Role of nuclear factor-κB in interleukin-1-induced collagen degradation by corneal fibroblasts
Role of nuclear factor-κB in interleukin-1-induced collagen degradation by corneal fibroblasts
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DOI：
10.1016/j.exer.2006.02.008
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发表时间：
2006-09-01
影响因子：
3.4
通讯作者：
Nishida, Teruo
中科院分区：
文献类型：
--
作者：
Lu, Ying;Fukuda, Ken;Nishida, Teruo
促炎细胞因子白细胞介素（IL）-1与角膜溃疡有关。研究了核因子（NF）-κ B在IL-1诱导的角膜成纤维细胞胶原降解中的作用，角膜成纤维细胞是角膜溃疡的基础。将兔角膜成纤维细胞培养在含有或不含有IL-1和柳氮磺胺吡啶（NF-κ B活化抑制剂）的I型胶原三维凝胶中。从培养上清液的酸热水解产生的羟脯氨酸的量评估胶原降解。通过免疫印迹分析和明胶酶谱法检测基质金属蛋白酶（MMPs）和金属蛋白酶组织抑制剂（TIMPs）释放到培养上清液中，并通过逆转录和实时聚合酶链反应分析确定MMP和TIMPmRNA的细胞丰度。通过免疫印迹分析检测NF-κ B抑制蛋白I κ B-α的磷酸化和降解。免疫荧光法和比色法分别检测NF-κ B B p65亚基的亚细胞定位和DNA结合活性。NF-κ B的反式激活活性用报告基因测定来评估。柳氮磺胺吡啶以浓度依赖性方式抑制IL-1诱导的角膜成纤维细胞胶原降解。它还以浓度依赖性方式抑制IL-1对各种MMPs合成或活化的刺激作用。IL-1诱导IKB-α的磷酸化和降解，核转位和NF-κ B的p65亚基的DNA结合活性的上调，以及NF-κ B的活化，其方式对柳氮磺胺吡啶敏感。这些结果表明，NF-κ B有助于IL-1诱导的角膜成纤维细胞胶原降解，因此是治疗角膜溃疡的潜在治疗靶点。(c)2006爱思唯尔有限公司版权所有。
The proinflammatory cytokine interleukin (IL)-1 is implicated in corneal ulceration. The role of nuclear factor (NF)-kappa B in the IL-1-induced degradation of collagen by corneal fibroblasts that underlies corneal ulceration was investigated. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen with or without IL-1 and sulfasalazine, an inhibitor of NF-kappa B activation. Collagen degradation was assessed from the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The release of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) into culture supernatants was examined by immunoblot analysis and gelatin zymography, and the cellular abundance of MMP and TIMP mRNAs was determined by reverse transcription and real-time polymerase chain reaction analysis. The phosphorylation and degradation of the NF-kappa B-inhibitory protein I kappa B-alpha were examined by immunoblot analysis. The subcellular localization and DNA binding activity of the p65 subunit of NF-kappa B were evaluated by immunofluorescence analysis and with a colorimetric assay, respectively. The transactivation activity of NF-kappa B was assessed with a reporter gene assay. Sulfasalazine inhibited IL-1-induced collagen degradation by corneal fibroblasts in a concentration-dependent manner. It also inhibited the stimulatory effects of IL-1 on the synthesis or activation of various MMPs in a concentration-dependent manner. IL-1 induced the phosphorylation and degradation of IKB-alpha, the nuclear translocation and up-regulation of the DNA binding activity of the p65 subunit of NF-kappa B, and the activation of NF-kappa B in a manner sensitive to sulfasalazine. These results suggest that NF-kappa B contributes to the IL-1-induced degradation of collagen by corneal fibroblasts and is therefore a potential therapeutic target for treatment of corneal ulcers. (c) 2006 Elsevier Ltd. All rights reserved.