HIF-1alpha, a Survival and Differentiation Factor for Cartilage

描述（由申请人提供）：氧（O2）不仅是各种酶促反应（包括线粒体呼吸）中不可或缺的代谢底物，而且是控制转录因子缺氧诱导因子-1 α（HIF-1α）稳定性和活性的调节信号，HIF-1α是细胞适应低氧张力（缺氧）的关键介质。胎儿生长板是一种独特的间充质组织，因为它是无血管的，尽管它需要血管生成开关才能被骨取代。多年来，我们已经证明，与其无血管性，胎儿生长板有一个内部缺氧区。我们已经提供了遗传学证据表明HIF-1α是体内缺氧软骨细胞的存活因子。我们已经证明，肢芽间充质凝聚也是缺氧的，肢芽间充质中缺乏HIF-1α会延迟体内间充质细胞向软骨细胞的分化。在这项研究中，我们建议确定介导HIF-1α在体内软骨中作为存活和分化因子的分子机制。沿着这些路线，我们已经报道了在HIF-1α无效生长板和肢芽的HIF-1α无效间充质凝聚物周围的活的软骨细胞比对照组明显更缺氧。此外，我们提供了遗传学证据，表明HIF-1α无效细胞的极端缺氧不是生长板O2可用性降低的结果。因此，我们假设这是氧气消耗增加的结果。我们的假设与HIF-1α在体外损害线粒体呼吸的能力是一致的。基于这些发现，我们现在提出HIF-1α的一个关键功能是减少由于O2可用性有限而已经缺氧的细胞中的O2消耗，以防止它们变得几乎缺氧，这是一种与细胞存活和分化不相容的状态。具体来说，我们假设HIF-1α是缺氧软骨细胞的生存和缺氧间充质细胞及时分化为软骨细胞通过负调节线粒体呼吸，从而线粒体O2消耗是必不可少的。我们将通过在体内和体外抑制HIF-1α无效软骨细胞（特异性目的I）和肢芽的HIF-1α无效间充质细胞（特异性目的II）中的线粒体呼吸来验证我们的假设。 此外，我们将确定HIF-1α是否降低体外软骨细胞的O2消耗（特定目标III）。我们的研究结果可能会导致一个范式的转变，如果我们确定，不同于已报道的情况下，良好的氧合组织，线粒体呼吸损伤是生存和缺氧软骨细胞的早期分化阶段的一个不可或缺的要求。

DESCRIPTION (provided by applicant): Oxygen (O2) is not only an indispensable metabolic substrate in various enzymatic reactions including mitochondrial respiration, but also a regulatory signal that controls stability and activity of the transcription factor Hypoxia Inducibl Factor-1α (HIF-1α), a key mediator of the cellular adaptation to low O2 tension (hypoxia). The fetal growth plate is a unique mesenchymal tissue because it is avascular, albeit it requires the angiogenic switch in order to be replaced by bone. Over the years, we have demonstrated that, consistent with its avascularity, the fetal growth plate has an inner hypoxic region. We have provided genetic evidence that HIF-1α is a survival factor for hypoxic chondrocytes in vivo. We have shown that mesenchymal condensations of the limb bud are also hypoxic, and lack of HIF-1α in limb bud mesenchyme delays differentiation of mesenchymal cells into chondrocytes in vivo. In this grant, we propose to identify the molecular mechanisms that mediate the role of HIF-1α as a survival and differentiation factor in cartilage in vivo. Along these lines, we have reported that viable chondrocytes at the periphery of HIF-1α null growth plates and HIF-1α null mesenchymal condensations of the limb bud are considerably more hypoxic than controls. Moreover, we have provided genetic evidence that the extreme hypoxia of HIF-1α null cells is not the consequence of reduced availability of O2 to the growth plate. Therefore, we hypothesized it had to be the consequence of increased O2 consumption. Our hypothesis is in line with the well- documented ability of HIF-1α to impair mitochondrial respiration in vitro. Based on these findings, we now propose that a key function of HIF-1α is to reduce O2 consumption in cells that are already hypoxic because of limited availability of O2, in order to prevent them from becoming virtually anoxic, a status that is not compatible with cell survival and differentiation. Specifically, we hypothesize that HIF-1α is essential for survival of hypoxic chondrocytes and for timely differentiation of hypoxic mesenchymal cells into chondrocytes by negatively regulating mitochondrial respiration, and thus mitochondrial O2 consumption. We will test our hypothesis by inhibiting mitochondrial respiration in HIF-1α null chondrocytes (Specific Aim I) and in HIF-1α null mesenchymal cells of the limb bud (Specific Aim II) in vivo and in vitro. Moreover, we will establish whether HIF-1α lowers O2 consumption in chondrocytes in vitro (Specific Aim III). Our findings may lead to a paradigm shift if we determine that, differently fro what has been reported in the context of well-oxygenated tissues, impairment of mitochondrial respiration is an indispensable requirement for survival and for early differentiation stages of hypoxic chondrocytes.