Abstract
Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor, plays a critical role in adaption to hypoxia, which is a major feature of diseases, including cancer. Protein disulfide isomerase (PDI) is up-regulated in numerous cancers and leads to cancer progression. PDI, a member of the TRX superfamily, regulates the transcriptional activities of several transcription factors. To investigate the mechanisms by which PDI affects the function of HIF-1alpha, the overexpression or knockdown of PDI was performed. The overexpression of PDI decreased HIF-1alpha expression in the human hepatocarcinoma cell line, Hep3B, whereas the knockdown of endogenous PDI increased its expression. NH4Cl inhibited the decrease in HIF-1alpha expression by PDI overexpression, suggesting that HIF-1alpha was degraded by the lysosomal pathway. HIF-1alpha is transferred to lysosomal membranes by heat shock cognate 70 kDa protein (HSC70). The knockdown of HSC70 abolished the decrease, and PDI facilitated the interaction between HIF-1alpha and HSC70. HIF-1alpha directly interacted with PDI. PDI exists not only in the endoplasmic reticulum (ER), but also in the cytosol. Hypoxia increased cytosolic PDI. We also investigated changes in the redox state of HIF-1alpha using PEG-maleimide, which binds to thiols synthesized from disulfide bonds by reduction. An up-shift in the HIF-1alpha band by the overexpression of PDI was detected, suggesting that PDI formed disulfide bond in HIF-1alpha. HIF-1alpha oxidized by PDI was not degraded in HSC70-knockdown cells, indicating that the formation of disulfide bond in HIF-1alpha was important for decreases in HIF-1alpha expression. To the best of our knowledge, this is the first study to show the regulation of the expression and redox state of HIF-1alpha by PDI. We also demonstrated that PDI formed disulfide bonds in HIF-1alpha 1–245 aa and decreased its expression. In conclusion, the present results showed that PDI is a novel factor regulating HIF-1alpha through lysosome-dependent degradation by changes in its redox state.
Highlights
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcription factor in the hypoxia response, which is common in ischemic diseases, including cancer
HIF-1alpha is a key factor in hypoxia, the relationship between HIF-1alpha and Protein disulfide isomerase (PDI) currently remains unclear; we investigated the effects of PDI on HIF-1alpha using the human hepatocarcinoma cell line, Hep3B
Since HIF-1alpha is a critical factor in cancer progression, we elucidated the relation between PDI and HIF-1alpha in cancer cells
Summary
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcription factor in the hypoxia response, which is common in ischemic diseases, including cancer. HIF-1alpha is up-regulated in various cancers and induces angiogenesis, alterations in metabolism, metastasis, migration, and invasion in cancer [1, 2]. HIF-1alpha has been shown to induce various angiogenetic factors, including vascular endothelial growth factor (VEGF) and angiopoietins [3, 4]. Angiogenesis enables cancer growth by supplying oxygen and nutrients [3, 4]. Matrix metalloproteinase-2 (MMP2) and MMP-9, which degrade components of the extracellular matrix (ECM), are induced in a HIF-1alpha-dependent manner [5]. The degradation of ECM by MMPs enables cancer invasion and metastasis [5]
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