Endothelial GPCRs Activate p38 MAPK Inflammatory Signaling Via Non‐canonical TAB1, 2 and 3‐dependent Pathways

Vasculature disease is a leading cause of morbidity and mortality worldwide. A hallmark of vascular inflammation is endothelial cell dysfunction, which is induced by vascular inflammatory mediators, many of which act through G protein‐coupled receptors (GPCRs). We previously showed that thrombin stimulation of endothelial protease‐activated receptor‐1 (PAR1) activates p38 MAPK through a non‐canonical pathway mediated by TAB1 (transforming growth factor‐β‐activated kinase binding protein‐1). TAB1 binds directly to p38α inducing a conformational change resulting in autophosphorylation and activation bypassing the requirement for upstream MAP2Ks. We also found that non‐canonical p38 activation induced by thrombin/PAR1 promotes endothelial barrier permeability in vitro and further showed that p38 is required for PAR1‐stimulated vascular leakage in vivo . While non‐canonical p38 activation induced by PAR1 is established, it remains unclear if other endothelial GPCRs also promote p38 activation through a TAB1‐dependent pathway and was examined. Here we report that multiple GPCRs agonists including histamine, PGE2, ADP and thrombin induce p38 activation via the non‐canonical TAB‐dependent pathway to promote cytokine production. We assessed GPCR‐induced p38 activation in macrovascular and microvascular endothelial cell lines including HUVEC, EA.hy926 and HDMECs. Using immunoblotting and qPCR, the expression of p38, TAB1, 2, 3 and MKK3,6 were quantified and appear to be comparable across the endothelial cell lines. To determine if p38 activation induced by GPCRs is mediated by autophosphorylation, we used the p38α/β SB203580 inhibitor which blocks p38 autophosphorylation but not phosphorylation mediated by upstream MAP2Ks. In HUVEC, EA.hy926 and HDMEC pretreated with SB203580, all four agonists including histamine, PGE2, ADP and thrombin failed to promote p38 phosphorylation, suggesting that endothelial GPCR‐induced p38 activation occurs through autophosphorylation. Next, we used combinations of TAB1, 2 and 3‐specific siRNAs to deplete endothelial cells of TAB proteins and found that all GPCR agonists require TAB expression to induce maximal p38 activation in endothelial cells. In addition, endothelial GPCR‐induced cytokine production was also dependent on p38 signaling and TAB expression. Taken together, these data support a critical role for TAB1‐dependent activation of p38 MAPK induced by endothelial GPCRs in inflammatory signaling. Support or Funding Information This work is funded by NIH/NIGMS R01 GM090689, R01 GM116597 and UC Tobacco‐related Disease Research Program High Impact Pilot Award 26IP‐0050 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .