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J. Rajaiya, N. Sadeghi, R. A. Astley, F. Shariati, J. Chodosh; Preferential NFB Subunit Activation for Chemokine Expression in Adenovirus Keratitis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):734.
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Corneal inflammation associated with ocular adenovirus infection manifests with multifocal leukocytic infiltrates in the subepithelial corneal stroma. We have shown that Ad19-infected human corneal fibroblasts (HCF) express the chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), and that these chemokines are respectively regulated by the signaling molecules extracellular-signal-regulated kinase (ERK1/2) and Jun-terminal kinase (JNK). Downstream regulation of these chemokines is centrally controlled by the NFΚB transcription factor family, including RELA (p65), RELB, c-REL, NFΚB1 (P50/100) and NFΚB2 (p52/105). The NFΚB proteins form specific homo- or heterodimers for transcriptional activation or repression of target genes in a cell-specific manner. The purpose of this study was to determine the molecular events immediately upstream of chemokine expression in adenovirus keratitis.
Cell lysates from Ad19- and mock-infected HCF pretreated with chemical inhibitors specific to ERK, JNK, p38 and Src or with DMSO control were subjected to immunoblotting or immunoprecipitation to analyze activation of NFΚB pathway components. Nuclear extracts from parallel experiments were used in mobility shift assays using IL-8 and MCP-1 probes containing specific NFΚB binding sequences. Localization of NFΚB-p65 and p50 was analyzed by confocal microscopy. Transient transfections with NFΚB-p65 siRNA and IL-8-luciferase constructs were performed and the cell lysates analysed for luciferase acitivity at various times post-infection.
: NFkB-p65 and p50 subunits translocate to the cell nucleus upon Ad19 infection, and nuclear translocation could be blocked by inhibitors to specific signaling pathways. EMSA analysis revealed that IL-8 and MCP-1 induction in Ad19-infected HCF is transactivated by NFΚB-p65 homodimers and NFΚB-p65 /p50 heterodimers respectively. Ad19 infection induced phosphorylation of IKKα/ß and NFΚB-p65. Finally, knock down of NFΚB-p65 by siRNA reduced the expression of IL-8.
These results indicate that Ad19 infection stimulates a classical NFΚB pathway in HCF via IKKα/ß molecules, and that the subunit NFΚB-p65 is essential for chemokine expression.
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