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

摘要 氧（O2）不仅是各种酶促反应中不可或缺的代谢底物， 线粒体呼吸，但也是一个调节信号，控制稳定性和活动的！“#$转录因子 缺氧诱导因子-1），细胞适应低氧张力（缺氧）的关键介质。的 胎儿生长板是一种独特的间充质组织，因为它是无血管的，尽管它需要血管生成开关， 被骨头代替。多年来，我们已经证明，与其无血管性一致，胎儿 生长板具有内部缺氧区。我们已经提供了基因证据表明HIF-1是一种生存因子， 体内缺氧软骨细胞。我们已经证明肢芽的间充质凝聚也是缺氧的， 在体内，肢芽间充质中缺乏HIF-1 <$会延迟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-1作为生存因子的分子机制。 和分化因子。沿着这些线索，我们已经报道了在软骨细胞中存活的软骨细胞， HIF-1-null生长板的外周和肢芽的HIF-1-null间充质凝聚是 比对照组缺氧程度更高。此外，我们已经提供了遗传证据表明，极端缺氧 HIF-1基因缺失细胞的减少不是生长板中O2可用性降低的结果。所以我们 假设这一定是O2消耗增加的结果。我们的假设与- HIF-1在体外损害线粒体呼吸的能力。基于这些发现，我们现在提出 HIF-1 <$的一个关键功能是减少已经缺氧的细胞中的O2消耗， 氧气的可用性，以防止它们变得几乎缺氧，这种状态与细胞不相容 生存和分化。具体地说，我们假设HIF-1 <$是缺氧的生存所必需的。 软骨细胞和及时分化缺氧间充质细胞向软骨细胞的负 调节线粒体呼吸，从而调节线粒体O2消耗。我们将测试我们的假设， 抑制HIF-1缺失软骨细胞（特异性目的I）和HIF-1缺失间充质细胞中的线粒体呼吸 肢芽细胞（特异性目标II）在体内和体外。此外，我们将确定HIF-1是否降低O2 体外软骨细胞中的消耗（特定目标III）。我们的发现可能会导致范式转变，如果我们 确定，与在氧合良好的组织中报道的不同， 线粒体呼吸受损是存活和早期 缺氧软骨细胞的分化阶段。 $

ABSTRACT 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 !"#$transcription factor Hypoxia Inducible Factor-1¿ (HIF-1¿), 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-1¿ 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-1¿ 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-1¿ as a survival and differentiation factor in cartilage in vivo. Along these lines, we have reported that viable chondrocytes at the periphery of HIF-1¿ null growth plates and HIF-1¿ 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-1¿ 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-1¿ to impair mitochondrial respiration in vitro. Based on these findings, we now propose that a key function of HIF-1¿ 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-1¿ 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-1¿ null chondrocytes (Specific Aim I) and in HIF-1¿ null mesenchymal cells of the limb bud (Specific Aim II) in vivo and in vitro. Moreover, we will establish whether HIF-1¿ lowers O2 consumption in chondrocytes in vitro (Specific Aim III). Our findings may lead to a paradigm shift if we determine that, differently from 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. $