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M. Hosseini, K. Song, M.J. Kelley, T.S. Acott; Involvement of JNK–MAP kinase pathway in TNF regulation of Matrix Metalloproteinases . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4380.
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Purpose:TNFα is a strong modulator of expression of trabecular meshwork matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Increasing MMP activity restores normal aqueous outflow facility. Studies were conducted to determine the role of jun N–terminal kinase (JNK) pathway in this process. Methods:Porcine trabecular meshwork cells (TM) were subjected to TNFα treatment for 5, 10, 15, 30, 60, 240 minutes and 24 hours. Western immunoblot analysis was used to examine the phosphorylation levels of MKK4 (Ser257/The261), JNK (Thr183/Tyr185) and c–jun (Ser63/Ser73). Treatment with JNK inhibitor II or transfection with dominant negative JNK was used to block the pathway. TM cells were co–transfected with a vector containing an alkaline phosphatase reporter gene under the control of MMP–3 promoter and another vector containing dominant negative JNK. Expression of secreted alkaline phosphatase was measured through chemiluminescence detection. Results:TNFα treatment significantly increased the phosphorylation and thus activation levels of MKK4, JNK and c–jun in a time dependent manner. MKK4 is phosphorylated at 5 minutes, with the highest activation level at 10 and 15 minutes. JNK (Thr183/Tyr185) is rapidly phosphorylated at 5 minutes, reaches a peak at 15 minutes and is maintained at moderate levels for 24 hours. Phosphorylation of c–jun at Ser63/Ser73 is significantly increased at 10 minutes, peaks at 60 minutes and is maintained at 10–20 fold for 24 hours. Inhibiting JNK completely blocks TNFα induction of myocilin and MMP–3 expression. Western blot studies show a reduction in c–jun phosphorylation at Ser63/Ser73 when cells are treated with JNK inhibitor II. The dominant negative JNK also blocked the effects of TNFα. Conclusions:The components of the JNK pathway are affected by TNFα treatment in TM cells. Activation of this pathway leads to the expression of metalloproteinases, which in turn lowers the intraocular pressure. Elucidation of these signaling pathways may lead to the discovery of potential new treatments for glaucoma.
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