Abstract | PURPOSE: 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 NFkappaB 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, NFkappaB 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 NFkappaB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFkappaB nuclear translocation. Western blot analysis revealed that activation of specific NFkappaB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFkappaB 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 NFkappaB p65 homodimer or NFkappaB p65/p50 heterodimer as early as 1 h post infection, whereas MCP-1 expression was dependent upon the NFkappaB cREL but not the p65 subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of NFkappaB p65 by short interfering RNA abrogated IL-8 but not MCP-1 expression after adenoviral infection. CONCLUSION: The kinetics of NFkappaB 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 NFkappaB subunits.
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Authors | Jaya Rajaiya, Neda Sadeghi, James Chodosh |
Journal | Molecular vision
(Mol Vis)
Vol. 15
Pg. 2879-89
(Dec 25 2009)
ISSN: 1090-0535 [Electronic] United States |
PMID | 20038977
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
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Chemical References |
- Chemokine CCL2
- Interleukin-8
- NF-kappa B
- Protein Subunits
- Transcription Factor RelA
- I-kappa B Kinase
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Topics |
- Adenoviridae
(physiology)
- Adenoviridae Infections
(complications, enzymology)
- Cell Nucleus
(metabolism)
- Chemokine CCL2
(genetics, metabolism)
- Chromatin Immunoprecipitation
- Enzyme Activation
- Humans
- I-kappa B Kinase
(metabolism)
- Interleukin-8
(genetics, metabolism)
- Keratitis
(complications, enzymology, metabolism, virology)
- Kinetics
- NF-kappa B
(antagonists & inhibitors, metabolism)
- Promoter Regions, Genetic
(genetics)
- Protein Binding
- Protein Subunits
(metabolism)
- Protein Transport
- Time Factors
- Transcription Factor RelA
(metabolism)
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