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J. Chodosh, J. Rajaiya, J. Xiao; Adenovirus Type 19 Activation of p38 MAPK Regulates Il-8 Expression in Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3167.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies showed that adenovirus type 19 (Ad19) infection of human corneal fibroblasts (HCF) induces activation of cSrc, and its downstream targets, the mitogen-activated protein kinases ERK1/2 and c-Jun-terminal kinase (JNK) which then differentially mediate IL-8 and MCP-1 expression, respectively. Here, we investigate the role of another mitogen-activated protein kinase, the p38 MAPK, and its downstream targets in adenoviral ocular pathogenesis.
Cell lysates from Ad19 and mock-infected HCF solubilized at various time points or pretreated with p38 specific inhibitor were subjected to immunoblot analysis with antibodies against components of the p38 MAPK pathway, or immunoprecipitated for p38 kinase assay. Similarly, expression of IL-8 mRNA and protein, and the subcellular localization of NFΚB-p65 were analyzed by RT-PCR, ELISA and confocal microscopy, respectively, and their specificity to the p38 pathway was determined by using p38-specific chemical and siRNA inhibitors.
Ad19 induced phosphorylation of MKK3/MKK6 and p38, along with the downstream effectors MAPKAP2, heat shock protein 27 (HSP27), ATF-2, and NFΚB-p65 in HCF at 30 minutes post-infection. Kinase activity of p38 was evident by its ability to phosphorylate exogenous ATF-2 at 15 and 30 minutes post infection. Inhibition of p38 with SB203580 or siRNA blocked nuclear localization of NFΚB-p65 and reduced IL-8 expression in Ad19-infected HCF, respectively. Finally, PP2, a specific inhibitor of Src, blocked phosphorylation of p38, ATF-2, and HSP27 upon subsequent infection.
The p38 MAPK cascade is rapidly activated and contributes to IL-8 expression in Ad19-infected HCF. Furthermore, when viewed in context with previous data from our laboratory, it appears that the activation of Src kinase upon Ad19 infection may regulate all three MAP kinase pathways.
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