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J. Xiao, J. Chodosh; Ad19 Infection of Human Corneal Fibroblasts Induces MCP–1 Expression Through JNK . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1018.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Our previous studies indicate that adenovirus type 19 (Ad19) induces expression of several pro–inflammatory molecules including IL–8 and MCP–1 in human corneal fibroblasts (HCF), and that the c–Src/extracellular signal–regulated kinase (ERK) cascade mediates IL–8 expression. In this context, we sought to investigate the potential role of the c–Jun N–terminal kinase (JNK) pathway in adenoviral ocular pathogenesis. Methods: Primary HCF were cultured from human donor corneas and infected at third passage with cesium–chloride gradient–purified Ad19 at a MOI of 50. Ad19– and mock–infected HCF were solubilized at various time points post–infection, and cell lysates subjected to SDS–PAGE followed by immunoblot analysis with a panel of antibodies against components of MKK4/7–JNK–c–Jun pathway, or immunoprecipitated for subsequent JNK kinase assay. The induction of chemokine mRNA and protein was determined by real–time PCR and ELISA, respectively. Results: Ad19 induced phosphorylation of MKK7, JNK, and the downstream transcription factor c–Jun at 15 min and 30 min post–infection. JNK kinase activity was demonstrated at 15 and 30 min post–infection using the GST– c–Jun fusion protein as a target substrate. SP600125, a specific pharmacological inhibitor of JNK, blocked MCP–1 but not IL–8 mRNA expression and protein production. Finally, PP2, a specific inhibitor of c–Src that inhibits ERK activation in Ad19–infected HCF, also blocked JNK phosphorylation after infection. Conclusions: The MKK7–JNK–c–Jun cascade is rapidly activated, and mediates MCP–1 expression in Ad19–infected HCF. Furthermore, the activation of c–Src upon Ad19 infection appears to regulate both the ERK and JNK–c–Jun pathways. Intracellular signaling molecules present novel therapeutic targets in ocular adenovirus infection.
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