ERK/AP-1途径在蛋白酶激活受体2促进肝癌细胞增殖中的作用

目的 探讨胰蛋白酶和蛋白酶激活受体2(PAR-2)激动剂SLIGKV-NH_2对肝癌细胞增殖的影响及其细胞内信号转导机制.方法 免疫荧光及逆转录(RT)-PCR法检测HepG2细胞PAR-2蛋白及mRNA的表达;用SLIGKV-NH_2、胰蛋白酶、反PAR-2激动肽VKGILS-NH_2及PD98059干预细胞生长.用流式细胞术检测细胞周期改变情况;噻唑蓝(MTT)法检测对细胞增殖能力的影响;RT-PCR法检测PAR-2、c-fos及增殖细胞核抗原(PCNA)mRNA表达变化;Western印迹检测c-fos和PCNA蛋白表达变化.结果 肝癌HepG2细胞存在PAR-2蛋白和mRNA的表达.50μmol/LSLIGKV-NH_2、25 nmol/L胰蛋白酶处理细胞后,PAR-2 mRNA表达量(PAR-2/β-肌动蛋白)分别为0.70±0.04,0.99±0.05,高于对照组(0.35±0.05,F=135.534,P<0.01).胰蛋白酶、SLIGKV-NH_2组G_0/G_1期比例均明显低于对照组[(56.11±0.85)%、(57.85±0.46)%比(79.12±0.67)%,均P<0.01],S期、G_2/M期细胞比例和细胞增殖指数(PI)则明显高于对照组(均P<0.01).胰蛋白酶、SLIGKV-NH_2可以诱导肝癌HepG2细胞增殖(F=319.287,P<0.01),上调c-fos、PCNA mRNA和蛋白的表达(均P<0.01),且这些作用可被ERK激活性蛋白激酶(MEK)抑制剂PD98059抑制(均P<0.01);反PAR-2激动肽VKGILS-NH_2对HepG2细胞增殖能力的影响不明显,与对照组相比差异无统计学意义(均P>0.05).结论 肝癌HepG2细胞表达PAR-2.胰蛋白酶、SLIGKV-NH_2可以通过激活PAR-2诱导HepG2细胞的增殖活性,且该过程部分由ERK/AP-1信号通路所介导.

Effect of ERK/AP-1 signaling pathway on proliferation of hepatoma cells induced by PAR-2 agonists

Objective To investigate the expression of protease activated receptor-2 (PAR-2) in human HepG2 hepatoma cells and elucidate the effects of trypsin and PAR-2 agonist peptide SLIGKV-NH_2 upon the proliferation of hepatoma cells and its intracellular signaling mechanism. Methods PAR-2 protein and mRNA expression were detected by immunofluorescence and RT-PCR. The cells were treated with SLIGKV-NH_2, trypsin, reverse PAR-2 agonist peptide VKGILS-NH_2 or PD98059. The changes of cell cycle distribution were evaluated by flow cytometry. The proliferative potential of HepG2 cells was estimated by MTT. The changes of PAR-2, c-fos and PCNA mRNA expression were detected by RT-PCR. The changes of c-fos and PCNA protein expression were detected by Western blotting. Results PAR-2 protein and mRNA were expressed in HepG2 cells. PAR-2 mRNA expression (PAR-2/β-actin) were 0.70±0.04 and 0.99± 0.05 respectively in cells treated with trypsin and SLIGKV-NH_2. They were both significantly higher than that in the control group (0.35±0.05, F=135.534, P<0.01). Percent G_0/G_1 phase of HepG2 cells treated with trypsin or SLIGKV-NH_2 were significantly lower than those in the control group [(56.11± 0.85)%、(57.85±0.46)% vs (79.12±0.67)%, both P<0.01] Percent S phase, G_2/M phase and proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH_2 were significantly elevated(P < 0.01). The proliferation-enhancing effects and the up-regulatian of mRNA and protein of c-fos and PCNA induced by trypsin or SLIGKV-NH_2 were significantly blocked by pretreatment with PD98059 (P<0.01). There was no statistical significance in proliferation of HepG2 cells between the reverse PAR-2 agonist peptide VKGILS-NH_2 and control group (P > 0.05). Conclusion PAR-2 is expressed in HepG2 hepatoma cells. PAR-2 activation induced by trypsin or SLIGKV-NH_2 promotes the proliferation of HepG2 cells partially via the ERK/AP-1 pathway.