AIM To investigate the regulatory mechanism of transforming growth factor-beta on activity of connective tissue growth factor promoter in mouse NIH/3T3 fibroblasts. METHODS The regulation fragment of the 5' flanking region of the human CTGF gene was linked to pGL3-Basic vector, a firefly luciferase reporter construct without promoter. The recombinant plasmid pCTGF-luc was transiently transfected to NIH/3T3 fibroblasts. The activity of CTGF promoter after treatment of TGF-beta(1) and MAPK pathway inhibitors were assayed with luciferase reporter gene assay system. RESULTS TGF-beta(1)-induced increase of CTGF promoter activity was concentration-dependent, with a plateau at 5 microg/L by 2.67-fold vs control (P<0.05). The TGF-beta(1) stimulation of CTGF promoter activity was time-dependent, too. After exposure to TGF-beta(1) (5 microg/L), the maximal level of luciferase activity was reached at 12 h and maintained to 24 h by 2.76- and 2.20-fold vs control, respectively (P<0.05). Blockade of MAPK pathway with PD98059 (10 micromol/L), the MAP kinase kinase 1 inhibitor, and SB203580 (10 micromol/L), the p38 MAP kinase inhibitor, decreased basal and TGF-beta (1)-induced activation of CTGF promoter. However, inhibition of c-Jun-N-terminal kinase/stress-activated protein kinase by SP600125 (20 micromol/L) was without effect. CONCLUSION TGF-beta(1) stimulated the transcriptional activity of CTGF gene promoter in NIH/3T3 fibroblasts in a dose- and time-dependent manner. MAPK pathway may play a role in the regulation of TGF-beta(1)-induced CTGF expression.

中文翻译：

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转化生长因子-β对小鼠NIH / 3T3成纤维细胞结缔组织生长因子基因启动子活性的影响。

目的探讨转化生长因子β对小鼠NIH / 3T3成纤维细胞结缔组织生长因子启动子活性的调控机制。方法将人类CTGF基因5'侧翼区的调控片段与pGL3-Basic载体连接，该载体是没有启动子的萤火虫荧光素酶报告基因构建体。将重组质粒pCTGF-luc瞬时转染至NIH / 3T3成纤维细胞。用萤光素酶报告基因检测系统检测TGF-β（1）和MAPK途径抑制剂治疗后CTGF启动子的活性。结果TGF-β（1）诱导的CTGF启动子活性增加是浓度依赖性的，相对于对照，稳定水平在5 microg / L时为2.67倍（P <0.05）。TGF-beta（1）刺激CTGF启动子活性也是时间依赖性的。暴露于TGF-beta（1）（5 microg / L）后，荧光素酶活性的最大水平在12 h达到，与对照组相比分别以2.76倍和2.20倍维持在24 h（P <0.05）。用PD98059（10 micromol / L）（MAP激酶激酶1抑制剂）和SB203580（10 micromol / L）（p38 MAP激酶抑制剂）阻断MAPK途径，降低基础和TGF-β（1）诱导的CTGF启动子激活。但是，SP600125（20 micromol / L）抑制c-Jun-N-末端激酶/应力激活蛋白激酶无效。结论TGF-beta（1）刺激了NIH / 3T3成纤维细胞中CTGF基因启动子的转录活性，呈剂量和时间依赖性。MAPK通路可能在调节TGF-beta（1）诱导的CTGF表达中起作用。0.05）。用PD98059（10 micromol / L）（MAP激酶激酶1抑制剂）和SB203580（10 micromol / L）（p38 MAP激酶抑制剂）阻断MAPK途径，降低基础和TGF-β（1）诱导的CTGF启动子激活。但是，SP600125（20 micromol / L）抑制c-Jun-N-末端激酶/应力激活蛋白激酶无效。结论TGF-beta（1）刺激NIH / 3T3成纤维细胞中CTGF基因启动子的转录活性呈剂量和时间依赖性。MAPK通路可能在调节TGF-beta（1）诱导的CTGF表达中起作用。0.05）。用PD98059（10 micromol / L）（MAP激酶激酶1抑制剂）和SB203580（10 micromol / L）（p38 MAP激酶抑制剂）阻断MAPK途径，降低基础和TGF-β（1）诱导的CTGF启动子激活。但是，SP600125（20 micromol / L）抑制c-Jun-N-末端激酶/应力激活蛋白激酶无效。结论TGF-beta（1）刺激NIH / 3T3成纤维细胞中CTGF基因启动子的转录活性呈剂量和时间依赖性。MAPK通路可能在调节TGF-beta（1）诱导的CTGF表达中起作用。SP600125（20 micromol / L）抑制c-Jun-N-末端激酶/应激激活蛋白激酶无效。结论TGF-beta（1）刺激NIH / 3T3成纤维细胞中CTGF基因启动子的转录活性呈剂量和时间依赖性。MAPK通路可能在调节TGF-beta（1）诱导的CTGF表达中起作用。SP600125（20 micromol / L）抑制c-Jun-N-末端激酶/应激激活蛋白激酶无效。结论TGF-beta（1）刺激NIH / 3T3成纤维细胞中CTGF基因启动子的转录活性呈剂量和时间依赖性。MAPK通路可能在调节TGF-beta（1）诱导的CTGF表达中起作用。