Specific NFkappaB subunit activation and kinetics of cytokine induction in adenoviral keratitis.
OBJECTIVE
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 (NFkappaB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFkappaB 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 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.


CONCLUSIONS
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.
Rajaiya
Jaya
J
Department of Ophthalmology, Howe Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.
Sadeghi
Neda
N
Chodosh
James
J
D000256:Adenoviridae / Q000502:physiology*; D000257:Adenoviridae Infections / Q000150:complications / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D007634:Keratitis / Q000150:complications / Q000201:enzymology / Q000378:metabolism* / Q000821:virology*; D007700:Kinetics; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism
D018932:Chemokine CCL2; D016209:Interleukin-8; D016328:NF-kappa B; D021122:Protein Subunits; D051996:Transcription Factor RelA; D051550:I-kappa B Kinase