Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression.

Introduction: Dysfunction of the endothelium impairs its' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete ß-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.

Calcium sensing receptor stimulates breast cancer cell migration via the Gßγ-AKT-mTORC2 signaling pathway.

Calcium sensing receptor, a pleiotropic G protein coupled receptor, activates secretory pathways in cancer cells and putatively exacerbates their metastatic behavior. Here, we show that various CaSR mutants, identified in breast cancer patients, differ in their ability to stimulate Rac, a small Rho GTPase linked to cytoskeletal reorganization and cell protrusion, but are similarly active on the mitogenic ERK pathway. To investigate how CaSR activates Rac and drives cell migration, we used invasive MDA-MB-231 breast cancer cells. We revealed, by pharmacological and knockdown strategies, that CaSR activates Rac and cell migration via the Gßγ-PI3K-mTORC2 pathway. These findings further support current efforts to validate CaSR as a relevant therapeutic target in metastatic cancer.

aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete, ß-arrestin-2-mediated SphK1-S1PR1-Akt signaling axis.

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via ß-arrestin-2 (ß-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a ß-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates ß-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on ß-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-ß-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, ß-arr2-driven signaling pathways in caveolae.

cAMP-dependent activation of the Rac guanine exchange factor P-REX1 by type I protein kinase A (PKA) regulatory subunits.

Regulatory subunits of protein kinase A (PKA) inhibit its kinase subunits. Intriguingly, their potential as cAMP-dependent signal transducers remains uncharacterized. We recently reported that type I PKA regulatory subunits (RIα) interact with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a chemotactic Rac guanine exchange factor (RacGEF). Because P-REX1 is known to be phosphorylated and inhibited by PKA, its interaction with RIα suggests that PKA regulatory and catalytic subunits may fine-tune P-REX1 activity or those of its target pools. Here, we tested whether RIα acts as a cAMP-dependent factor promoting P-REX1-mediated Rac activation and cell migration. We observed that Gs-coupled EP2 receptors indeed promote endothelial cell migration via RIα-activated P-REX1. Expression of the P-REX1-PDZ1 domain prevented RIα/P-REX1 interaction, P-REX1 activation, and EP2-dependent cell migration, and P-REX1 silencing abrogated RIα-dependent Rac activation. RIα-specific cAMP analogs activated P-REX1, but lost this activity in RIα-knockdown cells, and cAMP pulldown assays revealed that P-REX1 preferentially interacts with free RIα. Moreover, purified RIα directly activated P-REX1 in vitro We also found that the RIα CNB-B domain is critical for the interaction with P-REX1, which was increased in RIα mutants, such as the acrodysostosis-associated mutant, that activate P-REX1 at basal cAMP levels. RIα and Cα PKA subunits targeted distinct P-REX1 molecules, indicated by an absence of phosphorylation in the active fraction of P-REX1. This was in contrast to the inactive fraction in which phosphorylated P-REX1 was present, suggesting co-existence of dual stimulatory and inhibitory effects. We conclude that PKA's regulatory subunits are cAMP-dependent signal transducers.