Abstract 866: Rac1 Mediates Stat3 Activation in Vascular Endothelium Following Hypoxia-Reoxygenation
Tissue inflammation following ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) is dependent on upregulation of proinflammatory genes in vascular endothelium, mediated in part by activation of the transcription factor Stat3. The Jak-Stat pathway and the small GTPase Rac1 have been implicated in growth factor activation of Stat3. To better understand the mechanisms involved in Rac1-induced Stat3 activation after H/R, we performed a series of experiments in primary human umbilical vein endothelial cells. Overexpression of constitutively active (CA) Rac1 resulted in an increase in Stat3 activation, its binding to GAS elements (its DNA binding site), and expression of Stat-responsive genes, compared to control cells. Expression of dominant negative (DN) Rac1, or knockdown of Rac1 by specific siRNA had the opposite effect. We made the novel observation that Stat3 forms a multi-protein complex with Rac1 and PKC in an H/R-dependent manner that results in phosphorylation of Stat3 on S727. Direct binding between activated Rac1 and Stat3 was observed in yeast two-hybrid and in vitro binding assays using purified proteins. Confocal microscopic analysis of double immunofluorescent stained cells and cells expressing GFP-Rac1 revealed colocalization of CA Rac1 and Stat3 following exposure of cells to H/R, at the plasma membrane and unexpectedly, inside the nucleus. The amino acids necessary for Stat3 and Rac1 interaction were mapped to the coiled-coil domain of Stat3 and 40 N-terminal amino acids of Rac1. Employing specific pharmacological inhibitors as well as using in vitro phosphorylation assays, we found that a number of protein kinases, including PKC, Jak2 and the MAPK pathway involving JNK, ERK and p38 MAPK are involved in Rac1 activation of Stat3. These results reveal several important mechanisms of Rac1-induced Stat3 activation in vascular endothelial cells following H/R, and provide potential new therapeutic targets for reducing inflammation in post-ischemic tissues.