PKC和MAPK的激活在TGF-β1诱导人近端肾小管细胞发生细胞肥大中的作用-东南大学学报医学版

PKC和MAPK的激活在TGF-β1诱导人近端肾小管细胞发生细胞肥大中的作用

单位：东南大学附属中大医院,肾脏病研究所,江苏,南京,210009

分类号：R364.31

出版年·卷·期（页码）：2005·24·第二期（71-75）

摘要：

目的:研究细胞内蛋白激酶C(PKC)和有丝分裂原活化蛋白激酶(MAPK)途径的激活在转化生长因子-β1(TGF-β1)诱导人近端肾小管上皮细胞(HK2细胞)发生细胞肥大中的作用.方法:HK2细胞培养液中预先加入MAPK特异性抑制剂(MEK1)PD 98059或PKC特异性抑制剂STS,然后给予TGF-β1刺激,8h后用MTT法检测细胞增殖、[3H]-亮氨酸掺入试验检测细胞内蛋白水平、流式细胞仪检测细胞周期分布.结果:TGF-β1抑制细胞的增殖、促进细胞内蛋白的合成及促使细胞生长停滞在G0-G1期[(TGF-β1:0.719±0.032vs.对照组:1.199±0.022,P＜0.01);(TGF-β1:286.00±10.15vs.对照组:206.25±19.14,P＜0.01);(TGF-β1:81.633%±3.477%vs.对照组:60.497%±4.813%,P＜0.01)].PD98059和STS均能显著抑制TGF-β1诱导的细胞[3H]-亮氨酸掺入增加(TGF-β1+PD 98059:216.00±13.13,TGF-β1+STS:231.50±10.50vs.TGF-β1:286.00±10.15,P分别小于0.05和0.01).TGF-β1的以上作用均能被STS和PD 98059逆转(TGF-β1+STS:67.610%±3.732%,TGF-β1+PD98059:65.620%±5.625%vs.TGF-β1:81.633%±3.477%,均为P＜0.01;TGF-β1+STS:1.009±0.022;TGF-β1+PD 98059:0.947±0.022 vs.TGF-β1:0.719±0.032,均为P＜0.01).结论:PKC和MAPK的激活可能参与了TGF-β1诱导HK2细胞肥大的发生.

Objective To study the role of PKC and MAPK in TGF-β_1 stimulated cell hypertrophy in human proximal tubular cell. Methods All experiments were performed with human transformed proximal tubular epithelial cell line HK_2 under serum-free condition. Growth-arrested HK_2 cells were pre-treated with MEK_1 inhibitor, PD98059 or PKC inhibitor Staurosporine aglycone (STS) before adding TGF-β_1. After 8 hours, incorporation of [ 3H]-leucine was used to assess the level of protein synthesis and MTT was used to determine cell proliferation. Fluorescence-activated cell sorter (FACS) analysis was used to analyze cell cycle distribution.Results TGF-β_1 inhibited cell proliferation (TGF-β_1 group: 0.719±0.032 vs. Control: 1.199±0.022, P<0.01), enhanced incorporation of [ 3H]-leucine [TGF-β_1 group: 286.00±10.15 vs. Control: 206.25±19.14 cpm·(105 cell) -1, P<0.01] and arrested cells in the G_1 phase of the cell cycle (TGF-β_1 group: 81.633%±3.477% vs. Control: 60.497%±4.813% P<0.01). STS could potently attenuate TGF-β_1 induced incorporation of [ 3H]-leucine [TGF-β_1+PD 98059: 216.00±13.13; TGF-β_1+STS: 231.50±10.50 vs. TGF-β_1:286.00±10.15 cpm·(105 cell) -1,P<0.05, 0.01 respectively]. Both STS and PD98059 could reverse the inhibition of TGF-β_1 on cell proliferation in HK_2 cell (TGF-β_1+STS: 1.009±0.022; TGF-β_1+PD 98059: 0.947±0.022 vs. TGF-β_1: 0.719±0.032, P<0.01). FACS demonstrated TGF-β_1 arrested cell cycle at G_0/G_1 phrase, which was reversed when cells were exposed to serum-free medium containing STS+TGF-β_1 or PD98059+TGF-β_1 (Percentage of cells present in G_0/G_1 phrase: TGF-β_1+STS: 67.610%±3.732%; TGF-β_1+ PD 98059: 65.620%±5.625% vs. TGF-β_1: 81.633%±3.477% P<0.01). Conclusion Our results suggest that TGF-β_1induced cell hypertrophy in HK_2 cell may require activation of both PKC and MAPK.

参考文献：

[1] STRUTZ F, MuLLER G A. Interstitial pathomechnisms underlying progressive tubulointerstitial damage. 1999. doi:10.1159/000025911
[2] WOLF G, ZIYADEH F N. Molecular mechanism of diabetic renal hypertrophy. 1999. doi:10.1046/j.1523-1755.1999.00590.x
[3] RYAN M J, JOHNSON G, KIRK J. HK-2:an immortalized proximal tubule epithelial cell line from normal adult human kidney, 1994
[4] GILBERT R E, COOPER M E. The tubulointerstitium in progressive diabetic kidney disease:more than an aftermath of glomerular injury?. 1999
[5] CHEN Y J, BLOM I E, SA S. CTGF expression in mesangial cells:involvement of SMADs,MAP kinase,and PKC. 2002. doi:10.1111/j.1523-1755.2002.kid567.x
[6] KUCICH U, ROSENBLOOM J C, HERRICK D J. Signaling events required for transforming growth factor-β stimulation of connective tissue growth factor expression by cultured human lung fibroblasts, 2001
[7] KUTZ S M, HORDINES J, MCKEOWN-LONGO P J. TGFbetal-induced PAI-1 gene expression requires MEK activity and cell-to-substrate adhesion, 2001
[8] RODRIGUEZ-BARBERO A, OBREO J, YUSTE L. Transforming growth factor-beta1 induces collagen synthesis and accumulation via p38 mitogen-activated protein kinase (MAPK) pathway in cultured L(6)E(9) myoblasts. 2002(2-3)
[9] UTSUGI M, DOBASHI K, ISHIZUKA T. C-jun-NH2-terminal kinase mediates expression of connective tissue growth factor induced by transforming growth factor- [ beta } 1 in human lung fibroblasts. 2003(6). doi:10.1165/rcmb.4892
[10] YUE J, MULDER K M. Transforming growth factor-beta signal transduction in epithelial cells. 2001(1)
[11] WOLF G. Cell cycle regulation in diabetic nephropathy, 2000(zk)

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录