强力霉素通过Smad和MAPK信号通路抑制转化生长因子-β1诱导的人角膜上皮细胞中基质金属蛋白酶-9的表达。

Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells.

作者信息

Kim Hyun-Seung, Luo Lihui, Pflugfelder Stephen C, Li De-Quan

机构信息

Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA.

出版信息

Invest Ophthalmol Vis Sci. 2005 Mar;46(3):840-8. doi: 10.1167/iovs.04-0929.

DOI:10.1167/iovs.04-0929

PMID:15728539

Abstract

PURPOSE

To evaluate the effects of TGF-beta1 and doxycycline on production of gelatinase MMP-9 and activation of Smad, c-Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways in human corneal epithelial cells.

METHODS

Primary human corneal epithelial cells were cultured to confluence. The cells were treated with different concentrations of TGF-beta1 (0.1, 1, or 10 ng/mL), with or without TGF-beta1-neutralizing mAb (5 microg/mL), SP600125 (30 microM), PD98059 (40 microM), SB202190 (20 microM), or doxycycline (5-40 microg/mL) for different lengths of time. Conditioned media were collected from cultures treated for 24 to 48 hours to evaluate the MMP-9 production by zymography and activity assay. Total RNA was isolated from cells treated for 6 to 24 hours to evaluate MMP-9 expression by semiquantitative RT-PCR and Northern hybridization. Cells treated for 5 to 60 minutes were lysed in RIPA buffer for Western blot with phospho-specific antibodies against Smad2, JNK1/2, ERK1/2, or p38.

RESULTS

TGF-beta1 increased expression, production, and activity of MMP-9 by human corneal epithelial cells in a concentration-dependent fashion. TGF-beta1 also induced activation of Smad2, JNK1/2, ERK1/2, and p38 within 5 to 15 minutes, with peak activation at 15 to 60 minutes. Doxycycline markedly inhibited the TGF-beta1-induced production of MMP-9 and activation of the Smad, JNK1/2, ERK1/2, and p38 signaling pathways. Its inhibitory effects were of a magnitude similar to SP600125, PD98059, and SB202190, specific inhibitors of the JNK1/2, ERK1/2, and p38 pathways, respectively.

CONCLUSIONS

These findings demonstrated that doxycycline inhibits TGF-beta1-induced MMP-9 production and activity, perhaps through the Smad and MAPK signaling pathways. These inhibitory effects may explain the reported efficacy of doxycycline in treating MMP-9-mediated ocular surface diseases.

摘要

目的

评估转化生长因子-β1（TGF-β1）和强力霉素对人角膜上皮细胞中明胶酶基质金属蛋白酶-9（MMP-9）的产生以及Smad、c-Jun氨基末端激酶（JNK）、细胞外调节激酶（ERK）和p38丝裂原活化蛋白激酶（MAPK）信号通路激活的影响。

方法

将原代人角膜上皮细胞培养至汇合状态。用不同浓度的TGF-β1（0.1、1或10 ng/mL）处理细胞，同时或不同时添加TGF-β1中和单克隆抗体（5 μg/mL）、SP600125（30 μM）、PD98059（40 μM）、SB202190（20 μM）或强力霉素（5 - 40 μg/mL），处理不同时长。收集处理24至48小时的培养物的条件培养基，通过酶谱分析和活性测定评估MMP-9的产生。从处理6至24小时的细胞中分离总RNA，通过半定量逆转录聚合酶链反应（RT-PCR）和Northern杂交评估MMP-9的表达。将处理5至60分钟的细胞在RIPA缓冲液中裂解，用于使用针对Smad2、JNK1/2、ERK1/2或p38的磷酸化特异性抗体进行蛋白质印迹分析。

结果

TGF-β1以浓度依赖性方式增加人角膜上皮细胞中MMP-9的表达、产生和活性。TGF-β1还在5至15分钟内诱导Smad2、JNK1/2、ERK1/2和p38的激活，在15至60分钟时激活达到峰值。强力霉素显著抑制TGF-β1诱导的MMP-9产生以及Smad、JNK1/2、ERK1/2和p38信号通路的激活。其抑制作用的程度与JNK1/2、ERK1/2和p38信号通路的特异性抑制剂SP600125、PD98059和SB202190相似。