Yamane Kenichi, Ihn Hironobu, Tamaki Kunihiko
Department of Dermatology, University of Tokyo, Tokyo, Japan.
Arthritis Rheum. 2003 Jun;48(6):1652-66. doi: 10.1002/art.11029.
OBJECTIVE: Transforming growth factor beta receptors (TGFbetaRs) are known to be expressed at high levels in several fibrotic diseases, including systemic sclerosis. In the present study, we investigated the mechanism of up-regulation of TGFbetaR expression. METHODS: The levels of expression of TGFbetaR type II (TGFbetaRII) messenger RNA (mRNA), with or without stimulation by epidermal growth factor (EGF), were evaluated by Northern blot analysis, and the protein levels were determined by immunoblotting. The transcription activity of the TGFbetaRII gene was examined with luciferase assays using the -1670/+35 TGFbetaRII promoter luciferase construct. RESULTS: EGF up-regulates the expression of TGFbetaRII mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the EGF-mediated up-regulation of TGFbetaRII mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, EGF treatment did not significantly affect the TGFbetaRII mRNA half-life. EGF-mediated induction of TGFbetaRII expression was inhibited by treatment of fibroblasts with the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002, and Akt inhibitor also blocked EGF-induced expression of TGFbetaRII. In addition, EGF induced TGFbetaRII promoter activity, and this induction was significantly blocked by wortmannin, LY294002, or Akt inhibitor. Cotransfection with a dominant-negative mutant of p85 (the regulatory component of PI 3-kinase) or Akt significantly reduced the induction of TGFbetaRII promoter activity by EGF. Moreover, a constitutive active form of p110 (a catalytic component of PI 3-kinase) induced TGFbetaRII promoter activity. In addition, scleroderma fibroblasts expressed increased levels of TGFbetaRII but did not show further up-regulation of TGFbetaRII expression by EGF. CONCLUSION: These results indicate that EGF-mediated induction of TGFbetaRII expression occurs at the transcription level, does not require de novo protein synthesis, and involves the PI 3-kinase/Akt signaling pathway, and that abnormal activation of EGF-mediated signaling pathways, including PI 3-kinase or Akt, might play a role in the up-regulation of TGFbetaRII in scleroderma fibroblasts.
目的：已知转化生长因子β受体（TGFβRs）在包括系统性硬化症在内的多种纤维化疾病中高表达。在本研究中，我们调查了TGFβR表达上调的机制。 方法：通过Northern印迹分析评估有无表皮生长因子（EGF）刺激时II型TGFβ受体（TGFβRII）信使核糖核酸（mRNA）的表达水平，通过免疫印迹法测定蛋白水平。使用-1670/+35 TGFβRII启动子荧光素酶构建体，通过荧光素酶测定法检测TGFβRII基因的转录活性。 结果：EGF上调人皮肤成纤维细胞中TGFβRII mRNA和蛋白的表达。RNA合成抑制剂放线菌素D显著阻断EGF介导的TGFβRII mRNA表达上调，而蛋白合成抑制剂放线菌酮未阻断这种上调。此外，EGF处理对TGFβRII mRNA半衰期无显著影响。用选择性磷酸肌醇3激酶（PI 3激酶）抑制剂渥曼青霉素或LY294002处理成纤维细胞可抑制EGF介导的TGFβRII表达诱导，Akt抑制剂也可阻断EGF诱导的TGFβRII表达。此外，EGF诱导TGFβRII启动子活性，渥曼青霉素、LY294002或Akt抑制剂可显著阻断这种诱导。用p85（PI 3激酶的调节成分）的显性负突变体或Akt共转染可显著降低EGF对TGFβRII启动子活性的诱导。此外，p110（PI 3激酶的催化成分）的组成型活性形式可诱导TGFβRII启动子活性。此外，硬皮病成纤维细胞中TGFβRII表达水平升高，但EGF未进一步上调TGFβRII表达。 结论：这些结果表明，EGF介导的TGFβRII表达诱导发生在转录水平，不需要从头合成蛋白质，且涉及PI 3激酶/Akt信号通路，包括PI 3激酶或Akt在内的EGF介导信号通路的异常激活可能在硬皮病成纤维细胞中TGFβRII的上调中起作用。
Am J Physiol Cell Physiol. 2001-7
Curr Rheumatol Rep. 2005-4
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