Pivotal Role of Connective Tissue Growth Factor in Lung Fibrosis MAPK-Dependent Transcriptional Activation of Type I Collagen
Pivotal Role of Connective Tissue Growth Factor in Lung Fibrosis MAPK-Dependent Transcriptional Activation of Type I Collagen
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DOI：
10.1002/art.24620
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发表时间：
2009-07-01
影响因子：
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通讯作者：
Lindahl, Gisela E.
中科院分区：
文献类型：
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作者：
Ponticos, Markella;Holmes, Alan M.;Lindahl, Gisela E.
Objective.结缔组织生长因子（CTGF; CCN 2）在系统性硬化症（SSc）中过表达，并被假设为在这种疾病中经常观察到的肺纤维化的关键介质。CTGF由转化生长因子β（TGF β）诱导，并且是TGF β在体外的一些促纤维化作用的介质。本研究旨在探讨CTGF在博莱霉素诱导的肺纤维化中I型胶原表达增强的作用，并阐明CTGF在该小鼠模型和人肺成纤维细胞中对Col 1a 2（胶原基因I型α 2）的作用机制。将分别携带由Col 1a 2增强子/启动子和CTGF启动子驱动的荧光素酶和β-半乳糖苷酶报告基因的转基因小鼠注射博来霉素以诱导肺纤维化（或生理盐水作为对照），并将提取的肺成纤维细胞与CTGF阻断剂孵育。在体外，瞬时转染，启动子/报告基因构建和电泳迁移率变动分析用于确定CTGF在肺成纤维细胞中的作用机制。在小鼠肺组织中，CTGF表达和启动子活性在博来霉素攻击后1周达到峰值，而I型胶原表达和Col 1a 2启动子活性在攻击后2周达到峰值。博来霉素处理14天后，从小鼠肺部分离的成纤维细胞保留了促纤维化表达模式，其特征是I型胶原和CTGF蛋白水平大幅升高以及启动子活性增加。在体外，特异性小干扰RNA和中和抗体抑制CTGF可降低胶原蛋白表达和Col 1a 2启动子活性。此外，在体内，博来霉素攻击后应用的抗CTGF抗体显著降低了Col 1a 2启动子活性，降低幅度接近25%。博莱霉素处理的肺成纤维细胞中增强的Col 1a 2启动子活性部分依赖于Smad信号传导，而CTGF通过独立于Smad结合位点的机制作用于Col 1a 2启动子，而是依赖于ERK-1/2和JNK MAPK途径。CTGF的作用定位于CCAAT盒周围的近端启动子区域，可能涉及CREB和c-Jun。在人肺成纤维细胞中，人COL 1A 2启动子以类似的方式应答，其作用机制也涉及ERK-1/2和JNK信号。我们的研究结果清楚地定义了CTGF的直接促纤维化作用，并证明了其通过转录激活Colla 2对肺纤维化的贡献。阻断策略揭示了所涉及的信号机制。这些发现表明CTGF是治疗纤维化疾病如SSc的合理靶点。
Objective. Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor beta (TGF beta) and is a mediator of some profibrotic effects of TGF beta in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I alpha 2) in this mouse model and in human pulmonary fibroblasts.Methods. Transgenic mice that were carrying luciferase and beta-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts.Results. In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by similar to 25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycin-treated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling.Conclusion. Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation of Colla2. Blocking strategies revealed the signaling mechanisms involved. These findings show CTGF to be a rational target for therapy in fibrotic diseases such as SSc.