McGaha Tracy L, Le Maithao, Kodera Takao, Stoica Cristina, Zhu Jinfang, Paul William E, Bona Constantin A
Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
Arthritis Rheum. 2003 Aug;48(8):2275-84. doi: 10.1002/art.11089.
There is evidence that interleukin-4 (IL-4) plays a major role in the induction of extracellular matrix protein synthesis in fibrotic disease. We therefore examined the effect of IL-4 on collagen synthesis in primary fibroblasts isolated from normal and TSK/+ mice, which spontaneously develop a scleroderma-like syndrome characterized by diffuse cutaneous hyperplasia.
Expression of the IL-4 receptor was determined by flow cytometry and Western blotting. The IL-4 signal transduction cascade was analyzed by Western blotting. We assessed the role of signal transducer and activator of transcription 6 (STAT-6) in IL-4 induction of alpha2(I) collagen promoter activity and message levels via luciferase reporter assay and real-time polymerase chain reaction. The activation status of the transcription factors activator protein 1 (AP-1) and Sp-1 upon stimulation with IL-4 in normal and TSK/+ fibroblasts was examined by electrophoretic mobility shift assay.
Flow cytometry and Western blotting showed that IL-4 receptor alpha expression was elevated in TSK/+ fibroblasts compared with normal fibroblasts. After IL-4 stimulation, janus-activated kinase 1 (JAK-1) and JAK-2 were phosphorylated to a greater degree in TSK/+ fibroblasts than in C57BL/6 fibroblasts. TSK/+ fibroblasts appeared to be hyperresponsive to IL-4, displaying increased synthesis of alpha1(I) collagen messenger RNA (mRNA), collagen protein, and activity of a luciferase reporter construct containing the -300 to +54 murine alpha2(I) collagen promoter. Overexpression of STAT-6 enhanced this effect, whereas expression of a dominant-negative STAT-6 abrogated the ability of IL-4 to induce alpha1(I) collagen mRNA in TSK/+ fibroblasts. Moreover, IL-4 induced increased DNA binding activity of transcription factors that are important for collagen synthesis.
Our observations indicate that IL-4 has a profound effect on several factors that have been identified as playing major roles in the regulation of collagen synthesis and suggest that IL-4 increases the expression of type I collagen through a mechanism involving the activation of transcription factors that bind to and activate collagen promoter.
有证据表明白细胞介素-4（IL-4）在纤维化疾病中细胞外基质蛋白合成的诱导过程中起主要作用。因此，我们研究了IL-4对从正常小鼠和TSK/+小鼠分离的原代成纤维细胞中胶原蛋白合成的影响，TSK/+小鼠会自发发展出一种以弥漫性皮肤增生为特征的硬皮病样综合征。
通过流式细胞术和蛋白质免疫印迹法测定IL-4受体的表达。通过蛋白质免疫印迹法分析IL-4信号转导级联反应。我们通过荧光素酶报告基因测定和实时聚合酶链反应评估信号转导及转录激活因子6（STAT-6）在IL-4诱导α2（I）型胶原启动子活性和信使水平中的作用。通过电泳迁移率变动分析检测正常和TSK/+成纤维细胞经IL-4刺激后转录因子激活蛋白1（AP-1）和Sp-1的激活状态。
流式细胞术和蛋白质免疫印迹法显示，与正常成纤维细胞相比，TSK/+成纤维细胞中IL-4受体α的表达升高。IL-4刺激后，TSK/+成纤维细胞中janus激活激酶1（JAK-1）和JAK-2的磷酸化程度高于C57BL/6成纤维细胞。TSK/+成纤维细胞似乎对IL-4反应过度，表现为α1（I）型胶原信使核糖核酸（mRNA）、胶原蛋白合成增加，以及含有-300至+54小鼠α2（I）型胶原启动子的荧光素酶报告构建体的活性增加。STAT-6的过表达增强了这种效应，而显性负性STAT-6的表达消除了IL-4诱导TSK/+成纤维细胞中α1（I）型胶原mRNA的能力。此外，IL-4诱导了对胶原蛋白合成重要的转录因子的DNA结合活性增加。
我们的观察结果表明，IL-4对已被确定在胶原蛋白合成调节中起主要作用的几个因素有深远影响，并表明IL-4通过一种涉及激活与胶原启动子结合并激活的转录因子的机制增加I型胶原的表达。
