Corneal IL-8 Expression Following Adenovirus Infection Is Mediated by c-Src Activation in Human Corneal Fibroblasts

Abstract

Emerging evidence indicates that intracellular signaling cascades mediate entry of pathogenic adenoviruses into target host cells as well as some of the undesirable inflammatory responses to adenoviral gene vectors. We found that Ad19 infection of cultured human corneal fibroblasts induced IL-8 gene transcription independently of IL-1β, TNF-α, and viral gene expression, suggesting that intracellular signaling events might mediate early inflammatory events in adenovirus keratitis. Heat but not UV light inactivation of the virus abrogated the effect of infection on IL-8 mRNA and protein levels, consistent with a viral binding-mediated mechanism. The tyrosine kinase inhibitor herbimycin blocked Ad19-induced IL-8 expression. Western blot analysis revealed tyrosine phosphorylation of the functionally related kinases c-Src and extracellular signal-regulated kinase (ERK) 1/2 in corneal fibroblasts within 15 min after infection. Respective inhibitors of these kinases, PP2 and PD98059, also blocked Ad19-induced IL-8 mRNA and protein expression. Application of inhibitors to Src and ERK kinase assays suggested an upstream relationship of c-Src to ERK. Finally, DNA microarray studies performed 1 h after Ad19 or mock infection of corneal fibroblasts in the presence or absence of the Src-specific inhibitor PP2 confirmed a relationship between c-Src and IL-8 expression in Ad19-infected corneal cells. c-Src may act as a global regulator of early proinflammatory host responses to Ad19 infection of the human cornea.

Expression and Activity of Src Regulate Interleukin-8 Expression in Pancreatic Adenocarcinoma Cells: Implications for Angiogenesis

“…Both endogenous and ectopic c-Src expression and activity correlate with IL-8 protein expression, and inhibition of c-Src expression via siRNA or activity via the pharmacologic inhibitor PP2 significantly reduce IL-8 levels in cell culture supernatants. These data are consistent with studies in normal cell systems that show that Src mediates IL-8 expression downstream of diverse stimuli through many of the same signaling molecules used to regulate IL-8 expression in pancreatic cancer cells (24,26,39). Our data suggest that constitutive activation of Src in pancreatic tumor cells deregulates these pathways and leads to constitutive IL-8 expression and further that the extent of Src activation correlates with the amount of IL-8 produced.…”

Corneal Cell Survival in Adenovirus Type 19 Infection Requires Phosphoinositide 3-Kinase/Akt Activation

“…We have suggested that leukocyte infiltration into the human cornea after adenovirus type 19 infection depends upon an intracellular signaling cascade initiated by binding of the virus to cells within the superficial corneal stroma and culminating in the expression of leukocyte chemoattractants and adhesion molecules (9,36,37). The data presented in the present paper suggest that productive infection of corneal stromal fibroblasts by adenovirus type 19 further depends upon activation of the antiapoptotic PI3K/Akt signaling cascade.…”

“…Real-time PCR was performed as previously described (37) and DNase I (Promega) were used to prevent RNase action and remove contaminating DNA, respectively, followed by phenol-chloroform extraction, ethanol precipitation, and resuspension of the RNA. Spectrophotometric readings at 260 nm were used to determine RNA concentrations.…”

“…Our previous studies demonstrated phosphorylation and activation of cSrc in adenovirus type 19-infected HCF (37). Therefore, we wished to determine if cSrc plays a role in activation of PI3K.…”

“…We have previously shown that infection of primary human corneal fibroblasts (HCF) with adenovirus type 19 results in activation of focal adhesion kinase, cSrc, and ERK1/2 and that these signaling proteins mediate the expression of inflammatory mediators, such as interleukin-8, from virus-infected cells (36,37). Because PI3K has been reported to act in conjunction with cSrc to mediate adenoviral entry (30,31), we turned to the study of PI3K and Akt in adenovirus-infected human corneal cells.…”