p38/ERK激酶调控TGF-β_1诱导的HLF-02细胞Ⅰ型胶原表达及MMP-2活力

p38/ERK signal pathways regulating the expression of typeⅠcollagen and activity of MMP-2 in TGF-β_1-stimulated HLF-02 cells

目的探讨转化生长因子β1(TGF-β1)/丝裂原活化蛋白激酶(MAPK)通路在调控人肺成纤维细胞Ⅰ型胶原表达、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)活力中的作用。方法以人肺成纤维细胞HLF-02细胞系为研究对象,给予10 ng/ml的TGF-β1刺激不同时间;阻断实验以P38激酶特异性抑制剂(SB203580)、ERK激酶特异性抑制剂(PD98059)分别阻断p38通路和细胞外调控激酶(ERK)通路;采用逆转录聚合酶链反应(RT-PCR)检测细胞内Ⅰ型胶原mRNA的表达;运用Western印迹检测细胞上清液中Ⅰ型胶原蛋白表达;酶谱分析细胞上清液中MMP-2、MMP-9的活力。结果(1)TGF-β1刺激HLF-02细胞,244、8、72 h组的Ⅰ型胶原mRNA表达水平分别为1.33±0.07、2.46±0.09、2.39±0.08;Ⅰ型胶原蛋白表达水平分别为114.89±8.95、208.16±6.75、211.46±8.05;MMP-2的活力分别为190.33±5.86、214.33±8.39、212.67±11.59。(2)SB203580对TGF-β1诱导的Ⅰ型胶原mRNA水平、蛋白水平及MMP-2活力的抑制率分别为51%、24%、20%。(3)PD98059对TGF-β1诱导的Ⅰ型胶原mRNA水平、蛋白水平以及MMP-2活力的抑制率分别为42%、13%、16%。结论TGF-β1可促进HLF-02细胞Ⅰ型胶原的转录和蛋白表达,上调MMP-2活力;p38、ERK激酶通路在此过程中具有重要的调控作用。

Objective To investigate the role of TGF-β1/MAPK signaling pathways in the expression of type Ⅰ collagen and activity of MMP-2,9 in human lung fibroblasts. Methods Human lung fibroblasts cell line （HLF-02） was cultured and and then stimulated with 10 ng/ml TGF-β1 for different time; SB203580 or PD98059 was added into culture medium to block p38 or ERK kinase pathway before incubated with TGF-β1 ;the expression of type Ⅰ collagen was detected by Westerm blotting and RT-PCR; zymogram analysis was used to analyze the activity of MMP-2 and MMP-9. Results （ 1 ） In the process of stimulation by TGF-β1, the type Ⅰ collagen mRNA level of 24 h, 48 h and 72 h group was： 1.33 ± 0.07,2.46 ± 0.09 and 2.39 ± 0.08 respectivley; and the type Ⅰ collagen protein level of 24 h, 48 h and 72 h group was： 114.89 ± 8.95,208.16 ± 6.75 and 211.46 ± 8.05 respectively;and the activity of MMP-2 of 24 h, 48h and 72 h group was： 190.33 ± 5.86,214.33 ± 8.39 and 212.67 ± 11.59 respectively. （2）SB203580 significantly inhibited the TGF-β1-induced expression of type Ⅰ collagen tuRN A, protein and MMP-2 activity （ inhibition ratio： 51%, 24% and 20% ） ; （3） PD98059 also significantly attenuated the TGF-β1-induced expression of type Ⅰ collagen mRNA, protein and MMP-2 activity （inhibition ratio： 42 %,13 % and 16% ）. Conclusion TGF-β1 is capable of inducing the expression of type Ⅰ collagen mRNA and protein and up-regulating MMP-2 activity in HLF-02 cells, p38 and ERK kinase signaling pathways play important role in regulation and control for this process.