We recently found that deletion of the gulonolactone oxidase gene, which is involved in the synthesis of ascorbic acid (AA), was responsible for the fracture phenotype in spontaneous fracture mice. To explore the molecular mechanisms by which AA regulates osteoblast differentiation, we examined the effect of AA on osterix expression via Nrf1 (NF-E2-related factor-1) binding to antioxidant-responsive element (ARE) in bone marrow stromal (BMS) cells. AA treatment caused a 6-fold increase in osterix expression in mutant BMS cells at 24 h, which was unaffected by pretreatment with protein synthesis inhibitor. Sequence analyses of mouse osterix promoter revealed a putative ARE located at ؊1762 to ؊1733 upstream of the transcription start site to which Nrf potentially binds. A gel mobility shift assay revealed that nuclear proteins from AA-treated BMS cells bound to radiolabeled ARE much more strongly than nuclear extracts from AA-untreated cells. A chromatin immunoprecipitation assay with Nrf1 antibody confirmed the interaction of Nrf1 with the mouse osterix promoter.

Endochondral ossification plays an important role in the formation of the primary ossification centers (POCs) and secondary ossification centers (SOCs) of mammalian long bones. However, the molecular mechanisms that regulate POC and SOC formation are different. We recently demonstrated that Prolyl Hydroxylase Domain-containing Protein 2 (Phd2) is a key mediator of vitamin C effects on bone. We investigated the role of Phd2 on endochondral ossification of the epiphyses by conditionally deleting the Phd2 gene in osteoblasts and chondrocytes. We found that the deletion of Phd2 in osteoblasts did not cause changes in bone parameters in the proximal tibial epiphyses in 5 week old mice. In contrast, deletion of Phd2 in chondrocytes resulted in increased bone mass and bone formation rate (normalized to tissue volume) in long bone epiphyses, indicating that Phd2 expressed in chondrocytes, but not osteoblasts, negatively regulates secondary ossification of epiphyses. Phd2 deletion in chondrocytes elevated mRNA expression of hypoxia-inducible factor (HIF) signaling molecules including Hif-1α, Hif-2α, Vegfa, Vegfb, and Epo, as well as markers for chondrocyte hypertrophy and mineralization such as Col10, osterix, alkaline phosphatase, and bone sialoprotein. These data suggest that Phd2 expressed in chondrocytes inhibits endochondral ossification at the epiphysis by suppressing HIF signaling pathways.

To test if ephrin B1 overexpression enhances bone mass, we generated transgenic mice overexpressing ephrin B1 under the control of a 3.6 kb rat collagen 1A1 promoter (Col3.6-Tgefnb1). Col3.6-Tgefnb1 mice express 6-, 12- and 14-fold greater levels of full-length ephrin B1 protein in bone marrow stromal cells, calvarial osteoblasts, and osteoclasts, respectively. The long bones of both genders of Col3.6-Tgefnb1 mice have increased trabecular bone volume, trabecular number, and trabecular thickness and decreased trabecular separation. Enhanced bone formation and decreased bone resorption contributed to this increase in trabecular bone mass in Col3.6-Tgefnb1 mice. Consistent with these findings, our in vitro studies showed that overexpression of ephrin B1 increased osteoblast differentiation and mineralization, osterix and collagen 1A1 expression in bone marrow stromal cells. Interaction of ephrin B1 with soluble clustered EphB2-Fc decreased osteoclast precursor differentiation into multinucleated cells. Furthermore, we demonstrated that the mechanical loading-induced increase in EphB2 expression and newly formed bone were significantly greater in the Col3.6-Tgefnb1 mice than in WT littermate controls. Our findings that overexpression of ephrin B1 in bone cells enhances bone mass and promotes a skeletal anabolic response to mechanical loading suggest that manipulation of ephrin B1 actions in bone may provide a means to sensitize the skeleton to mechanical strain to stimulate new bone formation.

Based on our findings that PHD2 is a negative regulator of chondrocyte differentiation and that hypoxia signaling is implicated in the pathogenesis of osteoarthritis, we investigated the consequence of disruption of the Phd2 gene in chondrocytes on the articular cartilage phenotype in mice. Immunohistochemistry detected high expression of PHD2 in the superficial zone (SZ), while PHD3 and HIF-1α (target of PHD2) are mainly expressed in the middle-deep zone (MDZ). Conditional deletion of the Phd2 gene (cKO) in chondrocytes accelerated the transition of progenitors to hypertrophic (differentiating) chondrocytes as revealed by reduced SZ thickness, and increased MDZ thickness, as well as increased chondrocyte hypertrophy. Immunohistochemistry further revealed decreased levels of progenitor markers but increased levels of hypertrophy markers in the articular cartilage of the cKO mice. Treatment of primary articular chondrocytes, in vitro, with IOX2, a specific inhibitor of PHD2, promoted articular chondrocyte differentiation. Knockdown of Hif-1α expression in primary articular chondrocytes using lentiviral vectors containing Hif-1α shRNA resulted in reduced expression levels of Vegf, Glut1, Pgk1, and Col10 compared to control shRNA. We conclude that Phd2 is a key regulator of articular cartilage development that acts by inhibiting the differentiation of articular cartilage progenitors via modulating HIF-1α signaling.

roid hormone (TH) action is mediated through two nuclear TH receptors, THR␣ and THR␤. Although the role of THR␣ is well established in bone, less is known about the relevance of THR␤-mediated signaling in bone development. On ther basis of our recent finding that TH signaling is essential for initiation and formation of secondary ossification center, we evaluated the role of THRs in mediating TH effects on epiphysial bone formation. Two-day treatment of TH-deficient Tshr Ϫ/Ϫ mice with TH increased THR␤1 mRNA level 3.4-fold at day 7 but had no effect on THR␣1 mRNA level at the proximal tibia epiphysis. Treatment of serum-free cultures of tibias from 3-day-old mice with T3 increased THR␤1 expression 2.1-and 13-fold, respectively, at 24 and 72 h. Ten-day treatment of Tshr Ϫ/Ϫ newborns (days 5-14) with THR␤1 agonist GC1 at 0.2 or 2.0 g/day increased BV/TV at day 21 by 225 and 263%, respectively, compared with vehicle treatment. Two-day treatment with GC1 (0.2 g/day) increased expression levels of Indian hedgehog (Ihh) 100-fold, osterix 15-fold, and osteocalcin 59-fold compared with vehicle at day 7 in the proximal tibia epiphysis. Gel mobility shift assay demonstrated that a putative TH response element in the distal promoter of mouse Ihh gene interacted with THR␤1. GC1 treatment (1 nM) increased Ihh distal promoter activity 20-fold after 48 h in chondroctyes. Our data suggest a novel role for THR␤1 in secondary ossification at the epiphysis that involves transcriptional upregulation of Ihh gene. thyroid hormones; ossification; bone formation; osteoblasts; chondrocytes; hypothyroidism; Indian hedge hog ENDOCHONDRAL OSSIFICATION is one of two essential processes required for fetal development of the mammalian skeletal system and for fracture healing. Unlike intramembranous ossification, which is the other process by which mesenchymal cells from the cranial neural crest, sclerotomes, and lateral plate mesoderm migrate and proliferate, forming mesenchymal condensations, cartilage is present and replaced by trabecular bone during endochondral ossification (16,35). There are two centers of ossification for endochondral ossification, a primary and a secondary center. The primary ossification center (POC) usually appears in the diaphysis of the long bones or in the body of the irregular bones during prenatal development while the secondary ossification center (SOC) occurs in the epiphysis of long bones at the time of birth in mammals (11). Endochondral ossification at the POC is tightly regulated by a number of growth factors (PTHrp, IHH, IGF-I, BMP/TGF␤, Wnt, VEGF) and transcription factors (Sox9, Runx2, osterix, ␤-catenin) (3, 10, 21-23, 29, 42, 53). Dysregulation in the production and/or actions of any of the factors that regulate endochondral ossification can result in skeletal diseases including chondrodysplasias and osteoarthritis (21, 40). Although the processes leading to POC formation have been well established, signaling pathways that stimulate SOC formation are not well understood.Thyroid hormone (TH) is known ...

, a member of the tight junction family of proteins, is a negative regulator of RANKL-induced osteoclast differentiation and bone resorption (BR) in vivo. Since estrogen deficiency decreases bone mass in part by a RANKL-mediated increase in BR, we evaluated whether estrogen regulates Cldn-18 expression in bone. We found that Cldn-18 expression was reduced in the bones of estrogen deficient mice, whereas it was increased by estrogen treatment in osteoblasts and osteoclasts in vitro. We next evaluated the role of Cldn-18 in mediating estrogen-induced bone loss. Cldn-18 knockout (KO) and littermate wild-type (WT) mice were ovariectomized (OVX) or sham operated at 6 wk of age, and the skeletal phenotype was evaluated at 14 wk of age. PIXImus revealed that total body, femur, and lumbar BMD were reduced 8 -13% (P Ͻ 0.05) after 8 wk of OVX compared with sham in WT mice. As expected, total body, femur, and lumbar BMD were reduced 14 -21% (P Ͻ 0.05) in Cldn-18 KO sham mice compared with sham WT mice. However, ovariectomy failed to induce significant changes in BMD of total body, femur, or vertebra in the Cldn-18 KO mice. CT analysis of the distal femur revealed that trabecular (Tb) bone volume was decreased 50% in the OVX WT mice compared with sham that was caused by a 26% decrease in Tb number and a 30% increase in Tb separation (all P Ͻ 0.05). By contrast, none of the Tb parameters were significantly different in OVX Cldn-18 KO mice compared with sham KO mice. Histomorphometric analyses at the Tb site revealed that neither osteoclast surface nor osteoclast perimeter was increased significantly as a consequence of OVX in either genotype at the time point examined. Based on our findings, we conclude that the estrogen effects on osteoclasts may in part be mediated via regulation of Cldn-18 signaling.claudins; estrogen; bone resorption; bone density; osteoblasts; gene expression ESTROGEN DEFICIENCY IN MENOPAUSAL WOMEN is a serious health issue due to increased bone turnover, resulting in osteoporosis and increased fracture risk consequent to deterioration of microarchitectural bone structure (32). It is now recognized that estrogen prevents bone loss via multiple and complex effects on bone marrow and bone cells that result in decreased osteoclastogenesis, increased osteoclast apoptosis, and decreased capacity of mature osteoclasts to resorb bone (20). The canonical mechanism for osteoclast differentiation depends on activation of receptor activator of nuclear factor-B (RANK) by its ligand RANKL and tyrosine kinase receptor fms by macrophage colony-stimulating factor. Estrogen probably affects osteoclast differentiation in part indirectly through its modulation of osteoblast RANKL and osteoprotegerin expression (46) and directly by decreasing the responsiveness of osteoclast precursors to the RANKL (36). There is also evidence that estrogen blocks the production of the pro-osteoclastogenic cytokines IL-1, IL-6, and TNF (1, 18, 29). It has also been suggested that estrogen modulates osteoclast apoptosis and osteoclast acti...

We have recently demonstrated that mice with disruption of claudin‐18 (Cldn‐18) gene exhibited osteopenia due to increased bone resorption (BR). In this study, we found that gastric pH was significantly higher in Cldn‐18 knockout (KO) mice compared to heterozygous control mice at 10 weeks of age. To test the possibility that the increased BR in the Cldn‐18 KO mice fed a normal‐Ca diet is a consequence of decreased Ca absorption caused by increased stomach pH, we subjected KO and control mice to a normal‐Ca and high‐Ca diet at birth. Serum Ca levels were significantly lower in Cldn‐18 KO mice compared to control mice at a normal‐Ca diet but not at high‐Ca diet. Dual energy X‐ray absorptiometry revealed that a high‐Ca diet significantly increased lumbar bone mineral density (BMD), but had no effect on femur/tibia BMD in both Cldn‐18 KO and control mice compared to a normal‐Ca diet. While a high‐Ca diet did not affect volumetric BMD, trabecular, and cortical parameters of the lumbar vertebra (LV) as measured by μCT, the size of the LV did increase, in both genotypes due to reduced BR. Comparison of the skeletal phenotype of high‐Ca Cldn‐18 KO and control mice revealed that an osteopenia phenotype seen at a normal‐Ca diet was still maintained at different skeletal sites in the KO mice till 10 weeks of age. In conclusion, our findings suggest that increased BR is likely caused by direct effects of a lack of Cldn‐18 on osteoclasts rather than gastric pH changes.