Peroxisome proliferator-activated receptor-gamma (PPARgamma) regulates both glucose metabolism and bone mass. Recent evidence suggests that the therapeutic modulation of PPARgamma activity with antidiabetic thiazolidinediones elicits unwanted effects on bone. In this study, the effects of rosiglitazone on the skeleton of growing (1 month), adult (6 month), and aged (24 month) C57BL/6 mice were determined. Aging was identified as a confounding factor for rosiglitazone-induced bone loss that correlated with the increased expression of PPARgamma in bone marrow mesenchymal stem cells. The bone of young growing mice was least affected, although a significant decrease in bone formation rate was noted. In both adult and aged animals, bone volume was significantly decreased by rosiglitazone. In adult animals, bone loss correlated with attenuated bone formation, whereas in aged animals, bone loss was associated with increased osteoclastogenesis, mediated by increased receptor activator of nuclear factor-kappaB ligand (RANKL) expression. PPARgamma activation led to changes in marrow structure and function such as a decrease in osteoblast number, an increase in marrow fat cells, an increase in osteoclast number, and a loss of the multipotential character of marrow mesenchymal stem cells. In conclusion, rosiglitazone induces changes in bone reminiscent of aged bone and appears to induce bone loss by altering the phenotype of marrow mesenchymal stem cells.

Rosiglitazone (Rosi), a member of the thiazolidinedione class of drugs used to treat type 2 diabetes, activates the adipocyte-specific transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). This activation causes bone loss in animals and humans, at least in part due to suppression of osteoblast differentiation from marrow mesenchymal stem cells (MSC). In order to identify mechanisms by which PPARγ2 suppresses osteoblastogenesis and promotes adipogenesis in MSC, we have analyzed the PPARγ2 transcriptome in response to Rosi. A total of 4,252 transcriptional changes resulted when Rosi (1 μM) was applied to the U-33 marrow stromal cell line stably transfected with PPARγ2 (U-33/γ2) as compared to non-induced U-33/γ2 cells. Differences between U-33/γ2 and U-33 cells stably transfected with empty vector (U-33/c) comprised 7,928 transcriptional changes, independent of Rosi. Cell type-, time- and treatment-specific gene clustering uncovered distinct patterns of PPARγ2 transcriptional control of MSC lineage commitment. The earliest changes accompanying Rosi activation of PPARγ2 included effects on Wnt, TGFβ/BMP and G-protein signaling activities, as well as sustained induction of adipocyte-specific gene expression and lipid metabolism. While suppression of osteoblast phenotype is initiated by a diminished expression of osteoblast-specific signaling pathways, induction of the adipocyte phenotype is initiated by adipocyte-specific transcriptional regulators. This indicates that distinct mechanisms govern the repression of osteogenesis and the stimulation of adipogenesis. The co-expression patterns found here indicate that PPARγ2 has a dominant role in controlling osteoblast differentiation and suggests numerous gene-gene interactions that could lead to the identification of a “master” regulatory scheme directing this process.

Diet and nutritional status are critical factors that influences bone development. In this report we demonstrate that a mixture of phenolic acids found in the serum of young rats fed blueberries (BB) significantly stimulated osteoblast differentiation, resulting in significantly increased bone mass. Greater bone formation in BB diet-fed animals was associated with increases in osteoblast progenitors and osteoblast differentiation and reduced osteoclastogenesis. Blockade of p38 phosphorylation eliminated effects of BB on activation of Wnt signaling in preosteoblasts. Knocking down b-catenin expression also blocked the ability of serum from BB diet-fed rats to stimulate osteoblast differentiation in vitro. Based on our in vivo and in vitro data, we propose that the underlying mechanisms of these powerful bone-promoting effects occur through b-catenin activation and the nuclear accumulation and transactivation of TCF/LEF gene transcription in bone and in osteoblasts. These results indicate stimulation of molecular events leading to osteoblast differentiation triggered by P38 MAP kinase (MAPK)/b-catenin canonical Wnt signaling results in significant increases in bone growth in young rats consuming BB-supplemented diets. Liquid chromatography/mass spectrometry (LC/MS) characterization of the serum after BB feeding revealed a mixture of simple phenolic acids that may provide a basis for developing a new treatment to increase peak bone mass and delay degenerative bone disorders such as osteoporosis. ß

The mechanisms by which chronic ethanol intake induces bone loss remain unclear. In females, the skeletal response to ethanol varies depending on physiologic status (e.g., cycling, pregnancy, or lactation). Ethanol-induced oxidative stress appears to be a key event leading to skeletal toxicity. In this study, ethanol-containing liquid diets were fed to postlactational female Sprague-Dawley rats intragastrically for 4 weeks beginning at weaning. Ethanol consumption decreased bone mineral density (BMD) compared with control animals during this period of bone rebuilding following the end of lactation. Coadministration of the antioxidant N-acetylcysteine (NAC) was able to block bone loss and downregulation of the bone-formation markers alkaline phosphatase and osteocalcin in serum and gene expression in bone. Real-time array analysis of total RNA isolated from bone tissue revealed that the majority of Wnt signaling components were downregulated by chronic ethanol infusion. Real-time PCR confirmed downregulated gene expression in a subset of the Wnt signaling components by ethanol. However, the Wnt antagonist DKK1 was upregulated by ethanol. The key canonical Wnt signaling molecule β-catenin protein expression was inhibited, while glycogen synthase kinase-3-β was dephosphorylated by ethanol in bone and preosteoblastic cells. These actions of ethanol were blocked by NAC. Ethanol treatment inactivated TCF/LEF gene transcription, eliminated β-catenin nuclear translocation in osteoblasts, and reciprocally suppressed osteoblastogenesis and enhanced adipogenesis. These effects of ethanol on lineage commitment of mesenchymal stem cells were eliminated by NAC pretreatment. These observations are consistent with the hypothesis that ethanol inhibits bone formation through stimulation of oxidative stress to suppress Wnt signaling. © 2010 American Society for Bone and Mineral Research.