Research Article

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

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PNAS January 6, 2009 106 (1) 274-279; https://doi.org/10.1073/pnas.0807594106

  1. Edited by Charles T. Esmon, Oklahoma Medical Research Foundation, Oklahoma City, OK, and approved November 18, 2008 (received for review August 2, 2008)

Abstract

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 3β (GSK3β), 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 GSK3β, 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 Kd, ≈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 GSK3β, thereby contributing to APC's beneficial effects on cells.

Recombinant activated protein C (APC) reduces mortality in severe sepsis (1). APC, a well known plasma protein, can exert both anticoagulant and cytoprotective activities by distinctly different mechanisms (2–4). Because two potent anticoagulant plasma proteins, antithrombin and tissue factor pathway inhibitor, failed to reduce mortality in large phase III trials of severe sepsis (5, 6), one may infer that one or more of APC's cytoprotective actions contribute to mortality reduction by APC therapy. APC's cytoprotective actions include inter alii anti-apoptotic and anti-inflammatory activities, alterations of gene expression, pro-angiogenic activity, and endothelial barrier stabilization (4, 7, 8).

In vitro studies of the effects of APC on various cells support a paradigm for APC's cell signaling based on two key receptors, endothelial protein C receptor (EPCR) (9) and the G protein-coupled receptor protease activated receptor-1 (PAR1) (10), which mediate downstream cytoprotective effects (4). Central to this paradigm is the binding of APC's γ-carboxyglutamic acid-rich N-terminal domain to the EPCR with subsequent cleavage of PAR1 by APC's protease domain, followed by PAR1-induced direct effects of APC on cells. Strong support for the in vivo relevance of this mechanism for triggering cell signaling by pharmacologic doses of APC comes from studies of murine ischemic stroke, NMDA excitotoxicity injury, and severe sepsis models (11–14) .

Recently, two kinds of findings imply that the APC-EPCR-PAR1 paradigm is not always applicable and that alternative cell surface receptors might contribute to initiation of cell signaling by APC. First, for a few in vitro assays of APC's effects on certain cells, it appeared that APC-initiated cell signaling did not require PAR1 (15, 16) or EPCR (17). Second, in a report of APC-platelet interactions, an important role was posited for APC binding to apolipoprotein E receptor-2 [ApoER2; aka LDL receptor (LDLR)-related protein 8; LRP8] (18) and to glycoprotein Ibα (19). ApoER2 is a member of the LDLR family that includes, among others, the LDLR, the LDLR related protein (LRP), ApoER2, and the very low density lipoprotein receptor (VLDLR) (20–23). Various LDLR family members provide major endocytotic activities for cells whereas other family members also play prominent roles for signal transduction. LDLR family members can bind a spectrum of ligands on various cells, not limited to lipoproteins, and ligation of many members of this receptor family suffices to initiate cell signaling.

The physiologic neuronal Reelin signaling pathway involves Reelin ligation of both ApoER2 and VLDLR and subsequent phosphorylation of the adaptor protein disabled-1 (Dab1) involving the Src family kinases Src and Fyn, followed by phosphorylation of phosphatidylinositol 3-phosphate kinase (PI3K), Akt, and glycogen synthase kinase 3β (GSK3β) (20–23). Receptor-associated protein (RAP) (24) is an antagonist for ApoER2 ligand binding that blocks Reelin's effects on cells. Remarkably, murine genetic deficiencies of Reelin, ApoER2 plus VLDLR, Dab1, or Src plus Fyn produce mice with indistinguishable phenotypes (reeler, scrambler, yotari) (22, 23, 25–29) involving impaired motor coordination, compromised gait, tremors, and ataxia, plus abnormal patterns of neuronal positioning in the brain. Moreover, reeler adult mice exhibit learning defects and have neurons with compromised long-term potentiation functioning (30, 31).

Monocytic cell lines, such as the human leukemic monoblast U937 cell line, have been used to study APC's ability to modulate monocyte apoptosis, phagocytosis, inflammation, and tissue factor generation (4, 8, 32–34). To evaluate the potential relevance of ApoER2 for APC cell signaling mechanisms, APC-initiated signaling in U937 cells was assayed based on the hypothesis that APC ligation of ApoER2 would signal similarly to the Reelin signaling pathway wherein ApoER2 ligation by Reelin promotes signaling via phosphorylation of Dab1, which binds to an NPxY sequence of the intracellular C-terminal region of ApoER2 (21–23,26). Here we present data showing that APC binds to purified soluble (s) ApoER2, that U937 cells bind to immobilized APC, and that APC initiates RAP-sensitive phosphorylation of Dab1 with consequent PI3K-dependent signaling via phosphorylations of Akt and GSK3β. Our results indicate that APC initiates a Reelin-like signaling pathway and that ApoER2 should join EPCR and PAR1 as candidates for cell receptors or co-receptors that mediate APC's cell signaling activities.

Results

U937 Cells Contain Two Forms of ApoER2.

Because alternative splicing in various tissues determines the variable expression of ApoER2 in different tissues and different species (18, 35–38), we characterized the forms of ApoER2 mRNA in U937 cells [supporting information (SI) Fig. S1]. Two isoforms of ApoER2 were identified in U937 cells, both with deletions of exons 5 and 18 and one with an additional deletion of exon 15 that codes for the extracellular O-linked sugar region (Fig. S1C).

APC Causes Phosphorylation of Dab1, Akt, and GSK3β.

Addition of APC to U937 cells caused phosphorylation of tyrosine residues in Dab1 within 10 min (Fig. 1A). Based on immunoblotting by using antibodies specific for pTyr220, APC caused phosphorylation of Tyr-220 in Dab1 (data not shown). Phosphorylation of Tyr residues in Dab1 was not inhibited by a monoclonal antibody (RCR252) that blocks binding of APC to EPCR or by two anti-PAR1 monoclonal antibodies (ATAP2 and WEDE15) that block PAR1 signaling by APC. However, Dab1 phosphorylation was blocked by RAP, which blocks binding of ligands to LDLR family members such as ApoER2 (Fig. 1B). Knocking down ApoER2 by using siRNA ablated APC-induced Dab1 phosphorylation (Fig. 2 A and B), indicating that ApoER2 is required for Dab1 phosphorylation under these conditions. Similar to the Reelin signaling pathway that causes activation of Akt by phosphorylation of Ser-473 (39, 40) downstream of Dab1 phosphorylation, APC caused rapid phosphorylation of Ser-473 in Akt (Fig. 1C). Further similarly (39, 40), APC caused downstream phosphorylation of GSK3β on Ser-9 (Fig. 1D). The ratio of phosphorylated to total GSK3β (Fig. 1D) indicates that the relative amount of phosphorylated enzyme increased with time (Fig. 1E). Because PI3K mediates Dab1-dependent activation of Akt (39, 40), we used the PI3K inhibitor Ly294002 and showed that it blocked the APC-induced phosphorylation of GSK3β (Fig. 1F). When cells were preincubated with various receptor-blocking agents before APC addition and phosphorylation of Ser-9 in GSK3β at 5 min was determined, we found that only RAP blocked this reaction (Fig. 1G), showing that a RAP-sensitive receptor was required for APC-induced signaling. Remarkably, antibodies that block APC binding to EPCR or PAR1-dependent signaling had no significant effect on this action of APC (Fig. 1G).

Fig. 1.
Fig. 1.

APC stimulates RAP-sensitive phosphorylation of Dab-1 and GSK-3β. (A) Cells were incubated with or without APC (2 μg/mL) for 5 or 10 min at 37 °C. Dab1 was immunoprecipitated from cell lysates and was separated on SDS/PAGE and blotted with anti-phosphorylated Tyr (α-pY) or anti-Dab1 (α-Dab1) as described in Materials and Methods. (B) U937 cells were pre-treated with control, anti-EPCR mAb RCR252 (15 μg/mL), RAP (2 μM), or anti-PAR1 antibodies (ATAP2 plus WEDE15, 15 μg/mL each) for 15 min, then incubated with or without APC (2 μg/mL) for 10 min, and Dab1 tyrosine phosphorylation (pY) was examined as in A. (C) Cells were incubated with or without APC (2 μg/mL) for 5 min at room temperature, and then Akt was immunoprecipitated from cell lysates and analyzed by Western blot using anti-Akt-pSer473 or anti-Akt as described in Materials and Methods. (D) Cells were incubated with APC (2 μg/mL) for various times and cell lysates were directly subjected to Western blotting using anti-GSK3β-pSer9 or anti-GSK3β as described in Materials and Methods. (E) Band intensities (see D) were quantified using Un-Scan-IT (Silk Scientific), and the ratio of GSK3β-pSer9 to total GSK3β antigen was calculated for each time. (F) Cells were pre-treated with LY294002 (50 μM) or control buffer for 15 min at room temperature and then incubated with or without APC (2 μg/mL) for 5 min. Endogenous GSK3β-pSer9 and GSK3β were directly analyzed by Western blot. (G) Cells were pre-treated with anti-EPCR monoclonal RCR252 (15 μg/mL), RAP (2 μM), or anti-PAR1 antibodies (ATAP2 and WEDE15, 15 μg/mL each) for 15 min, then incubated with or without APC (2 μg/mL) for 5 min; GSK3β-pSer9 and GSK3β were then analyzed by Western blot. Western blots shown are representative of at least three independent experiments.

Fig. 2.
Fig. 2.

Knocking down ApoER2 expression by using siRNA ablates APC-induced Dab1 phosphorylation. (A) U937 Cells were transfected with ApoER2 siRNA or control siRNA for 48 h before incubation with or without APC (2 μg/mL) for 10 min at 37 °C. Dab1 was immunoprecipitated from cell lysates (IP:Dab1) containing equal amount of total protein and was separated on SDS/PAGE and immunoblotted with anti-phosphorylated Tyr (WB:α-pY) or anti-Dab1 (WB:α-Dab1) as described in Materials and Methods. (B) Knock-down of ApoER2 gene and protein expression was confirmed by semi-quantitative RT-PCR and Western blot analyses. Total RNA was isolated from U937 cells treated with ApoER2 siRNA or control siRNA and reverse-transcribed to cDNA as described in Fig. S1. β-Actin gene and protein expression was analyzed in the same samples to ensure specific knock-down of ApoER2. For protein expression analysis, ApoER2 was immunoprecipitated from whole-cell lysates containing equal amounts of total protein using rabbit polyclonal antibody raised against the C terminus of ApoER2 (Santa Cruz Biotechnology) and subjected to Western blot analysis using a mouse monoclonal antibody recognizing the N terminus of ApoER2 (Abcam). The same whole-cell lysates were also subjected to Western blotting using anti-β-actin antibody (Sigma). Data shown are representative of results from three independent experiments.

RAP Inhibits Binding of APC to sApoER2 But Not to sEPCR.

Previously, by using a non-equilibrium binding method, we showed that APC binds to sApoER2 immobilized on a microtiter plate (19). Here we used the equilibrium binding method of surface plasmon resonance (SPR) analysis to compare parameters for APC binding to sApoER2 and to sEPCR. sApoER2 bound with apparently higher affinity to APC (34 nM) than sEPCR (195 nM), and RAP completely blocked binding of sApoER2 to APC but not binding of sEPCR (Fig. 3). The kinetics of APC binding to sApoER2 and sEPCR showed remarkable differences, with much faster on-rates and off-rates for the latter receptor. No binding of the Reelin signaling pathway receptor sVLDLR to APC was detected by SPR (see Fig. 3A Inset) or by solid-phase binding assays (Fig. S2). Thus, RAP blocks high-affinity binding of APC to sApoER2, consistent with the hypothesis that the RAP-sensitive, APC-induced signaling in U937 cells is caused by APC ligation of ApoER2 and not by binding of APC to VLDLR.

Fig. 3.
Fig. 3.

Binding of sApoER2 and sEPCR to APC and effect of RAP on APC binding. WT APC with a V5 tag at its carboxyl terminus was captured on a CM5 chip using an immobilized anti-V5-antibody, and SPR was used to monitor binding of soluble receptor domains to APC and to determine the effects of RAP on receptor binding. (A) Binding of sApoER2 (117, 138, 159, 191, 255, and 308 nM) to APC was monitored by SPR. RAP (600 nM; dotted line) completely blocked binding of sApoER2 (300 nM) to APC. No binding of sVLDLR (705 nM) to APC was observed (Inset). (B) Binding of sEPCR (0.08, 0.17, 0.35, 0.7, and 1.4 μM) to APC was monitored. No change in binding of sEPCR (1.4 μM) to APC was observed in the presence of RAP (2 μM), as indicated by the dotted line that was indistinguishable from the solid line for sEPCR alone (1.4 μM). Rate constants kon and koff for binding of each soluble receptor to APC are shown (Inset), along with the calculated apparent dissociation constant KDapp.

RAP and Anti-EPCR Antibodies Inhibit Binding of U937 Cells to Immobilized APC.

To determine if RAP blocks binding of U937 cells to immobilized APC, cells were preincubated with RAP or an anti-EPCR antibody (RCR252) that blocks APC binding or control IgG, and then cells were incubated in chambers containing APC or fibronectin that had been immobilized on the surface. Following vigorous washing, the binding of cells to the surface was quantified by using phase-contrast microscopy. Cells were bound similarly to APC or fibronectin but not to BSA on the surface (Fig. 4A). RAP and the blocking anti-EPCR RCR252 antibody ablated binding of cells to immobilized APC but not to fibronectin (Fig. 4 A and B). Our data show that both a RAP-sensitive receptor and EPCR were required for static adhesion of U937 cells to immobilized APC after vigorous washing.

Fig. 4.
Fig. 4.

ApoER2 and EPCR mediate U937 cell binding to immobilized APC. U937 cells that were bound to Surface-coated APC or Surface-coated fibronectin (FN) were visualized (A) and quantified (B) using phase-contrast microscopy. Pre-treatment of cells with vehicle or RAP or an anti-EPCR antibody (RCR252) that blocks APC binding to EPCR was performed as described in Materials and Methods. Data are shown as mean ± SEM. In controls using vehicle with BSA, only minimal numbers of U937 cells were observed on BSA-coated wells (small gray bar; 2.8 ± 0.4 cells/mm2; n = 5; *, P < 0.01).

RAP and Anti-EPCR Antibodies Inhibit APC's Ability to Block LPS-Induced Pro-Coagulant Activity of U937 Cells.

To see if ApoER2 might contribute to APC's previously demonstrated ability to down-regulate tissue factor activity that appears following U937 cell stimulation with LPS (33), we pre-treated cells with various blocking antibodies or RAP and then treated cells with LPS and APC. When pro-coagulant activity (PCA) of reaction mixtures was measured at 6 h after LPS stimulation, APC diminished LPS-induced PCA, as expected (Fig. 5). RAP alone or the blocking anti-EPCR RCR252 antibody alone totally ablated APC's effects (Fig. 5). However, two antibodies (ATAP2 and WEDE15) that block APC activation of PAR1 had no effect on APC's ability to blunt LPS induction of tissue factor activity (Fig. 5). The observed ability of the blocking anti-EPCR antibody to prevent APC's down-regulation of tissue factor activity is similar to the findings of Shu et al. (33). Thus, both an RAP-sensitive receptor and EPCR were required for APC to down-regulate tissue factor PCA on LPS-stimulated cells.

Fig. 5.
Fig. 5.

RAP and anti-EPCR antibody (RCR252) blocked APC's ability to down-regulate LPS-induced PCA. Cells were incubated with buffer (□), LPS alone (25 ng/mL; ▨), or LPS (25 ng/mL) plus APC (1 μg/mL; ■) as described in Materials and Methods, and PCA was determined. Data are expressed as percentages of control PCA (cell PCA in the absence of LPS exposure or treatment with RAP or antibodies). Values represent mean ± SD for data from four independent experiments.

Discussion

APC has multiple signaling effects that may involve multiple receptors and cell signaling pathways. In equilibrium binding studies, APC bound with high affinity to the extracellular domain of sApoER2 (apparent Kd, ≈30 nM) but not to a closely related LDL receptor family member, sVLDLR. Several isoforms of ApoER2 are abundant in brain and testis (18) and also present in endothelial cells (41) and platelets (42), as well as the U937 monocytic cell line. We hypothesized that APC ligation of ApoER2 on U937 cells promotes Reelin-like signaling that is well described in neurons. Reelin ligation of ApoER2 causes phosphorylation of the adaptor protein Dab1 by Src family kinases (Src and Fyn), followed by activation of PI3K and Akt; and, in a key downstream step, GSK3β is phosphorylated and thereby inhibited by activated Akt (22, 23, 43–45).

Here we show that addition of APC to U937 cells initiated rapid tyrosine phosphorylation of Dab1 and of Ser-473 in Akt. APC also induced phosphorylation of Ser-9 in GSK3β, which was blocked by the PI3K inhibitor LY294002. RAP, an antagonist for ApoER2 ligand binding as well as a general antagonist for ligand binding to LDLR family members, inhibited APC-induced phosphorylation of Dab1 and GSK3β, whereas anti-EPCR or anti-PAR1 blocking antibodies did not. Moreover, binding of APC to sApoER2 in purified systems, like APC-induced signaling in U937 cells, was blocked by RAP, whereas RAP had no effect on binding of APC to sEPCR. Direct evidence of a required role for ApoER2 for APC's signaling came from siRNA studies showing that specific knock-down of ApoER2 ablated APC-induced Dab1 phosphorylation. Notably, neither EPCR nor PAR1 appeared necessary for APC's signaling effects via Dab1. Thus, the major hypothesis supported by our data is that APC ligation of ApoER2 causes Dab1-dependent signaling via the PI3K/Akt pathway in U937 cells.

Acting via an EPCR-dependent process, APC prevents elaboration of tissue factor activity on the surface of activated U937 cells (33). Remarkably, RAP effectively blocked APC's ability to inhibit endotoxin induction of tissue factor PCA of U937 cells, implicating a requirement for a RAP-sensitive receptor such as ApoER2. Anti-EPCR antibodies that block binding of APC to EPCR on U937 cells prevented APC's effects on tissue factor induction as reported (33), implying that binding of APC to both EPCR and ApoER2 on the cell surface is necessary for effective APC signaling needed to blunt tissue factor induction. Similarly, either RAP or anti-EPCR antibodies blocked binding of U937 cells to immobilized APC. As the assay for cell binding to immobilized APC involved vigorous washing steps, these data do not reflect a simple equilibrium binding reaction, and no definitive conclusion can be drawn regarding receptor-receptor interactions or regarding the energetic contributions of either individual receptor binding to APC. Although no data directly indicate that APC simultaneously binds to both ApoER2 and EPCR, such a situation is not inconsistent with data that suggest that EPCR binds to only APC's N-terminal Gla-domain (46, 47) whereas sApoER2 binds to Gla-domain-less APC (19). One possibility is that the distinct APC binding characteristics for Gla-domain-dependent EPCR binding (i.e., fast association) combined with Gla-domain-independent ApoER2 binding (i.e., slow dissociation) allow for the temporal retention of APC on the cell membrane beyond what can be accomplished by either receptor alone. It is also possible that EPCR binds APC to cells in a manner that permits APC-induced signaling via other currently unidentified receptors.

Depending on cell type, APC induces signaling via the MAPK pathway with phosphorylation of ERK-1/2 that is reported to be PAR-1-dependent and EPCR-dependent (48–51) or PAR1-dependent but EPCR-independent (17). Multiple members of the MAPK pathway can strongly influence a cell's fate for better or for worse, and there are multiple modes of regulating members of the MAPK pathway (52–57). In U937 cells, data here show that APC causes phosphorylation of Ser-473 in Akt, a key reaction for Akt activation (58), and that APC can induce signaling via ApoER2 and the PI3K-Akt pathway without any apparent requirement for EPCR or PAR1. Akt is a major node in the cell's signaling communications network with multiple major downstream targets that may modulate cell survival, proliferation, metabolism, cell cycle regulation, and angiogenesis (58, 59). Two major downstream Akt targets are GSK3β and endothelial NO synthase, and APC induces phosphorylation of GSK3β and of endothelial NO synthase in a PI3K-dependent manner (49). Thus, APC can cause alterations of at least two major downstream targets of the PI3K-Akt pathway, with some broad implications (58, 59). Studies indicate that the PI3K/Akt pathway activation can down-regulate LPS-induced PCA or tissue factor expression in monocytes (60, 61). These reports are consistent with the implication that ligation of ApoER2 by APC with subsequent activation of the PI3K/Akt pathway may help explain the mechanism for APC's ability to down-regulate LPS-induced PCA. It will be interesting to clarify the potential effects of APC signaling via ApoER2 and the PI3K-Akt pathway and how this might be integrated with EPCR/PAR1 pathway signaling.

In addition to reducing death by sepsis in humans and murine models (1, 14), APC has remarkable in vivo neuroprotective activities in murine brain injury model studies (11–13, 62, 63). Identification of an ApoER2-dependent, Reelin-like signaling pathway for APC on U937 cells that activates the PI3K-Akt pathway raises questions about potential roles for this new APC signaling mechanism for neurons and other brain cells. Interestingly, a number of studies implicate the Akt survival pathway as relevant for survival of neurons after ischemic stroke (reviewed in ref. 64). Also relevant to such future investigations of ApoER2-dependent effects of APC on neurons is the direct interaction of ApoER2 with the NMDA receptor (65, 66) which could provide an additional mechanism for APC's actions on neurons.

Engagement of receptors and signaling pathways that are stimulated by APC (e.g., G protein-coupled receptors such as PAR1 or PAR3 and the MAPK pathway or ApoER2 and a Reelin-like signaling pathway that includes Dab1 phosphorylation and the PI3K-Akt pathway) generates multiple signals that are interconnected components of complex systems whose output must be integrated for cellular outputs and survival. The net physiologic or pharmacological effects of APC's signaling must be ultimately determined by the context of each cell, each tissue, each blood vessel, and each animal. As shown here, the addition of a novel ApoER2-dependent signaling pathway to the list of potential mechanisms for APC's signaling activities should contribute significantly to efforts to decipher mechanisms that explain how APC can signal in various cells and tissues to prevent apoptosis, induce selective alterations of gene expression, reduce inflammation, stabilize endothelial barriers, provide neuroprotection following ischemic stroke, minimize damage in chronic neurodegenerative disease animal models, and reduce death from severe sepsis.

Materials and Methods

Recombinant Proteins.

Human GST-RAP, sApoER2, sEPCR, WT APC, and APC-V5 were prepared as described in SI Text.

Analysis of Dab1, Akt, and GSK-3β Phosphorylation.

U937 cells (ATCC) treated with or without APC were harvested in radio-immunoprecipitation assay buffer with a mixture of protease and phosphatase inhibitor cocktails (Pierce). Dab1 or Akt was immunoprecipitated from 100 μg lysate with goat polyclonal anti-Dab1 antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-Akt antibody (Cell Signaling). Equal amounts of immunoprecipitated samples or cell lysates were subjected to Western blot analysis using polyclonal or monoclonal antibodies directed against phosphorylated Tyr (pY) (Invitrogen), Dab-1 (Santa Cruz Biotechnology), Akt-pSer473 (Cell Signaling), Akt (Cell Signaling), GSK3β-pSer9 (Cell Signaling), GSK3β (Santa Cruz Biotechnology), or β-actin (Sigma). Appropriate secondary horseradish peroxidase-linked antibodies were used and developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposed to CL-XPosure film (Pierce).

RNA Interference.

ApoER2 expression in U937 cells was knocked down using a pool of three target-specific 20- to 25-nucleotide siRNAs (Santa Cruz Biotechnology) following the manufacturer's instructions. A non-targeting 20- to 25-nucleotide siRNA consisting of a scrambled sequence from the same company was used as a negative control.

SPR Analysis.

Binding was assessed by SPR using a BIAcore 3000 biosensor system. An anti-V5 tag antibody (V5-AB; ICL) was covalently immobilized on a CM5 sensor chip (BIAcore) and saturated with WT APC with a V5 tag at its −COOH terminal. The kinetics of APC binding to sApoER2 or sEPCR were determined as described in SI Text.

U937 Cell Adhesion Assays.

Polystyrene 24-well plates were incubated with APC or fibronectin (100 μg/mL) for 60 min at room temperature followed by washing and blocking with 2% fatty acid-free BSA for 60 min, then followed by washing with PBS solution. U937 cells (1 × 106/mL in Hepes buffered saline solution HBSS buffer containing 1% BSA) were incubated in APC-coated or fibronectin-coated wells for 60 min at 37 °C. Then, wells were washed with HBSS and fixed with paraformaldehyde for 10 min and the number of bound U937 cells was quantified via phase-contrast microscopy. In selected experiments, U937 cells were pre-treated for 10 min with RAP (40 μg/mL), a rat anti-EPCR mAb RCR-252 that blocks APC binding (50 μg/mL), or control rat IgG1 mAb (50 μg/mL) for 10 min.

Analysis of Tissue Factor PCA.

Reaction mixtures containing U937 cells suspended in RPMI were preincubated with RAP (2 μM), non-immune mouse IgG (10 μg/mL), anti-EPCR RCR252 antibody (10 μg/mL), or two anti-PAR-1 antibodies (ATAP2 and WEDE15, each at 10 μg/mL) for 30 min, and then 1 μg/mL APC was added and incubated for 20 min. Then the reaction mixtures containing U937 cells were stimulated with LPS (Sigma; Escherichia coli 055:B5) for 6 h. PCA in the reaction mixture was determined by a single-stage clotting assay as described (33, 67). Briefly, 50 μL of U937 suspension was mixed with 50 μL of 25 mM CaCl2. Clotting was initiated by the addition of 50 μL of normal human pooled plasma, and the clotting time at 37 °C was recorded using an Amelung KC4 coagulometer (Sigma). The PCA standard curve was calibrated using Innovin (Dade).

Acknowledgments

We thank Ms. Phuong M. Nguyen, Ms. Sarah K. Coit, and Ms. Tal Eshel for technical assistance. We thank Dr. D. Strickland (University of Maryland, Baltimore, MD) for the generous gift of sVLDLR and anti-sVLDLR antibodies, Dr. S. Gonias (University of California, San Diego, CA) for the human GST-RAP construct, and Dr. L. Brass (University of Pennsylvania, Philadelphia, PA) for anti-PAR-1 antibodies. This work was supported in part by National Institutes of Health Grants HL31950 and HL52246 (to J.H.G) and HL087618 (to L.O.M.), and American Heart Association Grant 0665512Z (to O.J.T.M.).

Footnotes

  • 1To whom correspondence should be addressed. E-mail: jgriffin{at}scripps.edu

  • Author contributions: X.V.Y., H.D., and J.H.G. designed research; X.V.Y., Y.B., J.A.F., H.D., X.X., L.O.M., T.C.W.-A., and O.J.T.M. performed research; R.T.U. and P.G.d.G. contributed new reagents/analytic tools; X.V.Y., Y.B., J.A.F., H.D., X.X., L.O.M., T.C.W.-A., and O.J.T.M. analyzed data; and X.V.Y. and J.H.G. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0807594106/DCSupplemental.

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Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells
Xia V. Yang, Yajnavalka Banerjee, José A. Fernández, Hiroshi Deguchi, Xiao Xu, Laurent O. Mosnier, Rolf. T. Urbanus, Phillip G. de Groot, Tara C. White-Adams, Owen J. T. McCarty, John H. Griffin
Proceedings of the National Academy of Sciences Jan 2009, 106 (1) 274-279; DOI: 10.1073/pnas.0807594106
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