Oxysterols Regulate Marrow Stromal Cell Differentiation

描述（由申请人提供）：骨质疏松性骨质流失在老龄化人群中引起显著的发病率和死亡率。刺激成骨细胞活动和骨形成的新型骨合成代谢剂对于改善骨质疏松症的干预是必要的。氧化甾醇是胆固醇氧化的自然产物，在体内和体外具有多种生物效应。我们之前的研究表明，特定的氧甾醇组合，即20(S)-羟胆固醇与22(R)-或22(S)-羟胆固醇，在体外诱导骨髓基质细胞（MSC）的成骨分化，单独或与外源性骨形态发生蛋白2（BMP2）协同。同时，这些成骨氧甾醇在体外抑制同一祖细胞的成脂分化，诱导Cbfal mRNA表达，并刺激Cbfal和SMAD DNA结合。此外，在体外培养的小鼠颅骨器官中，成骨氧甾醇刺激骨形成。我们提出的研究将通过实现以下具体目标，验证成骨氧甾醇通过调节谱系特异性分化的分子调节因子，对祖细胞间充质干细胞诱导新的促骨和抗脂肪作用的假设：1)通过考察氧甾醇对Cbfal、Osterix、SMAD、Msx-2等已知成骨分化调控因子和C/EBPa、PPARy、LXR等已知成脂分化调控因子的影响，阐明体外诱导祖细胞间充质干细胞成骨、抑制成脂分化的机制；2)通过评估氧甾醇对成骨转录因子（包括Cbfal和SMAD）及其协同相互作用的影响，以及对介导BMP2信号传导的信号转导分子的影响，研究氧甾醇对MSC对BMP2成骨反应的影响；3)通过组织形态学测量和微ct扫描测量骨形成的变化，研究氧甾醇对小鼠颅骨成骨和破骨分化以及骨形成的影响。这些研究将显著增强目前对成骨甾醇促进间充质干细胞成骨分化和骨形成机制的认识，并将为这些甾醇用于骨质疏松症合成代谢干预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.