Mohan Sumathy, Konopinski Ryszard, Yan Bo, Centonze Victoria E, Natarajan Mohan
Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229, USA.
Am J Physiol Cell Physiol. 2009 Jan;296(1):C182-92. doi: 10.1152/ajpcell.00575.2007. Epub 2008 Oct 22.
A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor kappaB kinases (IKK)alpha, beta, and gamma] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKbeta in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKbeta interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 +/- 0.65-fold) and translational (1.97 +/- 0.17-fold) expression as well as the catalytic activity of IKKbeta (2.45 +/- 0.4-fold). Both IKKbeta and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 microM) or Radicicol (20 microM) mitigated (0.45 +/- 0.04-fold and 0.93 +/- 0.16-fold, respectively) HG induced-IKKbeta activity (2.5 +/- 0.42-fold). Blocking of IKKbeta expression by IKK inhibitor II (15 microM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.
一氧化氮（NO）生物利用度的下降会导致内皮功能障碍，这是糖尿病的一个标志。血管中NO的可用性受内皮型一氧化氮合酶（eNOS）活性调节，并且热休克蛋白90（Hsp-90）参与eNOS活性调节已得到证实。Hsp-90已被证明可与非血管细胞中的上游激酶[抑制蛋白κB激酶（IKK）α、β和γ]相互作用。在本研究中，我们研究了高糖（HG）条件下内皮细胞中Hsp-90与IKKβ的相互作用，这可能是减少Hsp-90-eNOS相互作用的一种机制，而这种减少可能导致NO生物利用度降低。我们首次报道IKKβ与Hsp-90相互作用，并且在血管内皮细胞中，这种相互作用会被HG增强。HG还会增强IKKβ的转录表达（3.5±0.65倍）、翻译表达（1.97±0.17倍）以及催化活性（2.45±0.4倍）。IKKβ和eNOS均可与Hsp-90进行共免疫沉淀。用格尔德霉素（2μM）或放线菌酮（20μM）抑制Hsp-90可减轻HG诱导的IKKβ活性（分别为0.45±0.04倍和0.93±0.16倍，而HG诱导的IKKβ活性为2.5±0.42倍）。在HG条件下，用IKK抑制剂II（15μM水飞蓟宾）或小干扰RNA（siRNA）阻断IKKβ表达可改善Hsp-90-eNOS相互作用并增加NO生成。这些结果揭示了在HG条件下报道的eNOS活性下降的一种可能机制。
