Combinatorial Activation of STAT3 by EGF and Thrombin in Endothelial Cells

Endothelial cells line the luminal surface of blood vessels and form a regulatory interface between the bloodstream and underlying tissues. The endothelium responds to diverse, and potentially conflicting, environmental signals to regulate vessel growth, leukocyte adhesion, thrombogenicity, and vascular tone. Signaling pathways may interact, or crosstalk, in combinatorial signaling environments to enable cells to process disparate extracellular information at downstream signaling nodes and formulate appropriate biological responses based on combinations of extracellular stimuli. We have reported that simultaneous stimulation of endothelial cells with EGF and thrombin synergistically induces expression of immediate early genes (IEGs) associated with growth and angiogenesis. We sought to characterize the molecular mechanism of EGF receptor (EGFR) and thrombin receptor (protease-activated receptor-1, PAR-1) crosstalk that regulates synergistic IEG induction. Using a bioinformatic comparison of IEG promoter regions, we identified the transcription factor STAT3 as a critical integrator of EGF and thrombin-activated signaling pathways. Depletion of STAT3 by RNAi completely abrogates synergistic IEG induction following EGFR/PAR-1 activation. Analysis of upstream signaling pathways by phosphoproteomic, RNAi, and pharmacological approaches demonstrated that GSK-3╬▒/╬▓-dependent phosphorylation of STAT3 Ser727 is required for synergistic activation of the EGR1 promoter. Functionally, combinatorial EGFR/PAR-1 signaling suppresses EGF-induced proliferation and thrombin-induced leukocyte adhesion, and triggers a STAT3-dependent increase in endothelial cell migration. Next, we used quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) to examine the inducible flux and abundance of endogenous STAT3 phosphoforms. We identified and characterized a STAT3 phosphoform that is simultaneously modified at Thr714 and Ser727 by GSK-3╬▒/╬▓. Both Thr714 and Ser727 are required for STAT3-dependent