Hsp70 and CHIP Selectively Mediate Ubiquitination and Degradation of Hypoxia-inducible Factor (HIF)-1 alpha but Not HIF-2 alpha

From the Vascular Program, Institute for Cell Engineering.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2009; 285(6):3651-63. DOI: 10.1074/jbc.M109.068577
Source: PubMed


Hypoxia-inducible factors (HIFs) are transcription factors that mediate adaptive responses to reduced oxygen availability.
HIF-α subunits are stabilized under conditions of acute hypoxia. However, prolonged hypoxia leads to decay of HIF-1α but not
HIF-2α protein levels by unknown mechanisms. Here, we identify Hsp70 and CHIP (carboxyl terminus of Hsc70-interacting protein) as HIF-1α-interacting proteins. Hsp70, through recruiting the ubiquitin ligase CHIP, promotes the ubiquitination and
proteasomal degradation of HIF-1α but not HIF-2α, thereby inhibiting HIF-1-dependent gene expression. Disruption of Hsp70-CHIP
interaction blocks HIF-1α degradation mediated by Hsp70 and CHIP. Inhibition of Hsp70 or CHIP synthesis by RNA interference
increases protein levels of HIF-1α but not HIF-2α and attenuates the decay of HIF-1α levels during prolonged hypoxia. Thus,
Hsp70- and CHIP-dependent ubiquitination represents a molecular mechanism by which prolonged hypoxia selectively reduces the
levels of HIF-1α but not HIF-2α protein.

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    • "Despite the increased stability, HIF-1α protein can still be negatively regulated in hypoxia. For example, HSP70 interacts with HIF-1α and recruits the E3 ubiquitin ligase CHIP to HIF-1α, therefore leading to proteasomal degradation of HIF-1α in hypoxia [15]. Our results showed that SET9 interacts with HIF-1α and stabilizes HIF-1α by inhibiting the E3 ubiquitin ligase CHIP recruitment, suggesting that SET9 competes with HSP70 or CHIP for binding to HIF-1α, therefore protects HIF-1α from exposing to the proteasomal degradation system. "
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    ABSTRACT: HIF-1α is degraded by oxygen-dependent mechanisms but stabilized in hypoxia to form transcriptional complex HIF-1, which transactivates genes promoting cancer hallmarks. However, how HIF-1α is specifically regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase SET9 promotes HIF-1α protein stability in hypoxia and enhances HIF-1 mediated glycolytic gene transcription, thereby playing an important role in mediating cancer cell adaptation and survival to hypoxic stress. Specifically, SET9 interacts with HIF-1α and promotes HIF-1α protein stability in hypoxia. Silencing SET9 by siRNA reduces HIF-1α protein stability in hypoxia, and attenuates the hypoxic induction of HIF-1 target genes mediating hypoxic glycolysis. Mechanistically, we find that SET9 is enriched at the hypoxia response elements (HRE) within promoters of the HIF-1-responsive glycolytic genes. Silencing SET9 reduces HIF-1α levels at these HREs in hypoxia, thereby attenuating HIF-1-mediated gene transcription. Further, silencing SET9 by siRNA reduces hypoxia-induced glycolysis and inhibits cell viability of hypoxic cancer cells. Our findings suggest that SET9 enriches at HRE sites of HIF-1 responsive glycolytic genes and stabilizes HIF-1α at these sites in hypoxia, thus establishes an epigenetic mechanism of the metabolic adaptation in hypoxic cancer cells.
    Full-text · Article · Jan 2015 · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
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    • "In support of a VHL-independent role of USP52 in the HIF1A pathway, knockdown of USP52 in RCC4 cells caused a subsequent decrease in HIF1A levels (Figure 3B). In addition to VHL, several other proteins have been reported to be involved in targeting HIF1A for proteasomal degradation, such as RACK1 (receptor for activated C-kinase 1) [24], HAF (hypoxia-associated factor) [25] and CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] [26]. In order to exclude USP52 knockdown promoting HIF1A depletion through alternative degradation pathways, U2OS cells depleted of USP52 were treated with the proteasome inhibitor MG132. "
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    ABSTRACT: Hypoxia Inducible Factor-1 alpha (HIF1A) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. While the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. Here, we have identified the pseudoDUB/deadenylase USP52/PAN2 as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3'UTR-dependent, polyA-tail length-independent destabilization in HIF1A mRNA. Mass spectrometry analysis revealed an association of USP52 with several P-body components and we further confirmed that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.
    Full-text · Article · Feb 2013 · Biochemical Journal
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    • "The effect of MCM7 on HIF-1α (Figure 3F) and HIF-2α (Figure 3G) levels was abolished by treatment with the proteasome inhibitor MG132. HIF-1α proteasomal degradation occurs through two main pathways, a hydroxylation-dependent pathway mediated through the VHL ubiquitin ligase and hydroxylation-independent pathways mediated by RACK1, HAF, or CHIP/HSP70 (Liu et al., 2007; Koh et al., 2008; Luo et al., 2010). The effect of MCM7 on HIF-1α was abolished by mutation of P402/564, implicating the hydroxylation-dependent pathway (Figure 3H). "
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    ABSTRACT: MCM proteins are components of a DNA helicase that plays an essential role in DNA replication and cell proliferation. However, MCM proteins are present in excess relative to origins of replication, suggesting they may serve other functions. Decreased proliferation is a fundamental physiological response to hypoxia in many cell types, and hypoxia-inducible factor 1 (HIF-1) has been implicated in this process. Here, we demonstrate that multiple MCM proteins bind directly to the HIF-1α subunit and synergistically inhibit HIF-1 transcriptional activity via distinct O(2)-dependent mechanisms. MCM3 inhibits transactivation domain function, whereas MCM7 enhances HIF-1α ubiquitination and proteasomal degradation. HIF-1 activity decreases when quiescent cells re-enter the cell cycle, and this effect is MCM dependent. Exposure to hypoxia leads to MCM2-7 downregulation in diverse cell types. These studies reveal a function of MCM proteins apart from their DNA helicase activity and establish a direct link between HIF-1 and the cell-cycle machinery.
    Full-text · Article · Jun 2011 · Molecular cell
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