Circadian Factors Bmal1 And Ror Alpha Control Hif-1 Alpha Transcriptional Activity In Nucleus Pulposus Cells: Implications In Maintenance Of Intervertebral Disc Health

BMAL1 and ROR alpha 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 ROR alpha 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 ROR alpha. In addition, stable silencing of BMAL1 or inhibition of ROR alpha activity resulted in decreased HRE activation. Inhibition of ROR alpha also modulated HIF1 alpha-TAD activity. Interestingly, immunoprecipitation studies showed no evidence of BMAL1, CLOCK or ROR alpha binding to HIF-1 alpha in NP cells. Noteworthy, stable silencing of BMAL1 as well as inhibition of ROR alpha 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 ROR alpha 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.