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dc.contributor.authorRajaiya, Jayabarathy
dc.contributor.authorSadeghi, Neda
dc.contributor.authorChodosh, James
dc.date.accessioned2011-02-18T17:52:13Z
dc.date.issued2009
dc.identifier.citationRajaiya, Jaya, Neda Sadeghi, and James Chodosh. 2009. Specific NFκB subunit activation and kinetics of cytokine induction in adenoviral keratitis. Molecular Vision 15: 2879-2889.en_US
dc.identifier.issn1090-0535en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:4728143
dc.description.abstractPurpose: Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFκB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFκB in adenoviral infection of the cornea. Methods: Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFκB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay. Results: Upon adenoviral infection, NFκB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. 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 inhibited NFκB nuclear translocation. Western blot analysis revealed that activation of specific NFκB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFκB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis revealed that transactivation of IL-8 occurred with binding by the NFκB p65 homodimer or NFκB p65/p50 heterodimer as early as 1 h post infection, whereas MCP-1 expression was dependent upon the NFκB cREL but not the p65 subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of NFκB p65 by short interfering RNA abrogated IL-8 but not MCP-1 expression after adenoviral infection. Conclusion: The kinetics of NFκB subunit activation are partly responsible for the observed pattern of acute inflammation in the adenoviral-infected cornea. MAPKs differentially regulate chemokine expression in adenoviral keratitis by differential and time-dependent activation of specific NFκB subunits.en_US
dc.language.isoen_USen_US
dc.publisherMolecular Visionen_US
dc.relation.isversionofhttp://www.molvis.org/molvis/v15/a304/en_US
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797044/pdf/en_US
dash.licenseLAA
dc.titleSpecific NFκB Subunit Activation and Kinetics of Cytokine Induction in Adenoviral Keratitisen_US
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden_US
dc.relation.journalMolecular Visionen_US
dash.depositing.authorChodosh, James
dc.date.available2011-02-18T17:52:13Z
dash.affiliation.otherHMS^Ophthalmologyen_US
dash.contributor.affiliatedChodosh, James
dash.contributor.affiliatedRajaiya, Jaya


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