Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis.

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.

Lasofoxifene enhances vaginal mucus formation without causing hypertrophy and increases estrogen receptor beta and androgen receptor in rats.

OBJECTIVE: Lasofoxifene, a new selective estrogen-receptor modulator (SERM), shows efficacy in vaginal and vulvar atrophy in postmenopausal women. Here, we sought to explore the possible mechanisms of action for this effect in comparison with other SERMs using an immature ovariectomized rat model. DESIGN: SERMs (lasofoxifene, raloxifene, and tamoxifen) and 17alpha-ethinyl estradiol were administered orally to immature ovariectomized rats daily for 1 or 4 days. Vaginal and uterine tissues were weighed and processed for histomorphometric measurements, vaginal mucopolysaccharide staining, and immunohistochemistry of 5-bromo-2'-deoxyuridine and steroid receptors. Receptor quantification was determined by a novel ultrasensitive enzyme-linked immunosorbent assay method. RESULTS: Lasofoxifene and raloxifene showed a minimal increase in vaginal and uterine weight, epithelial cell proliferation, and epithelial thickness in comparison with estradiol and tamoxifen. Lasofoxifene significantly enhanced vaginal mucus formation in a dose-dependent manner. Vaginal progesterone receptor protein was increased fivefold by estradiol and all three SERMs tested. 17alpha-Ethinyl estradiol caused a significant decrease in estrogen receptor alpha, but no change with other treatments. Only lasofoxifene significantly increased vaginal estrogen receptor beta and androgen receptor protein levels. CONCLUSIONS: These results demonstrated that lasofoxifene stimulated vaginal mucus formation without causing cell proliferation in the rat reproductive tract. These effects may be due to the increased vaginal estrogen receptor beta and androgen receptor levels. This cellular and molecular profile of lasofoxifene in the vagina may account for its efficacy in the treatment of vaginal and vulvar atrophy in postmenopausal women.

A nonprostanoid EP4 receptor selective prostaglandin E2 agonist restores bone mass and strength in aged, ovariectomized rats.

UNLABELLED: CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia. INTRODUCTION: The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia. MATERIALS AND METHODS: CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined. RESULTS: Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls. CONCLUSIONS: CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces.

A new vitamin D analog, 2MD, restores trabecular and cortical bone mass and strength in ovariectomized rats with established osteopenia.

UNLABELLED: An orally active, highly potent analog of 1alpha,25-dihydroxyvitamin D3, 2MD, restores trabecular and cortical bone mass and strength by stimulating periosteal bone formation and decreasing trabecular bone resorption in OVX rats with established osteopenia. INTRODUCTION: The purposes of this study were to determine the effects of long-term treatment with 2-methylene-19-nor-(20S)-1alpha,25(OH)2D3 (2MD) on restoring bone mass and bone strength in ovariectomized (OVX) rats with established osteopenia and 2MD effects on bone formation and bone resorption on trabecular and cortical bone surfaces. MATERIALS AND METHODS: Sprague-Dawley female rats were sham-operated (sham) or OVX at 4 months of age. Beginning at 8 weeks after OVX, OVX rats were orally dosed with 2MD at 0.5, 1, 2.5, 5, or 10 ng/kg/day for 16 weeks. Serum calcium was measured at 6, 13, and 16 weeks after treatment, and bone mass and structure, bone formation, bone resorption, and bone strength were determined at the end of the study. RESULTS: Serum calcium did not change significantly with 2MD at 0.5 or 1 ng/kg/day, whereas it significantly increased at 2.5, 5, or 10 ng/kg/day. 2MD significantly and dose-dependently increased total body BMD, total BMC, and stiffness of femoral shaft (FS), maximal load and stiffness of femoral neck, and toughness of the fifth lumbar vertebral body (L5) at all doses compared with OVX controls. In 2MD-treated OVX rats, there was a dose-dependent increase in total BMD and total BMC of the distal femoral metaphysis (DFM), trabecular bone volume of L3, ultimate strength and stiffness of L5, and maximal load of FS compared with OVX controls at dosages>or=1 ng/kg/day. At dosages>2.5 ng/kg/day, most of the bone mass and bone strength related parameters were significantly higher in 2MD-treated OVX rats compared with sham controls. Bone histomorphometric analysis of L3 showed dose-dependent decreases in osteoclast number and osteoclast surface on trabecular bone surface and a dose-dependent increase in periosteal bone formation associated with 2MD treatment. CONCLUSIONS: 2MD not only restored both trabecular and cortical bone mass but also added bone to the osteopenic OVX rats beyond that of sham controls by stimulating bone formation on the periosteal surface and decreasing bone resorption on the trabecular surface. 2MD increased bone mass and strength at doses that did not induced hypercalcemia.

Long-term treatment of lasofoxifene preserves bone mass and bone strength and does not adversely affect the uterus in ovariectomized rats.

The purpose of this study was to determine the long-term effects of lasofoxifene, a new selective estrogen receptor modulator, on bone mass, bone strength, and reproductive tissues in ovariectomized (OVX) rats. Sprague Dawley female rats at 3.5 months of age were OVX and treated orally with lasofoxifene (60, 150, or 300 microg/kg x d) for 52 wk. The urinary deoxypyridinoline/creatinine ratio was significantly lower in all lasofoxifene-treated OVX rats compared with OVX controls at wk 26. Peripheral quantitative computerized tomography analysis of proximal tibial metaphysis showed that the significant loss in trabecular content and density induced by OVX was significantly prevented by lasofoxifene treatment. Proximal tibial and lumber vertebral trabecular bone histomorphometric analysis showed that all doses of lasofoxifene significantly reduced OVX-induced bone loss by decreasing bone resorption and bone turnover. The ultimate strength, energy, and toughness of the fourth lumbar vertebral body in OVX rats treated with all doses of lasofoxifene were significantly higher compared with those in OVX controls, and did not differ significantly from those in sham controls. Uterine weight in OVX rats treated with lasofoxifene was slightly, but significantly, higher when compared with that in OVX controls, but was still much less than that in sham controls. No abnormal finding associated with lasofoxifene was observed with uterine histology examination. In summary, long-term treatment with lasofoxifene preserves bone mass and bone strength and does not adversely affect the uterus in OVX rats. These data suggest that lasofoxifene is an effective antiosteoporosis agent, and its efficacy and safety can be maintained over an extended period of time.

Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass.

We have previously described osteoblast/osteocyte factor 45 (OF45), a novel bone-specific extracellular matrix protein, and demonstrated that its expression is tightly linked to mineralization and bone formation. In this report, we have cloned and characterized the mouse OF45 cDNA and genomic region. Mouse OF45 (also called MEPE) was similar to its rat orthologue in that its expression was increased during mineralization in osteoblast cultures and the protein was highly expressed within the osteocytes that are imbedded within bone. To further determine the role of OF45 in bone metabolism, we generated a targeted mouse line deficient in this protein. Ablation of OF45 resulted in increased bone mass. In fact, disruption of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the bone histomorphometric results, we also determined that OF45 knockout osteoblasts produced significantly more mineralized nodules in ex vivo cell cultures than did wild type osteoblasts. Osteoclastogenesis and bone resorption in ex vivo cultures was unaffected by OF45 mutation. We conclude that OF45 plays an inhibitory role in bone formation in mouse.