Activated protein C inhibits lipopolysaccharide-mediated acetylation and secretion of high-mobility group box 1 in endothelial cells.

Essentials APC elicits cytoprotective responses in endothelial cells via EPCR-dependent cleavage of PAR1. APC inhibits LPS-mediated translocation and extracellular secretion of HMGB1 in endothelial cells. Signaling activity of APC inhibits LPS-mediated acetylation of HMGB1 by epigenetic mechanisms. APC inhibits LPS-mediated HMGB1 expression in CD31-positive endothelial cells in cremaster muscle. SUMMARY: Background Activated protein C (APC) inhibits high-mobility group box 1 (HMGB1) signaling and its lipopolysaccharide (LPS)-mediated release by endothelial protein C receptor (EPCR)-dependent activation of protease-activated receptor 1 (PAR1) in endothelial cells. Post-translational acetylation is known to modulate the subcellular localization of HMGB1, and its hyperacetylated form is translocated to the cytoplasm of innate immune cells before being secreted into the extracellular space. Objective To determine whether APC inhibits LPS-mediated HMGB1 secretion from endothelial cells by modulating its acetylation status. Methods The subcellular localization of HMGB1 in LPS-treated endothelial cells was monitored in the absence and presence of APC by western blot analysis of fractionated cell lysates and confocal immunofluorescence microscopy. Results Both western blot and immunofluorescence data indicated that APC effectively inhibits LPS-mediated translocation of HMGB1 from the nucleus to the cytoplasm by EPCR-dependent and PAR1-dependent mechanisms. When EPCR was ligated by the Gla-domain of protein C/APC, thrombin also inhibited LPS-mediated HMGB1 translocation. Further studies revealed that APC inhibits the translocation of HMGB1 from the nucleus to the cytoplasm by inhibiting LPS-mediated hyperacetylation of HMGB1 by (de)acetylating enzymes. Furthermore, the translocated HMGB1 was found to be associated with lysosome-associated membrane protein 1 in LPS-treated endothelial cells. The in vivo relevance of these findings was investigated in the mouse cremaster muscle, and this demonstrated that both wild-type APC and a signaling-selective mutant of APC inhibit LPS-mediated HMGB1 expression and translocation in CD31-positive endothelial cells. Conclusion These results suggest that APC inhibits LPS-mediated cytoplasmic translocation and secretion of HMGB1 in endothelial cells by epigenetic mechanisms.

Cai X ，Biswas I ，Panicker SR ，Giri H ，Rezaie AR ... -  《-》

Occupancy of human EPCR by protein C induces β-arrestin-2 biased PAR1 signaling by both APC and thrombin.

Activation of protease-activated receptor 1 (PAR1) by activated protein C (APC) and thrombin elicits paradoxical cytoprotective and cytotoxic signaling responses in vascular endothelial cells through cleavage of the receptor at Arg-46 and Arg-41 protease recognition sites, respectively. It has been reported that unlike a disruptive G-protein-mediated PAR1 signaling by thrombin, APC induces a protective β-arrestin-2 biased PAR1 signaling by unknown mechanisms. We hypothesize that the occupancy of endothelial protein C receptor (EPCR) by the Gla-domain of protein C/APC is responsible for the β-arrestin-2 biased PAR1 signaling independent of the protease cleavage site. To test this hypothesis, we monitored the signaling specificity of thrombin in endothelial cells in response to lipopolysaccharide (LPS) with or without pretreatment of cells with protein C-S195A. The PAR1-dependent recruitment of β-arrestin-2 in response to LPS by both APC and thrombin was analyzed by functional, gene silencing, and signaling assays. Results indicate that similar to APC, thrombin exerts cytoprotective effects via β-arrestin-2 biased PAR1 signaling. Similar to APC, thrombin triggered β-arrestin-2-dependent recruitment of disheveled 2 (Dvl-2) in PC-S195A pretreated cells. Further studies in HeLa cells transfected with PAR1 constructs revealed that EPCR occupancy initiates β-arrestin-2 biased PAR1 signaling independent of the protease cleavage sites. We demonstrate that EPCR occupancy recruits G-protein coupled receptor kinase 5, thereby inducing β-arrestin-2 biased PAR1 signaling by both APC and thrombin. In support of a physiological relevance for these results, intraperitoneal administration of PC-S195A conferred a cytoprotective effect for thrombin in an in vivo inflammatory model.

Roy RV ，Ardeshirylajimi A ，Dinarvand P ，Yang L ，Rezaie AR ... -  《-》

A novel protein C-factor VII chimera provides new insights into the structural requirements for cytoprotective protease-activated receptor 1 signaling.

Essentials The basis of cytoprotective protease-activated receptor 1 (PAR1) signaling is not fully understood. Activated protein C chimera (APCFVII-82 ) was used to identify requirements for PAR1 signaling. APCFVII-82 did not initiate PAR1 signaling, but conferred monocyte anti-inflammatory activity. APC-specific light chain residues are required for cytoprotective PAR1 signaling. Background Activated protein C (APC) cell signaling is largely reliant upon its ability to mediate protease-activated receptor (PAR) 1 proteolysis when bound to the endothelial cell (EC) protein C (PC) receptor (EPCR). Furthermore, EPCR-bound PC modulates PAR1 signaling by thrombin to induce APC-like EC cytoprotection. Objective The molecular determinants of EPCR-dependent cytoprotective PAR1 signaling remain poorly defined. To address this, a PC-factor VII chimera (PCFVII-82 ) possessing FVII N-terminal domains and conserved EPCR binding was characterized. Methods Activated PC-FVII chimera (APCFVII-82 ) anticoagulant activity was measured with calibrated automated thrombography and activated FV degradation assays. APCFVII-82 signaling activity was characterized by the use of reporter assays of PAR1 proteolysis and EC barrier integrity. APCFVII-82 anti-inflammatory activity was assessed according to its inhibition of nuclear factor-κB (NF-κB) activation and cytokine secretion from monocytes. Results PCFVII-82 was activated normally by thrombin on ECs, but was unable to inhibit plasma thrombin generation. Surprisingly, APCFVII-82 did not mediate EPCR-dependent PAR1 proteolysis, confer PAR1-dependent protection of thrombin-induced EC barrier disruption, or limit PAR1-dependent attenuation of interleukin-6 release from lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, EPCR occupation by active site-blocked APCFVII-82 was, like FVII, unable to mimic EC barrier stabilization induced by PC upon PAR1 proteolysis by thrombin. APCFVII-82 did, however, diminish LPS-induced NF-κB activation and tumor necrosis factor-α release from monocytes in an apolipoprotein E receptor 2-dependent manner, with similar efficacy as wild-type APC. Conclusions These findings identify a novel role for APC light chain amino acid residues outside the EPCR-binding site in enabling cytoprotective PAR1 signaling.

Gleeson EM ，McDonnell CJ ，Soule EE ，Willis Fox O ，Rushe H ，Rehill A ，Smith OP ，O'Donnell JS ，Preston RJS ... -  《-》

Activated protein C inhibits high mobility group box 1 signaling in endothelial cells.

Bae JS ，Rezaie AR 《-》

Thrombin inhibits HMGB1-mediated proinflammatory signaling responses when endothelial protein C receptor is occupied by its natural ligand.

Bae JS ，Rezaie AR 《-》