Vascular System Research Center and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, Kangwon-do 200-701, Republic of Korea.
J Biol Chem. 2010 Oct 15;285(42):32116-25. doi: 10.1074/jbc.M110.131284. Epub 2010 Aug 19.
Carbon monoxide (CO) plays a significant role in vascular functions. We here examined the molecular mechanism by which CO regulates HIF-1 (hypoxia-inducible transcription factor-1)-dependent expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. We found that astrocytes stimulated with CORM-2 (CO-releasing molecule) promoted angiogenesis by increasing VEGF expression and secretion. CORM-2 also induced HO-1 (hemeoxygenase-1) expression and increased nuclear HIF-1α protein level, without altering its promoter activity and mRNA level. VEGF expression was inhibited by treatment with HIF-1α siRNA and a hemeoxygenase inhibitor, indicating that CO stimulates VEGF expression via up-regulation of HIF-1α protein level, which is partially associated with HO-1 induction. CORM-2 activated the translational regulatory proteins p70(S6k) and eIF-4E as well as phosphorylating their upstream signal mediators Akt and ERK. These translational signal events and HIF-1α protein level were suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K), MEK, and mTOR, suggesting that the PI3K/Akt/mTOR and MEK/ERK pathways are involved in a translational increase in HIF-1α. In addition, CORM-2 also increased stability of the HIF-1α protein by suppressing its ubiquitination, without altering the proline hydroxylase-dependent HIF-1α degradation pathway. CORM-2 increased HIF-1α/HSP90α interaction, which is responsible for HIF-1α stabilization, and HSP90-specific inhibitors decreased this interaction, HIF-1α protein level, and VEGF expression. Furthermore, HSP90α knockdown suppressed CORM-2-induced increases in HIF-1α and VEGF protein levels. These results suggest that CO stimulates VEGF production by increasing HIF-1α protein level via two distinct mechanisms, translational stimulation and protein stabilization of HIF-1α.
一氧化碳（CO）在血管功能中起着重要作用。我们在此研究了 CO 调节血管内皮生长因子（VEGF）表达的分子机制，VEGF 是一种重要的血管生成因子。我们发现，用 CORM-2（CO 释放分子）刺激星形胶质细胞可通过增加 VEGF 的表达和分泌来促进血管生成。CORM-2 还诱导 HO-1（血红素加氧酶-1）的表达，并增加核 HIF-1α 蛋白水平，而不改变其启动子活性和 mRNA 水平。用 HIF-1α siRNA 和血红素加氧酶抑制剂处理可抑制 VEGF 的表达，表明 CO 通过上调 HIF-1α 蛋白水平刺激 VEGF 的表达，这部分与 HO-1 的诱导有关。CORM-2 激活了翻译调节蛋白 p70(S6k)和 eIF-4E，并磷酸化其上游信号转导介质 Akt 和 ERK。这些翻译信号事件和 HIF-1α 蛋白水平被磷脂酰肌醇 3-激酶（PI3K）、MEK 和 mTOR 的抑制剂抑制，表明 PI3K/Akt/mTOR 和 MEK/ERK 途径参与了 HIF-1α 的翻译增加。此外，CORM-2 还通过抑制其泛素化来增加 HIF-1α 蛋白的稳定性，而不改变脯氨酰羟化酶依赖性 HIF-1α 降解途径。CORM-2 增加了 HIF-1α/HSP90α 的相互作用，这是 HIF-1α 稳定的原因，HSP90 特异性抑制剂降低了这种相互作用、HIF-1α 蛋白水平和 VEGF 的表达。此外，HSP90α 敲低抑制了 CORM-2 诱导的 HIF-1α 和 VEGF 蛋白水平的增加。这些结果表明，CO 通过两种不同的机制刺激 VEGF 的产生，增加 HIF-1α 蛋白水平，即翻译刺激和 HIF-1α 的蛋白稳定。
