Department of Biomedical Chemistry, School of Science and Technology, Kwansei Gakuin University, Gakuen, Sanda, Japan.
Program of Biomedical Science, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashihiroshima, Japan.
PLoS One. 2021 Feb 4;16(2):e0246531. doi: 10.1371/journal.pone.0246531. eCollection 2021.
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.
缺氧诱导因子-1α（HIF-1α）是一种转录因子，在适应缺氧中发挥关键作用，缺氧是包括癌症在内的许多疾病的主要特征。蛋白二硫键异构酶（PDI）在许多癌症中上调，并导致癌症进展。PDI 是 TRX 超家族的成员，调节几种转录因子的转录活性。为了研究 PDI 影响 HIF-1α 功能的机制，进行了 PDI 的过表达或敲低。PDI 的过表达降低了人肝癌细胞系 Hep3B 中的 HIF-1α 表达，而内源性 PDI 的敲低则增加了其表达。NH4Cl 抑制了 PDI 过表达导致的 HIF-1α 表达下降，表明 HIF-1α 通过溶酶体途径降解。HIF-1α 通过热休克同源 70kDa 蛋白（HSC70）转移到溶酶体膜上。HSC70 的敲低消除了这种下降，并且 PDI 促进了 HIF-1α 与 HSC70 之间的相互作用。HIF-1α 直接与 PDI 相互作用。PDI 不仅存在于内质网（ER）中，也存在于细胞质中。缺氧增加了细胞质中的 PDI。我们还使用 PEG-马来酰亚胺研究了 HIF-1α 的氧化还原状态的变化，PEG-马来酰亚胺与还原作用合成的来自二硫键的巯基结合。PDI 过表达检测到 HIF-1α 带的向上移动，表明 PDI 在 HIF-1α 中形成二硫键。在 HSC70 敲低细胞中，被 PDI 氧化的 HIF-1α 没有降解，表明 HIF-1α 中二硫键的形成对于 HIF-1α 表达的降低很重要。据我们所知，这是第一项表明 PDI 调节 HIF-1α 的表达和氧化还原状态的研究。我们还证明了 PDI 在 HIF-1α 1-245 aa 中形成二硫键并降低其表达。总之，本研究结果表明，PDI 是一种通过改变其氧化还原状态通过溶酶体依赖性降解来调节 HIF-1α 的新型因子。
