Circadian factors BMAL1 and RORα control HIF-1α transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health.

BMAL1 and RORα are major regulators of the circadian molecular oscillator. Since previous work in other cell types has shown cross talk between circadian rhythm genes and hypoxic signaling, we investigated the role of BMAL1 and RORα in controlling HIF-1-dependent transcriptional responses in NP cells that exist in the physiologically hypoxic intervertebral disc. HIF-1-dependent HRE reporter activity was further promoted by co-transfection with either BMAL1 or RORα. In addition, stable silencing of BMAL1 or inhibition of RORα activity resulted in decreased HRE activation. Inhibition of RORα also modulated HIF1α-TAD activity. Interestingly, immunoprecipitation studies showed no evidence of BMAL1, CLOCK or RORα binding to HIF-1α in NP cells. Noteworthy, stable silencing of BMAL1 as well as inhibition of RORα decreased expression of select HIF-1 target genes including VEGF, PFKFB3 and Eno1. To delineate if BMAL1 plays a role in maintenance of disc health, we studied the spinal phenotype of BMAL1-null mice. The lumbar discs of null mice evidenced decreased height, and several parameters associated with vertebral trabecular bone quality were also affected in nulls. In addition, null animals showed a higher ratio of cells to matrix in NP tissue and hyperplasia of the annulus fibrosus. Taken together, our results indicate that BMAL1 and RORα form a regulatory loop in the NP and control HIF-1 activity without direct interaction. Importantly, activities of these circadian rhythm molecules may play a role in the adaptation of NP cells to their unique niche

GLUT1 Is Redundant in Hypoxic and Glycolytic Nucleus Pulposus Cells of the Intervertebral Disc

Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose transporter, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and postnatal maintenance of the disc. Surprisingly, primary NP cells treated with 2 well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We showed in vitro that NP cells mitigated GLUT1 loss by rewiring glucose import through GLUT3. Of note, we demonstrated that substrates, such as glutamine and palmitate, did not compensate for glucose restriction resulting from dual inhibition of GLUT1/3, and inhibition compromised long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated 2 NP-specific knockout mice: Krt19CreERT Glut1fl/fl and Foxa2Cre Glut1fl/fl. There were no apparent defects in postnatal disc health or development and maturation in mutant mice. Microarray analysis verified that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence to our knowledge of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells

Lactate efflux from intervertebral disc cells is required for maintenance of spine health.

Maintenance of glycolytic metabolism is postulated to be required for health of the spinal column. In the hypoxic tissues of the intervertebral disc and glycolytic cells of vertebral bone, glucose is metabolized into pyruvate for ATP generation and reduced to lactate to sustain redox balance. The rise in intracellular H+ /lactate concentrations are balanced by plasma-membrane monocarboxylate transporters (MCTs). Using MCT4 null mice and human tissue samples, complimented with genetic and metabolic approaches, we determine that H+ /lactate efflux is critical for maintenance of disc and vertebral bone health. Mechanistically, MCT4 maintains glycolytic and TCA cycle flux and intracellular pH homeostasis in the nucleus pulposus compartment of the disc, where HIF-1α directly activates an intronic enhancer in SLC16A3. Ultimately, our results provide support for research into lactate as a diagnostic biomarker for chronic, painful disc degeneration

Insights into Glycolytic Metabolism and pH Homeostasis in the Hypoxic Intervertebral Disc

Low back pain and associated intervertebral disc degeneration are the leading causes of disability in the United States and largely contribute to the recent opioid epidemic. While mechanisms of disc degeneration have been studied in many contexts, the relationship between disc degeneration and low back pain remains elusive. Very recently, researchers validated intradiscal acidity as a biomarker for identifying pain-causing discs and severity of disc degeneration. The goal of this work was to elucidate how and why changes in acid/base metabolism and glucose consumption synergize with age-dependent disc degeneration. We therefore investigated the role and regulation of a network of pH regulatory enzymes, Carbonic Anhydrases (CAs), and plasma-membrane transporters, Monocarboxylate Transporters (MCTs) and Glucose Transporter 1 (GLUT1), in the physiologically hypoxic and acidic intervertebral disc niche. We hypothesized that loss of function of these key regulatory proteins may contribute to the pathogenesis of disc degeneration. Our studies confirm that the hypoxia-inducible and HIF-1-dependent CA9/12 isozymes and MCT4 isoform are critical for NP cell survival and maintenance of the aging spine, respectively. Mechanistically, CA9/12 are required for intracellular pH regulation through HCO3– recycling, and MCT4 is critical for lactic acid efflux from the cell, as lactate accumulation was shown to instigate metabolic and transcriptional reprogramming in NP cells. Furthermore, investigations into the role of CA3 revealed that it is, in fact, not involved in intracellular pH regulation, but rather acts as a robustly-expressed antioxidant that protcts NP cells from oxidative-stress induced apoptosis. Finally, we tested the hypothesis that glucose import into NP cells, via the NP phenotypic marker, GLUT1, was required for disc development and maintenance. However, using an NP-specific GLUT1 knock-out mouse model, we discovered that GLUT1 was not required for NP cell viability or disc development. Taken together, our work contributes to a new model of the pH regulatory network that enables NP cell survival in their harsh microenvironmental niche. In addition, we add nuanced mechanistic insights into the role and regulation of NP phenotypic markers, CA3 and GLUT1 which, in fact, are not required for pH regulation or maintenance of NP cell metabolism

Expression of Carbonic Anhydrase III, a Nucleus Pulposus Phenotypic Marker, is Hypoxia-responsive and Confers Protection from Oxidative Stress-induced Cell Death

Abstract The integrity of the avascular nucleus pulposus (NP) phenotype plays a crucial role in the maintenance of intervertebral disc health. While advances have been made to define the molecular phenotype of healthy NP cells, the functional relevance of several of these markers remains unknown. In this study, we test the hypothesis that expression of Carbonic Anhydrase III (CAIII), a marker of the notochordal NP, is hypoxia-responsive and functions as a potent antioxidant without a significant contribution to pH homeostasis. NP, but not annulus fibrosus or end-plate cells, robustly expressed CAIII protein in skeletally mature animals. Although CAIII expression was hypoxia-inducible, we did not observe binding of HIF-1α to select hypoxia-responsive-elements on Car3 promoter using genomic chromatin-immunoprecipitation. Similarly, analysis of discs from NP-specific HIF-1α null mice suggested that CAIII expression was independent of HIF-1α. Noteworthy, silencing CAIII in NP cells had no effect on extracellular acidification rate, CO2 oxidation rate, or intracellular pH, but rather sensitized cells to oxidative stress-induced death mediated through caspase-3. Our data clearly suggests that CAIII serves as an important antioxidant critical in protecting NP cells against oxidative stress-induced injury