Mapping of GPCR-p38 Signaling Reveals A Spatiotemporal Key for Regulating Vascular Inflammation.

Vascular dysfunction and inflammation drives the onset and progression of many diseases including acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The mitogen-activated protein kinase (MAPK) p38 is a master regulator of inflammation and vascular function, yet despite extensive and ongoing studies, p38-targeted therapeutics have failed. Their failure is in part due to the ubiquitous expression and dichotomous role of p38 in physiological and pathological signaling. Defining selective pathological p38 signaling pathways represents a novel therapeutic strategy to treat many diseases. Our prior studies demonstrate that G-protein coupled receptor (GPCR) stimulation activates an understudied atypical p38 pathway. Atypical p38 activity selectively induces pathological p38 signaling, via direct interaction between p38 and an essential adaptor protein TAB1, inducing p38 autophosphorylation. We hypothesized that a spatial bias in activated p38 differentially drives pathological vascular dysfunction. To test this, we developed a Forster's Resonance Energy Transfer (FRET) biosensors platform to spatially map subcellular p38 activity. We demonstrate that proinflammatory G protein-coupled receptor (GPCR)-mediated p38 activity is rapidly trafficked away from the plasma membrane, primarily occurring in the cytosol and more specifically the endosomal compartment, peaking at 15 minutes post-stimulation. Interestingly, these signaling dynamics are altered when receptor internalization is inhibited; where p38 activity is shifted into the nucleus, decreasing kinase activity at the cytoplasm and endosome. This shift towards a nuclear bias mirroring p38 signaling dynamics seen with classical MKK3/6-mediated signaling, which occurs primarily in the nucleus after osmotic stress. These data were validated with endogenous GPCRs in primary endothelial cells. Our preliminary data suggest that endosomal/cytosolically biased p38 signaling drives pathological atypical p38 signaling in vascular disease, regulated by a spatial axis at the endosome. In concurrent studies, we have also developed an array of inhibitory intrabodies that specifically block the critical p38-TAB1 interaction in atypical p38 signaling. Preliminary data suggests that these first generation intrabodies effectively block atypical p38 signaling and in conjunction with our biosensor platform will reveal how this endosomal axis drives vascular inflammation. Studies are ongoing to explore their capacity to block pathological signaling in vitro, and in future acute lung injury studies.