Heat Induction of the Unphosphorylated Form of Hypoxia-inducible Factor-1α Is Dependent on Heat Shock Protein-90 Activity

Abstract

Hypoxia-inducible factor (HIF)-1␣ is the oxygen-sensitive subunit of HIF-1, a transcriptional master regulator of oxygen homeostasis. Oxygen-dependent prolyl hydroxylation targets HIF-1␣ for ubiquitinylation and proteasomal degradation. Unexpectedly, we found that exposing mice to elevated temperatures resulted in a strong HIF-1␣ induction in kidney, liver, and spleen. To elucidate the molecular mechanisms responsible for this effect, HepG2 hepatoma cells were exposed to different temperatures (34 -42°C) under normoxic (20% O 2 ) or hypoxic (3% O 2 ) conditions. Heat was sufficient to stabilize mainly a phosphatase-resistant, low molecular weight form of HIF-1␣ (termed HIF-1␣ a ). Heat-induced HIF-1␣ a accumulated in the nucleus but neither bound to DNA nor trans-activated reporter or target gene expression, demonstrating the need for post-translational modifications for these functions. The protein banding pattern of heat-induced HIF-1␣ in immunoblot analyses was clearly distinct from the HIF-1␣ pattern after prolyl hydroxylase inhibition (by hypoxia or iron chelation/replacement) or following proteasome inhibition, suggesting that heat stabilizes HIF-1␣ by a novel mechanism. Inhibition of the ATP-dependent chaperone activity of HSP90 by novobiocin or geldanamycin prevented heatinduced as well as hypoxia-induced HIF-1␣ accumulation, indicating a common role of the HSP90 chaperone activity in HIF-1␣ stabilization by these two environmental parameters.The hypoxia-inducible factor-1 (HIF-1) 1 is a heterodimeric transcription factor composed of the two subunits HIF-1␣ and HIF-1␤, both belonging to the basic helix-loop-helix (bHLH)-Per/arylhydrocarbon receptor (AhR) nuclear translocator (ARNT)/Sim (PAS) protein superfamily (1). HIF-1␤ is identical to the previously described ARNT protein. HIF-1 is a critical regulator of the physiological adaptive response to hypoxia, since it activates genes regulating, among other processes, angiogenesis, erythropoiesis, and glucose metabolism. HIF-1 has also been described to be involved in tumor angiogenesis and ischemic diseases such as myocardial ischemia or stroke (reviewed in Refs. 2 and 3). Whereas ARNT is constitutively expressed, HIF-1␣ expression is induced in hypoxic cells with an exponential increase in expression as cells are exposed to decreased oxygen partial pressures. As determined in HeLa cells, the highest HIF-1␣ protein levels are reached at 0.5% oxygen (4, 5). Recent studies have shown that HIF-1␣ is modified by oxygendependent prolyl hydroxylation (6 -9), allowing the binding of the von Hippel-Lindau protein (pVHL), which targets HIF-1␣ for ubiquitinylation and proteasomal degradation (10 -17). Further activation of HIF-1 involves nuclear translocation, dimerization with ARNT, DNA binding, and recruitment of transcriptional co-activators. These processes are regulated at least in part by posttranslational modifications. It has been demonstrated that phosphorylation of HIF-1␣ via the Ras/Raf-MEK-p42/44 and the phosphatidylinositol 3-kinase-PTEN-Akt-GSK3 kinase...

Chrysin inhibits expression of hypoxia-inducible factor-1α through reducing hypoxia-inducible factor-1α stability and inhibiting its protein synthesis

“…Binding of HIF-1a with Hsp90 prevents HIF-1a from degradation (33,34). We found that chrysin inhibited binding of HIF-1a to Hsp90 (Fig.…”

“…2C. Hsp90, a chaperone protein, has been shown to stabilize HIF-1a (34,44). Chrysin was found to inhibit the binding of HIF-1a to Hsp90, suggesting that chrysin inhibits HIF-1a expression via interfering with the interaction between HIF-1a and Hsp90 (Fig.…”

Mitochondria: Oxygen sinks rather than sensors?

“…This makes the PHDs ideally suited to regulate HIFα protein levels over a wide range of physiologically relevant oxygen concentrations. Indeed, when we designed immunoblotting experiments for maximal sensitivity, it was possible to detect HIF-1α already under normoxic conditions, in line with a limited PHD activity [18]. Consistent with this observation, HIF-1α-deficient cells display reduced gene expression of HIF-1 target genes even when cultured under normoxic conditions, when HIF-1α protein levels normally are not detectable by immunoblotting [19][20][21].…”

Low concentration of GA activates a preconditioning response in HepG2 cells during oxidative stress—roles of Hsp90 and vimentin

“…Geldanamycin, a benzoquinone ansamycin antibiotic and natural product of Streptomyces geldanus, possesses antitumor activity and specifically binds with high affinity to ATP-binding sites of Hsp90 (Lewis et al 2000;Katschinski et al 2002;Nomura et al 2004). Previous studies have established that Hsp90 inhibitors are particularly toxic to tumor cells because of a presumed increased binding affinity for these drugs and an enhanced Hsp90 reliance of genetically unstable tumor cells.…”