Oxysterols Regulate Marrow Stromal Cell Differentiation

描述（由申请人提供）：骨质疏松性骨丢失导致老年人群的显著发病率和死亡率。刺激成骨细胞活性和骨形成的新型骨合成代谢剂对于改善骨质疏松症的干预是必要的。氧固醇是胆固醇氧化的天然产物，在体外和体内具有各种生物学效应。我们以前的研究表明，特定的氧化固醇组合，即20（S）-羟基胆固醇与22（R）-或22（S）-羟基胆固醇，诱导骨髓基质细胞（MSC）在体外成骨分化，单独和协同外源性骨形态发生蛋白2（BMP 2）。同时，这些成骨氧固醇在体外抑制相同祖细胞的成脂分化，诱导Cbfal mRNA表达，并刺激Cbfal和SMAD DNA结合。此外，成骨氧固醇刺激小鼠颅骨器官培养物中的骨形成。我们提出的研究将通过调节谱系特异性分化的分子调节剂，通过实现以下特定目的，测试成骨氧固醇对祖细胞MSC诱导新的促成骨和抗成脂作用的假设：1）阐明机制通过检测它们对已知的成骨调节因子的作用，在一些实施方案中，所述脂肪形成分化调节因子包括Cbfal、Osterix、SMAD和Msx-2，以及已知的脂肪形成分化调节因子包括C/EBPa、PPARy和LXR; 2）通过评估氧固醇对成骨转录因子（包括Cbfal和SMAD）及其协同相互作用的影响来检查氧固醇对MSC对BMP 2的成骨应答的影响，和对介导BMP 2信号传导的信号转导分子的影响;和3）检查氧甾醇对离体和体内小鼠颅骨中的成骨和成骨细胞分化以及骨形成的影响，如通过组织形态计量学和微CT扫描测量的骨形成的变化所确定的。这些研究应显着提高现有的知识的机制，通过成骨氧化固醇增强MSC成骨分化和骨形成，并将提供重要的新信息，这些氧化固醇的合成代谢干预骨质疏松症的潜在用途。

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.