A linear signal transduction pathway involving phosphatidylinositol 3-kinase, protein kinase Cε, and MAPK in mesangial cells regulates interferon-γ-induced STAT1α transcriptional activation

Abstract

Interferon-γ (IFN-γ) exerts an pleiotropic effect in mesangial cells in inflammatory glomerular diseases. The biologic effect of IFN-γ is mediated by STAT1α. The precise mechanism by which IFN-γ stimulates the transcriptional activity of STAT1α is poorly understood. I investigated the role of protein kinase C (PKC) ε in regulating the transcriptional activation of STAT1α in mesangial cells. IFN-γ increased PKCε activity in a time-dependent manner with a concomitant increase in STAT1α transcriptional activity. Expression of constitutively active PKCε mimicked the effect of IFN-γ on STAT1α-dependent transcription. Expression of dominant negative PKCε inhibited IFN-γ-induced STAT1α-dependent transcription. Ly294002, a pharmacological inhibitor of phosphatidylinositol (PI) 3-kinase, blocked IFN-γ-induced PKCε activity and resulted in inhibition of STAT1α transcriptional activity but had no effect on STAT1α tyrosine phosphorylation and STAT1α-DNA complex formation. A PKC inhibitor, H7, also had no effect on STAT1α tyrosine phosphorylation and DNA binding. However, Ly294002 and H7 blocked IFN-γ-induced serine phosphorylation of STAT1α. These data indicate that PI 3 kinase-dependent PKCε regulates STAT1α transcriptional activity in the absence of any effect on its DNA binding capability. In addition to activating PKCε IFN-γ increased MAPK activity, resulting in transcriptional activation of Elk-1, a nuclear target of MAPK. Ly294002 or a dominant negative PI 3-kinase significantly blocked IFN-γ-induced MAPK activity. On the other hand, ectopic expression of constitutively active PKCε significantly increased MAPK activity. IFN-γ-stimulated MAPK phosphorylated STAT1α in vitro. Inhibition of MAPK activity blocked IFN-γ-induced serine phosphorylation of STAT1α; but its tyrosine phosphorylation and DNA binding were partially inhibited. Finally, expression of dominant negative MAPK significantly inhibited IFN-γ-induced STAT1α-dependent transcription. These data provide the first evidence that IFN-γ stimulates PKCε in a PI 3-kinase-sensitive manner to activate MAPK, which regulates STAT1α transcriptional activity.