Department of Biochemistry, Faculty of Medicine, Justus-Liebig University, 35392 Giessen, Germany.
J Biol Chem. 2010 Apr 9;285(15):11638-51. doi: 10.1074/jbc.M109.045963. Epub 2010 Feb 8.
Coagulation factor XII (FXII) is a liver-derived serine protease involved in fibrinolysis, coagulation, and inflammation. The regulation of FXII expression is largely unknown. Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that has been linked to several pathological processes, including tissue fibrosis by modulating procoagulant and fibrinolytic activities. This study investigated whether TGF-beta1 may regulate FXII expression in human lung fibroblasts. Treatment of human lung fibroblasts with TGF-beta1 resulted in a time-dependent increase in FXII production, activation of p44/42, p38, JNK, and Akt, and phosphorylation and translocation into the nucleus of Smad3. However, TGF-beta1-induced FXII expression was repressed only by the JNK inhibitor and JNK and Smad3 antisense oligonucleotides but not by MEK, p38, or phosphoinositide 3-kinase blockers. JNK inhibition had no effect on TGF-beta1-induced Smad3 phosphorylation, association with Smad4, and its translocation into the nucleus but strongly suppressed Smad3-DNA complex formation. FXII promoter analysis revealed that the -299/+1 region was sufficient for TGF-beta1 to induce FXII expression. Sequence analysis of this region detected a potential Smad-binding element at position -272/-269 (SBE-(-272/-269)). Chromatin immunoprecipitation and streptavidin pulldown assays demonstrated TGF-beta1-dependent Smad3 binding to SBE-(-272/-269). Mutation or deletion of SBE-(-272/-269) substantially reduced TGF-beta1-mediated activation of the FXII promoter. Clinical relevance was demonstrated by elevated FXII levels and its co-localization with fibroblasts in the lungs of patients with acute respiratory distress syndrome. Our results show that JNK/Smad3 pathway plays a critical role in TGF-beta1-induced FXII expression in human lung fibroblasts and implicate its possible involvement in pathological conditions characterized by elevated TGF-beta1 levels.
凝血因子 XII（FXII）是一种肝脏来源的丝氨酸蛋白酶，参与纤溶、凝血和炎症反应。FXII 表达的调节很大程度上是未知的。转化生长因子-β1（TGF-β1）是一种多功能细胞因子，与多种病理过程有关，包括通过调节促凝和纤溶活性导致组织纤维化。本研究探讨了 TGF-β1 是否可能调节人肺成纤维细胞中的 FXII 表达。TGF-β1 处理人肺成纤维细胞导致 FXII 产生的时间依赖性增加，激活 p44/42、p38、JNK 和 Akt，并使 Smad3 磷酸化和转位入核。然而，TGF-β1 诱导的 FXII 表达仅被 JNK 抑制剂和 JNK 和 Smad3 反义寡核苷酸抑制，而不受 MEK、p38 或磷酯酰肌醇 3-激酶抑制剂的抑制。JNK 抑制对 TGF-β1 诱导的 Smad3 磷酸化、与 Smad4 的结合及其转位入核没有影响，但强烈抑制 Smad3-DNA 复合物的形成。FXII 启动子分析表明，-299/+1 区域足以诱导 TGF-β1 诱导 FXII 表达。该区域的序列分析在位置-272/-269（SBE-（-272/-269））检测到一个潜在的 Smad 结合元件。染色质免疫沉淀和链霉亲和素下拉实验证明 TGF-β1 依赖性 Smad3 结合到 SBE-（-272/-269）。SBE-（-272/-269）的突变或缺失显著降低了 TGF-β1 介导的 FXII 启动子的激活。急性呼吸窘迫综合征患者肺部 FXII 水平升高及其与成纤维细胞的共定位证实了临床相关性。我们的结果表明，JNK/Smad3 通路在人肺成纤维细胞中 TGF-β1 诱导的 FXII 表达中起关键作用，并暗示其可能参与以 TGF-β1 水平升高为特征的病理状态。
