Abstract
Abstract: :
Purpose: Activation of PI3–kinase by interaction with exogenous adenoviruses may mediate viral internalization into target cells. We sought to determine the role of AKT, the downstream substrate of PI3–kinase in the regulation of apoptosis and expression of inflammatory mediators in adenovirus type 19 (Ad19)–infected human corneal fibroblasts (HCF). Methods: Ad19 and mock infected HCF were solubilized at select time points, and cell lysates were subjected to SDS–PAGE followed by immunoblot analysis with phospho–specific AKT, GSK–3 beta, and BAD antibodies. TUNEL assays were performed after treatment of HCF with LY294002, a specific inhibitor of PI3–K activation, or transfection with dominant negative or constitutively active AKT mutants, followed by Ad19 infection. Expression of MCP–1 mRNA and protein was measured by real–time PCR and ELISA respectively. Results: Ad19 infection induced activation of PI3–kinase and AKT at 10 to 30 minutes post–infection, while phosphorylated GSK–3 beta and BAD, potential downstream substrates of AKT, were evident at 30 to 50 min post–infection. Inhibition of AKT activity by LY294002 or by a dominant negative AKT induced early viral cytopathic effect and premature apoptosis in Ad19–infected HCF. In contrast, over–expression of constitutively active AKT prevented early Ad19–induced apoptosis in LY294002–treated HCF. Pretreatment with LY294002 also reduced MCP–1 expression in Ad19–infected HCF at both mRNA and protein levels. Conclusions: In Ad19–infected human corneal cells, inhibition of the PI3–kinase/AKT intracellular signaling pathway induces premature apoptotic cell death, while over–expression of constitutively active AKT prevents apoptosis, even in the presence of a potent and specific PI3–kinase inhibitor. Apoptotic cells cannot sustain viral gene expression or packaging of the virus; activation of AKT by adenoviral infection may serve to maintain viability of the target cell to facilitate viral replication. The observed reduction in expression of MCP–1 mRNA and protein by a PI3–kinase inhibitor additionally suggests that MCP–1 expression may be mediated through PI3–K/AKT pathway. These findings are consistent with a central role for the PI3–kinase/AKT signaling pathway in adenoviral ocular pathogenesis.
Keywords: adenovirus • cornea: stroma and keratocytes • signal transduction