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 biomarke...