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

描述(申请人提供)：氧(O2)不仅是包括线粒体呼吸在内的各种酶反应中不可或缺的代谢底物，而且是控制转录因子低氧诱导因子-1α(HIF-1α)稳定性和活性的调节信号，HIF-1是细胞适应低氧压力(低氧)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，与胎儿生长板的无血管一致，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，证明HIF-1α是体内缺氧软骨细胞的生存因子。我们发现肢芽的间充质凝聚也是低氧的，在活体内，肢芽间充质中缺乏HIF-1α会延迟间充质细胞向软骨细胞的分化。在这项资助中，我们建议确定在体内介导缺氧诱导因子-1α作为软骨存活和分化因子的分子机制。沿着这些思路，我们已经报道，在肢体芽的HIF-1α缺失生长板和HIF-1α缺失的间充质凝聚体的周围，存活的软骨细胞比对照组低得多。此外，我们还提供了遗传学证据，证明HIF-1α缺失细胞的极度缺氧不是由于生长板中氧气供应减少的结果。因此，我们假设这一定是氧气消耗量增加的结果。我们的假设与HIF-1α在体外损害线粒体呼吸的已知能力是一致的。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的可获得性有限而已经处于缺氧状态的细胞的O2消耗，以防止它们变得几乎缺氧，这种状态与细胞的生存和分化不相容。具体地说，我们假设缺氧诱导因子-1α对缺氧软骨细胞的存活以及缺氧间充质细胞通过负性调节线粒体呼吸、从而线粒体耗氧而及时分化为软骨细胞是必不可少的。我们将在体内和体外通过抑制HIF-1α缺失的软骨细胞(特异性目标I)和HIF-1α缺失的肢芽间充质细胞(特异性目标II)的线粒体呼吸来验证我们的假设。 此外，我们将确定HIF-1α是否能降低体外培养的软骨细胞的耗氧量(特定目标III)。我们的发现可能会导致范式的转变，如果我们确定，与已报道的在氧气充足的组织背景下不同，线粒体呼吸障碍是缺氧软骨细胞生存和早期分化阶段不可或缺的条件。

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.