Role of HIF-1 in Intervertebral Disc Function

描述（由申请人提供）：退行性椎间盘疾病与背痛和其他脊柱病变的产生密切相关，对于数百万美国人来说，这是衰老过程中的一个困难部分。在椎间盘中，髓核（NP）的细胞在独特的环境生态位中发挥作用，其特征在于非常有限的血管供应，这对细胞施加代谢约束和缺氧状态。我们已经表明，在NP，HIF和PHD形成一个独特的调节回路：PHD 2控制HIF-1的降解？即使在缺氧条件下，PHD 3也能促进其转录活性。这是我们第一次证明HIF-2a对PHD介导的降解不敏感。相反，HIF-1？同源物以细胞类型特异性方式差异控制NP中的PHD表达。直接相关的椎间盘疾病，我们的试点数据表明，HIF-PHD电路可以解耦的炎性细胞因子TNF-？IL-1？基于这些信息，我们建议研究如何HIF控制PHD功能在缺氧NP龛，PHDs需要分子O2作为其催化功能的底物。我们将确定HIF-1的差异敏感性的机制？同源物PHD介导的降解，以及阐明的身份和功能的辅因子招募到HIF-1？在一个PHD依赖的方式。为了将这些信息与体内状态联系起来，我们将有条件地删除HIF-1？在脊索以及纳塔尔后的NP中，并分析突变小鼠的细胞增殖、存活和分化的变化。接下来，我们将研究 TNF-？IL-1？解耦HIF-PHD回路并检验PHD 3介导细胞因子对NP细胞的作用的假设。最后，我们将测试PHD 3失活减缓炎症条件下体内椎间盘退行性变化进展的假设。相反，我们将确定PHD 3缺失小鼠中HIF-PHD回路的扰动是否影响椎间盘中的年龄依赖性变化。综上所述，所提出的研究将因此测试椎间盘研究中的新假设，即HIF-PHD调节网络在维持NP细胞存活和功能中是至关重要的，并且通过炎性细胞因子使该回路解耦可能加剧退行性椎间盘疾病。这些研究的结果将导致新的药理学干预措施的发展，旨在保持椎间盘的结构完整性和减轻炎症反应。

DESCRIPTION (provided by applicant): Degenerative disc disease is closely linked to the generation of back pain and other spinal pathologies, which for millions of Americans represents a difficult part of the aging process. In the intervertebral disc, the cells of the nucleus pulposu (NP) function in a unique environmental niche, characterized by a very limited vascular supply that imposes metabolic constraints and a hypoxic state on cells. We have shown that in the NP, HIF and PHD form a unique regulatory circuit: PHD2 controls degradation of HIF-1? even under hypoxia, while PHD3 promotes its transcriptional activity. For the first time, we have demonstrated that HIF-2a is insensitive to PHD mediated degradation. Conversely, HIF-1? homologues differentially control PHD expression in the NP in a cell type specific manner. Directly relevant to disc disease, our pilot data suggests that the HIF-PHD circuit can be decoupled by inflammatory cytokines TNF-? and IL-1?. Based on this information, we propose to investigate how HIF controls PHD function in the hypoxic NP niche, given that PHDs require molecular O2 as a substrate for their catalytic function. We will determine mechanisms underlying differential sensitivity of HIF-1? homologues to PHD mediated degradation as well as elucidating the identity and function of co-factors that are recruited to HIF-1? in a PHD dependent fashion. To relate this information to the in vivo state, we will conditionally delete HIF-1? in the notochord as well as post natal NP and analyze the mutant mice for changes in cell proliferation, survival and differentiation. Next, we will investigate the mechanisms by which TNF-? and IL-1? decouple the HIF-PHD circuit and test the hypothesis that PHD3 mediates the effects of cytokines on NP cells. Lastly, we will test the hypothesis that PHD3 inactivation slows the progression of degenerative changes in the disc in vivo under inflammatory condition. Conversely, we will ascertain if perturbation of the HIF-PHD circuit in PHD3 null mice influences age dependent changes in the disc. Taken together, the proposed studies will thus test a novel hypothesis in disc research, namely, the HIF-PHD regulatory network is critical in maintenance of NP cell survival and function and decoupling of this circuit by inflammatory cytokines may exacerbate degenerative disc disease. Outcomes from these studies will lead to the development of new pharmacological interventions aimed at maintaining the structural integrity of the disc and mitigating the inflammatory response.