WO2008022182A1 - Methods for promoting coupling between bone formation and resorption - Google Patents

Abstract

Methods and compositions for promoting coupling between bone formation and bone resorption are provided. The method related to administering a therapeutically effective amount of a rho inhibitor, a Transforming Growth Factor ß receptor I (TßRI) inhibitor or a combination thereof to a subject. The methods and compositions are useful, for example, in a subject with a disorder linked to an imbalance between bone formation and bone resorption.

Description

METHODS FOR PROMOTING COUPLING BETWEEN BONE FORMATION

AND RESORPTION

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Serial No. 60/822,601, filed August 16, 2006, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The invention was supported by the National Institutes of Health, Grant No. DK057501. The government of the United States may have certain rights in this invention.

BACKGROUND

The skeleton is continuously being formed and resorbed through a process that starts in the embryo and continues throughout adult life. During development, the skeleton is continuously growing and bone formation exceeds bone resorption in a process known as bone modeling, in which bone formation occurs without previous bone resorption. In the adult, however, bone density has peaked and is maintained by balanced bone resorption and formation in a process known as bone remodeling. The balance between bone resorption and formation is accomplished by precise coordination between two cell types: osteoblasts, which deposit the calcified bone matrix; and osteoclasts, which resorb bone. The sequence of cycles in remodeling does not vary. Activation of osteoclast precursors and osteoclastic bone resorption always precedes the osteoblastic bone formation that repairs the defects. In remodeling, bone resorption is required for activation of bone formation, i.e., in bone remodeling, bone resorption and bone formation are coupled.

Many bone diseases are associated with defective coupling of bone resorption and formation. In diseases such as primary hyperparathyroidism, hyperthyroidism and Paget's disease, there is excessive bone formation after each cycle of osteoclastic bone formation. There also are a number of other diseases, including multiple myeloma, in which the osteoblast activity does not completely repair and replace the defect left by resorption. Camurati-Engelmann disease (CED) is also associated with a coupling disorder. CED is a form of hyperosteosis, characterized by a fusiform thickening of the diaphyseal and, occasionally, the metaphyseal cortex of the long bones. This leads to a narrowing of the medullary canal and sclerosis at the skull base and, in severe situations, the skeleton appears osteopenic.

A number of treatments have been developed and made available to patients suffering from skeletal diseases. These therapeutic approaches primarily are directed to increasing net bone formation and include hormone replacement therapy (HRT), selective estrogen receptor modulators (SERMs), bisphosphonates, and calcitonin. Methods designed to enhance bone coupling, however, are lacking in the art.

SUMMARY Methods of promoting coupling between bone formation and bone resorption are described. The method can comprise administering a therapeutically effective amount of a Transforming Growth Factor β receptor I (TβRI) inhibitor to a subject in need of such treatment. Thus, the method can comprise the step of selecting a subject in need of promotion of coupling between bone formation and bone resorption. Alternatively, the method comprises administering a therapeutically effective amount of a rho inhibitor to a subject in need of such treatment. The provided methods can also comprise administering combinations of TβRI and rho inhibitors to the subject. The subject with a disorder linked to an imbalance between bone formation and bone resorption benefits from the treatment. The method can result in a increase in bone formation or an increase in bone resorption in order to promote coupling and restore the balance between bone formation and bone resorption as desired. The method optimally comprises promoting coupling in cartilaginous bone.

Also described are compositions comprising rho inhibitors, TβRI inhibitors or a combination thereof. The compositions can be used to promote coupling between bone formation and bone resorption.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS Figure 1 shows osteoclastic bone resorption-conditioned medium (CM) induces mesenchymal stem cell (MSC) migration. Figure IA is a scanning electron microscopy of osteoclast formation from bone marrow precursors. Mouse bone marrow macrophages (BMMs) were cultured with (b) or without (a) M-CSF (22 ng/ml) plus RANKL (100 ng/ml). Multinucleated osteoclasts were shown by TRAP staining. BMMs were plated on dentin slices, and the cultures were treated with (d) or without (c) M-CSF plus RANKL. After osteoclasts formed, the cultures were continued for 5 more days to allow the cells to resorb bone. The dentin slices were harvested and subjected to scanning electron microscopy analysis. Figure IB is a schematic showing a type I collagen-coated Transwell assay. BMMs were isolated as described in Figure IA. Five different types of conditioned media (a'-e') were prepared and added individually to the lower chambers of the type I collagen-coated Transwell system. The upper chambers of the transwell were filled with MSC in serum-free DMEM. Cells that invaded the collagen gel and attached to the lower chamber of the Transwell were fixed and stained with hematoxylin. Figure 1C is a graph showing migrated cell numbers following the type I collagen-coated Transwell

4 assay. 4X10 MSCs in a serum- free DMEM medium were used to fill the upper chamber of the Transwell and the lower chamber was filled with each individual conditioned media (a'-e') as described in Figure IB. After incubation for 23 hours, cells that migrated through type I collagen gel and attached to the lower chamber of the Transwell were fixed and stained with hematoxylin. The filters were photographed (a"-e", left panel) and the cells counted (a"-e", right panel).

Figure 2 shows that TGF-βl is released during bone resorption and is required

4 for osteoprogenitor migration. 4X10 MSCs in a serum-free DMEM medium were used to fill the upper chamber of the Transwell and the lower chamber was filled with osteoclastic bone resorption conditioned medium (bone resorption CM) as described in Figure 1 in the presence or absence of neutralizing antibodies specific for TGF-βl, TGF-β2, TGF-β3, IGF-I, IGF-II or PDGF or noggin. After incubation for 23 hours, cells that migrated to the lower chamber of the Transwell were fixed, stained with hematoxylin and cells were counted. The neutralizing antibody specific for TGFβ 1 inhibited migration of MSCs induced by osteoclastic bone resorption CM. Figure 3 A shows an immunoblot of different types of conditioned medium probed as indicated with antibodies specific for the latent (LAP) form of TGF-βl or mature TGF-βl . Figure 3B is a graph showing the measurement of active TGFβl released in osteoclastic bone resorption CM by ELISA.

Figure 4 shows an immunoblot (lower panel) and a graph (upper panel) showing migrated cell counts following selected treatments. TGF-βl was depleted in the osteoclastic bone resorption-CM by repeated immunoprecipitation (IP) with the anti-TGF-βl antibody, and different amounts of recombinant human TGF-βl (rhTGF- βl) were then added into the depleted conditioned medium. The medium was also added to the lower chamber of the Transwell assays. After incubation for 23 hours, cells that migrated to the lower chamber of the Transwell were fixed and stained with hematoxylin. The cells were counted (upper panel). The media were immunobloted for mature TGF-βl or BSA (bottom panels).

Figure 5 A is a graph showing a low level of active TGFβl in bone resorption CM from TGFβl null mice (KO). Figure 5B is a graph showing that induction of MSC migration is reduced in bone resorption CM from TGFβl null mice (KO). Bone resoprtion CM was prepared using bone slices obtained from TGFβl knockout mice (Engle et al., Cancer Research 62:6362-6 (2002)) and assayed for migration of MSCs and active TGFβl .

Figures 6A and 6B are graphs showing Rho-Rock inhibitor stimulated TGFβl -induced MSC migration. MSCs in a serum-free DMEM medium in the presence or absence of Y-27632 were used to fill the upper chamber of the Transwell and the lower chamber was filled with osteoclastic bone resorption-conditioned medium (Figure 6A) or DMEM medium with or without rhTGF-βl (Figure 6B). After incubation for 23 hours, cells that migrated to the lower chamber were fixed, stained with hematoxylin and counted. Figure 7 is a graph of migrated cell numbers in the lower chamber of the

Transwell assay following over-expression of a dominant negative RhoA (Nl 9) or constitutive expression of RhoA (L63) in MSCs. Over-expression of a dominant negative mutant of RhoA enhanced migration whereas constitutive expression of RhoA inhibited migration of MSCs. Figures 8A, 8B, 8C and 8D show that TβRI inhibitor blocks MSC migration.

MSCs in a serum-free DMEM medium in the presence or absence of SB-431542 was used to fill the upper chamber of the Transwell and the lower chamber was filled with osteoclastic bone resorption-CM (Figure 8A) or DMEM medium with or without rhTGF-βl (Figure 8B). For Figure 8C, 0.1, 1, 5 or 10 μM of SB-431542 was added. After incubation for 23 hours, cells migrated to the lower chamber were fixed, stained with hematoxylin and counted. Figure 8C shows that inhibition of migration of MSCs induced by osteoclastic bone resorption CM is concentration dependent. Figure 8D shows that SB-431542 lost its inhibitory effect on migration of MSCs induced by osteoclastic bone resorption CM from bone slices of TGFβl null mice (KO).

Figure 9 shows TGF-β induces RhoA activation via inducing TβRII/GDIα binding. Figure 9 is a graph showing the interaction of TβRII with GDIα in yeast. The intracellular domain of TβRII cDNA was fused with the GAL4 DNA binding domain and transformed in yeast with GDIα cDNA or TβRII cDNA (positive control) in prey plasmid. The interactions were quantified by a liquid β-gal assay. The empty plasmids also were transformed in yeast as a negative control.

Figure 1OA is a schematic mapping of the TβRII interaction domains of GDIα. A series of truncated GDIα fragments were fused with the GAL4 activation domain and transformed in yeast with Intracellular domain of TβRII cDNA fused with the GAL4 DNA binding domain plasmids. The interactions were detected by β-gal assays. Figure 1OB is a schematic mapping of GDIα interaction domains of TβRII. A series of truncated TβRII fragments were fused with the Gal4 activation domain and transformed in yeast with intracellular domain of GDIα cDNA fused with the GAL4 DNA binding domain plasmids. The interactions were detected by β-gal assays.

Figure 11 shows GDIα is a critical regulator coordinating the TGF-β-induced RhoA activation pathway and the Smad signaling pathway. Figure 1 IA and 1 IB are graphs showing GDIα inhibits TGF-β signaling. SBE-luc (Figure 1 IA) and 3TP-luc (Figure 1 IB) luciferase reporters were co-transfected with GDIα plasmid in MvILu cells with or without TGF-β (2 ng/ml). The cells were lysed and the luciferase activity was measured. Luciferase values are representative of triplicate in at least three independent experiments.

Figure 12 is a graph showing that deletion of TGFβl Receptor II (TβRII) in primary mouse MSCs inhibits their migration induced by bone resorption CM.

TβRII^lox/lox mice were infected with adenovirus bearing either Cre or GFP to allow removal of the endogenous TβRII gene. MSCs were then isolated from the mice and subjected to migration assay.

Figure 13 shows TβRI inhibitor reduces bone formation in rats. Inhibitors or vehicle were injected into the proximal tibial metaphysis. Figure 13A shows X-ray and μCT images of tibia from no injection control, vehicle injection, and TβRI inhibitor injections. Cross and longitude sections were strained using Masson's trichrome and Von Kossa protocols. Figure 13B is a graph showing that the TβRI inhibitor reduces bone formation in a concentration dependent manner.

Figure 14 shows a RhoA inhibitor stimulates bone formation in rats. Inhibitors or vehicle were injected into the proximal tibial metaphysis. Figure 14A are X-ray and μCT images of tibia from no injection control, vehicle injection, and RhoA inhibitor injection. Cross and longitude sections were strained using Masson's trichrome and Von Kossa protocols. Figure 14B is a graph showing that the RhoA inhibitor stimulates bone formation in a concentration dependent manner. Figure 15 shows RhoA and TβRI inhibitors regulate bone formation by altering remodeling in the persisting surfaces. Figures 15A and 15B are graphs showing changes in bone formation by injection of RhoA or TβRI inhibitors. Rat proximal tibial metaphysic were injected with RhoA or TβRI inhibitors with 3 consecutive intraperitoneal administration of 3 consecutive different fluorochrome labels as described in Figure 6. The areas containing the first two labels (arrested area) and all three labels (persisting area) are active in bone remodeling. Areas without first label (induction area) represent modeling or bone growth only. Each of the three areas (arrested, induction and persisting) were measured and calculated with normalization of osteoclast and osteoblast surfaces. The results indicate that the changes of bone formation by injection of RhoA or TβRI inhibitors were primarily due to alteration of persisting surfaces, i.e., coupled bone remodeling area.

Figure 16 shows a graph indicating CED-derived TGF-βl mutations of the

4

LAP region cause premature release of active TGF-βl . 4X10 MSCs in a serum-free DMEM medium was used to fill the upper chamber of the Transwell and the lower chamber was filled with the conditioned medium collected from cells transfected with wild-type or CED-derived TGF-βl mutation constructs prepared as described in the Examples below. After incubation for 23 hours, cells that migrated to the lower chamber were fixed, stained with hematoxylin, photographed and counted.

Figures 17A and 17B show the effect of different doses of TβRI inhibitor on trabecular bone formation in vivo. Figure 17A are μCT images of cross section of rat tibia injected with different doses of TβRI inhibitor. Figure 17B is a graph showing bone formation in rat tibia injected with different doses of TβRI inhibitor. Low doses of TβRI inhibitor stimulated bone formation while higher dosέs of TβRI inhibitor reduced trabecular bone formation.

Figures 18A and 18B show uncoupled bone remodeling in TGFβl^"/"Rag2^"/" mice. Figure 18A shows normal trabecular bone volume, thickness and space in one- month old TGFβl^+/' and TGFβl^"'^' mice. Figure 18B shows reduced trabecular bone volume, thickness and increased trabecular bone space in three-month old TGFβl^+/" and TGFβl^"7' mice.

Figure 19 shows TβRI inhibitor blocks recruitment of BrdU labeled MSCs to osteoclastic bone resorption sites.

Figure 20 shows TβRI inhibitor restores coupling of bone resorption and formation in CED transgenic mice.

DETAILED DESCRIPTION

Before birth every skeleton bone appears as a fibrous membrane template (membranous bone) or a cartilaginous template (cartilaginous bone). These templates form the basic shapes that mature bone replaces. Membranous bone, also known as connective tissue bone, includes the brain vault, facial skeleton and parts of the clavicle and mandible. Axial bones, such as the vertebral column and rib bones, and the appendicular bones for the arms and legs are cartilaginous bones. In cartilaginous bone, osteoclasts resorb the cartilage while the osteoblasts lay down new bone.

Many osteotropic factors are found in the bone matrix. Among them, transforming growth factor-beta (TGF-β), platelet derived growth factor (PDGF), insulin-like growth factor I (IGFI) and insulin-like growth factor II (IGFII) are osteoblastotropic and, as they induce cell migration in vitro. The defect in the coupling mechanism underlying CED has not been elucidated; however, mutation analysis of TGF-β 1 in samples from nine CED families has revealed five different mutations. A recent study has reported that, of 100 patients in 24 CED families, all possess TGF-βl mutations and that, with one exception, all mutations identified are located in TGF-βl. TGF-βl is the factor that is required for directed osteoprogenitor migration during bone remodeling. The TGF-β family contains three closely related mammalian isoforms, TGF-βl, -β2 and -β3. Of the three isoforms, TGF-βl is the most abundant with the largest source of TGF-βl being bone (200μg/kg). It is synthesized as a large precursor molecule, which is then cleaved into mature TGF-βl and latency associated protein (LAP). The LAP remains non-covalently linked to the mature TGF-βl, rendering it inactive due to masking of the receptor-binding domains of the mature TGF-βl by the associated LAP. TGF-βl is thus secreted and deposited in the bone matrix as an inactive, latent complex.

TGF-βl is synthesized as three distinct components: the signal peptide, the latency-associated peptide (LAP), and the sequence representing the mature TGF-βl . Mature TGF-βl is released from the LAP during osteoclastic bone resorption. Smads are critical mediators of the TGF-β signaling pathway. In additional, Smad- independent pathways, TGF-β-activates mitogen-activated protein (MAP) kinases. The epithelial-mesenchymal transition is Smad-independent and utilizes the RhoA and phosphatidylinositol (PI) 3-kinase pathways. TGF-β induces interaction of TβRII with GDIα resulting in activation of RhoA (see Figures 3 and 4). TGF-βl stimulates Rho GTPase activity by inducing the interaction of transforming growth factor-beta receptor II (TβRII) with guanine nucleotide dissociation inhibitor-alpha (GDIα) thus promoting cell migration, which results in coupling of osteoblast precursor activity and osteoclastic bone resorption.

TGF-βl regulates a broad range of biologic processes, including cell proliferation, cell survival, cell differentiation, cell migration and production of the extracellular matrix (ECM). The combined actions of these cellular responses mediate the global effects of TGF-βl on immune responses, angiogenesis, wound healing, development, and bone formation. In the bone microenvironment, TGF-βl can both stimulate proliferation of osteoprogenitors and inhibit osteoblast differentiation; however, TGF-βl induces bone formation in vivo. TGF-βl also regulates osteoclastogenesis either directly or indirectly via osteoprogenitors. Brief exposure to low concentrations of TGF-βl stimulates migration of macrophages/monocytes, which are osteoclast precursors, but this migration is inhibited on extended exposure to, or at high concentrations of TGF-βl (see Figures 9A and 9B). In injury or inflammation, TGF-βl recruits macrophages/monocytes to the involved site by inducing their migration, whereas during bone resorption, a gradient of TGF-βl generated at the resorptive site inhibits migration of the macrophage/monocyte osteoclast precursors. Thus, TGF-βl has a role in osteoclastogenesis in the bone remodeling process.

TGF-βl has a stimulatory effect on bone formation through promotion of recruitment and proliferation of osteoblasts and enhancement of matrix deposition. During fracture healing, the initial recruitment of osteoblasts to the site of injury is followed by osteoblast proliferation and differentiation, secretion of bone matrix proteins and, finally, the organization of a higher ordered structure. Bone regulating factors including TGF-βl act as chemotactic factors. Directional cell migration, mediated by a variety of chemotactic factors, is a fundamental process in all organisms that is stringently regulated not only during tissue development but also throughout life. The process of cell migration is also evident during wound healing and bone remodeling. Migrating cells have a polarized morphology with an asymmetrical distribution of signaling molecules within the cyto skeleton. Microtubules are indispensable for the directional migration of certain cells. Recent studies have shown that Rho family GTPases regulate cell migration via interaction with microtubules and actin in the cytoskeleton. Rho family GTPases capture and stabilize microtubules through their effectors at the cell cortex, leading to a polarized microtubule array which in turn modulates the activities of Rho family GTPases. The resultant polarized microtubule array facilitates cell adhesion and migration. The Rho GTPase cycle is tightly regulated by three groups of proteins: (1) guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP to activate the GTPase; (2) GTPase-activating proteins (GAPs) negatively regulate the switch by enhancing its intrinsic GTPase activity; and (3) guanine nucleotide dissociation inhibitors (GDIs) are thought to block the GTPase cycle by sequestering and solubilizing the GDP -bound form. GDI preferentially interacts with the GDP- bound form and prevents it from being converted to the GTP-bound from by the action of each GEP and subsequent translocation to its target membrane. The function of Rho GDIα has been investigated in different types of cells. It causes disappearance of stress fibers and inhibition of cell motility. TGF-βl induces interaction of the TβRII cytoplasmic domain with GDIα. The TGF-βl -induced interaction of TβRII with GDIα frees GTPases from their GDIα-bound forms for further activation by other factors. Cell migration is a composite process involving RhoA, Rac and Cdc42, the three types of Rho GTPases.

A migrating cell performs a coordinated series of events to move. Activation of RhoA induces formation of focal adhesions and maturation of focal adhesion behind leading edge. Focal adhesions induced by Rho A produce necessary friction for cell migration. The tightness of focal adhesions also regulates the rate of cell migration. In addition, RhoA is thought to be involved in retraction at the rear of the cell. Cdc42 is involved in generating cell polarity. At the front of the cell, Racl and Cdc42 regulate the formation of ruffles and fϊlopodia, respectively, in concert with their effectors to promote cell migration. Protrusions are stabilized by the formation of adhesions.

An in vitro model has been developed in which the bone cells are cultured in conditioned medium. For in vivo studies, CED, as described above, serves as an ideal model for analysis of the mechanisms of TGF-βl function in bone.

During bone remodeling and healing, migration of osteoprogenitors to the fracture or resorption sites is the initial step for new bone formation. TGF-βl functions as a coupling factor for bone resorption and formation. TGF-βl is one of the most important factors in the bone environment, helping to retain the balance between the dynamic processes of bone resorption and bone formation.

Provided are methods of promoting coupling between bone formation and bone resorption in a subject comprising administering a therapeutically effective amount of a TβRI inhibitor to the subject. As described in the Examples below, TβRI inhibitors couples bone resorption and formation in vitro and in vivo. Also provided are methods of promoting coupling between bone formation and bone resorption in a subject comprising administering a therapeutically effective amount of a rho inhibitor to the subject. Optionally, a combination of TβRI and rho inhibitors can be administered to the subject. The method optimally comprises promoting coupling in cartilaginous bone. The subject in need of treatment has, for example, a disorder linked to an imbalance between bone formation and bone resorption. Thus, the subject can have a bone disease or disorder selected from the group consisting of Camurati-Engelmann disease (CED), osteoporosis, frailty, childhood idiopathic bone loss, alveolar bone loss, a bone defect, osteotomy, Paget's disease, osteoporotic fracture, osteogenesis imperfecta, spine injury, periodontal disease, osteopenia, bone fracture, osteolysis due to a prosthesis and bone necrosis. The subject can also have a bone defect selected from the group consisting of traumatic acetabular fracture, fϊbrodysplasia, spinal hyperostosis, myelopathy or spondylitis ankylosans and hip replacement. The subject can have a disorder characterized by heterotrophic ossification or undesired bone formation. Thus, the subject can have a disease selected from the group consisting of fibrodysplasia, acquired bone forming lesions such as spinal hyperostosis, myelopathy and spondylitis ankylosans.

The provided methods can further comprise the step of selecting a subject in need of promotion of coupling between bone formation and bone resorption. Thus, the provided methods can further comprise the step of selecting a subject with a disorder linked to an imbalance between bone formation or bone resorption. Thus, the provided methods can further comprise selecting a subject with one of the bone diseases or disorders recited herein. For example, the provided methods can further comprise the step of selecting a subject with Camurati-Engelmann disease (CED). The methods described herein can promote bone formation following bone surgery, wherein the bone surgery is selected from the group consisting of facial reconstruction, maxillary or mandibular reconstruction, fracture repair, bone graft, prosthesis implant, hip replacement and knee replacement. Bone disease, as used herein, refers to any bone disease or state which results in or is characterized by loss of health or integrity to bone and includes, but is not limited to, osteoporosis, osteopenia, faulty bone formation or resorption, Paget's disease, fractures and broken bones, bone metastasis, osteopetrosis, osteosclerosis and osteochondrosis. More particularly, bone diseases, which can be treated and/or prevented in accordance with methods described herein, include bone diseases characterized by a decreased bone mass relative to that of corresponding non-diseased bone (e.g., osteoporosis, osteopenia and Paget's disease), and bone diseases characterized by an increased bone mass relative to that of corresponding non- diseased bone (e.g., osteopetrosis, osteosclerosis and osteochondrosis). Treatment of bone disease or a symptom related to bone disease encompasses actively intervening after onset to slow down, ameliorate symptoms of, or reverse the disease or symptoms. More specifically, treating, as used herein, refers to a method that modulates bone mass to more closely resemble that of corresponding non-affected bone (that is a corresponding bone of the same type, e.g., long and vertebral) in a non- diseased or non-affected state. By way of example, following treatment post surgery, the bone mass would resemble healthy, non-surgically affected bone. The term therapeutically effective amount, as used herein, means that amount of the Rho A inhibitor, the TβRI inhibitor or combinations thereof that will elicit the desired effect or response or provide the desired benefit when administered in accordance with the desired treatment regimen. A preferred therapeutically effective amount relating to the promotion of coupling bone resorption and bone formation is the amount that restores the balance between bone resorption and bone formation.

As used herein the term coupling refers to the natural process involving bone formation and bone resoprtion.

As used herein the phrase promoting or to promote coupling of bone formation and bone resorption refers to promoting the balance between bone formation and bone resorption (i.e., show more characteristics of control bone). In other words, it is desirable to promote coupling between bone resorption and bone formation, if an abnormal amount of either bone resorption or bone formation is occurring. Thus the term can refer to stimulating bone formation to restore the balance between bone formation and bone resorption. The term can also refer to stimulating bone resorption to restore the balance between bone formation and bone resorption. An imbalance between bone formation and bone resorption occurs when there is either too much bone resorption or too much bone formation as compared to control bone (i.e., healthy, normal bone or bone without trauma or surgical intervention).

The provided methods comprise administering an agent that reduces or inhibits expression or activity of rhoA or TβRI. Reduction or inhibition of rhoA or

TβRI can comprise inhibiting or reducing expression of mRNA or protein, such as by administering antisense molecules, triple helix molecules, ribozymes and/or siRNA. Gene expression can also be reduced by inactivating the rhoA or TβRI gene or its promoter. The nucleic acids, ribozymes, siRNAs and triple helix molecules for use in the provided methods may be prepared by any method known in the art for synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramide chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the nucleic acid molecule. Such DNA sequences may be incorporated into a wide variety of vectors, which incorporate suitable RNA polymerase promoters. Antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

In addition, reduction or inhibition of rhoA or TβRI includes inhibiting the activity of the protein. Agents that inhibit activity are referred to herein as antagonists. Rho or TβRI antagonists include antibodies, soluble domains of rhoA or TβRI and polypeptides that interact with rhoA or TβRI to prevent protein activity. The nucleic acid and amino acid sequences of rhoA and TβRI are known in the art. For example, nucleic acid and amino acid sequences for TβRI can be found at GenBank Accession Nos. NP_004603.1 and NM_004612.2, respectively. The nucleic acid and amino acid sequences for rhoA can be found at GenBank Accession Nos. NP_001655.1 and NM_001664.2, respectively. Therefore, variants and fragments of rhoA or TβRI that act as antagonists can be prepared by any method known to those of skill in the art using routine molecular biology techniques. Numerous agents for modulating expression/activity of intracellular proteins in a cell are known. Any of these suitable for the particular system being used may be employed. Typical agents for inhibiting or reducing activity of proteins include mutant/variant polypeptides or fragments and small organic or inorganic molecules.

As used herein, rho inhibitors include compounds that inhibit the Rho A/Rho Kinase (ROK) pathway. Suitable rho inhibitors that can be used in the methods described herein include, but are not limited to, Y-27132, C3 transferase, HA- 1077, Y- 27632, Pasteurella multocida toxin (PMT), Wf-536, compounds disclosed in US

Publication No. 2005/0014783 to Dole, et al., isoquinoline compounds, (R)-trans-N- (pyridin-4-yl)-4-( 1 -aminoethyl)cyclohexanecarboxamide and (R)-(+)-N-( 1 H- pyrrolo[2,3-b]pyridin-4-yl)-4-(l-aminoethyl)-benzamide disclosed in WO 98/06433 and WO 00/09162, l-(5-isoquinolinesulfonyl)homopiperazine and l-(5- isoquinolinesulfonyl)-2-methylpiperazine disclosed in WO 97/23222, (1- benzylpyrrolidin-3-yl)-(lH-indazol-5-yl)amine disclosed in WO 01/56988, (1- benzylpiperidin-4-yl)-(lH-indazol-5-yl)amine disclosed in WO 02/100833, N-[2-(4- fluorophenyl)-6,7-dimethoxy-4-quinazolinyl] -N-( 1 H-indazol-5-yl)amine disclosed in WO 02/076976, N-4-(lH-indazol-5-yl)-6,7-dimethoxy-N-2-pyridin-4-yl-quinazolin- 2,4-diamine disclosed in WO 02/076977 and 4-methyl-5-(2-methyl-[l,4]diazepan-l- sulfonyl)isoquinoline disclosed in WO 99/64011. Suitable rho inhibitors also include, but are not limited to, Fasudil (hexahydro- l-(5 isoquinolinesulfonyl)-lH-l,4-diazepine), which can be obtained from commercial sources (e.g. from Asahi Kasei Corporation of Tokyo, Japan) or it can be synthesized according to conventional methods (U.S. Pat. No. 4,678,783). Other suitable inhibitors include derivatives and metabolites of fasudil such as, for example, hydroxyfasudil, the major active metabolite of fasudil.

Compounds can be assayed for activity as a rho-kinase inhibitor using a kinase activity assay such as that described by Amano et al. (1999) J. Biol. Chem. 274:32418-32424. Compounds are generally considered to be effective inhibitors if they have an IC₅₀ of 10 μM, 5 μM, 1 μM or any IC₅₀ less 10 μM, 5μM or lμM. Suitable TβRI inhibitors for use in the methods described herein include, but are not limited to, SB-431542, SB-505124, A-83-01, [3-(pyridine-2yl)-4-(4- quinonyl)]-lH pyrazole, 2-pyridinyl-[l,2,3]triazoles as described in Kim et al., Bioorg. Med. Chem. 12(9):2013-2020 (2004) and Kim et al., Bioorg. Med. Chem. Lett. 14(10): 2401-2405 (2004), SD208 and aryl-and heteroaryl-substituted pyrazole inhibitors described in Sawyer et al., J. Med. Chem. 46(19):3953-3956.

Inhibitors of rho A or TβRI include inhibitory peptides or polypeptides, As used herein, the term peptide, polypeptide, protein or peptide portion is used broadly herein to mean two or more amino acids linked by a peptide bond. Protein, peptide and polypeptide are also used herein interchangeably to refer to amino acid sequences. The term fragment is used herein to refer to a portion of a full-length polypeptide or protein. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more. Inhibitory peptides include dominant negative mutants of a rhoA or TβRI. Dominant negative mutations (also called antimorphic mutations) have an altered phenotype that acts antagonistically to the wild-type or normal protein. Thus, dominant negative mutants of a protein act to inhibit the normal protein. Such mutants can be generated, for example, by site directed mutagenesis or random mutagenesis. Proteins with a dominant negative phenotype can be screened for using methods known to those of skill in the art, for example, by phage display.

Nucleic acids that encode the aforementioned peptide sequences are also disclosed. These sequences include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. A wide variety of expression systems may be used to produce peptides as well as fragments, isoforms, and variants. Such peptides or proteins are selected based on their ability to reduce or inhibit expression or activity of rhoA or TβRI. Also provided herein are functional nucleic acids that inhibit expression of rhoA or TβRI. Such functional nucleic acids include but are not limited to antisense molecules, aptamers, ribozymes, triplex forming molecules, RNA interference (RNAi), and external guide sequences. Thus, for example, a small interfering RNA (siRNA) could be used to reduce or eliminate expression of rhoA or TβRI. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target nucleic acid molecule is designed to promote the destruction of the target nucleic acid molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target nucleic acid molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target nucleic acid molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target nucleic acid molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC.

Aptamers are molecules that interact with a target nucleic acid molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in, for example, U.S. Patent Nos. 5,476,766 and 6,051,698. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, hairpin ribozymes and tetrahymena ribozymes). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to U.S. Patent Nos. 5,807,718, and 5,910,408). Ribozymes may cleave RNA or DNA substrates. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in U.S. Patent Nos. 5,837,855; 5,877,022; 5,972,704; 5,989,906; and 6,017,756.

Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in U.S. Patent Nos. 5,650,316; 5,683,874; 5,693,773; 5,834,185; 5,869,246; 5,874,566; and 5,962,426. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in U.S. Patent Nos. 5,168,053; 5,624,824; 5,683,873; 5,728,521; 5,869,248; and 5,877,162.

Gene expression can also be effectively silenced in a highly specific manner through RNA interference (RNAi). Short Interfering RNA (siRNA) is a double- stranded RNA that can induce sequence-specific post- transcriptional gene silencing, thereby decreasing or even inhibiting gene expression. In one example, an siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA. Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer. siRNA can be chemically or in vzYrø-synthesized or can be the result of short double- stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs are generally designed using algorithms and a conventional DNA/RNA synthesizer. Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands). siRNA can also be synthesized in vitro using kits such as Ambion's SILENCER® siRNA Construction Kit (Ambion, Austin, TX).

Proteins that inhibit rhoA or TβRI include antibodies with antagonistic or inhibitory properties. The term antibody is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, fragments, chimeras, or polymers of immunoglobulin molecules are also useful in the methods taught herein, as long as they are chosen for their ability to inhibit rhoA or TβRI. The antibodies can be tested for their desired activity using in vitro assays, or by analogous methods, after which their in vivo therapeutic or prophylactic activities are tested according to known clinical testing methods.

The Rho inhibitors or TβRI inhibitors described herein may be contained in a composition comprising one or more pharmaceutically acceptable carriers or excipients. The inhibitors may be contained within the same or different compositions. The compositions may be formulated in any conventional manner for use in the methods described herein. Administration can be via any route known to be effective by a physician of ordinary skill. For example, the compositions are administered locally or systemically via oral or parenteral routes.

For oral administration, the compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

The compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain preferably a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also citric acid and its salts and sodium ethylenediaminetetraacetic acid (EDTA) can be used. In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl- paraben and chlorobutanol. Suitable pharmaceutical carriers are described in

Remington: The Science and Practice of Pharmacy, 21^st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005), which is incorporated by reference in its entirety at least for the material related to pharmaceutical carriers and compositions. The compositions may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The compositions can be applied to or embedded with bone prostheses, implants, surgical plates or pins or joint components prior to, concurrent with or after surgical implant.

Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

A composition for use in the methods described herein can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations may be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art. The compositions can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art may be readily selected for use with the compositions described herein. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, caplets, powders, that are adapted for sustained release can be used.

The compositions can be delivered by a controlled-release system. For example, the composition can be administered using intravenous infusion, an implantable osmotic pump, liposomes, or other modes of administration. A controlled release system can be placed in proximity of the target. For example, a micropump can deliver controlled doses directly into bone, thereby requiring only a fraction of the systemic dose (see e.g., Goodson, 1984, in Medical Applications of Controlled Release, vol. 2, pp. 115-138, which is incorporated by reference in its entirety at least for the material related to micropumps). In another example, a pharmaceutical composition of the invention can be formulated with a hydrogel (see, e.g., U.S. Pat. Nos. 5,702,717; 6,117,949; 6,201,072, which are incorporated by reference in their entirety at least for the material related to hydrogels).

It may be desirable to administer the composition locally, i.e., to the area in need of treatment. Local administration can be achieved, for example, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), injection, catheter, suppository, or implant. An implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

The compounds described herein can be formulated and administered to promote coupling between bone formation and bone resoprtion by any means that produces contact of the active ingredient with the agent's site of action. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. The dosage administered will be a therapeutically effective amount of the compound sufficient to result in promoting coupling between bone formation and bone resorption and will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. Dosing regimens for the inhibitors described herein include, but are not limited to, from about 1 to about 1000 mg or from about 10 to about 100 mg daily, optionally by multiple administrations times per day as necessary. For example, fasudil can be administered from about 10 mg to about 250 mg daily, with one or more administrations daily. Alternatively, the inhibitors can be administered at doses from about 0.1 to about 100 mg/kg or from about 1 to about 20 mg/kg or from about 1 to about 10 mg/kg. The inhibitors can be administered at doses at about any amount in between 0.1 and 100 mg/kg. Cellular concentrations of the inhibitors can be from about 1 μM to 100 μM or from about 5 μM to about 15 μM.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The instant compositions are also useful in combination with other agents. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the disease involved. Such agents that could be used in combination with rho inhibitors and TβRI inhibitors include, but are not limited to, the following an organic bisphosphonate, a cathepsin K inhibitor, an estrogen or an estrogen receptor modulator, an androgen receptor modulator, an inhibitor of osteoclast proton ATPase, an inhibitor of HMG-CoA reductase, an integrin receptor antagonist, an osteoblast anabolic agent, such as PTH, calcitonin, Vitamin D or a synthetic Vitamin D analogue and the pharmaceutically acceptable salts and mixtures thereof.

Thus, the provided compositions can be administered in combination with one or more other therapeutic or prophylactic regimens. As used throughout, a therapeutic agent is a compound or composition effective in ameliorating a pathological condition. Any of the aforementioned treatments can be used in any combination with the compositions described herein. Thus, for example, the compositions can be administered in combination with a chemotherapeutic agent and radiation. Other combinations can be administered as desired by those of skill in the art. Combinations may be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). Thus, the term combination is used to refer to either concomitant, simultaneous, or sequential administration of two or more agents.

Also provided are kits comprising one or more rho inhibitors, one or more TβRI inhibitors, one or more compositions comprising the inhibitors or any combinations thereof. The kit can further include instructions for use, one or more containers, one or more administrative means (e.g., a syringe), one or more other biologic components such as cells and the like. The inhibitors, compositions and other biologic components can be in one container or more than one container. Furthermore, the inhibitors, compositions or other biologic components may be contained within an administrative means. As used throughout, by a subject is meant an individual. Thus, the subject can include, for example, domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, and guinea pigs) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human.

Optional or optionally means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used herein, references to decreasing, reducing, or inhibiting include a change of 10, 20, 30, 40, 50 ,60, 70 ,80, 90 percent or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.

As used herein the terms treatment, treat or treating refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus in the disclosed method treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to control. Thus the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or any percent reduction in between 10 and 100 as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition or symptoms of the disease or condition.

There are a variety of sequences related to, for example, rhoA and TβRI that are disclosed on Genbank, at www.pubmed.gov and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made. Furthermore, when one characteristic or step is described it can be combined with any other characteristic or step herein even if the combination is not explicitly stated. Accordingly, other embodiments are within the scope of the claims. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention except as and to the extent that they are included in the accompanying claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for.

Examples Example 1. TGF-βl released during bone resorption is required for the induction of migration of Mesenchymal Stem Cells (MSCs).

Factors in bone resorption-conditioned medium (BRCM) induce migration of MSCs. An in vitro osteoclastic bone resorption assay was developed to identify the factor(s) released during bone resorption-coupled osteoblast activity, in which matrix proteins and minerals in the bone slice are resorbed by osteoclasts and released in the medium (Fig. IA). Macrophage/monocyte precursors isolated from mouse bone marrow differentiated into mature osteoclasts when cultured in the presence of RANK ligand and M-CSF as evidenced by their tartrate resistant acid posphatase (TRAP)- positive staining and multi-nuclear morphology, and activity in bone resorption (Fig. IA). The precursors cultured without RANK ligand and macrophage colony stimulating factor (M-CSF) did not differentiate into mature osteoclasts and did not exhibit bone resorption (Fig. IA). The effect of BRCM on migration of MSCs was examined in a type I collagen-coated transwell chamber with the lower chamber filled with different bone resorption conditioned media. As shown in Figs. IB and 1C, conditioned medium from osteoclasts cultured with bone slice induced 4-fold more cell migration than that induced by conditioned media from precursors cultured alone, precursors cultured with bone slice but not osteoclasts, or mature osteoclasts only.

These results confirm that factors released during bone resorption stimulate migration of osteoprogenitors. Similarly, BRCM induced migration of MSCs in a scratch assay. These results confirmed that factor(s) released during bone resorption are responsible for the induction of migration of MSCs and that these factors are present in an active form in the BRCM.

Example 2. TGF-βl in bone resorption-conditioned medium is required for induction of migration of MSCs.

Neutralizing antibodies were added into the bone resorption conditioned media during the migration assay to identify the specific factor(s) responsible for the induction of osteoprogenitor migration. Addition of a neutralizing antibody with specificity for TGF-βl blocked the migration completely. The addition of neutralizing antibodies against TGF-β2, TGF-β3, and other growth factors IGF-I, IGF-II, PDGF or noggin did not exert a significant effect on cell migration (Fig. 2). These results suggest that, of the factors that have been implicated in osteoprogenitor migration, TGF-βl appears to be the only factor that is required for cell migration, at least under the conditions of this assay.

The presence of the active form of TGFβl in the BRCM was confirmed by ELISA assay (Fig. 3). Active TGFβl was not detectable in the control CM in which osteoclast precursors or osteoclasts were cultured in the absence of bone slices. The inactive latent form of TGFβl was present in the control CM as well as the BRCM, however, suggesting that TGFβl is both released and activated during osteoclastic bone resorption.

Western blot analysis of active and latent TGF-βl in the conditioned medium indicated that significant amounts of active TGF-βl are present in conditioned medium harvested from mature osteoclasts cultured in the presence of bone slices contains (Fig. 3). The media harvested from osteoclasts alone did not have detectable levels of active TGF-βl, showing that the active TGF-βl was released by the mature osteoclasts during osteoclastic bone resorption. Similarly conditioned medium from the precursors, whether cultured with or without bone slices, did not contain detectable levels of active TGF-βl. The inactive latent form of TGF-βl was detectable in conditioned media from all cell types whether cultured in the presence or absence of bone. Taken together, these results demonstrate that osteoclasts and precursors secrete only the latent form of TGF-βl, and that the active form of TGF-βl is released from the bone during osteoclastic bone resorption (Fig. 3).

To verify these results, TGF-βl was depleted in the bone resorption- conditioned medium by repeated immunoprecipitation (IP) with the anti-TGF-βl antibody until there was no detectable active TGF-βl in the conditioned medium (Fig. 4). The depleted medium was no longer able to induce cell migration; however, readdition of TGF-βl restored the ability of the medium to induce cell migration in a concentration-dependent manner (Fig. 4). BRCM prepared using bone slices obtained from TGFβl knockout mice

(TGFβl ^v"Rag2^v") (Engle et al., Cancer Research 62:6362-6 (2002)). TGFβl ^v"Rag2^v" mice are also referred to herein as TGFβl^7" mice. The Rag2^"7' mutations prevent the early death of the TGFβl^7" mice due to organ failure associated with inflammatory disease. As shown in Figures 5 A and 5B, TGFβl^~/"Rag2^"/" mice did not contain active TGFβl and showed reduced induction of migration of MSCs. Taken together, these results indicated that active TGFβl released during osteoclastic bone resorption is essential for inducing migration of MSCs, at least under the conditions of this assay.

A TGF-β 1 gradient in a cell culture model was then established to examine cell migration and morphology. The MSCs migrated toward the TGF-βl gradient. Constitutively active TβRI stimulated cell migration, but the migration was not evenly directional, suggesting that random migration was stimulated. Staining with phalloidin demonstrated the formation of polarized microtubules, which facilitate cell migration, in the cells during migration in the TGF-β 1 gradient. Immunostaining of vinculin, which is a component of cell focal adhesions, showed that protrusion structures were formed at the leading edge of the cell with small adhesions. Taken together, these results indicate that active TGF-βl, which is released during osteoclastic bone resorption, is required for the migration of MSCs. Example 3. TGF-βl stimulates migration in osteoprogenitor cells in vitro and in vivo.

TGF-βl most likely activates at least one the members of the family of Rho small GTPases that are required for cell migration, RhoA, Cdc42 and Racl, in the osteoprogenitor cells. Cells were treated with or without TGF-βl (2ng/ml). The cell lysates were incubated with glutathione S-transferase-Rhotekin Rho binding domain (GST-TRBD) fusion protein beads and analyzed by Western blot with antibodies specific for RhoA, Racl and Cdc42. Western blot of cells treated with or without TGF-βl were also performed. The cell lysates were incubated with GST-TRBD fusion protein beads. Bound proteins were analyzed by Western blot for RhoA. Immunoblots were performed showing TGF-βl activation of cofilin. MSCs were treated with or without TGF-βl . The cell lysates were immunoblotted with antibodies specific for phosphorylated cofilin, total cofilin, phosphorylated Smad2 and total Smad2. To determined whether RhoA-Rock inhibitor blocked TGF-β 1 -induced cofilin phosphorylation, MSCs were treated with TGF-β 1 , Y27132 or vehicle. The cell lysates were immunobloted with antibodies for P-Cofϊlin, total Cofilin, P-Smad2 and total Smad2. TGF-βl was found to stimulate Rho A in a time-dependent manner in both MSCs and C3H10T1/2 cells. Activation of Cdc42 and Racl was not observed. Phosphorylation of cofillin, a Rho A downstream actin-binding factor required for the reorganization of actin filaments, was examined in the MSCs. TGF- βl stimulated phosphorylation of cofillin and Smad 2 in a time-dependent manner. Furthermore, addition of Y-27132, a Rho-ROCK inhibitor, blocked TGF-βl -induced cofillin phosphorylation, but did not inhibit phosphorylation of Smad2. To clarify the specific roles of RhoA and TβRI in cell migration, the effects of Y-27132 and SB- 431542, a specific inhibitor of TβRI, on cell migration were determined. The

RhoA/ROCK inhibitor stimulated migration of the MSCs (Fig. 6A and 6B). The TβRI inhibitor inhibited migration by either recombinant TGFβl or osteoclastic BRCM (Fig. 8 A and 8B). The level of inhibition was dependent on the concentration of TβRI inhibitor added to the osteoclastic BRCM (Fig. 8C). The TβRI inhibitor did not show an inhibitory effect on MSC migration in response to BRCM prepared from bone slices of TGFβl^"'^" mice (Fig. 8D). The results indicate that Rho A is activated by TGF-βl in parallel with R-Smad signaling, and that activation of these two pathways have distinct roles in TGF-βl -induced migration of MSCs.

Using the reverse approach, it was determined whether constitutive expression of the activated TβRI affected the migration of MSCs. Constitutive expression of activated TβRI was as effective as the addition of TGFβl and resulted in stimulated phosphorylation of Smad2. Moreover, when a cell culture in a TGFβl gradient was used to test the migration of MSCs, it was observed that the MSCs migrated toward the TGFβl gradient, whereas the cells with constitutively active TβRI exhibited random migration. Taken together, these results suggest that the TGFβl -induced migration of MSCs is mediated by TβRI.

Since Smad7 specifically binds to TβRI to inhibit phosphorylation of R- Smads, the effects of retro virus-mediated overexpression of Smad7 on the involvement of the TGFβl -Smads pathway in MSC migration was determined. It was observed that Smad7 effectively inhibited the migration of MSCs migration. Similarly, the effects of Smad4^lox/lox, which is the common partner for all R-Smads but is not directly phosphorylated by TβRI, on TGFβl -induced cell migration. MSCs isolated from Smad4 ° ^ox mice were infected with an adenovirus bearing either Cre or GFP. Mice infected with adenovirus bearing Cre resulted in deletion of the endogenous Smad4 gene. MSCs harboring the deleted Smad4 reduced migration in response to BRCM, suggesting that both phosphorylated R-Smad and Smad4 are required for TGFβl -induced migration.

A yeast-two hybrid library with mRNA isolated from MSCs was constructed to examine how RhoA is involved in osteoprogenitor migration. To examine the interaction of endogenous GDIα with TβRII, MSCs were treated with vehicle or TGF- β at different time. Cell extracts were immunoprecipitated with either preimmune antibody (Pre) or GDIα antibody, and the immunocomplex was detected TβRII. The expression levels of TβRII and GDIα were also determined with TβRII and GDIα antibodies. The TβRII cytoplasmic domain was used as bait for screening the two- hybrid library since TβRII is implied for non-Smad signaling pathways. More than twenty positive clones were identified, one of which was GDIα. GDIα is the central regulator of Rho GTPases and binds all three subfamily members (i.e., RhoA, Rac and Cdc42) that are required for cell migration. The interaction between TβRII and GDIα was confirmed in the yeast two-hybrid β-Gal assays (Fig. 9). The interaction between TβRII and GDIα in mammalian cells using immunoprecipitation was then examined. TGF-βl was found to induce the interaction in a time-dependent manner. The induction of an interaction of endogenous TβRII and GDIα by TGF-βl also was confirmed in HBMSCs. To analyze the interaction between TβRII and GDIα that mediates activation of Rho A for cell migration, the interaction domains for both proteins was mapped in yeast two-hybrid assays. The GDIα domain (20-154) with deletion of the first 20 amino acids at the N-terminal and the last 50 amino acids at the C-terminal interacts with TβRII (Fig. 10A). The kinase domain of TβRII mediates its interaction with GDIα (Fig. 10B). Structural analysis of GDIα indicates that the domain between the last C-terminal 147 amino acids is responsible for its binding to RhoA, showing that the mapped GDIα interaction domain with TβRII does not bind to RhoA. These data show that GDIα is a mediator between TβRII and RhoA in activation of this non-Smad signaling pathway in osteoprogenitors. GDIα is a central regulator that coordinates TGF-βl -mediated RhoA and

Smad activation. Since GDIα interacts with TβRII at its kinase domain, the TβRII/GDIα interaction was examined to determine if this interaction regulates the Smad-dependent signaling pathway. Two luciferase reporters containing Smad binding elements, p3TP-Lux or SBD-Luc reporter constructs, were co-transfected in MvILu cells with different amounts of GDIα expression plasmids in the presence or absence of TGF-βl, and luciferase activity was measured. GDIα inhibited TGF-βl - induced transcriptional activity in both luciferase reporter-transfected cells (Fig. 1 IA and HB). The inhibition for the Smad-specifϊc reporter SBD-Luc was much stronger than for the TGF-βl general reporter p3TP, suggesting a specific inhibition for Smad signaling as the p3TP reporter contains the AP-I response element (Fig. 1 IB). In a similar experiment, the effect of GDIα on Smad2 phosphorylation was examined using Western blot analysis. GDIα inhibited TGF-βl -induced phosphorylation of Smad2. These results show that GDIα coordinates TGF-βl -mediated Rho A and Smad activation via binding to TβRII or RhoA. Retro virus-mediated expression of this mapped GDIα interaction domain promoted migration of MSCs induced by BRCM in a similar way as dominant negative RhoA (N 19 RhoA, Fig. 7), consistent with the conclusion that regulation of RhoA is mediated through GDIα. This was confirmed using MSCs isolated from TβRII^Iox/loxmice and infected with adenovirus bearing either Cre or GFP. Mice infected with adenovirus bearing Cre resulted in deletion of the endogenous TβRII gene. The deletion of TβRII inhibited the migration of the MSCs in response to BRCM. Taken together, these results reveal that the interaction between GDIα and TβRII mediates TGFβl -induced activation of RhoA, and provide a role of RhoA in the migration of MSCs.

Example 4. RhoA inhibitor stimulates and TβRI inhibitor reduces bone formation. To determine the specific role of TGF-βl in coupling of bone resorption and bone formation in vivo through its activation of Rho A and Smad signaling, a Rho A/Rock inhibitor (Y-27132), and a TβRI inhibitor (SB-431542) was injected at the rat proximal tibia over a period of four weeks. The rats received three different fluorochrome labels by consecutive administration Lp.. The tibiae were then examined for bone mineral density and structure using X-ray, micro-computed tomography (μCT) analysis, and longitudinal sections of the proximal metaphysis were prepared for fluorescence microscopy. Significant trabecular bone formation was induced in rats injected with the RhoA inhibitor as demonstrated by X-ray, μCT, cross section analysis, longitudinal section analysis and Von Kossa straining (Fig. 14A). In contrast, injection of the TβRI inhibitor resulted in reduced trabecular bone formation (Fig. 13A). RhoA inhibitor stimulate bone formation in a dose-dependent manner (Fig. 14B). TβRI inhibitor reduced bone formation in a dose dependent manner (Fig. 13B). Immunostaining of tibia sections demonstrated that the TβRI inhibitor almost abolished the levels of phosphorylated Smad2/3. These results indicate the Smad signaling modulates bone formation in vivo.

To determine whether the changes in bone formation were due to disruption of coupling during remodeling, the labels of the trabecular surfaces by fluorochromes were assessed. Triple fluorochrome labeling utilizes consecutive labeling with three bone-specific fluorochrome labels, such as xylenol orange, tetracycline and calcein. In the adult animal, areas of the bone surface that are active in bone remodeling surfaces are indicated by retention of the first label or both the first and second labels (arrested surfaces) or all three labels (persisting surfaces). Bone surfaces that do not retain the first label, but are covered with the second and third labels (induction surfaces) are inactive in bone remodeling. Therefore, the areas containing first the two labels or all three labels are active in bone remodeling, i.e., cycled bone resorption and bone formation, whereas the areas in which the first label is absent representing modeling or bone growth only.

To investigate whether the changes in bone formation induced by TβRI inhibitor were due to stimulation of bone growth or alteration of coupling during bone remodeling, the rats were administered three bone-specific fluorochrome labels, xylenol orange, tetracycline and calcein, consecutively by i.p. injection. The labeling of the trabecular surfaces was then assessed. The significant stimulation of bone formation on injection of Rho A inhibitor in the rat tibia was associated primarily with an increase in persisting surfaces (Fig. 15B). The reduction in bone formation on injection of the rat tibia with the TβRI inhibitor was associated with a decrease in the persisting surfaces (Fig. 15A). These results indicate that modulation of TGF-βl- induced migration of osteoprogenitors regulates bone formation during remodeling, suggesting that TGF-βl couples bone resorption and bone formation in vivo.

To investigate whether inhibition of TβRI inhibit recruitment of MSCs to osteoclastic bone resorption sites in vivo, 4 month old rats were injected at the proximal tibia through the anterior-medial cortex 5 mm distal to the knee joint with 10 μl of TβRI inhibitor (SB-431542) or vehicle. The injection was repeated every 6 days and the rats were sacrificed at day 24 for analysis. TRAP staining of tibia from mice injected with the TβRI kinase inhibitor revealed an absence of preosteoblasts at the trabecular bone surface around osteoclasts (Fig. 19). The recruitment of MSCs in response to osteoclastic bone resorption was abrogated by the TβRI kinase inhibitor in vivo, which is consistent with our in vitro observations (Fig. 19). Taken together, these results suggest that the inhibitors of TβRI modulate TGFβl -induced recruitment of MSCs in response to bone resorption in vivo.

If TGFβl plays a key role in the coupling of bone remodeling, it would be expected that adult mice that are deficient in TGFβl would exhibit uncoupled bone formation resorption. In order to characterize adult TGFβl^7" null mice, it was necessary to cross the TGFβl^{" "} mice with immuno-deficient Rag2^~/~ mice to prevent the early death of the TGFβl^"7' null mice due to organ failure associated with inflammatory disease (Atti et al., Bone 31:675-84 (2002); Engle et al., Cancer Res. 62:6362-6 (2002); Zhao et al., Genes Dev. 10:1657-69 (1996)). The overall size of the young (1 -month-old) TGFβl^"7" mice approximated that of their wild-type littermates. At this developmental stage, there were only slight differences in trabecular bone volume, thickness and space as measured by microcomputed tomography (μCT) with construction of a 3D skeleton (Fig. 18A). However, adult (3- month-old) TGFβl^"7" mice, significantly differed from their wild-type littermates in their smaller size, reduced trabecular bone volume and thickness, and greater trabecular bone space (Fig. 18B).

Histologic staining of femur sections showed irregular trabecular bone in three-month old TGFβl^"7' mice and a lack of preosteoblasts at the surface of trabecular bone. Moreover, TRAP staining of mature osteoclasts confirmed an absence of preosteoblasts around the mature osteoclasts on the surface of trabecular bone. Toluidine-blue staining revealed remnants of mineralized calcified cartilage as evidenced with dark blue staining within mature trabecular bone which indicates defects in bone remodeling. Unresorbed mineralized calcified cartilage in bone is a typical pathological histology of osteopetrosis with decreased osteoclastic bone resorption and bone density is increased. However, in this case, bone density was also decreased. The observation showed that the decreased osteoclast activity in three-month old TGFβl^"'^" mice is a result of reduced bone remodeling. Bone histomorphormetry analyses indicated that there was a significant difference in the parameters of osteoblasts and osteoclasts when three-month-old TGFβl^"'^" mice were compared with their wild-type littermates, but these parameters in one-month-old TGFβl^{" "} mice were not significantly different from those of their wild-type littermates. Coupling involves coordinating the balance between osteoblast and osteoclast activity. Downregulation of osteoclast activity together with decreased osteoblast activity in TGFβl^"7 mice reflects the coupling function of TGFβl for osteoclastic bone resorption. Example 5. CED-derived TGF-βl mutations of LAP cause premature release of active TGF-βl. CED is an inherited bone disease associated with mutations of the TGF-βl gene in the region encoding LAP; thus, it is an ideal model to study the function of TGF-βl in osteoprogenitor migration in bone remodeling. Six different TGF-βl expression constructs were generated with different CED-derived point mutations. The wild-type and the six TGF-βl mutation constructs were transfected individually into 293T cells. The cells transfected with CED-derived mutants express similar levels of the latent form of TGF-βl as those transefcted with wild-type TGF- βl . Active TGF-βl was detectable in cells transfected with most of the CED mutants and transfection with at least two of the mutants (Y81H and H222D) exhibit significantly high level of active TGF-βl, whereas cell transfected with the wild-type molecule do not express detectable active TGF-βl . This indicates that mature, active TGF-βl is more readily released from the latent form in cells that carry CED-derived TGF-βl mutants. To confirm these observations, the efficiency of the TGF-βl mutant-induced phosphorylation of Smad2 was examined. Cells transfected with most of the CED-derived TGF-βl mutants exhibited higher levels of Smad2 phosphorylation than cells transfected with wild-type TGF-βl . Consistently, most of the TGF-βl mutants were more potent in inducing migration of MSCs (Fig. 16). To determine whether the premature release of active TGF-βl affects bone formation in vivo, two bone tissue-specific transgenic mice were generated: wild-type TGF-βl (TGF-βl WT) and CED-derived TGF-βl with the H222D mutation. The 2.3 kb type I collagen promoter was used in both types of mice. The CED TGF-βl mutant transgenic mice are only about one-half the size of TGF-βl WT mice. LTBP- 3 knockout mice in which TGF-βl cannot be deposited in the bone matrix also have a much smaller size. The CED TGF-βl mutant transgenic mice exhibit diaphyseal dysplasia typical of CED patients. In addition, CED TGF-βl mutant transgenic mice exhibit bone defects located in active remodeling areas. Thus, CED TGF-βl mutant transgenic mice exhibit uncoupled bone resorption and formation. TβRI inhibitor (SB-431542) was injected at the rat proximal tibia to determine effects of TβRI inhibitors. As shown in Figure 20, TβRI inhibitor restores coupling between bone resorption and formation in CED TGF-βl mutant transgenic mice. These results demonstrate TGF-βl is a factor that is required for osteoprogenitor migration and is one of the most important factors in the bone environment, helping to retain the balance between the dynamic processes of bone resorption and bone formation. Many other bone diseases are superimposed on coupling bone resorption and bone formation. In diseases such as primary hyperparathyroidism, hyperthyroidism and Paget' s disease, there is extra bone formation after each cycle of osteoclastic bone resorption during remodeling. Bone resorption and formation are therefore not well coupled and coordinated in these diseases. In addition, there are a number of diseases, including multiple myeloma, in which osteoblast activity does not completely repair and replace the defect left by previous resorption. Thus, the present application provides methods for restoring the balance between bone resorption and formation, which may be used to treat such diseases.

Example 6. Low doses of TβRI inhibitor stimulates bone formation while higher doses reduce bone formation in rats.

TβRI inhibitor (SB-431542) was injected at the rat proximal tibia to determine dose effects of TβRI inhibitors. The rats received three different fluorochrome labels by consecutive administration i.p. The tibiae were then examined using X-ray, micro- computed tomography (μCT) analysis, and longitudinal sections of the proximal metaphysis were prepared for fluorescence microscopy. Injection of the TβRI inhibitor resulted in increased bone formation at low doses and reduced trabecular bone formation at higher doses (Fig. 17A and 17B). These results show that low doses of TβRI inhibitors can be used to promote coupling by stimulating bone formation while higher doses of TβRI inhibitors can be used to promote coupling by inhibiting or reducing bone formation.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of promoting coupling between bone formation and bone resorption in a subject comprising administering a therapeutically effective amount of a Transforming Growth Factor β receptor I (TβRI) inhibitor to the subject.

2. The method of claim 1, wherein the TβRI inhibitor is selected from the group consisting of SB-431542, [3-(pyridine-2yl)-4-(4-quinonyl)]-lH pyrazole, SD208, SB-505124, A-83-01, 2-pyridinyl-[l,2,3]triazoles and aryl-and heteroaryl- substituted pyrazole inhibitors.

3. The method of claim 1, wherein the TβRI inhibitor is selected from the group consisting of an antisense molecule, a triple helix molecule, a ribozyme and an siRNA.

4. The method of claim 1, wherein the TβRI inhibitor is administered systemically.

5. The method of claim 1, wherein the TβRI inhibitor is administered locally.

6. The method of any one of claims 1 to 5, wherein the subject has a disease or disorder linked to an imbalance between bone formation and bone resorption.

7. The method of any one of claims 1 to 5, wherein the disease or disorder is selected from the group consisting of Camurati-Engelmann disease (CED), osteoporosis, frailty, childhood idiopathic bone loss, alveolar bone loss, a bone defect, osteotomy, Paget's disease, osteoporotic fracture, osteogenesis imperfecta, spine injury, periodontal disease, osteopenia, bone fracture, osteolysis due to a prosthesis and bone necrosis.

8. The method of any one of claims 1 to 5, wherein the method promotes bone formation following bone surgery.

9. The method of claim 8, wherein the bone surgery is selected from the group consisting of facial reconstruction, maxillary or mandibular reconstruction, fracture repair, bone graft, prosthesis implant, hip replacement and knee replacement.

10. The method of any one of claims 1 to 5, wherein the subject has osteoporosis.

11. The method of any one of claims 1 to 5, wherein the subject has heterotrophic ossification or undesired bone formation.

12. The method of any one of claims 1 to 5, wherein the bone defect is selected from the group consisting of traumatic acetabular fracture, fibrodysplasia, spinal hyperostosis, myelopathy, spondylitis ankylosans and hip replacement.

13. The method of any one of claims 1 to 5, wherein the subject has a disorder selected from the group consisting of fibrodysplasia, acquired bone forming lesions such as spinal hyperostosis, myelopathy and spondylitis ankylosans.

14. The method of any one of claims 1 to 5, further comprising administering to the subject an agent selected from the group consisting of an organic bisphosphonate, a cathepsin K inhibitor, an estrogen or an estrogen receptor modulator, an androgen receptor modulator, an inhibitor of osteoclast proton ATPase, an inhibitor of HMG-CoA reductase, an integrin receptor antagonist, an osteoblast anabolic agent, such as PTH, calcitonin, Vitamin D and a synthetic Vitamin D analogue.

15. The method of any one of claims 1 to 5, further comprising administering a rho inhibitor to the subject.

16. The method of claim 15, wherein the rho inhibitor is selected from the group consisting of Y-27132, C3 transferase, Y-27632, HA- 1077, Y-27632, Wf-536, isoquinoline compounds, (R)-trans-N-(pyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide, (R)-(+)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4- (l-aminoethyl)-benzamide, l-(5-isoquinolinesulfonyl)homopiperazine l-(5- isoquinolinesulfonyl)-2-methylpiperazine, ( 1 -benzylpyrrolidin-3 -yl)-( 1 H-indazol-5 - yl)amine, (l-benzylpiperidin-4-yl)-(lH-indazol-5-yl)amine, N-[2-(4-fluorophenyl)- 6,7-dimethoxy-4-quinazolinyl]-N-(lH-indazol-5-yl)amine, N-4-(lH-indazol-5-yl)- 6,7-dimethoxy-N-2-pyridin-4-yl-quinazolin-2,4-diamine, 4-methyl-5-(2-methyl- [l,4]diazepan-l-sulfonyl)isoquinoline, fasudil (hexahydro-l-(5 isoquinolinesulfonyl)- lH-l,4-diazepine) and hydro xyfasudil.

17. A method of promoting coupling between bone formation and bone resorption in a subject comprising administering a therapeutically effective amount of a rho inhibitor to the subject.

18. The method of claim 17, wherein the rho inhibitor is selected from the group consisting of Y-27132, C3 transferase, Y-27632, HA-1077, Y-27632, Wf-536, isoquinoline compounds, (R)-trans-N-(pyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide, (R)-(+)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4- (l-aminoethyl)-benzamide, l-(5-isoquinolinesulfonyl)homopiperazine l-(5- isoquinolinesulfonyl)-2-methylpiperazine, ( 1 -benzylpyrrolidin-3-yl)-( 1 H-indazol-5- yl)amine, (l-benzylpiperidin-4-yl)-(lH-indazol-5-yl)amine, N-[2-(4-fluorophenyl)- 6,7-dimethoxy-4-quinazolinyl]-N-(lH-indazol-5-yl)amine, N-4-(lH-indazol-5-yl)- 6,7-dimethoxy-N-2-pyridin-4-yl-quinazolin-2,4-diamine, 4-methyl-5-(2-methyl- [l,4]diazepan-l-sulfonyl)isoquinoline, fasudil (hexahydro-l-(5 isoquinolinesulfonyl)- lH-l,4-diazepine) and hydroxyfasudil.

19. The method of claim 17, wherein the rho inhibitor is fasudil.

20. The method of claim 17, wherein the rho inhibitor is Y-27132.

21. The method of claim 17, wherein the rho inhibitor is selected from the group consisting of an antisense molecule, a triple helix molecule, a ribozyme and an siRNA.

22. The method of claim 17, wherein the rho inhibitor is administered systemically.

23. The method of claim 17, wherein the rho inhibitor is administered locally.

24. The method of any one of claims 17 to 23, wherein the subject has a disease or disorder linked to an imbalance between bone formation and bone resorption.

25. The method of any one of claims 17 to 23, wherein the disease or disorder is selected from the group consisting of Camurati-Engelmann disease (CED), osteoporosis, frailty, childhood idiopathic bone loss, alveolar bone loss, a bone defect, osteotomy, Paget's disease, osteoporotic fracture, osteogenesis imperfecta, spine injury, periodontal disease, osteopenia, bone fracture, osteolysis due to a prosthesis and bone necrosis.

26. The method of any one of claims 17 to 23, wherein the method promotes bone formation following bone surgery.

27. The method of claim 26, wherein the bone surgery is selected from the group consisting of facial reconstruction, maxillary or mandibular reconstruction, fracture repair, bone graft, prosthesis implant, hip replacement and knee replacement.

28. The method of any one of claims 17 to 23, wherein the subject has osteoporosis.

29. The method of any one of claims 17 to 23, wherein the subject has heterotrophic ossification or undesired bone formation.

30. The method of any one of claims 17 to 23, wherein the bone defect is selected from the group consisting of traumatic acetabular fracture, fibrodysplasia, spinal hyperostosis, myelopathy, spondylitis ankylosans and hip replacement.

31. The method of any one of claims 17 to 23 , wherein the subj ect has a disorder selected from the group consisting of fibrodysplasia, acquired bone forming lesions such as spinal hyperostosis, myelopathy and spondylitis ankylosans.

32. The method of any one of claims 17 to 23, further comprising administering to the subject an agent selected from the group consisting of an organic bisphosphonate, a cathepsin K inhibitor, an estrogen or an estrogen receptor modulator, an androgen receptor modulator, an inhibitor of osteoclast proton ATPase, an inhibitor of HMG-CoA reductase, an integrin receptor antagonist, an osteoblast anabolic agent, such as PTH, calcitonin, Vitamin D and a synthetic Vitamin D analogue.

33. The method of any one of claims 17 to 23, further comprising administering a TβRI inhibitor to the subject.

34. The method of claim 33, wherein the TβRI inhibitor is selected from the group consisting of SB-431542, [3-(pyridine-2yl)-4-(4-quinonyl)]-lH pyrazole, SD208, SB-505124, A-83-01, 2-pyridinyl-[l,2,3]triazoles and aryl-and heteroaryl- substituted pyrazole inhibitors.