Apolipoprotein E Receptor 2 Mediates Activated Protein C-Induced Endothelial Akt Activation and Endothelial Barrier Stabilization.

OBJECTIVE: Activated protein C (APC), a plasma serine protease, initiates cell signaling that protects endothelial cells from apoptosis and endothelial barrier disruption. Apolipoprotein E receptor 2 (ApoER2; LRP8) is a receptor known for mediating signaling initiated by reelin in neurons. ApoER2 contributes to APC-initiated signaling in monocytic U937 cells. The objective was to determine whether ApoER2 is required for APC's beneficial signaling in the endothelial cell surrogate EA.hy926 line. APPROACH AND RESULTS: We used small interfering RNA and inhibitors to probe requirements for specific receptors for APC's antiapoptotic activity and for phosphorylation of disabled-1 by Src family kinases and of Akt. When small interfering RNA for ApoER2 or endothelial cell protein C receptor or protease activated receptor 1 was used, APC's antiapoptotic activity was ablated, indicating that each of these receptors was required. In EA.hy926 cells, APC induced a 2- to 3-fold increased phosphorylation of Ser473-Akt and Tyr232-disabled-1, a phosphorylation known to trigger disabled-1-mediated signaling in other cell types. Ser473-Akt phosphorylation was inhibited by ApoER2 small interfering RNA or by inhibitors of Src (PP2), phosphatidylinositol-3 kinase (LY303511), and protease activated receptor 1 (SCH79797). ApoER2 small interfering RNA blocked the ability of APC to prevent thrombin-induced endothelial barrier disruption in TransEndothelial Resistance assays. Binding studies using purified APC and purified immobilized wild-type and mutated ApoER2 ectodomains suggested that APC binding involves Lys49, Asp50, and Trp64 on the surface of the N-terminal LA1 domain of ApoER2. CONCLUSIONS: ApoER2 contributes cooperatively with endothelial cell protein C receptor and protease activated receptor 1 to APC-initiated endothelial antiapoptotic and barrier protective signaling.

Oxysterols direct B-cell migration through EBI2.

EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein-Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450 pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C.

Activated protein C (APC) reduces mortality of severe sepsis patients but increases the risk of serious bleeding. APC exerts anticoagulant activity by proteolysis of factors Va/VIIIa. APC also exerts antiinflammatory and antiapoptotic effects and stabilizes endothelial barrier function by APC-initiated cell signaling that requires two receptors, endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR1). The relative importance of APC's various activities for efficacy in sepsis is unknown. We used protein engineering of mouse APC and genetically altered mice to clarify mechanisms for the efficacy of APC in mouse sepsis models. Mortality reduction in LPS-induced endotoxemia required the enzymatic active site of APC, EPCR, and PAR-1, highlighting a key role for APC's cytoprotective actions. A recombinant APC variant with normal signaling but <10% anticoagulant activity (5A-APC) was as effective as wild-type APC in reducing mortality after LPS challenge, and enhanced the survival of mice subjected to peritonitis induced by gram-positive or -negative bacteria or to polymicrobial peritoneal sepsis triggered by colon ascendens stent implantation. Thus, APC's efficacy in severe sepsis is predominantly based on EPCR- and PAR1-dependent cell signaling, and APC variants with normal cell signaling but reduced anticoagulant activities retain efficacy while reducing the risk of bleeding.

Identification of structural motifs critical for epstein-barr virus-induced molecule 2 function and homology modeling of the ligand docking site.

Epstein-Barr virus-induced molecule 2 (EBI2) (also known as G-protein-coupled receptor 183) is a G-protein-coupled receptor (GPCR) that is best known for its role in B cell migration and localization. Our recent deorphanization effort led to the discovery of 7α,25-dihydroxycholesterol (7α,25-OHC) as the endogenous ligand for EBI2, which provides a tool for mechanistic studies of EBI2 function. Because EBI2 is the first GPCR known to bind and to be activated by an oxysterol, the goal of this study was to understand the molecular and structural bases for its ligand-dependent activation; this was achieved by identifying structural moieties in EBI2 or in 7α,25-OHC that might affect receptor-ligand interactions. By using a series of chemically related OHC analogs, we demonstrated that all three hydroxyl groups in 7α,25-OHC contributed to ligand-induced activation of the receptor. To determine the location and composition of the ligand binding domain in EBI2, we used a site-directed mutagenesis approach and generated mutant receptors with single amino acid substitutions at selected positions of interest. Biochemical and pharmacological profiling of these mutant receptors allowed for structure-function analyses and revealed critical motifs that likely interact with 7α,25-OHC. By using a hybrid ß(2)-adrenergic receptor-C-X-C chemokine receptor type 4 structure as a template, we created a homology model for EBI2 and optimized the docking of 7α,25-OHC into the putative ligand binding site, so that the hydroxyl groups interact with residues Arg87, Asn114, and Glu183. This model of ligand docking yields important structural insight into the molecular mechanisms mediating EBI2 function and may facilitate future efforts to design novel therapeutic agents that target EBI2.

Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells.

Binding of activated protein C (APC) to cells triggers multiple beneficial cytoprotective activities that suppress apoptosis, inflammation, and endothelial barrier breakdown. One paradigm for APC's signaling emphasizes its binding to endothelial cell protein C receptor (EPCR) and subsequent protease activated receptor (PAR)-1 activation. Here we used human monocytic-like U937 cells to evaluate apolipoprotein E receptor 2 (ApoER2)-dependent signaling by APC and found that APC initiated rapid phosphorylation of Tyr-220 in the adaptor protein disabled-1 (Dab1) and of Ser-473 in Akt. APC also induced phosphorylation of Ser-9 in glycogen synthase kinase 3beta (GSK3beta), which was blocked by the PI3K inhibitor LY294002. Receptor-associated protein (RAP), a general antagonist for binding of ligands to LDL receptor family members, inhibited APC-induced phosphorylation of Dab1 and GSK3beta, whereas anti-EPCR or anti-PAR1 blocking antibodies did not. Knocking down ApoER2 by using siRNA-ablated APC induced Dab1 phosphorylation, suggesting that RAP-sensitive APC-induced signaling requires ApoER2. In surface plasmon resonance equilibrium binding studies, APC bound with high affinity to soluble (s) ApoER2 (apparent K(d), approximately 30 nM) but not to soluble very low density lipoprotein receptor. RAP blocked APC binding to sApoER2 but not to sEPCR. RAP blocked binding of U937 cells to immobilized APC. RAP also blocked APC's ability to inhibit endotoxin-induced tissue factor pro-coagulant activity of U937 cells. Thus, we propose that ligation of ApoER2 by APC signals via Dab1 phosphorylation and subsequent activation of PI3K and Akt and inactivation of GSK3beta, thereby contributing to APC's beneficial effects on cells.

Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant.

Activated protein C (APC) reduces mortality in severe sepsis patients. APC exerts anticoagulant activities via inactivation of factors Va and VIIIa and cytoprotective activities via endothelial protein C receptor and protease-activated receptor-1. APC mutants with selectively altered and opposite activity profiles, that is, greatly reduced anticoagulant activity or greatly reduced cytoprotective activities, are compared here. Glu149Ala-APC exhibited enhanced in vitro anticoagulant and in vivo antithrombotic activity, but greatly diminished in vitro cytoprotective effects and in vivo reduction of endotoxin-induced murine mortality. Thus, residue Glu149 and the C-terminal region of APC's light chain are identified as functionally important for expression of multiple APC activities. In contrast to Glu149Ala-APC, 5A-APC (Lys191-193Ala + Arg229/230Ala) with protease domain mutations lacked in vivo antithrombotic activity, although it was potent in reducing endotoxin-induced mortality, as previously shown. These data imply that APC molecular species with potent antithrombotic activity, but without robust cytoprotective activity, are not sufficient to reduce mortality in endotoxemia, emphasizing the need for APC's cytoprotective actions, but not anticoagulant actions, to reduce endotoxin-induced mortality. Protein engineering can provide APC mutants that permit definitive mechanism of action studies for APC's multiple activities, and may also provide safer and more effective second-generation APC mutants with reduced bleeding risk.

Activated protein C mutant with minimal anticoagulant activity, normal cytoprotective activity, and preservation of thrombin activable fibrinolysis inhibitor-dependent cytoprotective functions.

Activated protein C (APC) reduces mortality in severe sepsis patients and exhibits beneficial effects in multiple animal injury models. APC anticoagulant activity involves inactivation of factors Va and VIIIa, whereas APC cytoprotective activities involve the endothelial protein C receptor and protease-activated receptor-1 (PAR-1). The relative importance of the anticoagulant activity of APC versus the direct cytoprotective effects of APC on cells for the in vivo benefits is unclear. To distinguish cytoprotective from the anticoagulant activities of APC, a protease domain mutant, 5A-APC (RR229/230AA and KKK191-193AAA), was made and compared with recombinant wild-type (rwt)-APC. This mutant had minimal anticoagulant activity but normal cytoprotective activities that were dependent on endothelial protein C receptor and protease-activated receptor-1. Whereas anticoagulantly active rwt-APC inhibited secondary-extended thrombin generation and concomitant thrombin-dependent activation of thrombin activable fibrinolysis inhibitor (TAFI) in plasma, secondary-extended thrombin generation and the activation of TAFI were essentially unopposed by 5A-APC due to its low anticoagulant activity. Compared with rwt-APC, 5A-APC had minimal profibrinolytic activity and preserved TAFI-mediated anti-inflammatory carboxypeptidase activities toward bradykinin and presumably toward the anaphlatoxins, C3a and C5a, which are well known pathological mediators in sepsis. Thus, genetic engineering can selectively alter the multiple activities of APC and provide APC mutants that retain the beneficial cytoprotective effects of APC while diminishing bleeding risk due to reduction in APC's anticoagulant and APC-dependent profibrinolytic activities.