摘要
目的探讨转化生长因子β1(TGF-β1)对人近端肾小管上皮细胞系HK-2中结缔组织生长因子(CTGF)基因启动子活性的调控作用,以及丝裂原激活蛋白激酶(MAPK)途径对该生长因子作用的影响。方法构建含有人类CTGF基因启动子的报告基因pCTGF-luc,将其瞬时转染HK-2细胞。通过检测荧光素酶的活性观察TGF-β1和MAPK途径抑制剂对CTGF基因启动子活性的影响。结果TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子的活性。最佳刺激浓度是5ng/ml,最佳刺激时间为12h,荧光素酶相对活性分别为对照组的1.82倍和2.10倍(P<0.05)。应用PD98059、SB203580和SP600125分别特异性抑制MAPK途径的胞外信号调节蛋白激酶(ERK)、蛋白激酶p38(p38MAPK)和c-Jun-氨基末端激酶(JNK)通路,对TGF-β1上调CTGF启动子活性的作用有不同影响。PD98059显著增加HK-2中pCTGF-luc的基础活性,并在一定浓度范围内(0.5-10μmol/L)促进TGF-β1的上调作用。SB203580对pCTFGF-luc基础活性无影响,但以剂量依赖方式显著抑制TGF-β1的激活效应。而SP600125对基础状态和TGF-β1刺激下CTGF基因启动子活性无影响。结论TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子活性,在转录水平调节CTGF表达。MAPK途径的ERK和p38MAPK通路可影响TGF-β1的这一调控作用。
Objective To investigate the effect of transforming growth factor-β1 （TGF-β1） on the activity of connective tissue growth factor（CTGF） gene promoter in human renal proximal tubular epithelial cell line HK-2. Methods The regulation fragment of 5＇ flanking region of human CTGF gene was linked to pGL3-Basic vector. The recombinant plasmid pCTGF-luc was transient transfected to HK-2 ceils. The activity of CTGF promoter after treatment of TGF-β1 and mitogen-activated prontein kinases （MAPK） pathway inhibitors was assayed by means of luciferase reporter gene assay system. Results TGF-β1-induced increase of CTGF promoter activity was concentration-dependent, with a plateau at 5 ng/ml by 1.82-fold vs control （P 〈 0.05）. The TGF-β1 stimulation of CTGF promoter activity was time-dependent, too. After exposure to TGF-β1 （5 ng/ml）, the maximal level of luciferase activity was reached at 12 h by 2.10-fold vs control （P 〈 0.05）. Blockade of MAPK pathway with PD98059,the extracellular signal-regulated protein kinase （ERK） inhibitor, significantly increased basal activation of CTGF promoter in HK-2 and that of TGF-β1-induced in a range of concentration （0.5-10 μmol/L）. SB203580, the p38MAP kinase （p38MAPK） inhibitor, markedly decreased TGF-β1-induced activation of CTGF promoter in a dose-dependent manner. However, inhibition of c-Jan-N-terminal kinase（JNK） of MAPK pathway by SP600125 was without effect. Conclusions TGF-β1 stimulates the transcriptional activity of CTGF gene promoter in HK-2 ceils in a dose- and time-dependent manner. MAPK pathway （ERK and p38MAPK）may play a role in the regulation of TGF-β1-induced CTGF expression.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2005年第8期453-457,共5页
Chinese Journal of Nephrology
基金
上海市卫生局医学领先专业项目(983009)上海市教委发展基金(02BK12)上海市科委重点学科基金国家自然科学基金(30270613)
