Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.