Activation of Stat3 in endothelial cells following hypoxia–reoxygenation is mediated by Rac1 and protein kinase C

Stat3 is an important transcription factor that regulates both proinflammatory and anti-apoptotic pathways in the heart. This study examined the mechanisms of activation of Stat3 in human endothelial cells following hypoxia/reoxygenation (H/R). By expression of constitutively active Rac1 mutant protein, and by RNA silencing of Rac1, we found that Stat3 Y705 and S727 phosphorylation following H/R is dependent on Rac1. Reactive oxygen species produced during H/R, and direct physical association with Rac1 both contribute to Stat3 activation. Stat3 forms a multiprotein complex with Rac1 and PKC in an H/R-dependent manner, which at least in part, appears to regulate Stat3 S727 phosphorylation. Selective inhibition of PKC with calphostin C produces a marked suppression of Stat3 S727 phosphorylation. The association of Stat3 with Rac1 occurs predominantly at the cell membrane, but also inside the nucleus, and occurs through the binding of the coiled-coil domain of Stat3 to the 54 NH2-terminal residues of Rac1. Transfection with a peptide comprising the NH2-terminal 17 amino acid residues of Rac1 inhibits Stat3 S727 phosphorylation after H/R. Thus, Stat3 is activated in endothelial cells by H/R through Rac1-dependent signaling pathways resulting in physical association between Rac1 and Stat3 and the formation of a novel multiprotein complex with PKC.

► Stat3 activation by hypoxia/reoxygenation is mediated by both Rac1 and PKC. ► Activation of Stat3 occurs through a multiprotein complex with Rac1 and PKC. ► Stat3 binds through its coiled-coil domain to the 54 NH2-terminal residues of Rac1. ► A peptide of the 17 NH2-terminal amino acids of Rac1 can inhibit Stat3 activation.