WO2000038707A1

WO2000038707A1 - Method for the treatment of bone loss

Info

Publication number: WO2000038707A1
Application number: PCT/AU1999/001154
Authority: WO
Inventors: John Allan Eisman; Edith Margaret Gardiner
Original assignee: Garvan Institute Of Medical Research
Priority date: 1998-12-24
Filing date: 1999-12-24
Publication date: 2000-07-06
Also published as: AUPP794898A0

Abstract

The present invention relates to methods for the treatment or prevention of bone loss based on specific activation of mature osteoblastic cells. The present invention also relates to methods of screening for compounds which reduce bone loss.

Description

Method for the treatment of bone loss

FIELD OF THE INVENTION

The present invention relates to methods for the treatment or prevention of bone loss, and to methods of screening for compounds which reduce bone loss.

BACKGROUND OF THE INVENTION

Osteoporosis is a condition in which fractures occur with minimal trauma due to underlying bone fragility. Peak bone mass and optimum bone structure achieved during early adulthood and the subsequent age and menopause-related bone loss are key determinants of this condition. Bone loss at menopause relates to increased osteoclastic resorption, predominantly in trabecular bone, whereas the more gradual but continuous loss which occurs with aging is thought to be the result of decreased osteoblastic bone formation [Parfitt, 1995] and primarily affects cortical bone [Rico, 1997]. In both situations, there is gradual deterioration of the bone microarchitecture as well as the overall loss of bone mass.

Effective osteoporosis therapies minimise bone loss, essentially by reducing resorption [McClung, 1996; Vedi, 1996]. Therapeutic use of the active la-hydroxylated forms of vitamin D (e.g., 1,25-dihydroxyvitamin D2 and D3 forms, referred to collectively as 1,25-dihydroxyvitamin D) has yielded somewhat disparate clinical results [Gallagher, 1990; Lips, 1996; Ott, 1989; Tilyard, 1992], presumably because of diverse actions on the osteoblastic and osteoclastic cell lineages as well as on other tissues such as intestine, parathyroid and kidney.

1,25-Dihydroxyvitamin D directly inhibits osteoblastic differentiation, but stimulates mineralised matrix formation by mature osteoblasts [Marie, 1985]; however, it also indirectly stimulates osteoclastic recruitment and differentiation, acting through immature cells of the osteoblastic lineage via the vitamin D receptor (VDR) [Martin, 1994]. This stimulatory pathway, in which resorption is regulated by cells of the bone forming lineage, is a link by which the opposing actions of these two cell lineages are coordinated in healthy bone. This tight linkage was ini tally proposed as a mechanism to explain maintenance of overall bone mass and calcium homeostasis [Rodan, 1982]. This model, however, is inherently incomplete as it does not explain how bone resorption is restrained once homeostatic requirements have been met. Clearly, the molecular pathways that allow a l,25-(OH)₂ D response are present in osteoblastic and osteoclastic lineages, but dissection of these responses in intact animals is difficult. In order to overcome these difficulties the present inventors have developed a transgenic mouse with elevated l,25-(OH)₂ D responsiveness in mature osteoblasts to provide a model in which to address the stimulatory effect of the hormone specifically in osteoblasts, distinct from its effects in osteoclasts and other tissues. This transgenic mouse was produced by a process as described in WO 96/05299.

SUMMARY OF THE INVENTION

The present inventors have now identified a specific osteoclastic inhibitory mechanism which acts via mature osteoblasts and which counterbalances osteoclastogenic signals from immature osteoblasts and osteoblastic stromal cells. This finding indicates that a therapeutic approach which specifically enhances the activity of mature osteoblastic cells may be an ideal strategy for osteoporosis treatment.

Accordingly, in a first aspect the present invention provides a method for treating or preventing bone loss in a subject which method comprises increasing the activity of mature osteoblastic cells relative to the activity of immature osteoblastic cells and/or osteoclastic cells in the subject, such that the level of osteoclastic bone resorption is reduced.

In the context of the first aspect, the activity of the mature osteoblastic cells may be increased by any suitable means. For example, mature osteoblastic cells may be selectively stimulated by administration of a vitamin D analogue which targets mature osteoblastic cells but does not target immature osteoblastic or osteoclastic cells.

In a second aspect the present invention provides a method for treating or preventing bone loss in a subject which method comprises increasing the responsiveness of mature osteoblastic cells in the subject to vitamin D or a vitamin D analogue.

In one preferred embodiment of the second aspect, the method involves administering to the subject an expression vector comprising a sequence encoding a vitamin D receptor, wherein the sequence encoding a vitamin D receptor is operably linked to at least one control sequence which allows expression of the vitamin D receptor in mature osteoblastic cells but not in immature osteoblastic or osteoclastic cells. In a preferred embodiment, the vector comprises control sequences derived from the 5' and 3' regions of the osteocalcin gene. The control regions may be derived from the pGOSCAS vector described herein.

Methods for delivering DNA molecules to target cells in vivo will be well known to those skilled in the field. Suitable methods are described, for example, in W. French Anderson, 1992, "Human gene therapy" Science 256:808-813; Danks, D.M., "Cancer gene therapy", Today's Life Science, July 1995: 18-21; and "Gene transfer technology in therapy: Current applications and future goals" by Romano, G, Pacilio, C. and Giordano, A. in: Stem Cells 17(4): 191-202 (1999).

Suitable delivery systems include viral delivery systems, examples of which include retroviral and adenoviral vectors as well as adeno-associated viruses. Other suitable viral vector systems are based on lentivirus, HEN, simian virus 40 (SN40), Sindbis virus and Sendai virus constructs.

Suitable non-viral delivery systems employ lipids, polycations, or 'polyplexes' which allow a high level of tissue specificity.

In vivo delivery of vector constructs may also be achieved by direct injection of DΝA at the target site in the subject.

In another embodiment of the second aspect, the method involves ex vivo gene therapy. Preferably, the method involves removing bone marrow cells from the subject, transfecting the cells with an expression vector encoding a vitamin D receptor, wherein the expression vector comprises control regions which allow expression of the vitamin D receptor in mature osteoblastic cells but not in immature osteoblastic or osteoclastic cells, and returning the transfected bone marrow cells to the subject. A suitable procedure for the removal, culture and transplant of transgenic bone marrow cells is described in Hou et al, 1999. In a preferred embodiment of the first and second aspects, the method does not significantly alter the activity of non-bone tissue cells in the subject. In a third aspect the present invention provides a method of screening for compounds which reduce bone loss, the method comprising exposing mature osteoblastic cells to a compound suspected of reducing bone loss; and detecting the activity of the mature osteoblastic cells wherein an increase in activity of the mature osteoblastic cells following exposure to the compound is indicative that the compound is capable of reducing bone loss.

In a preferred embodiment of the third aspect, the method further comprises the steps of exposing immature osteoblastic and/or osteoclastic cells to the compound; and detecting the activity of the immature osteoblastic or osteoclastic cells wherein no significant increase in activity of the immature osteoblastic and/or osteoclastic cells following exposure to the compound is a further indication that the compound is capable of reducing bone loss.

In one embodiment of the third aspect, the method may be conducted in vivo. For example, the method may be conducted by administering the compound to a transgenic animal, wherein mature osteoblastic cells of the transgenic animal comprise a heterologous reporter gene. Preferably, the activity of the mature osteoblastic cells is detected by monitoring the expression of the reporter gene in the transgenic animal.

The present inventors have also found that the bones of transgenic mice expressing the human vitamin D receptor in mature osteoblastic cells exhibit an increased cortical bone response to mechanical loading.

Experimental results showed that cortical bone mass was significantly increased in response to loading in transgenic mice, but this effect was not observed in non-transgenic mice. This finding suggests that transgenic animals which express a vitamin D receptor in mature osteoblastic cells provide a sensitive assay means for screening for compounds which increase bone mass in response to weight bearing.

Accordingly, in a fourth aspect the present invention provides a method of screening for compounds which cause an increase in bone mass, the method comprising (a) administering a compound suspected of increasing bone mass to a transgenic animal, the transgenic animal comprising an expression vector encoding a vitamin D receptor , wherein the expression vector comprises one or more control sequences which allow expression of the vitamin D receptor in mature osteoblastic cells; (b) subjecting bones of the transgenic animal to mechanical loading or relieving the bones of the transgenic animal of mechanical loading; and (c) determining the relationship between load bearing and change in bone mass caused by administration of the compound.

It will be appreciated that the methods of the present invention will we useful for the treatment of conditions such as osteoporosis, osteomalacia, Paget's disease, sports injury, rheumatoid and osteoarthritis, plastic surgery, orthodontics/dentistry, orthopaedics, and counteracting age-related bone loss.

Specific applications of the methods of the present invention in the field of orthopaedics may include enhancing bone consolidation after osteotomy or bone graft; enhancing repair in cases of nonunion or delayed union; stabilising prosthetic joints; enhancing spinal fusion; improving bone formation after distraction osteogenesis; use in orthopaedic tissue engineering, osteoinduction; and improving bone healing after fracture, sports injury or plastic surgery.

Specific applications of the methods of the present invention in the field of rheumatology may include preventing or repairing bone erosion due to osteoarthritis, or cartilage erosion due to rheumatoid arthritis; suppressing inflammatory response in rheumatoid arthritis by delivery of anti- inflammatory cytokines to bone surfaces.

Specific applications of the methods of the present invention in the field of orthodontics/dentistry may include stabilisation of teeth; enhancing orthodontic tooth movement and/or stabilising final placement of teeth.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described by reference to the following Figures and non-limiting examples.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. transgene expression increases osteoblastic VDR levels. Total VDR protein was elevated in OSV9 and OSV3 bones. Values are means +_ SE. Figure 2. Tibiae from two lines of transgenic mice were stronger than non- transgenic tibiae. (A) Tibial peak load, a measure of the maximum bending force withstood by bone prior to fracture, was greater in OSV9 and OSV3 mice, as was tibial stiffness (B). Cortical area moment of inertia (C), a measure of the distribution of bone mass around the central axis and determinant of bone strength, was significantly greater in OSV9 and OSV3 mice as was tibial cortical area (D). Tibial cortical area and peak load were correlated (E). Dashed lines indicate mean values for FNB/N, OSV9, and OSV3 in order from left to right. Peak load = 1.6 x tibial cortical area xlO^" + 1. 7, R^z = 0.41, p < 0.0001. FNB/N (O), OSV9 (Δ) and OSV3 (■). Tibial diameter was significantly greater in OSV9 and OSV3 mice (F). Values are means ± SE. Significant differences from FVB/N are indicated by asterisks above individual lines (p<0.05).

Figure 3. Cellular responses to the OSVDR transgene in femoral cortical and vertebral cancellous bone. Femoral width (A) was greater than FVB/N in OSV3 (7%) but not in OSV9 mice. Periosteal mineral appositional rates were elevated (66 - 130%) in both transgenic lines (B). Values are means ± SE. Significant differences from FVB/N are indicated by asterisks above individual lines (p< 0.05).

Figure 4. The OSVDR transgene increases trabecular bone volume by reducing resorption. Photographs of mid-saggital sections of fourth caudal vertebrae from four month old mice (A) show more abundant mineralised tissue in OSV3 bones. Vertebral trabecular bone volume (B) was 17 - 20% greater in both transgenic lines with similar increases (14 - 17%) in vertebral trabecular thickness (C) but not trabecular number (D). Bone formation rate (E) was not affected by the transgene. Osteoclast surface (F) in vertebral bone was reduced (> 30%) in both transgenic lines. Trends to reduced osteoclast number (G) of 15% for OSV3 and 9% for OSV9, were not significant. In the femoral metaphyseal region, trabecular bone showed a trend for greater bone volume (H) and reduced osteoclast surface (I) with no change in the epiphysis (J). Values are means ± SE. Significant differences from FVB/N are indicated by asterisks above individual lines (p<0.05). EXAMPLES

Materials and Methods

Transgenic mice. pOSVDR was generated by inserting 2.1 kb EcoRI fragment from phVDRl/3 [McDonnell, 1989 ] into pGOSCAS [Sims, 1997], followed by SV40 small t antigen splice and polyadenylation signals (Promega Corporation, Madison WI) immediately downstream. Transgenic mice were generated by pronuclear injection of FNB/N embryos. The OSV9 and OSV3 lines were independently derived. Hemizygous experimental animals, bred by mating homozygous males to FVB/N females, were studied. Age-matched female non-transgenic and transgenic mice were mixed and group housed at weaning. This study was conducted twice. The first study included FVB/N and OSV9; the repetition included these lines plus OSV3. Data shown are from the second study. A third line, OSV8 line, showed inconsistent growth characteristics and was excluded from statistical analyses as the level of VDR protein in its bones was not significantly different from the FVB/N level (p = 0.35). Four weeks prior to collection, the mice were randomly assigned to two groups and changed from standard laboratory chow (0.9% calcium) to semi-synthetic diets [Reeves, 1993] with moderate (0.5%) or low (0.1%) calcium content. Vitamin D was supplied (1000 IU/kg) in all diets. Mice were injected with the fluorescent tetracycline compounds calcein and demeclocycline (Sigma Chemical Company, St. Louis), each at 15mg/kg, 10 and 3 days prior to collection. Tibiae were collected and stored for mechanical testing at -20C in phosphate buffered saline. Femora and vertebrae were collected, fixed in 4% paraformaldehyde and prepared for histomorphometry. Calvaria, radius, kidney, liver, brain, muscle, heart, lung and spleen were collected for molecular analyses.

Analysis of transgene expression. Total RNA was prepared from tissues of 8 week old mice and analysed by northern blot as previously described [Sims, 1997]. Filters were probed with a random primed α- P- dCTP (Amersham, Buckinghamshire, England) labelled mouse VDR cDNA fragment which was cloned after reverse transcription-polymerase chain reaction (RT-PCR) from non-transgenic mouse kidney RNA, using primers derived from the human cDNA sequence (forward primer 5'-CGGAATTCTCATTCTGACAGATGAGGAAGTGC-3' and reverse primer 5'-AACTGCAGTCCTGGTATCATCTTAGCAAAGCC-3'). The filter was stripped and reprobed for osteocalcin using a radiolabelled insert from pOC918 rat osteocalcin cDNA (Harris et al) and for GAPDH using a radiolabelled PCR product. Relative signals were quantitated by phosphorimager (Molecular Dynamics 445SI, Sunnyvale, CA). Total VDR protein was measured by ELISA from long bones of six to eight 9 month old mice for each line, with equal numbers of mice from the low and moderate calcium diet groups.

Biochemistry. Serum 1,25-dihydroxyvitamin D was measured by radioimmunoassay (RIA) [Hollis, 1996]. PTH was also measured by RIA (Immutopics, San Clemente, CA), as was serum osteocalcin, using the method of Gundberg et al [Gundberg, 1992] except 50ml samples sizes were assayed. Primary antibody and osteocalcin standards were generously provided by Dr. C. Gundberg. Iodinated osteocalcin was purchased from Biomedical Technologies, Inc. (Stoughton, MA) and donkey anti-goat IgG secondary antibody from Sigma.

Total VDR protein was measured by ELISA [Uhland-Smith, 1996] using antibodies, generously provided by Dr. H. DeLuca, and commerically supplied biotin-conjugated alkaline phosphatase (Bio-Rad, Hercules CA) and purifed VDR protein standards (Pan Vera, Madison, WI). Nuclear protein extracts for VDR assay were prepared using a protocol adapted from Pierce et al 1987 [Pierce, 1987]. Whole bones were homogenised initially using a Polytron Homogeniser and subsequently by Dounce Homogeniser. Total protein levels were determined by Bradford colorimetric assay (Bio-Rad). Values are means ± SE.

Histology. For histology, animals were treated with a single intraperitoneal injection of 1,25-dihydroxyvitamin D3 (Tetrionics Inc.,

Madison WI) at a dose of 2mg per kg body weight. Femora were collected 6 hours later, prepared and analysed as previously [Sims, 1997]. In situ hybridisation used antisense and sense human VDR cDNA riboprobes generated from linearised pGhVcEBx, which contains 400 bp of 5' sequence from the human VDR cDNA. The antisense riboprobe detects both mouse and transgenic human transcripts. Immunohistochemistry on sections of the same specimens used a human-specific anti-VDR antibody [Tuohimaa, 1992] .

Histomorphometry. The fourth caudal vertebra and the distal half of the right femur from each animal were fixed and embedded undecalcified in K-Plast resin (Medim-Medizinische Diagnostik, Giessen, Germany) and 5mm saggital sections were analysed (Bioquant, R&M Biometrics Inc., Nashville, TN). Femoral width was measured using bright field microscopy, and periosteal mineral appositional rate and vertebral bone formation rate (BFR= double labelled surface x MAR) by fluorescence microscopy (Leica,

Heerbrugg, Switzerland). Sections were stained for mineralised bone [Page, 1977], and trabecular bone volume (BV/TV), thickness (Tb.Th) and number (Tb.N) were quantitated [Parfitt, 1983]. For measurements of osteoclast surface and number, sections were stained for tartrate-resistant acid phosphatase activity as described previously [Sims, 1997].

Mechanical testing and morphometry. The biomechanical and physiological consequences of osteoblastic and osteocytic VDR elevation were evaluated in four and nine month-old female mice. Tibiae were dissected free of remaining soft tissue prior to 3-point bending tests using an MTS 858 Bionix Testing Machine (MTS Systems Corporation, Minneapolis, MN) at 2 mm per minute until failure. Samples were tested immersed in normal saline at room temperature with a support span of 10 mm. After mechanical testing, tibiae were imaged at the fracture site at 4x magnification using the Leica Quantimet 500 stereo microscope.

Measurements and analyses were completed using the Bioquant System (R&M Biometrics Inc., Nashville, TN). Cortical moments of inertia were calculated [Bak, 1992].

Bone organ cultures. Fetal metatarsal and metacarpal organ cultures were established using a protocol adapted from Minkin and Yu [Minkin, 1991]. Briefly, pregnant females were injected intraperitoneally with 30mCi 45Ca as aqueous calcium chloride solution (Amersham) on day 16 of gestation, and fetal bones were taken one day later. Bones were cultured aat 37C on a rocking platform for 24 hours without 1,25-dihydroxyvitamin D3, then transferred to fresh medium containing 10-8M 1,25-dihydroxyvitamin D3 or vehicle. After a further 48 hours radioactivity in culture medium and bone fragments was measured by liquid scintillation.

Statistics. Statistical analyses were performed by one-way analysis of variance (ANOVA) within age groups with linear contrasts selected a priori to compare results from each of the transgenic lines with those from the FVB/N control line, * p<0.05 (SPSS for Macintosh v. 4.02; SPSS Inc., Chicago, IL). The effects of transgenic line and dietary calcium group were examined by two-way ANOVA. As there were no significant interactions between transgenic line and diet, diet groups were combined, n = 17-19 mice per line per age group. For analysis of organ culture data, FVB/N and OSV3 mean values were compared using unpaired t-tests.\

Results

Transgenic VDR expression was detected in the bones of adult mice from two OSV transgenic lines (OSV3 and OSV9), whereas VDR transcripts were not detected in the bones of normal FVB/N mice. VDR expression was undetectable in transgenic or non-transgenic bones by immunohistochemistry or in situ hybridization. However, injection of mice with 1,25-dihydroxyvitamin D3 six hours prior to tissue collection elevated VDR expression in cuboidal and flattened osteoblasts and osteocytes, as detected by in situ hybridisation, and in hypertrophic chondrocytes of transgenic but not non-transgenic bones. This 1,25-dihydroxyvitamin D3 response was expected based on previous studies of the transgenic human osteocalcin promoter [Sims, 1997]. Immunohistochemistry with an antibody which recognises human but not mouse VDR [Tuohimaa, 1992] detected VDR protein in osteoblasts and osteocytes of bones from treated transgenic but not non-transgenic mice. Elevation of VDR protein in the transgenic lines was also apparent by ELISA measurement (Figure 1), with a three-fold greater level in OSV3 bones relative to FVB/N (7.0 ± 0.6 vs. 1.8 ± 0.5 fmol / mg protein), and intermediate elevation in OSV9 bones (5.7 + 0.7). Transgenic animals were healthy and phenotypically normal, with no consistent transgene-associated effect on body weight, bone length (tibia), or serum levels of calcium or calcium homeostatic hormones (Table 1). Serum osteocalcin was lower in transgenic mice at 9 months but not at 4 months (Table 1) or in older animals. Northern blot analysis revealed a reduction in osteocalcin gene expression in 8 week old animals.

Table 1 Morphometric parameters and calcium homeostatic hormones were not affected by the transgene in female mice.

Mouse line FVB/N OSV9 OSV3 FVB/N OSV9 OSV3 Age (months) 4 9

Body Weight 34.0 ±0.9 34.3 ±1.0 36.7 ±1.2 35.0 ±1.3 35.9 ±1.5 38.3 ±1.2

(8)

Tibial Length 18.2 ±0.1 18.3 ±0.1 18.2 ±0.1 18.0 ±0.1 18.1 ±0.1 18.4 ±0.1

(mm)

Serum Ca 9.5 ±0.2 9.1 ±0.1 9.1 ±0.2 11.4 ±0.2 11.0 ±0.3 11.8 ±0

(mg/dl)

Serum OC 168 ±8 148 ±7 160 ±9 140 ±6 117 ±7 105 ±7 *

(ng/ml)

Serum 1.25 D 64 ±6 58 ±5 58 ±8 61 ±2 58 ±3 66 ±2

(pg/ml)

Serum PTH 60 ±5 77 ±6 81 ±6 * 109 ±8 100 ±8 106 ±6

(ng/ml)

Body weight, tibial length and serum concentrations of total calcium (Ca), osteocalcin (OC) and the calcium homeostatic hormones

1,25-dihydroxyvitamin D (1,25 D) and parathyroid hormone (PTH) were not consistently affected by transgene status, although some differences were detected at single ages (see text). Asterisks denote significant differences between FVB/N and both OSV lines (p<0.05). Analysis was by ANOVA within age groups with post hoc linear contrasts.

Cortical bone.

Tibiae from transgenic females were significantly stronger in a three- point bending test than non-transgenic bones (mean peak load for OSV9 and OSV3, 16% higher than FVB/N at 4 months and 25% higher at 9 months; Figure 2A). Transgenic bones were also stiffer than their wildtype counterparts (up to 24%, Figure 2B). Similar differences in peak load and stiffness were also observed for males at the same ages, with mean peak loads up to 27% higher than FVB/N males (16.3 ± 0.3, OSV3 vs 12.8 ± 0.3, FVB/N at nine months) and stiffness values elevated by up to 30% (78.0 ± 2.2, OSV3 vs 62.7 ± 1.7, FVB/N at nine months).

Cortical area moments of inertia (a measure of bone geometry and determinant of bone strength [Bak, 1992]) of the female tibiae were greater in OSV9 and OSV3 than in FVB/N mice by 8 and 11%, respectively at 4 months, and by 23 and 34% at 9 months (Figure 2C). There were similar differences in cortical areas at both ages, with OSV3 tibial cortical area up to 18% larger than FVB/N area (Figure 2D). The greater cross-sectional area of the transgenic tibiae was positively correlated with their greater strength in the three-point bending test (Figure 2E). The increases in moments of inertia and cross-sectional areas were associated with increased cortical diameter (Figure 2F), consistent with an increase in periosteal bone formation. This parameter was therefore investigated in femora from these mice.

By histomorphometry, the OSV3 femora were also wider than those of FVB/N femora, with diameter 7% greater at 4 and 9 months (Figure 3A). Femoral periosteal mineral appositional rate (MAR), indicated by greater separation of tetracycline labels (as shown at four months, Figure 3B) was elevated in both transgenic lines at 4 months (130% increase in OSV3, 66% in OSV9), and showed a similar pattern at 9 months (Figure 3C). These MAR increases are consistent with the greater cortical dimensions of the OSV3 bones. Endocortical mineral apposition rate was not altered in the transgenic femora (data not shown).

Trabecular bone.

Trabecular bone volume, measured in the fourth caudal vertebral body (Fig. 4A), was significantly greater in OSV3 and OSV9 mice than FVB/N mice at 4 and 9 months (Fig. 4B). This difference was associated with thicker trabeculae without a change in trabecular number (Fig. 4C-D). In contrast to the pattern observed in cortical bone, however, this difference in trabecular thickness was not attributable to greater bone formation in the transgenic mice (Fig. 4E). The observed increase in trabecular bone volume was, instead, associated with a reduction in bone resorption. Osteoclast surface was reduced by 33% on the trabecular surfaces of vertebrae from transgenic animals at both ages (Fig. 4F). An apparent transgene-related reduction in osteoclast number was not significant (Fig. 4G). A similar pattern of increased trabecular bone volume and reduction in osteoclast surface was also apparent at four months in the femoral metaphysis but not the epiphysis (Fig. 4H-J).

Organ cultures.

Serum mediators of calcium homeostasis, i.e. PTH and 1,25-dihydroxyvitamin D, were not consistently different between wildtype and OSV mice, indicating that the transgene effect on osteoclast surface was mediated locally rather than systemically. Bone resorption was evaluated in organ cultures to test this possibility. In vitro release of ⁴⁵Ca from in vivo- labelled OSV3 fetal metatarsals and metacarpals in response to 1,25-dihydroxyvitamin D3 treatment tended to be lower than FVB/N levels after three days of culture, although this effect was not significant (1.3 ± 0.3- fold induction by 1,25-dihydroxyvitamin D3 in FVB/N cultures vs 1.1 ± 0.2 for OSV3, one-tailed p = 0.10). Calcium release from calvarial cultures did not show this trend.

Discussion

The consequences of elevated vitamin D receptor in mature osteoblasts and osteocytes of transgenic mice differed according to bone type. In long bones the transgene effect was most apparent in cortical bone and was evidenced as elevated bone formation with greater periosteal mineral appositional rates. The amount of trabecular bone, as measured in caudal vertebrae, was also greater in the transgenic mice. In this site, however, the thicker trabeculae and elevated trabecular bone volume were associated with decreased bone resorption rather than increased formation. This is the first demonstration in vivo that 1,25-dihydroxyvitamin D directly stimulates bone forming activity by mature osteoblasts, and also the first detection of a specific osteoclastic inhibitory pathway from mature osteoblasts.

The increased mineral apposition rate reflects an increase in the anabolic ability of individual osteoblasts, rather than a change in osteoblastic proliferation or survival [Parfitt, 1995]. Interestingly, age-related deficiencies in the anabolic potential of osteoblasts has been suggested as a major contributor to age-related bone loss in humans [Thomsen, 1996] . This increased cellular activity may be most simply explained by a transgene- enhanced ability of 1,25-dihydroxyvitamin D to regulate expression of bone structural or regulatory genes [Murakami, 1998; Yasuda, 1998; Zhang, 1997, Lian, 1997]. The change in osteoblast activity was associated with wider tibiae and femora in OSVDR mice. As a consequence of these transgenic differences, the transgenic tibiae were stronger and stiffer than non-transgenic bones. This cortical effect was envelope specific, being evident only on the periosteal surface of the long bones, and not the endosteal or trabecular surfaces. This suggests transgene interaction with local factors in the bone microenvironment. The modest increases in bone deposition result in substantial gains in long bone strength because area moment of inertia increases with the fourth power of the radius of the bone [Bak, 1992]. Material properties of the transgenic bones, which may also contribute to the increased OSVDR bone strength and/or stiffness, are currently being investigated.

In caudal vertebrae, the thicker trabeculae and reduced bone resorption occurred despite the maintenance of normal or slightly elevated levels of serum 1,25-dihydroxyvitamin D and PTH in the transgenic mice. This surprising result was not predicted by the large number of studies which have shown that 1,25-dihydroxyvitamin D acts through immature osteoblasts and stromal cells to stimulate osteoclastic recruitment and activity by direct and indirect mechanisms ) [Martin, 1994]. Rather, the inhibition of bone resorption apparently results from the elevated sensitivity of mature osteoblasts to normal endogenous levels of 1,25-dihydroxyvitamin D. The present approach allowed the specific responses of mature osteoblasts to be assessed without altering the sensitivity of immature osteoblastic and stromal cells, in contrast to earlier studies in which intact animals, mixed cell populations in culture, or tissues were treated with active vitamin D compounds. Given the dominance of the pro-resorptive response of immature osteoblastic and stromal cells to 1,25-dihydroxyvitamin D, it is unlikely that this counter-regulatory pathway would be detected in vivo without an experimental enhancement of the mature osteoblast response. Indeed, the transgene inhibitory effect was barely evident in vitro in OSVDR bone organ cultures, presumably because of the strong positive response by immature osteoblastic and stromal cells which do not express the transgene Possible mechanisms.

Importantly, the levels of the circulatory factors 1,25-dihydroxyvitamin D and PTH were not consistently changed and certainly were not reduced in the OSVDR mice, indicating that cortical and trabecular transgene effects are paracrine rather than endocrine. Paracrine pathways may act via soluble mediators such as growth factors or cytokines, or via cell-cell or cell-matrix interactions. A network of tumor necrosis factor (TNF) family members has recently been shown to regulate osteoclastogenesis including the NFk-B receptor activator RANK on osteoclast precursors, its ligand RANKL on immature osteoblastic cells, and the soluble decoy receptor osteoprotegerin (OPG)[Suda, 1999]. As RANKL and OPG are regulated by 1,25-dihydroxyvitamin D, a transgene-associated decrease in the local RANKL/OPG ratio could reduce osteoclastic recruitment/activation. Another cytokine which may participate in similar regulatory systems is OCIL, a recently described cytokine which inhibits osteoclastogenesis, is expressed by mature osteoblastic cells and is upregulated by 1,25-dihydroxyvitamin D [Zhou, 1999]. Preliminary analyses of RNA from mineralising primary osteoblastic cultures do not support involvement of RANKL or OPG (GPT, M. Malakelis and G. Nicholson, unpublished data).

Extracellular matrix composition can alter bone cell biology and gene expression [Gerstenfeld, 1999] and thus may contribute to the OSVDR bone phenotype, as 1,25-dihydroxyvitamin D is a common regulator of bone matrix protein genes. A reduction in osteocalcin protein, as suggested by Northern blots, could contribute to the increase in periosteal mineral apposition rate, as it has been suggested that osteocalcin is an inhibitor of bone formation [Price, 1982; Ducy, 1996]. There is, however, no evidence that a decrease in osteocalcin protein would causally relate to the decrease in trabecular resorption observed in the OSVDR mice, and there is some evidence to refute this possibility [Price, 1982].

Given that VDR levels are elevated in the mechano-transducing osteocytes and osteoblastic lining cells in OSVDR mice, response to loading or unloading may be altered in the OSVDR bones; both cortical and trabecular bone changes in these mice support this hypothesis. Interestingly, the transgene effect on trabecular osteoclast surface appears to be stronger in unloaded bone, as it was evident in the fourth caudal vertebrae and the femoral metaphyseal region but not the heavily loaded [Westerlind, 1997] femoral epiphysis. These local differences in trabecular bone response to mechanical loading could relate to differential expression of regulatory genes which are expressed by osteocytes, regulated by 1,25-dihydroxyvitamin D and change levels in response to mechanical loading, such as c-fos or osteopontin [Candeliere, 1996; Inaoka, 1995; Terai, 1999; Lian, 1997].

These findings in the OSVDR mice highlight a specific osteoclastic inhibitory mechanism. It acts via mature osteoblasts, where the transgene is expressed, and counterbalances osteoclastogenic signals from immature osteoblasts and osteoblastic stromal cells. Such a coupling of pathways may provide a system for local control of bone turnover and maintenance of microarchitectural integrity. It will be important to define the molecular and physiological mechanisms underlying this novel negative regulatory activity, which may also be controlled by other calcium homeostatic regulators such as PTH, interleukins, and prostanoids. A therapeutic approach which specifically enhances mature osteoblastic responses to endogenous or exogenous agents could constitute an ideal strategy for osteoporosis treatment, decreasing trabecular bone resorption and increasing cortical bone formation, thereby reversing the typical patterns of osteoporotic bone loss. The publications referred to above are incorporated in their entirety herein by reference.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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Claims

1. A method for treating or preventing bone loss in a subject which method comprises increasing the activity of mature osteoblastic cells relative to the activity of immature osteoblastic cells and/or osteoclastic cells in the subject, such that the level of osteoclastic bone resorption is reduced.

2. A method for treating or preventing bone loss in a subject which method comprises increasing the responsiveness of mature osteoblastic cells in the subject to vitamin D or a vitamin D analogue.

3. A method as claimed in claim 2 which comprises administering to the subject an expression vector comprising a sequence encoding a vitamin D receptor, wherein the sequence encoding a vitamin D receptor is operably linked to at least one control sequence which allows expression of the vitamin D receptor in mature osteoblastic cells but not in immature osteoblastic or osteoclastic cells.

4. A method as claimed in claim 6 in which the sequence encoding a vitamin D receptor is operably linked to control sequences derived from the 5' and 3' regions of the osteocalcin gene.

5. A method as claimed in claim 4 in which the control sequences are derived from the pGOSCAS vector described herein.

6. A method for treating or preventing bone loss in a subject which method comprises removing bone marrow cells from the subject, transfecting the bone marrow cells with an expression vector comprising a sequence encoding a vitamin D receptor, wherein the sequence encoding a vitamin D receptor is operably linked to at least one control sequence which allows expression of the vitamin D receptor in mature osteoblastic cells but not in immature osteoblastic or osteoclastic cells, and returning the transfected bone marrow cells to the subject.

7. A method of screening for compounds which reduce bone loss, the method comprising exposing mature osteoblastic cells to the agent; and detecting the activity of the mature osteoblastic cells wherein an increase in activity of the mature osteoblastic cells following exposure to the agent is indicative that the agent is capable of reducing bone loss.

8. A method as claimed in claim 8 which further comprises the steps of exposing immature osteoblastic and/or osteoclastic cells to the agent; and detecting the activity of the immature osteoblastic or osteoclastic cells wherein no significant increase in activity of the immature osteoblastic and/or osteoclastic cells following exposure to the agent is a further indication that the agent is capable of reducing bone loss.

9. A method as claimed in claim 7 or claim 8 in which the method is conducted in vivo.

10. A method of screening for compounds which cause an increase in bone mass, the method comprising

(a) administering a compound suspected of increasing bone mass to a transgenic animal, the transgenic animal comprising an expression vector encoding a vitamin D receptor , wherein the expression vector comprises one or more control sequences which allow expression of the vitamin D receptor in mature osteoblastic cells;

(b) subjecting bones of the transgenic animal to mechanical loading or relieving the bones of the transgenic animal of mechanical loading; and

(c) determining the relationship between load bearing and change in bone mass caused by administration of the compound.