Osteoblast-targeted overexpression of PPARγ inhibited bone mass gain in male mice and accelerated ovariectomy-induced bone loss in female mice.

PPARγ has critical role in the differentiation of mesenchymal stem cells into adipocytes while suppressing osteoblastic differentiation. We generated transgenic mice that overexpress PPARγ specifically in osteoblasts under the control of a 2.3-kb procollagen type 1 promoter (Col.1-PPARγ). Bone mineral density (BMD) of 6- to 14-week-old Col.1 - PPARγ male mice was 8% to 10% lower than that of their wild-type littermates, whereas no difference was noticed in Col.1-PPARγ female mice. Col.1-PPARγ 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-PPARγ male mice compared with their wild-type littermates. Interestingly, osteoclast number and surface also were decreased by 40% and 58%, respectively, in Col.1-PPARγ 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-PPARγ male mice, and OPG/RANKL ratio was reduced in stromal cells from Col.1-PPARγ male mice. Although there was no significant difference in BMD in Col.1-PPARγ female mice up to 30 weeks, bone loss was accelerated after ovariectomy compared with wild-type female mice (-3.9% versus -6.8% at 12 weeks after ovariectomy, p < .01), indicating that the effects of PPARγ overexpression becomes more evident in an estrogen-deprived state in female mice. In conclusion, in vivo osteoblast-specific overexpression of PPARγ negatively regulates bone mass in male mice and accelerates estrogen-deficiency-related bone loss in female mice.

Wnt inhibitory factor (WIF)-1 inhibits osteoblastic differentiation in mouse embryonic mesenchymal cells.

Wnt inhibitory factor (WIF)-1 belongs to the members of secreted modulators of Wnt proteins. Secreted frizzled-related proteins (sFRPs), another member of Wnt modulators, have been shown to play differential roles in Wnt signaling depending on the subtypes and cell models. This study was undertaken to investigate the functional role of WIF-1 in osteoblastic differentiation of mouse mesenchymal C3H10T1/2 cells. C3H10T1/2 cells express endogenous WIF-1 and its expression level decreases during osteoblastogenesis. Treatment of C3H10T1/2 cells with WIF-1 significantly reduced alkaline phosphatase (ALP) activities induced by either osteogenic medium (OM, ascorbic acid and beta-glycerophosphate) or Wnt-3a conditioned medium (CM) in a dose-dependent manner. In contrast, the expression level of endogenous WIF-1 increased during adipogenesis and WIF-1 treatment resulted in increased adipogenesis. C3H10T1/2 cells transduced with WIF-1 retrovirus also exhibited reduced ALP activity and decreased mRNA expression of Runx2, collagen type 1, ALP and osteocalcin during osteoblastic differentiation compared to empty virus-transduced cells. Moreover, treatment with WIF-1 dose-dependently attenuates beta-catenin/T-cell factor (TCF) transcriptional activity in this cell line. Finally, knockdown of WIF-1 in C3H10T1/2 cells by RNA interference leads to increase in ALP activities. Collectively, these results indicate that WIF-1 plays as a negative regulator of osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells in vitro.

Differential effects of secreted frizzled-related proteins (sFRPs) on osteoblastic differentiation of mouse mesenchymal cells and apoptosis of osteoblasts.

Secreted frizzled-related proteins (sFRPs) are modulators of Wnt signaling. This study was undertaken for definitive assessment of contribution of different sFRPs in osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. Treatment of C3H10T1/2 cells with sFRP-2 at concentrations of 10, 50, and 100nM and sFRP-4 at low concentrations (5nM) significantly increased Wnt-3A-induced alkaline phosphatase (ALP) activities, whereas sFRP-1 or 3 did not. Retroviral transduction of the sFRP-2 but not other sFRPs also significantly enhanced ALP activity induced by beta-glycerophosphate and ascorbic acid. Furthermore, transfection of all the sFRP expression vectors significantly increased beta-catenin/TCF reporter activity and the effects were most prominent with sFRP-2 and -4. In osteoblast apoptosis assay, only sFRP-3 increased etoposide-induced apoptosis in MC3T3-E1 mouse osteoblasts. In conclusion, we found that different repertoires of sFRPs exert differential effects on osteoblastic differentiation of mouse mesenchymal cells and cellular apoptosis of mouse osteoblasts in vitro.

CCAAT/enhancer-binding protein delta activates the Runx2-mediated transcription of mouse osteocalcin II promoter.

CCAAT/enhancer-binding proteins (C/EBPs) are involved in the regulation of cell proliferation, differentiation, and control of metabolic function. Although the roles of C/EBPs in osteoblasts are largely unknown, both C/EBPbeta and -delta have been shown to enhance rat osteocalcin promoter activity through the synergistic activation of Runx2 at the C/EBP element. Here we show that in the mouse, C/EBPdelta increases the expression of osteocalcin whereas C/EBPbeta does not. This increased expression was found to occur at the transcriptional level, as demonstrated by the increased transcriptional activity from mouse osteocalcin II (OG2) promoter by C/EBPdelta. Although we found three putative C/EBP sites in the -637/+/-34 region of the OG2 promoter, none of these sites showed binding activity with in vitro translated C/EBP proteins. Notably, we show that C/EBPdelta physically interacts with Runx2 and that C/EBPdelta overexpression increases binding between the Runx2-C/EBPdelta complex and the OSE2 element, a critical osteoblast-specific cis-acting element in the OG2 promoter. Consistent with these DNA binding data, a mutation in OSE2 abrogated the stimulatory effect of C/EBPdelta on this promoter activity. Finally, chromatin immunoprecipitation analysis in MC3T3-E1 cells showed in vivo occupancy of the OG2 promoter by Runx2 and C/EBPdelta. In conclusion, C/EBPdelta was found to regulate mouse osteocalcin OG2 promoter activity indirectly by interacting with Runx2 in the context of the OSE2 element and this subsequently resulted in the cooperative activation of the OG2 promoter.

Retrovirus-mediated gene transfer of receptor activator of nuclear factor-kappaB-Fc prevents bone loss in ovariectomized mice.

Postmenopausal osteoporosis is characterized by increased bone resorption due to estrogen deficiency. Receptor activator of nuclear factor-kappaB-Fc (RANK-Fc), a fusion protein that specifically blocks receptor activator of nuclear factor ligand binding to RANK, has been known to be efficient and well tolerated in animal models of osteoporosis. Here we show that cell-based gene therapy with RANK-Fc effectively prevented bone loss in ovariectomized (OVX) mice. Thirty-one young adult female C57Bl/6 mice were used, and repeated intraperitoneal injection of mesenchymal stem cells (MSCs) transduced with retrovirus was performed as follows: 1) Sham-operated mice (n = 8); 2) OVX mice treated with phosphate-buffered saline (OVX-PBS; n = 8); 3) OVX mice injected with MSCs transduced with control retrovirus (OVX-green fluorescent protein [GFP]; n = 7); and 4) OVX mice injected with MSCs transduced with RANK-Fc (OVX-RANK-Fc; n = 8). Cellular expression of RANK-Fc was confirmed by Western blot analysis of cell lysates and conditioned medium and also by enzyme-linked immunosorbent assay for the mice serum. Measurement of bone mineral density (BMD) by dual-energy x-ray absorptiometry (PIXImus) revealed that the OVX-RANK-Fc group gained significantly higher BMD than either the OVX-PBS group or OVX-GFP group after 8 weeks. The expression of GFP, which is coexpressed with RANK-Fc, was detected by polymerase chain reaction analysis of DNA isolated from femur and intra-abdominal fat, whereas no GFP signal was identified in liver, brain, heart, lung, or bone marrow aspirates. These suggest that expression of RANK-Fc by genetically modified MSCs may be a feasible option for the prevention of bone loss induced by ovariectomy.

Dominant negative N-cadherin inhibits osteoclast differentiation by interfering with beta-catenin regulation of RANKL, independent of cell-cell adhesion.

UNLABELLED: We studied the effects of dominant negative N-cadherin (NCadDeltaC) expression in ST2 cells on their ability to support osteoclastogenesis. Expression of NCadDeltaC in ST2 cells did not decrease cell-to-cell adhesion but significantly reduced osteoclast formation when co-cultured with BMMs. NCadDeltaC inhibited beta-catenin/TCF signaling, resulting in decreased RANKL expression, which could contribute to the reduced osteoclast formation. INTRODUCTION: Cadherin is a calcium-dependent cell adhesion molecule that plays major roles during embryonic development and morphogenesis. Classic cadherins interact with beta-catenin, which is also involved in the Wnt signaling pathway. We tested whether disruption of N-cadherin function in stromal cells by dominant negative N-cadherin affects their ability to support osteoclastogenesis by altering heterotypic interaction with osteoclast precursors. MATERIALS AND METHODS: ST2 cells were transduced with retrovirus encoding extracellular domain-truncated, dominant negative N-cadherin (NCadDeltaC) and co-cultured with bone marrow macrophages (BMMs) to study the ability to support osteoclastogenesis. As a downstream target of NCadDeltaC, beta-catenin/T-cell factor (TCF) transcriptional activity was analyzed using TOPflash reporter construct. Real-time RT-PCR analysis and RANKL-luciferase reporter assays were performed to study the effects of NCadDeltaC on the osteoprotegerin (OPG)/RANKL system. RESULTS: Immunoblotting analysis showed that primary bone marrow stromal cells, ST2 cells, and BMMs expressed N-cadherin. Retroviral expression of NCadDeltaC in ST2 cells did not significantly inhibit cell adhesion but markedly impaired the formation of TRACP(+) osteoclasts (>40%) when co-cultured with BMMs. However, the inhibition of osteoclastogenesis was not reproduced by neutralizing antibody against N-cadherin. Expression of NCadDeltaC, however, strongly suppressed beta-catenin/TCF transcriptional activity in ST2 cells, which was rescued by constitutively active beta-catenin adenovirus (Ad DeltaN46 beta-catenin) or constitutively active TCF mutant (pCS2-VP16DeltabetaXTCF-3). As a potential downstream target of Wnt signaling, we found that the expression of RANKL was reduced in ST2 cells expressing NCadDeltaC. Moreover, Wnt-3A, Ad DeltaN46 beta-catenin, and VP16DeltabetaXTCF-3 increased the expression of RANKL and enhanced the transcriptional activity of mouse RANKL promoter in ST2 cells. CONCLUSIONS: Our data suggest that expression of dominant negative N-cadherin in ST2 cells suppressed osteoclastogenesis by interfering with beta-catenin regulation of RANKL independent of cell-cell adhesion.

Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells.

Ghrelin is a 28-amino-acid peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) that strongly stimulates the release of growth hormone at the hypothalamus and pituitary level. Although GHS-Rs are expressed in a variety of peripheral tissues, little is known about its effect on bone independent of GH/IGF-1 axis. This study was undertaken to investigate whether ghrelin exerts a direct effect on osteoblasts. We identified mRNA and protein expression of GHS-R in primary osteoblasts as well as a number of osteoblastic cell lines, including MC3T3-E1, ROS 17/2.8, UMR-106, MG63, and SaOS2 cells. Treatment of ghrelin (10(-11) to 10(-7) M) to MC3T3-E1 cells showed dose-dependent stimulation of proliferation, which was abrogated by treatment with [d-Lys]-GHRP-6 (10(-3) M), a selective antagonist of the ghrelin receptor. Ghrelin activated ERK1/2 MAPK and pretreatment with MAPK kinase inhibitors, PD98059 attenuated the ghrelin-induced cell proliferation. Ghrelin also inhibited TNFalpha-induced apoptosis and suppressed caspase-3 activation that occurs in response to TNFalpha as well as during in vitro differentiation process. Moreover, ghrelin treatment enhanced in vitro osteoblast differentiation as evidenced by matrix mineralization, alkaline phosphatase activity, and osteoblast-specific gene expression. These results suggest that ghrelin promotes proliferation and differentiation and inhibits apoptosis of osteoblasts.

Ectopic overexpression of adipogenic transcription factors induces transdifferentiation of MC3T3-E1 osteoblasts.

Osteoblasts and adipocytes originate from common mesenchymal progenitor cells. We have investigated whether mouse osteoblastic MC3T3-E1 cells can be induced to transdifferentiate into mature adipocytes by the ectopic expression of adipogenic transcription factors, PPARgamma, C/EBPalpha, or both. Retrovirus-mediated overexpression of PPARgamma alone or both PPARgamma and C/EBPalpha resulted in reduced alkaline phosphatase activity and osteoblast-specific gene expression. Moreover, foci of adipocytes were identified in conditions favoring osteoblastic maturation. Upon treatment with insulin, dexamethasone, and IBMX, cells overexpressing PPARgamma alone or both PPARgamma and C/EBPalpha showed marked transdifferentiation to mature adipocytes expressing molecular markers of adipocytes. Cells expressing both PPARgamma and C/EBPalpha showed more robust phenotype of adipocytes than the cells expressing PPARgamma alone. Overexpression of C/EBPalpha alone did not result in adipogenesis. These results suggest that PPARgamma is a key molecular switch for the transdifferentiation to adipocytes whereas C/EBPalpha may differentiate MC3T3-E1 cells into osteoblasts and adipocytes.

Nordihydroguaiaretic acid, an antioxidant, inhibits transforming growth factor-beta activity through the inhibition of Smad signaling pathway.

Transforming growth factor-beta (TGF-beta) and its family are potent and multi-functional cytokines that affect various fundamental biological events. TGF-beta has a unique signaling pathway that is carried by Smad family, and many recent studies showed the extensive crosstalk between Smad pathway and other signaling pathway. There were also clear evidences for the involvement of oxidative events in TGF-beta signaling pathway. To elucidate the role of oxidative events in carrying TGF-beta signals, we examined the effect of various antioxidants on TGF-beta activities in osteoblastic cell line. Among the examined compounds, we found nordihydroguaiaretic acid (NDGA) has a unique and strong inhibitory effect on various TGF-beta activities. Since the majority of TGF-beta activities are mediated by Smad, we questioned whether NDGA blocks the Smad signaling pathway. The result showed that NDGA inhibits the translocation of Smad2 to the nucleus. Further study revealed the strong inhibitory effect of NDGA on the phosphorylation of Smad2. This result may be important for designing chemical modulators of TGF-beta and its family related events and may provide new insights into the action mechanism of antioxidant.