexity of the curve precludes this, the curves are split into adjacent portions each of which is approximated by low degree polynomials called B-splines, which connect well at the split points (Piegl and Tiller, 1997). If the degree of the approximating polynomial can be kept lower than five, we use polynomials for the whole curve; otherwise we use B-splines in x, y and z. The degree is increased until the goodness-to-fit exceeds an r² of 0.98. It is anticipated that a degree between 4 and 7 suffices. The boundaries of the nucleus and of the fluorescent region are approximated by surfaces as described for the cell membrane.

B. 3D virtual rendering of fluorescence movement within cell

[0175] Algorithms are written to perform the following tasks: (a) the 3D modeling surfaces are plotted; (b) the volume of each surface equation is computed and the volumetric center (called ceatroid) of each fluorescent region is computed by integration; (c) the equation(s) of each fluorescent region path are computed by interpolation of the centers of the fluorescent regions by means of low degree polynomials that pass through the desired points, called splines (Piegl and Tiller, 1997) as described for the Preliminary Work. Note that in (c), interpolation is appropriate to preserve the experimental data point information. At this point, for each of the 5-stacks of each of the 5 imaged cells per group we have: a 3D geometric description through B-splines of relevant cell membrane, nucleus boundary and fluorescent regions and a 3D geometric description through splines of volumes and centers of relevant cell membrane, nucleus boundary, and fluorescent regions.

C. 4D virtual rendering of Wnt/β-catenin pathway in cell

[0176] An algorithm is written to organize the 3D modeling B-splines from the 5-stacks into a movie that in terms of time shows the 3D movement of fluorescent region's from z- stack to z-stack. This is the 4D rendering of Wnt/β-catenin pathway in each cell. Specifically, 3D modeling splines from the 5-stacks are plotted in terms of z-stack number (1 through 5), that is in terms of time, to describe the path of β-catenin through the estimated centers of fluorescent regions. The total fluorescent volume relative to the cell and to the nucleus volume is estimated from the modeling surface for each cell in terms of time. The results of all the calculation are then added to the original Access data file.

D. General 4D virtual rendering of Wnt/β-catenin pathway

[0177] The calculated information is then analyzed to evaluate patterns and ranges of variables in order to prepare a 4D rendering that encompasses the ranges of parameters derived from the individual cell models for diabetic and non diabetic groups separately. The analysis focuses on coefficients and degrees of B-splines and plane splines and of values of relative volumes and location of fluorescent regions among the analyzed data of the 5 imaged cells. To do so, it may be necessary to rotate the modeling volumetric images first with Maple (the relative equations would be accordingly transformed) because, even though cells of similar size are selected, their orientation to the confocal light source/point of image acquisition may not be exactly the same as that for tracking fluorescence movement in each cell relative to the cell itself. Then, the equations of a geometrical object of dimension greater than or equal to 4 (that mathematicians call "variety") are formulated by writing equations that contain not only the current x, y, z, and t (time) variables, but possibly additional variables that specify to the original equations for specific ranges of the original variables or specific values of the additional variables (for a lower dimensional example, see Ascenzi M.- G., 1986 and 1988). The dimension of the higher dimensional object equals 4+n where 4 is the number of the original variables and n is the number of the additional variables. Here, we compute the general model of minimum dimension. If the behavior of β-catenin is relatively consistent from cell to cell, the dimension of the general model is expected to be close to 5.

E. Comparison between diabetic and non-diabetic pathways

[0178] The patterns and parameters' values and ranges obtained for diabetic cells are confirmed to be distinct from the patterns and parameters' values obtained for the non- diabetic cell. Degrees, coefficients and ranges of both B-splines and regular splines are compared. We proceed carefully to see if values of parameters are distributed homogeneously and if ranges overlap. Any behavior that differentiates each cell from pairs of remaining cells is critically examined. We then compare the degrees and geometrical characteristics of the two 4D virtual renderings, one per each of the two cell groups.

[0179] Expected Results: The patterns and ranges of the 4D rendering of Wnt/β-catenin are expected to differ for MSC cells from diabetic and non-diabetic rats. Fluorescent regions are expected to be less numerous and/or smaller in the GK rat in comparison with the control group as diabetes is hypothesized to have a negative effect on Wnt. The results are discussed in relation to other Wnt-dependent events, associations with E-cadherin, and possible influence on nuclear function.

[0180] If the paths of β-catenin within a cell are numerous and a point detection approach is not advisable, machine learning techniques (Hastie et al., 2001) and computer vision technology that recognizes and models image patterns (2hu et al., 1997; Wu et al., 2000, 2002, 2004) are employed.

[0181] AU references cited and/or discussed in this specification are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference and incorporated particularly with respect to their subject matter discussed herein.

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[0182] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof -will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

Claim 1. A method for determining the capacity for bone repair or formation in a mammal, said method comprising characterizing the amount or intracellular localization of β-catenin in a multipotent mesenchymal cell from the mammal whereby said capacity is determined.

Claim 2. The method of claim 1 , wherein the multipotent mesenchymal cell is isolated from bone marrow.

Claim 3. The method of claim 1, wherein the multipotent mesenhcymal cell is isolated from blood.

Claim 4. The method of claim 1 , wherein the mammal is a rat, mouse or human.

Claim 5. The method of claim 1, wherein the total amount of β-catenin in the mesenchymal cell is determined.

Claim 6. The method of claim 1, wherein the physical distribution of β- catenin in the mesenchymal cell is determined.

Claim 7. The method of claim 1 , wherein the relative localization of β- catenin in the nucleus and the cytoplasm is compared.

Claim 8. The method of claim 1, wherein the relative localization is expressed as a ratio.

Claim 9. The method of claim 1, wherein the β-catenin is detected using a fluorescently labeled monoclonal antibody.

Claim 10. The method of claim 1, wherein the B-catenin is fused to a fluorescent protein and the fluorescence of the fluorescent protein is determined.

Claim 11. The method of claim 1, wherein the subject has osteoporosis or altered bone mineral density.

Claim 12. The method of claim 1, wherein the subject has Type 2 diabetes.

Claim 13. The method of claim 1, wherein the subject is insulin resistant.

Claim 14. The method of claim 1, wherein the B-catenin is detected and its intracellular localization tracked using scanning microscopy or multiphoton microscopy.

Claim 15. The method of claim 14, wherein the microscopy is a confocal microscopy.

Claim 16. The method of claim 1, wherein the location of intracellular β- catenin molecules are tracked over time in a single cell using scanning microscopy or multiphoton microscopy.

Claim 17. The method of claim 16, wherein the microscopy is confocal microscopy.

Claim 18. The method of any one of claims 1 to 17, wherein the amount of β-catenin or nuclear localization of β-catenin is lower than that for a comparison population of the mammal and the mammal is thereby identified or diagnosed as having reduced ability to repair or form bone tissue with respect to the comparison population.

Claim 19. A method of identifying a modulator of bone repair or formation, said method comprising the steps of: performing a first determination of the amount or intracellular distribution of β-catenin in a mammalian multipotent mesenchymal cell, osteoclast, or osteoblast; contacting the cell with the agent and performing a second determination of the amount or the intracellular distribution of β-catenin in the cell; and comparing the amount or intracellular distribution of β-catenin determined in the first determination to that of the second determination, wherein changes in the amount or nuclear localization of β-catenin identifies the agent to be a modulator of bone repair or formation.

Claim 20. A method of claim 19, wherein contacting the agent increases the total amount or nuclear localization of β-catenin and the agent is identified as being useful in promoting bone repair, formation, osteroporosis, or pathological alterations of bone density and structure.

Claim 21. A method of claim 19, wherein the first determination and second determination monitor the amount or intracellular localization of β-catenin over time and the amount and movement of the β-catenin is compared over time.

Claim 22. A method of claim 19, wherein both the amount and intracellular localization is determined.

Claim 23. A method of claim 19, wherein the modulator is a GSK3 inhibitor or a WNT modulator.

Claim 24. A method of claim 19, wherein the mammalian multipotent mesenchymal cell is a human multipotent mesenchymal cell.

Claim 25. The method of claim 19, wherein the β-catenin is fused to a fluorescent protein and the fluorescence of the fusion protein is monitored to determine the amount or localization of B-catenin in the cell.

Claim 26. A method of claim 19, wherein the multipotent mesenchymal cell is obtained from a subject having Type 2 diabetes, insulin resistance, osteoporosis, a bone fracture, a bone healing disorder, a bone pathology, or a metabolic disorder that affects bone tissue repair or formation.

Claim 27. A method of characterizing mechanisms promoting bone repair or formation in a cell selected from the group consisting of a multipotent mesenchymal cell, an osteoblast, or an osteoclast, said method comprising tracking the amount, location or movement of fluorescently labeled β-catenin in the cell using scanning microscopy or multiphoton microscopy.

Claim 28. The method of claim 27, wherein the microscopy is confocal microscopy.

Claim 29. The method of claim 27, wherein the β-catenin is fused to a fluorescent protein.

Claim 30. Use of the GK rat as an animal model for studying local effects of Type 2 diabetes on bone tissue.

Claim 31. A method of characterizing the pathogenesis of local tissue in bone from a mammal, said method comprising a histomorphometry of the local tissue in bone and an evaluation of the response to Wnt or β-catenin amounts and intracellular localization of MSC from the local tissue.

Claim 32. The method of claim 31 , wherein the mammal is rat, mouse, or human.

Claim 33. The method of claim 31 , wherein the bone is from the tibia.

Claim 34. The method of claim 31 , wherein the mammal has Type 2 diabetes.

Claim 35. The method of claim 31, wherein histomorphometry is performed on bone microstructures from a region and bone marrow from the region.

Claim 36. The method of claim 31, wherein the histomorphometry comprises a histological examination of cartilage, bone and marrow, bone surface area, and osteoblast number, density, surface are and shape.

Claim 37. A method of characterizing the MSC response to Wnt in a subject having a bone pathology , said method comprising: obtaining a first sample of MSC from a first mammal having a bone pathology and culturing the first sample of MSC in the presence of Wnt to obtain a first culture and staining the first culture with an osteogenic stain to determine the number of osteoblasts therein , wherein the number of osteoblasts in the first culture provides a first measure of the differentiation of MSC into osteoblasts; obtaining a second sample of MSC from a second mammal which is different from the first mammal in not having the bone pathology and culturing the second sample of MSC in the presence of Wnt to obtain a second culture and staining the second culture with an osteogenic stain to determine the number of osteoblasts therein , wherein the number of osteoblasts in the second culture provides a second measure of the differentiation of MSC into osteoblasts; comparing the first and second measure to characterize the response of bone to Wnt signaling in the bone pathology.

Claim 38. The method of claim 37, wherein the first subject has Type 2 diabetes and the second subject does not.

Claim 39. The method of claim 37, wherein the Wnt is Wnt 3a, Wntl 1 , or Wnt 5.

Claim 40. The method of claim 37, wherein the stain is alizarin red or an alkaline phosphatase assay.

Claim 41. A method of treating a pathology of bone, said method . comprising administering a modulator as identified in Claim 19