Hosseini Mojgan, Rose Anastasia Y, Song Kaili, Bohan Cynthia, Alexander J Preston, Kelley Mary J, Acott Ted S
Casey Eye Institute, Oregon Health & Science University, Portland, Oregon 97239-4197, USA.
Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1469-76. doi: 10.1167/iovs.05-0451.
The cytokines TNF and IL-1 mediate the MMP-3 increase that occurs in response to trabecular meshwork (TM) treatment by laser trabeculoplasty. This MMP-3 increase appears to play a key role in the efficacy of this treatment for open-angle glaucoma. Protein kinase Cmu and the Erk mitogen-activated protein (MAP) kinases are essential signaling components in transducing MMP-3 increases produced by treatment of TM cells with these cytokines. Here, the involvement of the JNK-MAP kinase pathway in this process was evaluated.
Porcine TM cells were treated with TNFalpha, IL-1alpha, or IL-1beta. Changes in MMP-3 and MMP-9 protein levels in the media were then determined by Western immunoblot. The effect of JNK inhibitor 2 was evaluated. Changes in the level of phosphorylation of JNK, c-Jun, ATF-2, MKK4, and MKK7 were also determined at various times after TNFalpha or IL-1alpha treatment. A 2.3-kb MMP-3 promoter fragment was cloned into a secreted alkaline phosphatase reporter vector. This reporter construct was cotransfected into TM cells with a mammalian expression vector containing a dominant-negative mutant of JNK. The involvement of JNK activity in the TNFalpha and IL-1alpha induction of MMP-3 expression was then evaluated.
TNFalpha, IL-1alpha, and IL-1beta increase media MMP-3 and MMP-9 protein levels, and JNK inhibitor 2 blocks these increases. JNK1/2, MKK4, c-Jun, and ATF-2 phosphorylation levels increase in response to TNFalpha and IL-1alpha treatment. JNK inhibitor 2 pretreatment blocks these c-Jun and ATF-2 phosphorylation increases. Dominant-negative JNK dramatically reduces the MMP-3 promoter-driven reporter activity induced by these cytokines.
JNK activity is necessary for the induction of MMP-3 and MMP-9 by TNFalpha, IL-1alpha, or IL-1beta in TM cells. Phosphorylation of components of the JNK signaling pathway and of the transcription factors c-Jun and ATF-2 support a role for this pathway in the induction of MMP-3 and MMP-9 in the TM in response to these cytokines. Thus, at least three separate signal transduction pathways are necessary in this signaling event in TM cells.
细胞因子TNF和IL-1介导了小梁网（TM）经激光小梁成形术治疗后MMP-3的增加。这种MMP-3的增加似乎在开角型青光眼的这种治疗效果中起关键作用。蛋白激酶Cμ和Erk丝裂原活化蛋白（MAP）激酶是转导这些细胞因子处理TM细胞所产生的MMP-3增加的重要信号成分。在此，评估了JNK-MAP激酶途径在该过程中的参与情况。
用TNFα、IL-1α或IL-1β处理猪TM细胞。然后通过Western免疫印迹法测定培养基中MMP-3和MMP-9蛋白水平的变化。评估JNK抑制剂2的作用。在TNFα或IL-1α处理后的不同时间，还测定了JNK、c-Jun、ATF-2、MKK4和MKK7的磷酸化水平变化。将一个2.3 kb的MMP-3启动子片段克隆到一个分泌型碱性磷酸酶报告载体中。将该报告构建体与含有JNK显性负性突变体的哺乳动物表达载体共转染到TM细胞中。然后评估JNK活性在TNFα和IL-1α诱导MMP-3表达中的作用。
TNFα、IL-1α和IL-1β增加培养基中MMP-3和MMP-9蛋白水平，而JNK抑制剂2可阻断这些增加。JNK1/2、MKK4、c-Jun和ATF-2的磷酸化水平在TNFα和IL-1α处理后升高。JNK抑制剂2预处理可阻断这些c-Jun和ATF-2磷酸化的增加。显性负性JNK显著降低了这些细胞因子诱导的MMP-3启动子驱动的报告活性。
JNK活性对于TNFα、IL-1α或IL-1β在TM细胞中诱导MMP-3和MMP-9是必需的。JNK信号通路成分以及转录因子c-Jun和ATF-2的磷酸化支持了该通路在TM中响应这些细胞因子诱导MMP-3和MMP-9中的作用。因此，在TM细胞的这一信号事件中至少需要三个独立的信号转导通路。
