Specific NFκB subunit activation and kinetics of cytokine induction in adenoviral keratitis

[摘要] Purpose: Corneal inflammation associatedwith ocular adenoviral infection is caused by leukocytic infiltrationof the subepithelial stroma in response to expression of interleukin-8(IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infectedcorneal cells. We have shown that these two chemokines are activated bythe mitogen-activated protein kinases (MAPKs) extracellularsignal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase(JNK) for MCP-1. It is also well established that transcription of eachof these chemokines is tightly controlled by the nuclear factor kappa B(NFκB) transcription factor family. Therefore, we sought to betterunderstand the differential regulation of chemokine expression by NFκBin adenoviral infection of the cornea. Methods: Primary keratocytes derivedfrom human donor corneas were treated with signaling inhibitors andsmall interfering RNA specific to MAPKs, and infected with adenovirusfor different time periods before analysis. Activation of specific NFκBsubunits was analyzed by western blot, confocal microscopy,electromobility shift assay, and chromatin immunoprecipitation, andchemokine expression was quantified by enzyme-linked immunosorbentassay. Results: Upon adenoviral infection, NFκBp65, p50, and cREL subunits translocate to the nucleus. Thistranslocation is blocked by inhibitors of specific MAPK signalingpathways. Confocal microscopy showed that inhibitors of the p38, JNK,and ERK pathways differentially inhibited NFκB nuclear translocation,while PP2, an inhibitor of Src family kinases, completely inhibitedNFκB nuclear translocation. Western blot analysis revealed thatactivation of specific NFκB subunits was time dependent followinginfection. Chromatin immunoprecipitation experiments indicated thatbinding of NFκB p65 and p50 subunits to the IL-8 promoter upon viralinfection was differentially reduced by chemical inhibitors of MAPKs.Electromobility shift assay and luciferase assay analysis revealed thattransactivation of IL-8 occurred with binding by the NFκB p65 homodimeror NFκB p65/p50 heterodimer as early as 1 h post infection, whereasMCP-1 expression was dependent upon the NFκB cREL but not the p65subunit, and occurred 4 h after IL-8 induction. Finally, knockdown ofNFκB p65 by short interfering RNA abrogated IL-8 but not MCP-1expression after adenoviral infection. Conclusion: The kinetics of NFκB subunitactivation are partly responsible for the observed pattern of acuteinflammation in the adenoviral-infected cornea. MAPKs differentiallyregulate chemokine expression in adenoviral keratitis by differentialand time-dependent activation of specific NFκB subunits.

[学科分类] 生物化学/生物物理