Makondo Kennedy, Kamikawa Akihiro, Ahmed Mohamed, Terao Akira, Saito Masayuki, Kimura Kazuhiro
Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Eur J Pharmacol. 2008 Mar 17;582(1-3):110-5. doi: 10.1016/j.ejphar.2007.12.018. Epub 2007 Dec 28.
Previously, we demonstrated that hepatocyte growth factor (HGF) potently stimulates endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production through a calcium- and Akt-mediated phosphorylation at Ser-1179 (Ser-1177 human) in bovine aortic endothelial cells. The regulation of eNOS, however, also involves interaction with chaperone proteins such as heat shock protein (HSP) 90, which can be enhanced by agonist stimulation of the enzyme. In the present work, the role of HSP90 in HGF stimulation of eNOS was examined in an endothelial cell culture system. Treatment of endothelial cells with geldanamycin, a commonly used HSP90 inhibitor, augmented HGF-stimulated eNOS phosphorylation at Ser-1179, while it did not alter eNOS phosphorylation at Thr-497. However, other HSP90 inhibitors, namely 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and radicicol, did not possess similar effects. Neither HGF nor geldanamycin treatment, independently or in combination, altered HSP90/eNOS interaction in endothelial cells. In addition, geldanamycin treatment did not enhance the HGF-induced phosphorylation of Akt, ERK1/2 and p38MAPK. Src kinase inhibition by PP2 also failed to block the geldanamycin effects. These results suggest that geldanamycin, but neither 17-AAG nor radicicol, may enhance HGF-mediated eNOS Ser-1179 phosphorylation by some as yet unknown mechanisms independently of HSP90 inhibition.
此前，我们证明肝细胞生长因子（HGF）可通过钙和Akt介导的牛主动脉内皮细胞中Ser-1179（人类为Ser-1177）位点磷酸化，有效刺激内皮型一氧化氮合酶（eNOS）活性及一氧化氮（NO）生成。然而，eNOS的调节还涉及与伴侣蛋白如热休克蛋白（HSP）90的相互作用，该相互作用可被该酶的激动剂刺激增强。在本研究中，我们在内皮细胞培养系统中检测了HSP90在HGF刺激eNOS过程中的作用。用常用的HSP90抑制剂格尔德霉素处理内皮细胞，可增强HGF刺激的eNOS在Ser-1179位点的磷酸化，而不改变其在Thr-497位点的磷酸化。然而，其他HSP90抑制剂，即17-烯丙基氨基-17-去甲氧基格尔德霉素（17-AAG）和雷迪西醇，不具有类似作用。单独或联合使用HGF和格尔德霉素处理均未改变内皮细胞中HSP90/eNOS的相互作用。此外，格尔德霉素处理并未增强HGF诱导的Akt、ERK1/2和p38MAPK的磷酸化。PP2抑制Src激酶也未能阻断格尔德霉素的作用。这些结果表明，格尔德霉素而非17-AAG或雷迪西醇，可能通过一些尚未明确的机制独立于HSP90抑制作用来增强HGF介导的eNOS Ser-1179磷酸化。
