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

Abstract

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.

RNA Sequencing Reveals a Role of TonEBP Transcription Factor in Regulation of Pro-inflammatory Genes in Response to Hyperosmolarity in Healthy Nucleus Pulposus Cells

“…ical loading of the healthy disc would activate an inflammatory program. It is, therefore, more likely that the acute nature of CCL2 induction may be tied to diurnal loading of the disc, considering that genes controlling circadian rhythm are essential for disc homeostasis (35,36). It is noteworthy that in other cell types, CCL2 promotes survival (37), proliferation (38), and phosphorylation of Akt, ERK, and STAT3 (39), molecules critical for NP function (22, 40 -43).…”

The embryonic and evolutionary boundaries between notochord and cartilage: a new look at nucleus pulposus-specific markers

“…To date, circadian time has been shown to affect the expression of >600 genes of IVDs, and this pattern may be affected by natural aging, exposure to interleukin-1b 97 , and/or passive exposure to cigarette smoke 98 . Of the proposed NP-specific markers, HIF-1a expression is indirectly controlled by the BMAL1 (brain and muscle ARNT-like1) and RORa (retinoic acid receptorrelated orphan receptor-a) proteins, two important regulators of clock gene dynamics 99 . Thus, caution is required when exploring the specificity of NP phenotypic makers; it is certainly possible that the expression thereof is downregulated or even turned off at specific times.…”

Bicarbonate Recycling by HIF-1–Dependent Carbonic Anhydrase Isoforms 9 and 12 Is Critical in Maintaining Intracellular pH and Viability of Nucleus Pulposus Cells

“…(35) NP cells adapt to their unique hypoxic environment through the essential activities of the HIF homologues, (4) which are regulated in a unique fashion in the NP. (5,(36)(37)(38)(39)(40) HIF-1a, in particular, regulates the expression of many genes critical to the survival of NP cells-including enzymes controlling glycolysis. (10) Likewise, HIF-1 is implied to control CA12 expression in NP cells; however, in vivo evidence and mechanistic insights of this regulation are lacking.…”