TNF-α signals through PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability.
TNF-α signals through PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability.
复制标题
DOI：
10.2337/db09-1606
复制
发表时间：
2010-11
期刊：
影响因子：
7.7
通讯作者：
Antonetti DA
中科院分区：
文献类型：
--
作者：
Aveleira CA;Lin CM;Abcouwer SF;Ambrósio AF;Antonetti DA
肿瘤坏死因子-α（TNF-α）和白细胞介素-1 β（IL-1β）在糖尿病患者的玻璃体和糖尿病大鼠的视网膜中升高，与视网膜血管通透性增加相关。然而，这些细胞因子诱导的视网膜血管通透性的分子机制知之甚少。本研究比较了IL-1β和TNF-α对视网膜内皮细胞通透性的影响，并阐明了TNF-α增加细胞通透性的分子机制。在牛视网膜内皮细胞（BRECs）和大鼠视网膜中测量细胞因子诱导的视网膜血管通透性。Western印迹，定量实时PCR和免疫细胞化学进行，以确定紧密连接蛋白的表达和定位。IL-1β和TNF-α均能增加BREC的通透性，且TNF-α作用更强。TNF-α降低了紧密连接蛋白ZO-1和claudin-5的蛋白和mRNA含量，并改变了这些紧密连接蛋白的细胞定位。地塞米松通过糖皮质激素受体的反式激活和核因子-κ B（NF-κB）的反式抑制来阻止TNF-α诱导的细胞通透性。用抑制剂κB激酶（IKK）化学抑制剂或抑制剂κB α（IκBα）的腺病毒过表达阻止NF-κB活化可降低TNF-α刺激的渗透性。最后，在细胞培养物和大鼠视网膜中，使用肽和新型化学抑制剂抑制蛋白激酶C zeta（PKC β）可降低NF-κB活化，并完全阻止TNF-α诱导的紧密连接复合物和细胞通透性的改变。这些结果表明，PKC β可能为预防以TNF-α升高为特征的视网膜疾病（包括糖尿病视网膜病变）的血管通透性提供特异性治疗靶点。
Tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) are elevated in the vitreous of diabetic patients and in retinas of diabetic rats associated with increased retinal vascular permeability. However, the molecular mechanisms underlying retinal vascular permeability induced by these cytokines are poorly understood. In this study, the effects of IL-1β and TNF-α on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-α increases cell permeability were elucidated. Cytokine-induced retinal vascular permeability was measured in bovine retinal endothelial cells (BRECs) and rat retinas. Western blotting, quantitative real-time PCR, and immunocytochemistry were performed to determine tight junction protein expression and localization. IL-1β and TNF-α increased BREC permeability, and TNF-α was more potent. TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins. Dexamethasone prevented TNF-α–induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-κB) transrepression. Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α–stimulated permeability. Finally, inhibiting protein kinase C zeta (PKCζ) using both a peptide and a novel chemical inhibitor reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α in cell culture and rat retinas. These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.