Disrupted type II collagenolysis impairs angiogenesis, delays endochondral ossification and initiates aberrant ossification in mouse limbs.

Cartilage remodelling and chondrocyte differentiation are tightly linked to angiogenesis during bone development and endochondral ossification. To investigate whether collagenase-mediated cleavage of the major cartilage collagen (collagen II) plays a role in this process, we generated a knockin mouse in which the mandatory collagenase cleavage site at PQG775↓776LAG, was mutated to PPG775↓776MPG (Col2a1Bailey). This approach blocked collagen II cleavage, and the production of putative collagen II matrikines derived from this site, without modifying matrix metalloproteinase expression or activity. We report here that this mouse (Bailey) is viable. It has a significantly expanded growth plate and exhibits delayed and abnormal angiogenic invasion into the growth plate. Deeper electron microscopy analyses revealed that, at around five weeks of age, a small number of blood vessel(s) penetrate into the growth plate, leading to its abrupt shrinking and the formation of a bony bridge. Our results from in vitro and ex vivo studies suggest that collagen II matrikines stimulate the normal branching of endothelial cells and promote blood vessel invasion at the chondro-osseous junction. The results further suggest that failed collagenolysis in Bailey leads to expansion of the hypertrophic zone and formation of a unique post-hypertrophic zone populated with chondrocytes that re-enter the cell cycle and proliferate. The biological rescue of this in vivo phenotype features the loss of a substantial portion of the growth plate through aberrant ossification, and narrowing of the remaining portion that leads to limb deformation. Together, these data suggest that collagen II matrikines stimulate angiogenesis in skeletal growth and development, revealing novel strategies for stimulating angiogenesis in other contexts such as fracture healing and surgical applications.

Deleting Suppressor of Cytokine Signaling-3 in chondrocytes reduces bone growth by disrupting mitogen-activated protein kinase signaling.

OBJECTIVE: To investigate the impact of deleting Suppressor of Cytokine Signaling (SOCS)-3 (SOCS3) in chondrocytes during murine skeletal development. METHOD: Mice with a conditional Socs3 allele (Socs3fl/fl) were crossed with a transgenic mouse expressing Cre recombinase under the control of the type II collagen promoter (Col2a1) to generate Socs3Δ/Δcol2 mice. Skeletal growth was analyzed over the lifespan of Socs3Δ/Δcol2 mice and controls by detailed histomorphology. Bone size and cortical bone development was evaluated by micro-computed tomography (micro-CT). Growth plate (GP) zone width, chondrocyte proliferation and apoptosis were assessed by immunofluorescence staining for Ki67 and TUNEL. Fibroblast growth factor receptor-3 (FGFR3) signaling in the GP was assessed by immunohistochemistry, while the effect of SOCS3 overexpression on FGFR3-driven pMAPK signaling in HEK293T cells was evaluated by Western blot. RESULTS: Socs3Δ/Δcol2 mice of both sexes were consistently smaller compared to littermate controls throughout life. This phenotype was due to reduced long bone size, poor cortical bone development, reduced Ki67+ proliferative chondrocytes and decreased proliferative zone (PZ) width in the GP. Expression of pMAPK, but not pSTAT3, was increased in the GPs of Socs3Δ/Δcol2 mice relative to littermate controls. Overexpression of FGFR3 in HEK293T cells increased Fibroblast Growth Factor 18 (FGF18)-dependent Mitogen-activated protein kinase (MAPK) phosphorylation, while concomitant expression of SOCS3 reduced FGFR3 expression and abrogated MAPK signaling. CONCLUSION: Our results suggest a potential role for SOCS3 in GP chondrocyte proliferation by regulating FGFR3-dependent MAPK signaling in response to FGF18.

Biomechanical testing of the calcified metacarpal articular surface and its association with subchondral bone microstructure in Thoroughbred racehorses.

BACKGROUND: Palmar/plantar osteochondral disease (POD) and third metacarpal/-tarsal condylar fractures are considered fatigue injuries of subchondral bone (SCB) and calcified cartilage due to repetitive high loads in racehorses. In combination with adaptive changes in SCB in response to race training, the accumulation of SCB fatigue is likely to result in changes of joint surface mechanical properties. OBJECTIVES: To determine the spatial relationship and correlation of calcified articular surface biomechanical properties with SCB microstructure and training history in the distal palmar metacarpal condyle of Thoroughbred racehorses. STUDY DESIGN: Cross-sectional study. METHODS: Third metacarpal condyles were examined from 31 Thoroughbred horses with micro-computed tomography (microCT). Hyaline cartilage was removed and reference point indentation (RPI) mechanical testing of the calcified articular surface was performed. Training histories were obtained from trainers. The association among indentation distance increase (IDI, an inverse RPI measure of toughness), and microCT and training variables was assessed using a mixed-effects generalised linear model. RESULTS: Untrained horses had higher IDI than horses that had commenced training (P<0.001). Death as a result of musculoskeletal bone fatigue injury (P = 0.044) and presence of POD (P = 0.05) were associated with higher IDI. The microCT variables connectivity density and trabecular pattern factor were positively (P = 0.002) and negatively (P<0.001) correlated with IDI respectively. MAIN LIMITATIONS: The application of RPI to the calcified articular surface is novel and there is a potential for measurement variability with surface unevenness. CONCLUSION: Commencement of race training is associated with altered material properties of the calcified articular surface in horses. Reduced articular surface material properties can also be detected in horses that have fatigue injuries of the distal metacarpus and at other sites in the skeleton. Measures of SCB connectivity and trabecular surface shape may be more important determinants of resistance to failure of the calcified articular surface than traditional measures such as SCB volume and density.

Parthenolide inhibits pro-inflammatory cytokine production and exhibits protective effects on progression of collagen-induced arthritis in a rat model.

OBJECTIVES: Progressive destruction of synovial joint cartilage and bone occurs in pathological conditions such as rheumatoid arthritis (RA) because of the overproduction of pro-inflammatory cytokines and activation of nuclear factor kappa B (NF-κB). Through the screening of NF-κB inhibitors by a luciferase reporter gene assay, we identified parthenolide (PAR) as the most potent NF-κB inhibitor, among several PAR analogue compounds. This study was undertaken to determine whether PAR inhibits pro-inflammatory cytokine production, cartilage degradation, and inflammatory arthritis. METHOD: The mRNA levels of pro-inflammatory cytokines were examined by real-time polymerase chain reaction (PCR). Proteoglycan content and release were determined by measuring glycosaminoglycan (GAG) levels using the dimethylmethylene blue (DMMB) dye-binding assay. The potential role of PAR in treatment of arthritis was studied using a collagen-induced arthritis (CIA) model. RESULTS: We established that PAR, as a prototype compound, suppressed lipopolysaccharide (LPS)- and tumour necrosis factor (TNF)-α-induced increases in matrix metalloproteinase (MMP)-1, MMP-3, inducible nitric oxide synthase (iNOS), and interleukin (IL)-1ß mRNA in chondrocytes. In addition, PAR prevented proteoglycan degradation triggered by pro-inflammatory cytokines. PAR treatment at the onset of CIA symptoms significantly reduced synovitis, inflammation, and pannus formation scores. Reduced synovial inflammation after PAR treatment was also reflected in significantly less bone erosion and cartilage damage. CONCLUSIONS: These data indicate a protective effect of PAR on the catabolic insults of pro-inflammatory cytokines on chondrocyte metabolism and GAG release in vitro and in CIA. PAR had anti-inflammatory and structure-modifying effects on experimental arthritis, suggesting that PAR may be useful as a potential alternative or adjunct therapy for inflammatory arthritis.

Hematopoietic stem cell mobilizing agents G-CSF, cyclophosphamide or AMD3100 have distinct mechanisms of action on bone marrow HSC niches and bone formation.

The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide (CYP) as adjuvant to granulocyte colony-stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. It has recently emerged that G-CSF mediates HSC mobilization and inhibits bone formation via specific bone marrow (BM) macrophages. We compared the effect of these three mobilizing agents on BM macrophages, bone formation, osteoblasts, HSC niches and HSC reconstitution potential. Both G-CSF and CYP suppressed niche-supportive macrophages and osteoblasts, and inhibited expression of endosteal cytokines resulting in major impairment of HSC reconstitution potential remaining in the mobilized BM. In sharp contrast, although AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, endosteal cytokine expression or reconstitution potential of HSC remaining in the mobilized BM. In conclusion, although G-CSF, CYP and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches and bone formation are distinct with both G-CSF and CYP targeting HSC niche function and bone formation, whereas AMD3100 directly targets HSC without altering niche function or bone formation.

Proteinase-activated receptor-2 is required for normal osteoblast and osteoclast differentiation during skeletal growth and repair.

Proteinase-activated receptor-2 (PAR(2)) is a G-protein coupled receptor expressed by osteoblasts and monocytes. PAR(2) is activated by a number of proteinases including coagulation factors and proteinases released by inflammatory cells. The aim of the current study was to investigate the role of PAR(2) in skeletal growth and repair using wild type (WT) and PAR(2) knockout (KO) mice. Micro computed tomography and histomorphometry were used to examine the structure of tibias isolated from uninjured mice at 50 and 90 days of age, and from 98-day-old mice in a bone repair model in which a hole had been drilled through the tibias. Bone marrow was cultured and investigated for the presence of osteoblast precursors (alkaline phosphatase-positive fibroblastic colonies), and osteoclasts were counted in cultures treated with M-CSF and RANKL. Polymerase chain reaction (PCR) was used to determine which proteinases that activate PAR(2) are expressed in bone marrow. Regulation of PAR(2) expression in primary calvarial osteoblasts from WT mice was investigated by quantitative PCR. Cortical and trabecular bone volumes were significantly greater in the tibias of PAR(2) KO mice than in those of WT mice at 50 days of age. In trabecular bone, osteoclast surface, osteoblast surface and osteoid volume were significantly lower in KO than in WT mice. Bone marrow cultures from KO mice showed significantly fewer alkaline phosphatase-positive colony-forming units and osteoclasts compared to cultures from WT mice. Significantly less new bone and significantly fewer osteoclasts were observed in the drill sites of PAR(2) KO mice compared to WT mice 7 days post-surgery. A number of activators of PAR(2), including matriptase and kallikrein 4, were found to be expressed by normal bone marrow. Parathyroid hormone, 1,25 dihydroxyvitamin D(3), or interleukin-6 in combination with its soluble receptor down-regulated PAR(2) mRNA expression, and fibroblast growth factor-2 or thrombin stimulated PAR(2) expression. These results suggest that PAR(2) activation contributes to determination of cells of both osteoblast and osteoclast lineages within bone marrow, and thereby participates in the regulation of skeletal growth and bone repair.

Severe developmental bone phenotype in ClC-7 deficient mice.

Bone development is dependent on the functionality of three essential cell types: chondrocytes, osteoclasts and osteoblasts. If any of these cell types is dysfunctional, a developmental bone phenotype can result. The bone disease osteopetrosis is caused by osteoclast dysfunction or impaired osteoclastogenesis, leading to increased bone mass. In ClC-7 deficient mice, which display severe osteopetrosis, the osteoclast malfunction is due to abrogated acidification of the resorption lacuna. This study sought to investigate the consequences of osteoclast malfunction on bone development, bone structure and bone modeling/remodeling in ClC-7 deficient mice. Bones from wildtype, heterozygous and ClC-7 deficient mice were examined by bone histomorphometry and immunohistochemistry. ClC-7 deficient mice were found to have a severe developmental bone phenotype, characterized by dramatically increased bone mass, a high content of cartilage remnants, impaired longitudinal and radial growth, as well as lack of compact cortical bone development. Indices of bone formation were reduced in ClC-7 deficient mice; however, calcein labeling indicated that mineralization occurred on most trabecular bone surfaces. Osteoid deposition had great regional variance, but an osteopetrorickets phenotype, as observed in oc/oc mice, was not apparent in the ClC-7 deficient mice. A striking finding was the presence of very large abnormal osteoclasts, which filled the bone marrow space within the ClC-7 deficient bones. The development of these giant osteoclasts could be due to altered cell fate of the ClC-7 deficient osteoclasts, caused by increased cellular fusion and/or prolonged osteoclast survival. In summary, malfunctional ClC-7 deficient osteoclasts led to a severe developmental bone phenotype including abnormally large and non-functional osteoclasts. Bone formation paremeters were reduced; however, bone formation and mineralization were found to be heterogenous and continuing.

Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes.

The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.

Communication between ephrinB2 and EphB4 within the osteoblast lineage.

Members of the ephrin and Eph family are local mediators of cell function through largely contact-dependent processes in development and in maturity. Production of ephrinB2 mRNA and protein are increased by PTH and PTHrP in osteoblasts. Both a synthetic peptide antagonist of ephrinB2/EphB4 receptor interaction and recombinant soluble extracellular domain of EphB4 (sEphB4), which is an antagonist of both forward and reverse EphB4 signaling, were able to inhibit mineralization and the expression of several osteoblast genes involved late in osteoblast differentiation. The findings are consistent with ephrinB2/EphB4 signaling within the osteoblast lineage having a paracrine role in osteoblast differentiation, in addition to the proposed role of osteoclast-derived ephrinB2 in coupling of bone formation to resorption. This local regulation might contribute to control of osteoblast differentiation and bone formation at remodeling sites, and perhaps also in modeling.

Regulatory pathways revealing new approaches to the development of anabolic drugs for osteoporosis.

The understanding of cell interactions and genetic controls of bone cells has provided new approaches to drug development for osteoporosis. Current emphasis in the development of new anabolic therapies is directed at modifying the effects of Wnt signalling on osteoblast differentiation and bone formation. Local signalling that results in bone formation during remodelling takes place in several ways. Growth factors released from resorbed bone matrix can contribute to preosteoblast differentiation and bone formation. Osteoclasts in the bone multicellular units (BMUs) might also generate activity that contributes to bone formation. The preosteoblasts themselves, growing in the resorption space, can communicate through cell contact and paracrine signalling mechanisms to differentiate. Osteocytes can sense the need for bone repair by detecting damage and pressure changes, and signalling to surface cells to respond appropriately. These recent insights into cell communication, together with discoveries from human and mouse genetics, have opened new pathways to drug development for osteoporosis. With the anabolic effect of parathyroid hormone on the skeleton having been established, human genetics revealed the major role of Wnt signalling in bone formation, and this has become the target of activity. Current approaches include activation at any of several points in the Wnt pathway, and neutralization of sclerostin, the protein product of the SOST gene that is produced in osteocytes as a powerful inhibitor of bone formation.

Mechanisms involved in skeletal anabolic therapies.

Since parathyroid hormone (PTH) is the only proven anabolic therapy for bone, it becomes the benchmark by which new treatments will be evaluated. The anabolic effect of PTH is dependent upon intermittent administration, but when an elevated PTH level is maintained even for a few hours it initiates processes leading to new osteoclast formation, and the consequent resorption overrides the effects of activating genes that direct bone formation. Identification of PTH-related protein (PTHrP) production by cells early in the osteoblast lineage, and its action through the PTH1R upon more mature osteoblastic cells, together with the observation that PTHrP+/- mice are osteoporotic, all raise the possibility that PTHrP is a crucial paracrine regulator of bone formation. The finding that concurrent treatment with bisphosphonates impairs the anabolic response to PTH, adds to other clues that osteoclast activity is necessary to complement the direct effect that PTH has in promoting differentiation of committed osteoblast precursors. This might involve the generation of a coupling factor from osteoclasts that are transiently activated by receptor activator of nuclear factor-kappaB ligand (RANKL) in response to PTH. New approaches to anabolic therapies may come from the discovery that an activating mutation in the LRP5 gene is responsible for an inherited high bone mass syndrome, and the fact that this can be recapitulated in transgenic mice, whereas inactivating mutations result in severe bone loss. This has focused attention on the Wnt/frizzled/beta-catenin pathway as being important in bone formation, and proof of the concept has been obtained in experimental models.

The increased bone mass in deltaFosB transgenic mice is independent of circulating leptin levels.

Transgenic mice overexpressing deltaFosB, a naturally occurring splice variant of FosB, develop an osteosclerotic phenotype. The increased bone formation has been shown to be due, at least in part, to autonomous effects of deltaFosB isoforms on cells of the osteoblast lineage. However, abdominal fat and marrow adipocytes are also markedly decreased in deltaFosB mice, leading to low serum leptin levels. Increased bone mass has been linked to the absence of leptin and leptin receptor signaling in ob/ob and db/db mice. Thus, in addition to affecting directly osteoblastogenesis and bone formation, deltaFosB isoforms might increase bone mass indirectly via a decrease in leptin. To test this hypothesis, we restored normal circulating levels of leptin in deltaFosB mice via sc implanted osmotic pumps. Complete histomorphometric analysis demonstrated that trabecular bone volume as well as dynamic parameters of bone formation was unchanged by this treatment in both deltaFosB transgenic mice and control littermates. This demonstration that restoring circulating levels of leptin in deltaFosB transgenic mice failed to rescue the bone phenotype further indicates that the marked increase in bone formation is autonomous to the osteoblast lineage.

Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-beta in bone remodeling in females but not in males.

To determine the contributions of estrogen receptor (ER)alpha and ERbeta in bone growth and remodeling in male and female mice, we generated and analyzed full knockouts for each receptor, and a double ER knockout. Although suppression of the ligand to the ERs (i.e., estradiol) after menopause or gonadectomy in females led to a catastrophic increase in bone turnover and concomitant bone loss, deletion of one or both ERs failed to show such an effect. Complete deletion of ERalpha led to a decrease, not an increase, in bone turnover and an increase, not a decrease, in trabecular bone volume in both male and female animals. Deletion of ERbeta led to different responses in males, where bone was unaffected, and in females, where bone resorption was decreased and trabecular bone volume increased. In contrast, deletion of both ERs led to a profound decrease in trabecular bone volume in females, which was associated with a decrease, not an increase, in bone turnover. Finally, deletion of ERalpha, but not ERbeta, led to major changes in circulating levels of estradiol and/or testosterone, indirectly affecting bone remodeling and bone mass. Thus, only ERalpha was shown to regulate bone remodeling in males, whereas in females both receptor subtypes influenced this process and could, at least under basal knockout conditions, compensate for each other.

Regulating DeltaFosB expression in adult Tet-Off-DeltaFosB transgenic mice alters bone formation and bone mass.

The DeltaFosB isoforms are naturally occurring AP-1 family members that increase bone volume via a cell-autonomous effect on osteoblastic bone formation. Mice overexpressing DeltaFosB demonstrate a very high level of bone formation, resulting in a progressive osteosclerosis. Despite the linkage of bone formation and resorption in physiological systems, no alteration in bone resorption was detected in mice overexpressing DeltaFosB. To determine whether altering DeltaFosB expression can regulate bone formation independently of bone resorption in adult mice, we used the Tet-Off-inducible transgene system to induce or block transgenic DeltaFosB overexpression and thereby regulate bone formation in vivo. Overexpression of DeltaFosB after skeletal maturity increased trabecular bone volume by increasing bone formation, again without altering bone resorption, indicating that developmental DeltaFosB overexpression is not required for the osteosclerotic phenotype. Similarly, switching off DeltaFosB overexpression after osteosclerosis had developed led to a marked decrease in bone formation and loss of bone mass such that trabecular bone volume approached normal levels. Despite this dramatic reduction, no alteration in bone resorption was detected. These results clearly demonstrate that DeltaFosB regulates bone formation and bone mass in adult mice with no effect on bone resorption.

Activated parathyroid hormone/parathyroid hormone-related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone.

Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. To assess the role of the PTH/PTH-related protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansen's metaphyseal chondrodysplasia. In these transgenic mice, osteoblastic function was increased in the trabecular and endosteal compartments, whereas it was decreased in the periosteum. In trabecular bone of the transgenic mice, there was an increase in osteoblast precursors, as well as in mature osteoblasts. Osteoblastic expression of the constitutively active PPR induced a dramatic increase in osteoclast number in both trabecular and compact bone in transgenic animals. The net effect of these actions was a substantial increase in trabecular bone volume and a decrease in cortical bone thickness of the long bones. These findings, for the first time to our knowledge, identify the PPR as a crucial mediator of both bone-forming and bone-resorbing actions of PTH, and they underline the complexity and heterogeneity of the osteoblast population and/or their regulatory microenvironment.

Bone homeostasis in growth hormone receptor-null mice is restored by IGF-I but independent of Stat5.

Growth hormone (GH) regulates both bone growth and remodeling, but it is unclear whether these actions are mediated directly by the GH receptor (GHR) and/or IGF-I signaling. The actions of GH are transduced by the Jak/Stat signaling pathway via Stat5, which is thought to regulate IGF-I expression. To determine the respective roles of GHR and IGF-I in bone growth and remodeling, we examined bones of wild-type, GHR knockout (GHR(-/-)), Stat5ab(-/-), and GHR(-/-) mice treated with IGF-I. Reduced bone growth in GHR(-/-) mice, due to a premature reduction in chondrocyte proliferation and cortical bone growth, was detected after 2 weeks of age. Additionally, although trabecular bone volume was unchanged, bone turnover was significantly reduced in GHR(-/-) mice, indicating GH involvement in the high bone-turnover level during growth. IGF-I treatment almost completely rescued all effects of the GHR(-/-) on both bone growth and remodeling, supporting a direct effect of IGF-I on both osteoblasts and chondrocytes. Whereas bone length was reduced in Stat5ab(-/-) mice, there was no reduction in trabecular bone remodeling or growth-plate width as observed in GHR(-/-) mice, indicating that the effects of GH in bone may not involve Stat5 activation.

Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage.

The microarchitecture of bone is regulated by complex interactions between the bone-forming and resorbing cells, and several compounds regulate both actions. For example, vitamin D, which is required for bone mineralization, also stimulates bone resorption. Transgenic mice overexpressing the vitamin D receptor solely in mature cells of the osteoblastic bone-forming lineage were generated to test the potential therapeutic value of shifting the balance of vitamin D activity in favor of bone formation. Cortical bone was 5% wider and 15% stronger in these mice due to a doubling of periosteal mineral apposition rate without altered body weight or calcium homeostatic hormone levels. A 20% increase in trabecular bone volume in transgenic vertebrae was also observed, unexpectedly associated with a 30% reduction in resorption surface rather than greater bone formation. These findings indicate anabolic vitamin D activity in bone and identify a previously unknown pathway from mature osteoblastic cells to inhibit osteoclastic bone resorption, counterbalancing the known stimulatory action through immature osteoblastic cells. A therapeutic approach that both stimulates cortical anabolic and inhibits trabecular resorptive pathways would be ideal for treatment of osteoporosis and other osteopenic disorders.

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

Overexpression of DeltaFosB transcription factor(s) increases bone formation and inhibits adipogenesis.

Members of the AP-1 family of transcription factors participate in the regulation of bone cell proliferation and differentiation. We report here a potent AP-1-related regulator of osteoblast function: DeltaFosB, a naturally occurring truncated form of FosB that arises from alternative splicing of the fosB transcript and is expressed in osteoblasts. Overexpression of DeltaFosB in transgenic mice leads to increased bone formation throughout the skeleton and a continuous post-developmental increase in bone mass, leading to osteosclerosis. In contrast, DeltaFosB inhibits adipogenesis both in vivo and in vitro, and downregulates the expression of early markers of adipocyte differentiation. Because osteoblasts and adipocytes are thought to share a common precursor, it is concluded that DeltaFosB transcriptionally regulates osteoblastogenesis, possibly at the expense of adipogenesis.