Role of HIF-1 in Intervertebral Disc Function

在椎间盘中，髓核(NP)细胞驻留在一个独特的缺氧区，它施加代谢 对单元格的约束。NP细胞表现出HIF-1的强阳性表达，并存在对其非规范调控 脯氨酸羟基酶(PhDS)的周转和活性。根据我们最近的发现， 研究目的是评估HIF-PHD轴控制NP细胞代谢和IF的机制 持续的低氧诱导因子-1活性可延缓与年龄相关的椎间盘代谢和退行性改变。在目标1中，我们 将检验HIF-PHD轴是低氧中NP细胞功能的主要调节者这一假设 通过对其代谢状态的复杂控制来控制微环境。我们将确定HIF-1如何控制 GLUT-1和关键糖酵解酶通过ChIP-Seq转录、突变和沉默或过表达 接近了。HIF-1在控制代谢流中的作用将通过测定[1-2-]的命运来描述。 [13C2]-葡萄糖和[U-13C5]-谷氨酰胺在幼年和老年大鼠的NP细胞内。我们将使用NP删除GLUT-1 特异性FOXA2-Cre和Shh-CreERT2-Cre小鼠。最后，我们将使用从退化的人类中分离出来的NP细胞 确定疾病严重程度如何改变GLUT-1和HIF-1依赖的代谢表达 目标。在目标2中，我们将验证糖酵解NP细胞的pH动态平衡受HIF-1-1调节的假设。 由乳酸转运蛋白MCT4、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依赖于 CA9和12的表达在通过HCO3循环维持胞液pH中起着关键作用。我们会 确定HIF-1调控MCT4和basigin表达的机制。我们将描绘出功能 MCT4在糖酵解终末产物乳酸和H+胞浆清除中的作用使用MCT4基因敲除小鼠，我们 将确定pH动态平衡的扰动是否会随着年龄的增长而损害椎间盘的健康。最后，使用人类 我们将确定HIF-1活性和疾病严重程度如何改变MCT4的表达， 在目标3中，我们将检验以下假设：增加HIF-1活性可以将NP细胞从 维持糖酵解代谢和pH动态平衡所致的年龄依赖性椎间盘退行性变。我们 结果表明，PHD3调控HIF-1活性，体内缺乏PHD3可促进NP变性。我们会 PHD3-/-小鼠NP中条件性过表达缺氧诱导因子-1及视盘的增龄变化 表型。我们将研究恢复HIF-1活性对关键代谢和pH表达的影响 体内平衡调节器。最后，我们将确定HIF-1的过度表达是否单独减缓了进展 与年龄相关的椎间盘退行性变。这些研究在光盘研究领域是第一次，并将提供 深入了解HIF-PhD电路对NP细胞的独特代谢控制。调查将产生 健康和退化的NP细胞的代谢生物标记物以及新的可药物靶点。结果是 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理论基础。

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.