ROLE OF HYPOXIA IN DIFFERENTIATION

描述（由申请人提供）：细胞对低氧（缺氧）的适应是一个重要的生物学问题，不仅与癌症和缺血性疾病等病理状况有关，而且与正常胎儿发育和细胞分化有关。我们的研究表明，软骨细胞是一个很好的模型来学习细胞如何适应，并在低氧环境中分化。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骼取代。我们最近已经证明，胎儿生长板有一个向外的氧合梯度与中央，缺氧区域。此外，我们发现转录因子缺氧诱导因子-1a （Hif-1a）是细胞适应缺氧的主要介质，控制软骨内骨发育的关键步骤。事实上，到目前为止，我们的工作表明，Hif-1a的作用对软骨细胞的存活和分化的调节至关重要；这次更新，将努力理解Hif-1a的这些重要作用。缺乏Hif-1a的软骨细胞发生大量细胞死亡，特别是在发育中的生长板中心。因此，Hif-1a是一种存活因子，至少对于软骨细胞等间充质细胞是如此。值得注意的是，在胎儿生长板中，这种“中心细胞死亡表型”通过血管内皮生长因子A （VEGF）的遗传消融来模拟，VEGF是Hif-1a的直接下游靶点。为了开始剖析Hif-1a作为存活因子下游的分子机制，在本拨款申请的Aim I中，我们将研究VEGF是否能够至少部分地挽救在Hif-1a缺乏的胎儿生长板中观察到的细胞活力丧失，从而我们将进一步了解VEGF作为Hif-1a在胎儿生长板中存活功能的介质之一的作用。对软骨中Hif-1a的体内功能缺失模型的详细分析提供了强有力的证据，表明该转录因子在控制间充质细胞向软骨细胞分化方面也具有非冗余功能，因为肢体芽间充质中缺乏Hif-1a会大大延迟软骨原基的形成。此外，初步数据显示，缺氧和Hif-1a调节软骨细胞分化的终末阶段。总的来说，这些发现表明，低氧张力远不是有害的，而是通过上调Hif-1a转录活性来形成软骨的“必需”。在Aim II中，我们将研究Hif-1a在软骨细胞分化中的重要作用，并验证缺氧和Hif-1a调节脯氨酸-4-羟化酶II的表达的假设，脯氨酸-4-羟化酶II控制软骨细胞中胶原的翻译后羟基化。公共卫生相关性。如果成功，这一建议将显著推进我们对细胞适应缺氧和间充质细胞向软骨细胞分化过程的分子机制的认识。因此，它在器官发生和间充质干细胞生物学中都具有很高的相关性。

DESCRIPTION (provided by applicant): Cellular adaptation to low oxygen (hypoxia) is an important biological problem not only in relation to pathological conditions such as cancer and ischemic diseases, but also in normal fetal development and in cell differentiation. Our studies have shown that chondrocytes are an excellent model to learn how cells adapt to, and differentiate in a low oxygen environment. The fetal growth plate is a unique mesenchymal tissue since it is avascular, though it requires the angiogenic switch in order to be replaced by bone. We have recently demonstrated that the fetal growth plate has an out-in gradient of oxygenation with a central, hypoxic region. Moreover, we have discovered that the transcription factor Hypoxia-inducible factor-1a (Hif-1a), which is a major mediator of the cellular adaptation to hypoxia, controls critical steps of endochondral bone development. Our work thus far, in fact, suggests that the actions of Hif-1a are central to the regulation of survival and differentiation of chondrocytes; this renewal, will endeavor to understand these vital roles of Hif-1a. Chondrocytes lacking Hif-1a undergo massive cell death, particularly in the center of the developing growth plate. Hif-1a is thus a survival factor, at least for cells of mesenchymal origin such as chondrocytes. Notably, in the fetal growth plate this "central cell death phenotype" is mimicked by genetic ablation of Vascular Endothelial Growth Factor A (VEGF), a direct downstream target of Hif-1a. To start dissecting out the molecular mechanisms downstream of Hif-1a as a survival factor, in Aim I of this grant proposal, we will investigate whether VEGF is able to rescue, at least in part, the loss of cell viability observed in fetal growth plate deficient in Hif-1a, and we will thus gain further insights into the role of VEGF as one of the mediators of the survival function of Hif-1a in the fetal growth plate. Detailed analysis of an in vivo loss-of-function model of Hif-1a in cartilage has provided strong evidence that this transcription factor has also non-redundant functions in controlling differentiation of mesenchymal cells into chondrocytes, as lack of Hif-1a in limb bud mesenchyme considerably delays the formation of the cartilaginous primordia. In addition, preliminary data have shown that hypoxia and Hif-1a regulate terminal stages of chondrocyte differentiation. Collectively, these findings suggest that low oxygen tension, far from being detrimental, is 'required' for formation of cartilage, by up regulating Hif-1a transcriptional activity. In Aim II, we will study the essential role of Hif-1a in chondrocyte differentiation, and we will test the hypothesis that hypoxia and Hif-1a regulate expression of prolyl-4-hydroxylase II, which controls post-translational hydroxylation of collagens in chondrocytes. PUBLIC HEALTH RELEVANCE. If successful this proposal will significantly advance our knowledge of the molecular mechanisms underlying the cellular adaptation to hypoxia and the differentiation process of mesenchymal cells into chondrocytes. Thus, its relevance is high for both organogenesis and mesenchymal stem cell biology.