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J. Rajaiya, N. Sadeghi, J. Chodosh; Differential Regulation of IL-8 and MCP-1 by NFB Transcription Factors in Adenovirus Keratitis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3460.
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Human Adenovirus type 19 (HAdV-19) infection of human keratocytes induces expression of several pro-inflammatory mediators including IL-8 and MCP-1.We have shown that chemokine expression in HAdV-19 infection is differentially regulated: extracellular signal regulated kinase (ERK) and p38 mitogen activated protein kinase (MAPK) mediate IL-8 expression, while Jun terminal kinase (JNK) activation controls MCP-1 expression. Each of these kinases activates NFΚB proteins to form specific homo- or heterodimers for transcriptional activation of target genes in a cell specific manner. However, the role of downstream NFΚB subunits and their kinetics of activation in the specificity of chemokine expression in adenovirus keratitis is not known.
Cell lysates from HAdV-19 and mock infected keratocytes with or without pretreatment with specific kinase inhibitors were subjected to immunoblot analysis with antibodies against components of the NFΚB pathway. Chromatin immunoprecipitation was performed at different time points post infection using NFΚB p65, p50, and cRel antibodies and analyzed for their IL-8 and MCP-1 promoter binding activity. ELISA was performed for IL-8 and MCP-1 at various time points post infection. SiRNA methodology was applied to dissect the role of NFΚB in the specificity of chemokine induction.
NFkB p65, p50, and cRel subunits were activated upon HAdV-19 infection. Specific activation events were blocked by chemical inhibitors to p38, ERK, JNK, and Src signaling pathways. Chromatin immunoprecipitation assay showed that p65 and cRel binding to the IL-8 promoter was increased at one hour relative to binding at the MCP-1 promoter. Knock down of NFΚB p65 by siRNA reduced IL-8 expression induced by infection, but did not affect MCP-1 induction.
These results indicate that HAdV-19 infection of keratocytes activates the classical NFΚB pathway, and that the subunit NFΚB-p65 is essential for IL-8 but not MCP-1 production. HAdV-19 induction of IL-8 occurs as an initial response to infection followed by MCP-1 expression later in infection.
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