Role of HIF-1 in Intervertebral Disc Function

在椎间盘中，髓核（NP）细胞存在于一个独特的缺氧环境中， 对细胞的限制。NP细胞表现出HIF-1 α的稳健表达，并且其表达存在非典型控制。 脯氨酰羟化酶（PHDs）的周转和活性。根据我们最近的发现， 研究的目的是评估HIF-PHD轴控制NP细胞代谢的机制， 持续的HIF-1 α活性延缓椎间盘中年龄依赖性代谢和退行性变化。在目标1中， 我们将检验HIF-PHD轴是缺氧条件下NP细胞功能的主要调节因子这一假设。 微环境通过复杂的控制他们的代谢状态。我们将确定HIF-1 α如何控制 GLUT-1和关键糖酵解酶通过ChIP-Seq转录、诱变和沉默或过表达 接近。HIF-1在控制代谢通量中的作用将通过测量[1-2- 3]的命运来描述。 13 C2]-葡萄糖和[U-13 C5]-谷氨酰胺。我们将使用NP删除GLUT-1 特异性FoxA 2-Cre和Shh-CreERT 2-Cre小鼠。最后，我们将使用从人类退化的骨髓中分离的NP细胞， 以确定疾病严重程度如何改变GLUT-1和HIF-1依赖性代谢产物的表达。 目标的在目标2中，我们将检验糖酵解NP细胞中pH稳态受HIF-1 α调节的假设。 依赖性分子回路，包括乳酸转运蛋白MCT 4、其辅助蛋白basigin和 质膜相关碳酸酐酶（CA）9和12。我们已经证明，HIF-1依赖性 CA 9和12的表达在通过HCO 3-再循环维持细胞溶质pH中起关键作用。我们将 确定HIF-1控制MCT 4和basigin表达的机制。我们将描述功能 MCT 4在糖酵解终产物、乳酸盐和H+的胞质清除中的作用。使用MCT 4敲除小鼠，我们 将确定pH稳态的扰动是否会随着年龄的增长而损害椎间盘健康。最后，利用人类 我们将确定HIF-1 α活性和疾病严重程度如何改变MCT 4的表达， basigin和CA 9/12。在目标3中，我们将检验增加HIF-1 α活性拯救NP细胞免于凋亡的假设。 通过维持糖酵解代谢和pH稳态来治疗年龄依赖性椎间盘退变。我们 显示PHD 3控制HIF-1 α活性，体内缺乏PHD 3促进NP变性。我们将 在PHD 3-/-小鼠的NP中条件性过表达HIF-1 α，并检查椎间盘中的年龄依赖性变化。 表型。我们将研究恢复的HIF-1 α活性对关键代谢和pH表达的影响。 自我平衡调节器最后，我们将确定是否HIF-1 β过度表达单独减缓进展， 年龄依赖性椎间盘退变这些研究是光盘研究领域的首创， 深入了解HIF-PHD回路对NP细胞的独特代谢控制。调查将产生 健康和退化的NP细胞的代谢生物标志物以及新的可药用靶标。成果 将为使用PHD抑制剂（在治疗贫血的临床试验中）控制椎间盘疾病提供依据。

In the intervertebral disc, nucleus pulposus (NP) cells reside in a unique hypoxic niche that imposes metabolic constraints on cells. NP cells exhibit a robust expression of HIF-1 and there is non-canonical control of its turnover and activity by prolyl hydroxylases (PHDs). Based on our recent findings, a major goal of the investigation is to evaluate the mechanisms by which HIF-PHD axis controls NP cell metabolism and if sustained, HIF-1 activity retards age-dependent metabolic and degenerative changes in the disc. In Aim 1 we will test the hypothesis that the HIF-PHD axis is the master regulator of NP cell function in the hypoxic microenvironment through intricate control of their metabolic state. We will determine how HIF-1 controls GLUT-1 and key glycolytic enzyme transcription by ChIP-Seq, mutagenesis and silencing or overexpression approaches. The role of HIF-1 in controlling metabolic flux will be delineated by measuring the fate of [1-2- 13C2]-glucose and [U-13C5]-glutamine in NP cells from young and old rats. We will delete GLUT-1 using NP specific FoxA2-Cre and Shh-CreERT2-Cre mice. Finally, we will use NP cells isolated from human degenerated tissues to determine how disease severity alters the expression of GLUT-1 and HIF-1 dependent metabolic targets. In Aim 2, we will test the hypothesis that pH homeostasis in glycolytic NP cells is regulated by HIF-1- dependent molecular circuit comprising the lactate transporter, MCT4, its accessory protein basigin and plasma membrane associated carbonic anhydrase (CA) 9 and 12. We have shown that HIF-1-dependent expression of CA9 and 12 play a critical role in cytosolic pH maintenance through HCO3- recycling. We will determine mechanisms by which HIF-1 controls MCT4 and basigin expression. We will delineate the functional role of MCT4 in cytosolic clearance of glycolytic end products, lactate and H+. Using MCT4 knockout mice, we will ascertain if perturbation of pH homeostasis compromises disc health with aging. Finally, using human degenerated tissues we will determine how HIF-1 activity and disease severity alters expression of MCT4, basigin and CA9/12. In Aim 3 we will test the hypothesis that increasing HIF-1 activity rescues NP cells from age-dependent disc degeneration through maintenance of glycolytic metabolism and pH homeostasis. We showed that PHD3 controls HIF-1 activity and lack of PHD3 in vivo promotes NP degeneration. We will conditionally overexpress HIF-1 in the NP of PHD3-/- mice and examine the age dependent changes in disc phenotype. We will study the influence of restored HIF-1 activity on expression of key metabolic and pH homeostatic regulators. Finally, we will determine if HIF-1 overexpression alone slows down the progression of age-dependent disc degeneration. The studies are first-of-a-kind in field of disc research and will provide insights into the unique metabolic control of NP cells by the HIF-PHD circuit. The investigations will generate metabolic biomarkers of healthy and degenerating NP cells as well as novel druggable targets. The outcomes will provide rationale for use of PHD inhibitors (in clinical trials for treating anemia) to control disc disease.