Selective inhibition of activin receptor-like kinase 5 signaling blocks profibrotic transforming growth factor β responses in skin fibroblasts
Selective inhibition of activin receptor-like kinase 5 signaling blocks profibrotic transforming growth factor β responses in skin fibroblasts
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DOI：
10.1002/art.20658
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发表时间：
2004-12-01
影响因子：
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通讯作者：
Varga, J
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文献类型：
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作者：
Mori, Y;Ishida, W;Varga, J
目标。转化生长因子β(TGFbeta)细胞因子超家族成员在动态平衡和疾病中发挥重要作用。鉴于它们的促纤维化作用，这些分子被认为与纤维化的发病机制有关。在成纤维细胞中，TGFbeta通过激活素受体样激酶5(ALK-5)I型TGFbeta发出信号，并触发Smad和MAP激酶信号通路。由于靶向TGFbeta信号转导是治疗系统性硬化症(SSC)和其他纤维化疾病的潜在途径，我们研究了第一个ALK-5小分子抑制剂SB431542对细胞内TGFbeta信号转导的调节作用。通过Western印迹分析和共聚焦免疫细胞化学检测配体诱导的人真皮成纤维细胞Smad信号通路的激活。采用Northern印迹分析、瞬时转染实验和共聚焦显微镜观察促纤维蛋白原基因表达的调控。用酶联免疫吸附试验检测TGFβ的诱生作用。SB431542抑制TGFbeta诱导的内源性Smad2/3和Smad4的磷酸化和核输入，并抑制Smad3和Smad2依赖的基因转录。SB431542可阻止TGFbeta对胶原、纤维连接蛋白、纤溶酶原激活物抑制物1、结缔组织生长因子基因表达、TGFbeta自身诱导和肌成纤维细胞转分化的刺激，即使在TGFbeta之后加入该药也能逆转刺激。相反，SB431542不能阻止STAT-6介导的对IL-13诱导的胶原基因表达的刺激，这表明阻断ALK-5依赖的信号转导具有特异性。此外，与其对受体激活的Smad激活的影响相比，SB431542不能阻止TGFbeta诱导的MAP激活。结果表明，SB431542通过选择性干扰ALK-5介导的Smad激活和Smad依赖的转录反应，有效地抑制了正常成纤维细胞内的TGF13信号转导。因此，SB431542可作为一种新的实验工具，用于详细了解SSC中正常和异常的TGFbeta信号。此外，作为一种抗转化生长因子β的药物，SB431542可能代表着一种潜在的治疗纤维化的新途径。
Objective. Members of the transforming growth factor beta (TGFbeta) cytokine superfamily play critical roles in both homeostasis and disease. In light of their profibrotic effects, these molecules are implicated in the pathogenesis of fibrosis. In fibroblasts, TGFbeta signals through the activin receptor-like kinase 5 (ALK-5) type I TGFbeta and triggers Smad and MAP kinase signaling pathways. Because targeting of TGFbeta signaling represents a potential approach to the treatment of systemic sclerosis (SSc) and other fibrotic disorders, we investigated the modulation of intracellular TGFbeta signal transduction by SB431542, the first small-molecule inhibitor of ALK-5 to be described.Methods. Ligand-induced activation of the Smad signaling pathway in human dermal fibroblasts was examined by Western blot analysis and confocal immunocytochemistry. Modulation of profibrotic gene expression was investigated using Northern blot analysis, transient transfection assays, and confocal microscopy. Induction of TGFbeta production was evaluated by enzyme-linked immunosorbent assay.Results. SB431542 abrogated TGFbeta-induced phosphorylation and nuclear importation of endogenous Smad2/3 and Smad4, and inhibited Smad3- and Smad2-dependent gene transcription. Treatment with SB431542 prevented TGFbeta-induced stimulation of collagen, fibronectin, plasminogen activator inhibitor 1, and connective tissue growth factor gene expression, TGFbeta autoinduction, and myofibroblast transdifferentiation, and it could reverse stimulation even when added to the cultures after TGFbeta. In contrast, STAT-6-mediated stimulation of collagen gene expression induced by interleukin-13 was not prevented by SB431542, indicating the specificity of blockade for ALK-5-dependent signaling. Furthermore, in contrast to its effects on receptor-activated Smad activation, SB431542 failed to prevent TGFbeta-induced activation of MAP kinases.Conclusion. The results indicate that SB431542 is a potent inhibitor of intracellular TGF13 signaling in normal fibroblasts through selective interference with ALK-5-mediated Smad activation and Smad-dependent transcriptional responses. Therefore, SB431542 is useful as a novel experimental tool for gaining a detailed understanding of normal and aberrant TGFbeta signaling in SSc. Furthermore, as an anti-TGFbeta agent, SB431542 may represent a potential new approach to the treatment of fibrosis.