Doxycycline inhibits TGF-β1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells
Doxycycline inhibits TGF-β1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells
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DOI：
10.1167/iovs.04-0929
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发表时间：
2005-03-01
影响因子：
4.4
通讯作者：
Li, DQ
中科院分区：
文献类型：
--
作者：
Kim, HS;Luo, LH;Li, DQ
目的.观察TGF-β 1和强力霉素对人角膜上皮细胞明胶酶MMP-9的表达及Smad、c-Jun N-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）信号通路的影响。原代人角膜上皮细胞培养至汇合。用不同浓度的TGF-β 1（0.1、1或10 ng/mL）、有或没有TGF-β 1-中和mAb（5 μ g/mL）、SP 600125（30 μ M）、PD 98059（40 μ M）、SB 202190（20 μ M）或多西环素（5-40 μ g/mL）处理细胞不同的时间长度。从处理24至48小时的培养物中收集条件培养基，以通过酶谱和活性测定来评估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RT-PCR和北方杂交评估MMP-9表达。用RIPA缓冲液裂解细胞5 ~ 60 min，用抗Smad 2、JNK 1/2、ERK 1/2或P38的磷酸化特异性抗体进行Western印迹。TGF-β 1以浓度依赖性方式增加人角膜上皮细胞MMP-9的表达、产生和活性。TGF-β 1还在5至15分钟内诱导Smad 2、JNK 1/2、ERK 1/2和p38的活化，在15至60分钟达到活化峰值。强力霉素显著抑制TGF-β 1诱导的MMP-9的产生以及Smad、JNK 1/2、ERK 1/2和P38信号通路的激活。其抑制作用与JNK 1/2、ERK 1/2和P38通路特异性抑制剂SP 600125、PD 98059和SB 202190相似。这些发现表明，强力霉素抑制TGF-β 1诱导的MMP-9的生产和活性，可能通过Smad和MAPK信号通路。这些抑制作用可以解释报道的多西环素治疗MMP-9介导的眼表疾病的疗效。
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 mug/mL), SP600125 (30 muM), PD98059 (40 muM), SB202190 (20 muM), or doxycycline (5-40 mug/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.