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J. Rajaiya, R. A. Astley, F. Shariati, R. V. S. Rajala, J. Chodosh; Hsp27-Mediated p38/NFB-p65 Association: Effect on Interleukin-8 Expression in Adenovirus Infected Keratocytes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):823.
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Our earlier studies showed that human adenovirus serotype 19 (HAdV-19) infection of human keratocytes induces expression of the pro-inflammatory mediator interleukin-8 (IL-8). We have also shown that virus infection causes activation of the p38 mitogen-associated protein kinase (MAPK), and its downstream target heat shock protein 27 (Hsp27), and also induces p38 MAPK dependent NFΚB-p65 activation. Inhibition of p38 MAPK activity with p38-specific siRNA reduced subsequent IL-8 expression. However, the molecular mechanisms of p38 MAPK activation in HAdV-19 infection and the possible roles of Hsp27 and NFΚB-p65 in subsequent chemokine expression remain unclear.
Mock and HAdV-19 infected keratocytes that had been transfected with either control siRNA, Hsp27 siRNA, or NFΚB-p65 siRNA were analyzed using Western blot, immunoprecipitation, and confocal microscopy for activation of signaling molecules within the NFΚB and p38 MAPK pathways. Nuclear extracts from parallel experiments were used in mobility shift assays using IL-8 probe containing specific NFΚB-p65 binding sequence. ELISA was performed to measure IL-8 protein levels.
Hsp27 and NFΚB-p65 were phosphorylated within 30 minutes of HAdV-19 infection. Immunoprecipitation assays revealed an association between NFΚB-p65 and p38 in virus infected cells, but not in mock infected cells or in cells transfected with Hsp27 specific siRNA. Confocal analysis showed the presence of p38 MAPK in the nucleus of virus infected cells as compared to mock infected cells. Nuclear translocation of p38 MAPK could be inhibited by transfection with NFΚB-p65 siRNA. By ELISA, IL-8 expression was reduced in cells that had been transfected with Hsp27 siRNA.
Our results strongly suggest that Hsp27-dependent activation of the p38 MAPK/NFΚB-p65 pathway is essential to IL-8 expression in HAdV-19 infected keratocytes. The data further suggest possible roles for a physical association between p38 MAPK and NFΚB-p65 and subsequent nuclear translocation of p38 MAPK in IL-8 expression in adenoviral keratitis.
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