Role of HIF-1 in Intervertebral Disc Function

描述(申请人提供)：退行性腰椎间盘疾病与背部疼痛和其他脊柱疾病的产生密切相关，对数百万美国人来说，这是衰老过程中的一个困难部分。在椎间盘中，髓核(NP)的细胞在一个独特的环境中发挥作用，其特征是非常有限的血管供应，对细胞施加代谢限制和低氧状态。我们发现，在NP中，HIF和PHD形成了一个独特的调节回路：PHD2控制HIF-1？即使在低氧条件下，PHD3也促进其转录活性。我们首次证明了HIF-2a对PHD介导的降解不敏感。相反，HIF-1？同源物以一种细胞类型特异的方式在NP中差异控制PhD的表达。与椎间盘疾病直接相关，我们的试点数据表明，炎症细胞因子TNF-？？可以解偶联HIF-PHD回路。和IL-1？基于这些信息，我们建议研究HIF如何控制缺氧NP生态位中的PHD功能，因为PHD需要分子O2作为其催化功能的底物。我们将确定HIF-1差异敏感性的潜在机制。与PHD介导的降解同源，以及阐明招募到HIF-1的辅助因子的身份和功能？以一种依赖博士的方式。为了将这一信息与体内状态联系起来，我们将有条件地删除HIF-1？在脊索以及出生后的NP中分析突变小鼠细胞增殖、存活和分化的变化。接下来，我们将研究通过哪些机制 肿瘤坏死因子-？那么IL-1呢？解偶联HIF-PHD电路，并测试PHD3介导细胞因子对NP细胞影响的假设。最后，我们将在炎症条件下验证PHD3失活减缓体内椎间盘退行性改变进展的假说。相反，我们将确定PHD3基因缺失小鼠的HIF-PHD电路的扰动是否会影响椎间盘中年龄相关的变化。综上所述，拟议的研究将检验一个新的假说，即HIF-PhD调节网络在维持NP细胞的生存和功能方面至关重要，而炎性细胞因子对该回路的解偶联可能会加剧退行性腰椎间盘疾病。这些研究的结果将导致开发新的药理干预措施，旨在维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.