Farnesoid X receptor (FXR) is a nuclear receptor that functions as a bile acid sensor controlling bile acid homeostasis. We investigated the role of FXR in regulating bone metabolism. We identified the expression of FXR in calvaria and bone marrow cells, which gradually increased during osteoblastic differentiation in vitro. In male mice, deletion of FXR (FXR À/À ) in vivo resulted in a significant reduction in bone mineral density by 4.3% to 6.6% in mice 8 to 20 weeks of age compared with FXR þ/þ mice. Histological analysis of the lumbar spine showed that FXR deficiency reduced the bone formation rate as well as the trabecular bone volume and thickness. Moreover, tartrateresistant acid phosphatase (TRACP) staining of the femurs revealed that both the osteoclast number and osteoclast surface were significantly increased in FXR À/À mice compared with FXR þ/þ mice. At the cellular level, induction of alkaline phosphatase (ALP) activities was blunted in primary calvarial cells in FXR À/À mice compared with FXR þ/þ mice in concert with a significant reduction in type I collagen a1(Col1a1), ALP, and runt-related transcription factor 2 (Runx2) gene expressions. Cultures of bone marrow-derived macrophages from FXR À/À mice exhibited an increased number of osteoclast formations and protein expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). In female FXR À/À mice, although bone mineral density (BMD) was not significantly different from that in FXR þ/þ mice, bone loss was accelerated after an ovariectomy compared with FXR þ/þ mice. In vitro, activation of FXR by bile acids (chenodeoxycholic acid [CDCA] or 6-ECDCA) or FXR agonists (GW4064 or Fexaramine) significantly enhanced osteoblastic differentiation through the upregulation of Runx2 and enhanced extracellular signal-regulated kinase (ERK) and b-catenin signaling. FXR agonists also suppressed osteoclast differentiation from bone marrow macrophages. Finally, administration of a farnesol (FOH 1%) diet marginally prevented ovariectomy (OVX)-induced bone loss and enhanced bone mass gain in growing C57BL/6J mice. Taken together, these results suggest that FXR positively regulates bone metabolism through both arms of the bone remodeling pathways; ie, bone formation and resorption.

Osteoblasts are derived from mesenchymal progenitors. Differentiation to osteoblasts and adipocytes is reciprocally regulated. Transcriptional coactivator with a PDZ-binding motif (TAZ) is a transcriptional coactivator that induces differentiation of mesenchymal cells into osteoblasts while blocking differentiation into adipocytes. To investigate the role of TAZ on bone metabolism in vivo, we generated transgenic mice that overexpress TAZ under the control of the procollagen type 1 promoter (Col1-TAZ). Whole body bone mineral density (BMD) of 6- to 19-week-old Col-TAZ mice was 4% to 7% higher than that of their wild-type (WT) littermates, whereas no difference was noticed in Col.1-TAZ female mice. Microcomputed tomography analyses of proximal tibiae at 16 weeks of age demonstrated a significant increase in trabecular bone volume (26.7%) and trabecular number (26.6%) with a reciprocal decrease in trabecular spacing (14.2%) in Col1-TAZ mice compared with their WT littermates. In addition, dynamic histomorphometric analysis of the lumbar spine revealed increased mineral apposition rate (42.8%) and the serum P1NP level was also significantly increased (53%) in Col.1-TAZ mice. When primary calvaria cells were cultured in osteogenic medium, alkaline phosphatase (ALP) activity was significantly increased and adipogenesis was significantly suppressed in Col1-TAZ mice compared with their WT littermates. Quantitative real-time polymerase chain reaction analyses showed that expression of collagen type 1, bone sialoprotein, osteocalcin, ALP, osterix, and Runx2 was significantly increased in calvaria cells from Col1-TAZ mice compared to their WT littermates. In vitro, TAZ enhanced Runx2-mediated transcriptional activity while suppressing the peroxisome proliferator-activated receptor gamma signaling pathway. TAZ also enhanced transcriptional activity from 3TP-Lux, which reflects transforming growth factor-beta (TGF-β)-mediated signaling. In addition, TAZ enhanced TGF-β-dependent nuclear translocation of Smad2/3 and Smad4. Taken together, these results suggest that TAZ positively regulates bone formation in vivo, which seems to be mediated by enhancing both Runx2 and TGF-β signaling.

PPARg has critical role in the differentiation of mesenchymal stem cells into adipocytes while suppressing osteoblastic differentiation. We generated transgenic mice that overexpress PPARg specifically in osteoblasts under the control of a 2.3-kb procollagen type 1 promoter (Col.1-PPARg). Bone mineral density (BMD) of 6-to 14-week-old Col.1 À PPARg male mice was 8% to 10% lower than that of their wildtype littermates, whereas no difference was noticed in Col.1-PPARg female mice. Col.1-PPARg male mice exhibited decreased bone volume (45%), trabecular thickness (23%), and trabecular number (27%), with a reciprocal increase in trabecular spacing (51%). Dynamic histomorphometric analysis also revealed that bone-formation rate (42%) and mineral apposition rate (32%) were suppressed significantly in Col.1-PPARg male mice compared with their wild-type littermates. Interestingly, osteoclast number and surface also were decreased by 40% and 58%, respectively, in Col.1-PPARg male mice. In vitro whole-marrow culture for osteoclastogenesis also showed a significant decrease in osteoclast formation (approximately 35%) with the cells from Col.1-PPARg male mice, and OPG/RANKL ratio was reduced in stromal cells from Col.1-PPARg male mice. Although there was no significant difference in BMD in Col.1-PPARg female mice up to 30 weeks, bone loss was accelerated after ovariectomy compared with wild-type female mice (À3.9% versus À6.8% at 12 weeks after ovariectomy, p < .01), indicating that the effects of PPARg overexpression becomes more evident in an estrogen-deprived state in female mice. In conclusion, in vivo osteoblast-specific overexpression of PPARg negatively regulates bone mass in male mice and accelerates estrogen-deficiency-related bone loss in female mice. ß

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Umbilical cord blood (UCB) has recently been recognized as a new source of mesenchymal stem cells (MSCs) for use in stem cell therapy. We studied the effects of systemic injection of human UCB-MSCs and their conditioned medium (CM) on ovariectomy (OVX)-induced bone loss in nude mice. Ten-week-old female nude mice were divided into six groups: Sham-operated mice treated with vehicle (Sham-Vehicle), OVX mice subjected to UCBMSCs (OVX-MSC), or human dermal fibroblast (OVX-DFB) transplantation, OVX mice treated with UCB-MSC CM (OVX-CM), zoledronate (OVX-Zol), or vehicle (OVX-Vehicle). Although the OVX-Vehicle group exhibited significantly less bone mineral density (BMD) gain compared with the Sham-Vehicle group, transplantation of hUCB-MSCs (OVX-MSC group) has effectively prevented OVX-induced bone mass attenuation. Notably, the OVX-CM group also showed BMD preservation comparable to the OVX-MSC group. In addition, microcomputed tomography analysis demonstrated improved trabecular parameters in both the OVX-MSC and OVX-CM groups compared to the OVX-Vehicle or OVX-DFB group. Histomorphometric analysis showed increased bone formation parameters, accompanied by increased serum procollagen type-I N-telopeptide levels in OVX-MSC and OVX-CM mice. However, cell-trafficking analysis failed to demonstrate engraftment of MSCs in bone tissue 48 h after cell infusion. In vitro, hUCB-MSC CM increased alkaline phosphatase (ALP) activity in human bone marrow-derived MSCs and mRNA expression of collagen type 1, Runx2, osterix, and ALP in C3H10T1/2 cells. Furthermore, hUCB-MSC CM significantly increased survival of osteocyte-like MLO-Y4 cells, while it inhibited osteoclastic differentiation. To summarize, transplantation of hUCB-MSCs could effectively prevent OVX-mediated bone loss in nude mice, which appears to be mediated by a paracrine mechanism rather than direct engraftment of the MSCs.