Involvement of the Erk-MAP kinase pathway in TNFalpha regulation of trabecular matrix metalloproteinases and TIMPs.
OBJECTIVE
TNFalpha is a strong modulator expression of trabecular meshwork (TM) matrix metalloproteinase (MMP) and tissue inhibitor (TIMP). Laser trabeculoplasty appears to rely on this process to restore normal aqueous humor outflow facility. Thus, studies were conducted to determine whether the extracellular signal-regulated kinase (Erk)-mitogen-activated protein (MAP) kinase signal-transduction pathway is involved.


METHODS
Porcine TM cells were treated with TNFalpha, and changes in MMPs and TIMPs were evaluated by zymography and Western immunoblot assay. Phosphospecific antibodies to proteins from the Erk pathway were used to evaluate responses to treatment with TNFalpha. Inhibitors of Mek, the kinase that activates Erk, and of protein kinase C (PKC) isoforms were used to define pathway involvement.


RESULTS
Treatment with TNFalpha increased MMP-1, -3, and -9 and TIMP-1, whereas expression of MMP-2 was not affected and expression of TIMP-2 was decreased. Erk and Mek were rapidly phosphorylated after treatment with TNFalpha, and c-Raf-1 showed a significant bandshift. A specific inhibitor of Mek blocked the TNFalpha induction of the MMPs and TIMPs and the phosphorylation of Erk. An inhibitor of the PKC- micro isoform, which also blocks the effects of MMP-TIMP of TNFalpha, did not affect phosphorylation of Erk.


CONCLUSIONS
The components of this MAP kinase pathway in the TM are dramatically affected by TNFalpha and inhibition of Erk's phosphorylation blocks the changes in MMP and TIMP expression. PKC micro, which is also required in this transduction process, does not appear to be upstream from Erk in the signaling cascade. Manipulation of this and related TM signal-transduction pathways may provide targets for developing improved glaucoma treatments.
Alexander
J Preston
JP
Casey Eye Institute, Oregon Health and Sciences University, 3375 SW Terwilliger, Portland, OR 97201, USA.
Acott
Ted S
TS
D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism*; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D014409:Tumor Necrosis Factor-alpha; D020928:Mitogen-Activated Protein Kinases; D020782:Matrix Metalloproteinases; D013755:Tetradecanoylphorbol Acetate