Oxysterols Regulate Marrow Stromal Cell Differentiation

描述(由申请人提供)：骨质疏松性骨丢失在老龄人口中会导致严重的发病率和死亡率。刺激成骨细胞活性和骨形成的新型骨合成代谢药物对于改善对骨质疏松症的干预是必要的。氧化甾醇是胆固醇氧化的天然产物，在体内外具有多种生物效应。我们以前的研究表明，特定的氧固醇组合，即20(S)-羟基胆固醇与22(R)-或22(S)-羟基胆固醇在体外单独或与外源性骨形态发生蛋白2协同诱导骨髓基质细胞成骨分化。同时，这些成骨的氧化甾醇在体外抑制同一祖细胞的成脂分化，诱导Cbfal基因的表达，并刺激Cbfal和SmadDNA结合。此外，成骨氧固醇还能促进体外培养的小鼠颅骨器官的骨形成。我们的研究将验证这一假说，即成骨性氧化甾醇通过调节谱系特异性分化的分子调节因子而对前体MSC诱导新的促成骨和抗成脂作用，从而达到以下特定目的：1)通过检测已知的成骨分化调节因子Cbfal、Osterix、SMAD和MSX-2以及已知的成脂分化调节因子C/EBPA、PARy和LXR，阐明氧化甾醇在体外诱导前体MSC成骨和抑制成脂分化的机制(S)；2)通过评估氧化甾醇对成骨转录因子(包括Cbfal和SMAD)及其协同作用的影响，以及对介导BMP2信号的信号转导分子的影响，检测氧化甾醇对MSC对BMP2成骨反应的影响；以及3)通过组织形态计量学和显微CT扫描检测氧化甾醇对小鼠颅盖骨的成骨和破骨分化以及体内成骨的影响。这些研究将显著提高目前对成骨氧合固醇促进MSC成骨分化和骨形成的机制的认识，并将为这些氧化甾醇在骨质疏松症的合成代谢干预中的潜在应用提供重要的新信息。

DESCRIPTION (provided by applicant): Osteoporotic bone loss causes significant morbidity and mortality in the aging population. Novel bone anabolic agents that stimulate osteoblast activity and bone formation are necessary for improved interventions in osteoporosis. Oxysterols are naturally occurring products of cholesterol oxidation with various biologic effects in vitro and in vivo. Our previous studies demonstrated that specific oxysterol combinations, namely 20(S)-hydroxycholesterol with either 22(R)- or 22(S)-hydroxycholesterol, induced the osteogenic differentiation of bone marrow stromal cells (MSC) in vitro, alone and in synergy with exogenous bone morphogenetic protein 2 (BMP2). In parallel, these osteogenic oxysterols inhibited the adipogenic differentiation of the same progenitor cells in vitro, induced Cbfal mRNA expression, and stimulated Cbfal and SMAD DNA binding. Moreover, the osteogenic oxysterols stimulated bone formation in mouse calvaria organ cultures ex vivo. Our proposed studies will test the hypothesis that osteogenic oxysterols induce novel pro-osteogenic and antiadipogenic effects on progenitor MSC by modulating molecular regulators of lineage-specific differentiation, by achieving the following Specific Aims: 1) Elucidate the mechanism(s) by which oxysterols induce the osteogenic and inhibit the adipogenic differentiation of progenitor MSC in vitro by examining their effects on known regulators of osteogenic differentiation, including, Cbfal, Osterix, SMAD, and Msx-2, and known regulators of adipogenic differentiation including C/EBPa, PPARy, and LXR; 2) Examine the effects of oxysterols on osteogenic response of MSC to BMP2 by assessing their effects on osteogenic transcription factors, including Cbfal and SMAD and on their cooperative interactions, and on signal transduction molecules that mediate BMP2 signaling; and 3) Examine the effects of oxysterols on osteogenic and osteoclastic differentiation and bone formation in mouse calvaria ex vivo and in vivo, as determined by changes in bone formation measured by histomorphometry and micro-CT scanning. These studies should significantly enhance current knowledge of the mechanisms by which osteogenic oxysterols enhance MSC osteogenic differentiation and bone formation, and will provide important new information relevant to the potential use of these oxysterols for anabolic interventions in osteoporosis.