Tonicity enhancer binding protein (TonEBP) and hypoxia-inducible factor (HIF) coordinate heat shock protein 70 (Hsp70) expression in hypoxic nucleus pulposus cells: Role of Hsp70 in HIF-1α degradation

Abstract

The objective of our study was to examine the regulation of hypoxic expression of Hsp70 in nucleus pulposus cells and to determine if Hsp70 promoted HIF-1α degradation. Rat nucleus pulposus cells were maintained in culture in either 21% or 1% oxygen. To determine the regulation of Hsp70 expression by Tonicity enhancer binding protein (TonEBP) and HIF-1/2, loss- and gain-of-function experiments and mutational analysis of Hsp70 promoter were performed. Hypoxia increased Hsp70 expression in nucleus pulposus cells. Noteworthy, hypoxia increased TonEBP transactivation and mutation of TonE motifs blocked hypoxic induction of Hsp70 promoter. In contrast, mutation of HRE motifs coupled with loss of function experiments suggested that HIF-1 and HIF-2 suppressed Hsp70 promoter activity and transcription. Interestingly, HIF-α interferes with TonEBP function and suppresses inductive effect of TonEBP on Hsp70 promoter. In terms of Hsp70 function, when treated with Hsp70 transcriptional inhibitor, KNK437, there was an increase in HIF-1α protein stability and transcriptional activity. Likewise, when Hsp70 was overexpressed, the stability of HIF-1α and its transcriptional activity decreased. Hsp70 interacted with HIF-1α under hypoxic conditions and evidenced increased binding when treated with MG132, a proteasomal inhibitor. These results suggest that Hsp70 may promote HIF-1α degradation through proteasomal pathway in nucleus pulposus cells. In hypoxic and hyperosmolar nucleus pulposus cells, Hsp70, TonEBP and HIFs form a regulatory loop. We propose that the positive regulation by TonEBP and negative regulation of Hsp70 by HIF-1 and HIF-2 may serve to maintain Hsp70 levels in these cells, whereas Hsp70 may function in controlling HIF-1α homeostasis.

Understanding Nucleus Pulposus Cell Phenotype: A Prerequisite for Stem Cell Based Therapies to Treat Intervertebral Disc Degeneration

“…Careful work by Risbud and coworkers has shown that NP cells constitutively express HIF-1α and HIF-2α under both normoxia and hypoxia [92, 93]. Consistent with this observation, alternative oxygen-independent means of HIF-α degradation and activity have been shown to impact protein levels in NP cells [94-96]. Taken together, unique regulation of HIF protein turnover and activity in NP cells suggests its importance in NP physiology.…”

Hypoxia-inducible Factor (HIF)-1α and CCN2 Form a Regulatory Circuit in Hypoxic Nucleus Pulposus Cells

“…regulation of Hsp70 and GlcAT-I promoters in nucleus pulposus cells (6,11). Related to this discussion, one possibility is that hypoxia may exert its effects on CCN2 through the modulation of Fli1 or Ets-1, members of the Ets family of transcription factors.…”

“…HIF is a basic helix-loop-helix transcription factor comprising a constitutively expressed ␤ subunit and an ␣ subunit that undergoes degradation by both oxygen-dependent and -independent pathways (5,6). When stabilized, the ␣ subunit dimerizes with the ␤ subunit and binds to hypoxia response elements (HREs) in the promoter of target genes.…”

Toll-like Receptor Agonists and Febrile Range Hyperthermia Synergize to Induce Heat Shock Protein 70 Expression and Extracellular Release

“…HSF1 trimerization and activation of its transactivation domain are regulated by multiple serine and threonine phosphorylation events. Transcription of Hsps is also influenced by other transcription factors, including STAT-1, STAT-3, and NF-IL-6 (22)(23)(24), tonicity enhancer-binding protein and hypoxia-inducible factor 1 (25) and NFB (26). We previously showed that HSF1 trimerization and Hsp70 transcription were activated in A549 cells by exposure to mild hyperthermia (38.5°C), that Hsp70 transcription increased as the exposure temperature increased from 38.5°C to 42°C, and that increased transcription was associated with additional HSF1 phosphorylation but not additional HSF1 trimerization (27).…”