Bone is continuously remodeled throughout life by a tightly coupled process involving absorption by osteoclasts and formation by osteoblasts. Dysregulation of this coupled remodeling can lead to diseases such as osteoporosis (18,19). The precursors of osteoblasts are pluripotent cells known as mesenchymal stem cells (MSCs) (3,7,36). MSCs are viewed as potential tools for therapeutic intervention in diseases related to impaired function of osteoblasts because they can be derived from bone marrow, manipulated in culture, and administered back to donor individuals (12,13,24,39,40). However, the mechanistic pathways that drive differentiation of MSCs along the osteoblast lineage are not completely understood. Therefore, elucidation of molecular mechanisms underlying osteogenesis not only is important for our understanding of bone development but also may advance strategies for bone repair. Targeting therapeutic molecules to bone in order to enhance the bone-forming activity of osteoblast precursors may aid in the treatment of bone disease.Osteoinductive factors are required to drive the lineagespecific differentiation of MSCs into osteoblastic cells in culture. Osteoblast differentiation is influenced by multiple signaling pathways, including transforming growth factor ␤1, Hedgehog, Wnt, fibroblast growth factors, insulin-like growth factor 1, and bone morphogenetic proteins (BMPs) (8,20,25,46,49). Strategies employing BMPs have been successfully used with animals and humans to regenerate bones (16,22,27). However, the high cost and supraphysiologic doses of BMPs necessary to achieve osteoinductive activity illustrate the need for additional strategies for the stimulation of osteoblast differentiation and bone formation in vivo (23,32,48). Recent studies of zebrafish have validated the concept of employing small molecules to modulate BMP activity in vivo (50). Similarly, certain oxysterols have been shown to activate sonic hedgehog (SHH) and to stimulate osteoblastic differentiation and bone formation (1, 15). However, smallmolecule BMP stimulators that are able to bypass the need for high doses of BMP and induce bone formation remain to be identified.Here we show that the small molecule phenamil, a derivative of the diuretic amiloride, induces osteoblastic differentiation and mineralization of mouse MSCs. Phenamil and BMPs show additive effects on the expression of BMP target genes, osteogenic markers, and matrix mineralization in M2-10B4 (M2) MSCs as well as in calvarial organ cultures. We show that phenamil acts, at least in part, by inducing the expression of tribbles homolog 3 (Trb3), a previously identified positive regulator of BMP signaling (9,33,51). We further show that phenamil reduces the protein level of SMAD ubiquitin regulatory factor 1 (Smurf1) and induces expression of SMAD, the critical transcription factor in BMP signaling. These results suggest that phenamil or related small molecules may represent a novel strategy for increasing BMP activity in the clinical setting.

Specific oxysterols have been shown to be pro-osteogenic and anti-adipogenic. However, the molecular mechanism(s) by which oxysterols inhibit adipogenic differentiation is unknown. We show that the anti-adipogenic effects of osteogenic oxysterol, 20(S)-hydroxycholesterol, are mediated through a hedgehogdependent mechanism(s) and are associated with inhibition of PPAR␥ expression.Introduction: Multipotent bone marrow stromal cells (MSCs) are common progenitors of osteoblasts and adipocytes. A reciprocal relationship between osteogenic and adipogenic differentiation may explain the increased adipocyte and decreased osteoblast formation in aging and osteoporosis. We have previously reported that specific oxysterols stimulate osteogenic differentiation of MSCs while inhibiting their adipogenic differentiation. Materials and Methods:The M2-10B4 (M2) murine pluripotent bone MSC line was used to assess the inhibitory effects of 20(S)-hydroxycholesterol (20S) and sonic hedgehog (Shh) on peroxisome proliferatoractivated receptor ␥ (PPAR␥) and adipogenic differentiation. All results were analyzed for statistical significance using ANOVA. Results and Conclusions: Treatment of M2 cells with the osteogenic oxysterol 20S completely inhibited adipocyte formation induced by troglitazone after 10 days. PPAR␥ mRNA expression assessed by RT-qPCR was significantly induced by Tro after 48 (5-fold) and 96 h (130-fold), and this induction was completely inhibited by 20S. In contrast, 20S did not inhibit PPAR␥ transcriptional activity in M2 cells overexpressing PPAR␥ and retinoid X receptor (RXR). To elucidate the molecular mechanism(s) by which 20S inhibits PPAR␥ expression and adipogenic differentiation, we focused on the hedgehog signaling pathway, which we previously showed to be the mediator of osteogenic responses to oxysterols. The hedgehog signaling inhibitor, cyclopamine, reversed the inhibitory effects of 20S and Shh on troglitazone-induced adipocyte formation in 10-day cultures of M2 cells by 70% and 100%, respectively, and the inhibitory effect of 20S and Shh on troglitazone-induced PPAR␥ expression was fully reversed at 48 h by cyclopamine. Furthermore, 20S and Shh greatly inhibited PPAR␥2 promoter activity induced by CCAAT/enhancer-binding protein ␣ overexpression. These studies show that, similar to the induction of osteogenesis, the inhibition of adipogenesis in murine MSCs by the osteogenic oxysterol, 20S, is mediated through a hedgehog-dependent mechanism(s).

By using molecular methods for the identification and quantification of methanogenic archaea in adult chicken ceca, 16S rRNA genes of 11 different phylotypes, 10 of which were 99% similar to Methanobrevibacter woesei, were found. Methanogen populations, as assessed by cultivation, and the 16S rRNA copy number were between 6.38 and 8.23 cells/g (wet weight) and 5.50 and 7.19 log 10 /g (wet weight), respectively.

We previously reported that specific oxysterols stimulate osteogenic differentiation of pluripotent bone marrow stromal cells (MSCs) through activation of hedgehog (Hh) signaling and may serve as potential future therapies for intervention in osteopenia and osteoporosis. In this study we report that the osteogenic oxysterol 20(S)-hydroxycholesterol (20S) induces the expression of genes associated with Notch signaling. Using M2-10B4 (M2) MSCs, we found that 20S significantly induced HES-1, HEY-1, and HEY-2 mRNA expression compared with untreated cells, with maximal induction after 48 hours, whereas the nonosteogenic oxysterols did not. Similar observations were made when M2 cells were treated with sonic hedgehog (Shh), and the specific Hh pathway inhibitor cyclopamine blocked 20S-induced Notch target gene expression. 20S did not induce Notch target genes in Smo−/− mouse embryonic fibroblasts, further confirming the role of Hh signaling in 20S-induced expression of Notch target genes. Despite the inability of liver X-receptor (LXR) synthetic ligand TO901317 to induce Notch target genes in M2 cells, LXR knockdown studies using siRNA showed inhibition of 20S-induced HEY-1 but not HES-1 expression, suggesting the partial role of LXR signaling in MSC responses to 20S. Moreover, 20S-induced Notch target gene expression was independent of canonical Notch signaling because neither 20S nor Shh induced CBF1 luciferase reporter activity or NICD protein accumulation in the nucleus, which are hallmarks of canonical Notch signaling activation. Finally, HES-1 and HEY-1 siRNA transfection significantly inhibited 20S-induced osteogenic genes, suggesting that the pro-osteogenic effects of 20S are regulated in part by HES-1 and HEY-1. © 2010 American Society for Bone and Mineral Research

Hedgehog (Hh) signaling is indispensable in embryonic development, and its dysregulated activity results in severe developmental disorders as shown by genetic models of naturally occurring mutations in animal and human pathologies. Hh signaling also functions in postembryonic development and adult tissue homeostasis, and its aberrant activity causes various human cancers. Better understanding of molecular regulators of Hh signaling is of fundamental importance in finding new strategies for pathway modulation. Here, we identify liver X receptors (LXRs), members of the nuclear hormone receptor family, as previously unrecognized negative regulators of Hh signaling. Activation of LXR by specific pharmacological ligands, TO901317 and GW3965, inhibited the responses of pluripotent bone marrow stromal cells and calvaria organ cultures to sonic Hh, resulting in the inhibition of expression of Hh-target genes, Gli1 and Patched1, and Gli-dependent transcriptional activity. Moreover, LXR ligands inhibited sonic Hh-induced differentiation of bone marrow stromal cells into osteoblasts. Elimination of LXRs by small interfering RNA inhibited ligand-induced inhibition of Hh target gene expression. Furthermore, LXR ligand did not inhibit Hh responsiveness in mouse embryonic fibroblasts that do not express LXRs, whereas introduction of LXR into these cells reestablished the inhibitory effects. Daily oral administration of TO901317 to mice after 3 d significantly inhibited baseline Hh target-gene expression in liver, lung, and spleen. Given the importance of modulating Hh signaling in various physiological and pathological settings, our findings suggest that pharmacological targeting of LXRs may be a novel strategy for Hh pathway modulation.