Shelton Melissa D, Distler Anne M, Kern Timothy S, Mieyal John J
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4965, USA.
J Biol Chem. 2009 Feb 20;284(8):4760-6. doi: 10.1074/jbc.M805464200. Epub 2008 Dec 12.
Protein S-glutathionylation is a reversible redox-dependent post-translational modification. Many cellular functions and signal transduction pathways involve proteins whose cysteine-dependent activities are modulated by glutathionylation. Glutaredoxin (Grx1) plays a key role in such regulation because it is a specific and efficient catalyst of deglutathionylation. We recently reported an increase in Grx1 in retinae of diabetic rats and in rat retinal Müller glial cells (rMC-1) cultured in high glucose. This up-regulation of Grx1 was concomitant with NFkappaB activation and induction of intercellular adhesion molecule-1 (ICAM-1). This proinflammatory response was replicated by adenoviral-directed up-regulation of Grx1 in cells in normal glucose. The site of regulation of NFkappaB was localized to the cytoplasm, where IkappaB kinase (IKK) is a master regulator of NFkappaB activation. In the current study, inhibition of IKK activity abrogated the increase in ICAM-1 induced by high glucose or by adenoviral-directed up-regulation of Grx1. Conditioned medium from the Müller cells overexpressing Grx1 was added to fresh cultures of Müller or endothelial cells and elicited increases in the Grx1 and ICAM-1 proteins in these cells. These effects correlate with a novel finding that secretion of interleukin-6 was elevated in the cultures of Grx overexpressing cells. Also, pure interleukin-6 increased Grx1 and ICAM-1 in the rMC-1 cells. Thus, Grx1 appears to play an important role in both autocrine and paracrine proinflammatory responses. Furthermore, IKKbeta isolated from Müller cells in normal glucose medium was found to be glutathionylated on Cys-179. Hence Grx-mediated activation of IKK via deglutathionylation may play a central role in diabetic complications in vivo where Grx1 is increased.
蛋白质S-谷胱甘肽化是一种可逆的依赖氧化还原的翻译后修饰。许多细胞功能和信号转导途径涉及的蛋白质，其依赖半胱氨酸的活性受谷胱甘肽化调节。谷氧还蛋白（Grx1）在这种调节中起关键作用，因为它是去谷胱甘肽化的特异性高效催化剂。我们最近报道，糖尿病大鼠视网膜以及在高糖环境中培养的大鼠视网膜穆勒胶质细胞（rMC-1）中Grx1增加。Grx1的这种上调与NFκB激活和细胞间黏附分子-1（ICAM-1）的诱导同时发生。正常葡萄糖环境下细胞中通过腺病毒介导Grx1上调可复制这种促炎反应。NFκB的调节位点定位于细胞质，其中IκB激酶（IKK）是NFκB激活的主要调节因子。在本研究中，抑制IKK活性可消除高糖或腺病毒介导的Grx1上调所诱导的ICAM-1增加。将过表达Grx1的穆勒细胞的条件培养基添加到穆勒细胞或内皮细胞的新鲜培养物中，可引起这些细胞中Grx1和ICAM-1蛋白增加。这些效应与一个新发现相关，即过表达Grx的细胞培养物中白细胞介素-6的分泌升高。此外，纯白细胞介素-6可增加rMC-1细胞中的Grx1和ICAM-1。因此，Grx1似乎在自分泌和旁分泌促炎反应中均起重要作用。此外，发现在正常葡萄糖培养基中从穆勒细胞分离的IKKβ在半胱氨酸179处发生了谷胱甘肽化。因此，Grx通过去谷胱甘肽化介导的IKK激活可能在体内Grx1增加的糖尿病并发症中起核心作用。
