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Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2017 March 01. Published in final edited form as: Arterioscler Thromb Vasc Biol. 2016 March ; 36(3): 518–524. doi:10.1161/ATVBAHA.115.306795.
Apolipoprotein E Receptor 2 mediates Activated Protein Cinduced endothelial Akt activation and endothelial barrier stabilization
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Ranjeet K. Sinha, Xia V. Yang*, José A. Fernández, Xiao Xu, Laurent O. Mosnier, and John H. Griffin Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037 USA
Abstract 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.
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Approach and Results—We used siRNA and inhibitors to probe requirements for specific receptors for APC’s anti-apoptotic activity and for phosphorylation of disabled-1 (Dab1) by Src family kinases and of Akt. When siRNA for ApoER2 or endothelial cell protein C receptor or protease activated receptor 1 was employed, APC’s anti-apoptotic activity was ablated, indicating that each of these receptors was required. In EA.hy926 cells, APC induced a 2 to 3-fold increase phosphorylation of Ser473-Akt and Tyr232-Dab1, a phosphorylation known to trigger Dab1mediated signaling in other cell types. Ser473-Akt phosphorylation was inhibited by ApoER2 siRNA or by inhibitors of Src (PP2), PI3K (LY303511), and protease activated receptor 1 (SCH79797). ApoER2 siRNA 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.
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Conclusion—ApoER2 contributes cooperatively with endothelial cell protein C receptor and protease activated receptor 1 to APC-initiated endothelial anti-apoptotic and barrier protective signaling.
Corresponding author: John H. Griffin, Phone (858) 784 8220, [email protected]. *Present address: Regulus Therapeutics. 3545 John Jay Hopkins Ct., Suite 210. San Diego, CA 92121-1121 Disclosure of Conflicts of Interest None.
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Introduction The normal blood serine protease, activated protein C (APC), provides multiple beneficial physiologic and pharmacologic effects, including antithrombotic, cytoprotective, and regenerative actions1–5 and proteolytic detoxification of extracellular histones.6 Multiple receptors on various cell types have been implicated as required for APC’s cytoprotective cell signaling with most reports emphasizing a role for the APC-EPCR-PAR1 signaling paradigm in which biased signaling by cleavage of PAR1 at Arg46 may be central.7, 8 Although direct molecular interactions of purified APC have been well documented for EPCR, PAR1, PAR3, and apolipoprotein E receptor 2 (ApoER2) (also known as LRP8),1–4, 6, 7, 9, 10 the multiplicity of cell types and biologic activities linked to APC’s actions leaves many unanswered questions about mechanisms for APC’s beneficial cell signaling activities.
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One major pharmacologic activity of APC-induced signaling involves its ability to reduce vascular leakage induced by edemagenic mediators (e.g., thrombin, VEGF, P. aeruginosaderived toxins, etc.) that involves increased endothelial Rac1 activation and decreased RhoA activation in vitro and in vivo.1, 5, 7–9, 11–15 On monocytic-like U937 cells, ApoER2 promotes APC-induced activation of this receptor’s adaptor protein, disabled-1 (Dab1), and of Akt signaling, linked to APC anti-inflammatory actions.10 ApoER2 is central to the reelin pathway that mediates reelin-induced signaling and neuronal pattern development.16–22 Reelin binds ApoER2 and triggers phosphorylation of Dab1 involving the Src family kinases (SFK), Src and Fyn. This ligation of ApoER2 causes activation of phosphatidylinositol-3 phosphate kinase (PI3K) and phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β) in neurons. Engagement of endothelial ApoER2 by apoE results in suppression of metastatic endothelial recruitment by melanoma cells, implying that ligation of endothelial ApoER2 by one of its ligands, apo E, triggers beneficial signaling.23 Here we used the human EA.hy926 transformed endothelial cell line to assess APC’s ability to initiate reelin-like signaling and cytoprotection via ApoER2. The results show that APC induces phosphorylation of endothelial Dab-1 and that ApoER2 is required for APC-induced phosphorylation and activation of the PI3K-Akt pathway, activation of endothelial Rac1, and vasculoprotection against thrombin-induced vascular leakage and cytotoxicity.
Materials and Methods Materials and Methods are available in the online-only Data Supplement
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Results ApoER2 mediates protective activity of APC against apoptosis Endothelial cells and other cells contain alternatively spliced ApoER2 variants.10, 21, 24 When the EA.hy926 cells were analyzed for ApoER2 variants by PCR and sequencing (see Methods, Fig. SI), we identified 4 isoforms that are schematically indicated in Fig. SIC with deletions of exons (i) 5, (ii) 5 and 15, (iii) 5, 15 and 18, and (iv) 5 and 18. The N-terminal ligand binding region includes LDL receptor class A (LA) modules, each containing ~ 40
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residues. Given the absence of exon 5 from all endothelial ApoER2 variants, these data indicate that endothelial ApoER2 generally lacks LA domains 4–6 (Fig. SIC).
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Studies of human umbilical vein endothelial cells and EA.hy926 cells showed that APC similarly reduced staurosporine-induced apoptosis for each cell line,25 and we first showed that this endothelial cell cytoprotection required EPCR and PAR1 for its anti-apoptotic activity.26 To determine if ApoER2 is also required, siRNA was used to knock down each of these three receptors in EA.hy926 cells prior to apoptosis assays. When two different proapoptotic stimuli, staurosporine and TNFα, were used and apoptosis was monitored using Apopercentage dye uptake (Fig. 1A) or TUNEL staining (Fig. 1B), respectively, the results showed that knocking down any one of these three receptors ablated APC’s anti-apoptotic activity (Figs. 1A, 1B) (p < 0.0002). Because soluble ectodomains of EPCR and ApoER2 bind APC,10 they were studied as inhibitors of APC-initiated anti-apoptotic activity in each apoptosis model. When sApoER2 or sEPCR was preincubated with APC prior to the apoptosis assays, each one caused loss of APC’s anti-apoptotic activity (Figs. 1C, 1D). Predictably, the negative controls, sE86A-EPCR and soluble very low density lipoprotein receptor (VLDLR) which do not bind APC27, 28 did not cause loss of activity. When cells subjected to TNFα-induced apoptosis were stained with TUNEL or Hoechst dye or their combination, the cell images show that APC’s anti-apoptotic activity was inhibited by its prior incubation with sApoER2 but not with sVLDLR (Fig. 1E). These data support the hypothesis that each one of EPCR, PAR1 and ApoER2 was required for APC’s antiapoptotic activity under the conditions employed. APC requires ApoER2, PAR1, Src kinases and PI3-kinase to induce Akt phosphorylation in endothelial cells
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To determine requirements for ApoER2 for APC’s activation of Akt in endothelial cells,7 cells were treated with ApoER2 siRNA or control siRNA prior to APC. Studies to quantify mRNA levels showed that ApoER2 mRNA was knocked down by > 80% (Fig. SID), and Western blots showed similar reduction of ApoER2 antigen while EPCR antigen levels remained normal (Fig. SID). The ability of APC to cause phosphorylation of Ser473 in Akt was significantly inhibited by knocking down ApoER2 (Fig. 2A), indicating that this phosphorylation required ApoER2. Addition of APC to endothelial cells caused phosphorylation of Tyr232 in Dab1 that was sustained over 5 to 60 min, and this phosphorylation was significantly inhibited by the Src inhibitor PP2 but not by the control compound, PP3. When the requirements for SFK, PAR1, and PI3K for APC-induced Akt phosphorylation were probed using their inhibitors (PP2, SCH79797, and LY294002, or respective control compounds), the results showed that each of these proteins was required for the typical pattern of APC-induced Akt phosphorylation over the 90 min time course (Figs. 2C–E and Fig. SII A–C). Inhibition of either PAR1 or PI3K completely ablated Akt phosphorylation over 90 min (Fig. 2D–E and Fig. SII B–C). Interestingly, although PP2 treatment of endothelial cells completely ablated Dab-1 phosphorylation at up to 90 min (Fig. 2B), it did not reduce the level of Akt phosphorylation seen at 90 min, although it retarded this reaction over 5 to 60 min (Fig. 2C). This suggests that some pathways of APCinitiated signaling which are less efficient without ApoER2-mediated contributions can
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eventually augment Akt activation. In summary, these results show that ApoER2 is required for APC-initiated signaling in endothelial cells. ApoER2 is required for endothelial barrier stabilization by APC
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The vasculoprotective actions of APC for protecting against thrombin-induced vascular leakage on endothelial cells were described for human pulmonary artery endothelial cells using Transendothelial Electric Resistance (TER) assays11 and for EA.hy.926 human endothelial cells based on fluorescent dye leakage assays.12 In both those studies, a requirement for the S1P receptor, S1P1, was shown using siRNA, and a requirement for PI3K was also demonstrated.11 Additional data showed that EPCR and PAR1 were required for APC’s vasculoprotective effects.1 Therefore, here we used both TER and dye (FITCdextran) leakage bioassays to probe the requirement for ApoER2. Endothelial cells were pretreated with either ApoER2 siRNA or control siRNA and then exposed to either thrombin alone or thrombin plus APC. First, without APC addition, data showed that the extensive amount of thrombin-induced endothelial barrier leakage was the same for cells exposed to ApoER2 siRNA or control siRNA (Fig, 3A). Second, APC treatment completely ablated thrombin-induced leakage in controls, whereas ApoER2 siRNA treatment significantly diminished by > 50% the ability of APC to prevent thrombin-induced endothelial barrier leakage (Fig. 3A). When TER assays were used for defining thrombin-induced barrier disruption, the conclusions were the same as for FITC-Dextran leakage studies (Figs.3B, 3C). For cells treated with control siRNA, APC reduced by 50 % thrombin-induced barrier disruption (Fig. 3B) whereas APC had no protective effects when ApoER2 was knocked down (Fig. 3C). Rac1 activation mediates APC’s prevention of thrombin-induced vascular leakage.8, 11 When the effects of knocking down ApoER2 on Rac1 activation was studied, ApoER2 was absolutely essential for the observed APC-induced Rac1 activation (Fig. 3D). Thus, ApoER2 is required for APC’s vasculoprotective activity against thrombin-induced endothelial barrier disruption. APC and reelin share an LA1 domain binding site in ApoER2
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Endothelial ApoER2, similar to other LDL receptor family members, binds its ligands via LA domains.18, 21, 22, 29 A binding site for reelin on ApoER2’s LA1 domain was delineated using mutagenesis, protein structural information, and binding studies.29 We constructed and purified recombinant wild type and variant sApoER2 species comprising LA domains 1, 2, 3 and 7 plus EGF domains A and B with the LA1 domain mutations, K49P/D50S, W64A, or E68G (numbering according to Pro-protein sequence). The mutated 49, 50, and 68 residues involved replacement of ApoER2 residues with those of the highly homologous VLDLR. Binding studies using these purified sApoER2 preparations showed that data for each sApoER2 construct fit a binding isotherm for a 1:1 stoichiometry (Fig. 4A). Mutations of K49P and D50S or of W64A decreased the binding affinity by 75 to 92 % compared to control wild type sApoER2 (Fig. 4A) whereas the E68G mutation in ApoER2 caused no significant change in binding affinity (Fig. 4A). Thus, LA1 domain residues 49, 50 and 64 are essential for normal binding of APC to sApoER2.
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Discussion While neuronal ApoER2 is widely recognized in the neuroscience literature for neuronal and brain cell signaling and biologic outcomes,21, 30–33 there is a dearth of data for biological roles for endothelial ApoER2.23, 24, 34–37 To explore the importance of endothelial ApoER2 for APC’s cytoprotective actions, we used EA.hy926 cells as an endothelial cell surrogate38, 39 to study some of APC’s various activities. Extensive data in this report show that ApoER2 is required for APC’s cytoprotective and signaling activities in this cell line.
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In two distinct apoptosis assays that employed either staurosporine or TNFα, data here show that ApoER2 was required for APC’s anti-apoptotic activity in endothelial cells. Activation of the “Akt cell survival pathway” can provide an important mechanism to reduce apoptosis. APC-induced signaling caused phosphorylation of both Dab1, a known mediator of ApoER2-dependent signaling, and of Akt. The Src inhibitor, PP2, prevented phosphorylation of Dab1 and significantly delayed the phosphorylation of Akt, thereby implicating the known Dab1 signaling partner Src as mediating APC’s signaling. APC-induced Akt phosphorylation was blocked by a PI3K inhibitor and a PAR1 antagonist, indicating that this kinase and this receptor were required for Akt activation. These requirements are partially reminiscent of reelin-initiated activation of Akt downstream of Dab1 phosphorylation that involves Src and PI3K in neurons.17–19, 21, 22 Moreover, Akt phosphorylation in our studies was significantly inhibited by knocking down ApoER2. These data indicate that ApoER2 contributes to APC-induced cytoprotective signaling under the conditions of this study.
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An important protective effect of APC for stressed endothelial cells is its ability to stabilize endothelial barrier function that involves Rac1 activation which is known to require EPCR and PAR1.7, 8, 11, 12, 40 When TER was used to monitor the time course for thrombininduced barrier disruption in the absence or presence of APC, the data showed that knocking down of ApoER2 ablated the ability of APC to prevent endothelial barrier disruption. Thus, ApoER2 is required for APC’s vasculoprotective activity against thrombin-induced endothelial barrier disruption. This discovery is consistent with reports that, in neurons, reelin activates Rac1 via Dab-1, SFKs and PI3K. 20
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Studies of the binding of purified sApoER2 molecular variants to APC here indicated that LA1 domain residues 49, 50 and 64 are essential for normal binding of APC to sApoER2. A model of this LA1 domain face containing these residues shows that the surface which putatively binds APC (black oval in Fig. 4B) overlaps the domain surface previously identified as binding reelin (yellow oval in Fig. 4B).29 It remains to be defined how ligation of this LA1 domain surface mechanistically contributes to intracellular signaling mediated by ApoER2. APC’s cell signaling activities generally require EPCR and APC’s proteolytic GPCR target, namely PAR1, as recently reviewed.5, 41–43 However, other receptors such as PAR3, S1P1, Mac-1, EGFR, Tie-2 and/or other receptors may also contribute, directly or indirectly, to APC’s beneficial signaling by transactivation or crosstalk. Previously we showed that ApoER2 contributes to APC’s actions in U937 cells.10 Here we demonstrate that ApoER2 is required for APC’s antiapoptotic activity and endothelial barrier stabilization. Moreover, we
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found that both ApoER2 and PAR1 were necessary for the normal pattern of APC-induced Akt activation in EA.hy926 cells. These requirements are schematically depicted in Fig. 5 to indicate the complexity and diversity for receptors that bind APC and directly initiate APCinduced signaling. Localization of EPCR, PAR1 and ApoER2 to caveolae44–46 enables localization of the depicted signal initiations that must be integrated for APC’s benefits. Specifically, ligation of ApoER2 promotes intracellular interactions between Dab1 that is bound to ApoER2 and Src kinases resulting in phosphorylation of Dab1. In turn, phosphorylated Dab1 may serve as a scaffold that promotes additional protein – protein interactions that ultimately enhance Akt phosphorylation and activation (Fig. 5). In summary, the message of this paper is that ApoER2, which directly binds APC via its LA1 domain, should be considered as a potential mediator of APC’s actions on endothelial and possibly other cells not yet studied.
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Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments Ranjeet K Sinha, Xia V. Yang Jose A. Fernandez and Xiao Xu performed experiments. Ranjeet K. Sinha, Laurent Mosnier, Xia Yang and John H. Griffin designed the study. Ranjeet K Sinha, Jose A. Fernandez and John H. Griffin wrote the paper. Sources of Funding This work was supported in part by the National Heart, Lung, and Blood Institute (grants HL087618 and HL104165 (L.O.M.) and grants HL031950 and HL052246 (J.H.G.).
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Abbreviations
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APC
activated protein C
ApoER2
apolipoprotein E receptor 2
EPCR
endothelial protein C receptor
PAR
protease activated receptor
LDL
low density lipoprotein
MoAb
monoclonal antibody
PI3K
phosphatidylinositol-3 kinase
Dab-1
disabled-1
s
soluble
S1P1
sphingosine 1-phosphate receptor 1
Rac1
ras-related C3 botulinum toxin substrate 1
SFK
Src family kinase
GPCR
G protein coupled receptor
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Significance
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Activated protein C (APC) provides multiple beneficial physiologic and pharmacologic effects via its antithrombotic and cell signaling actions. Multiple receptors have been implicated to mediate APC’s cytoprotective cell signaling. Many but not all reports emphasize a role for the APC-Endothelial Cell Protein C Receptor (EPCR)-protease activated receptor-1 (PAR1) signaling paradigm. Here we used the human EA.hy926 endothelial cell line to assess the requirement for Apolipoprotein E Receptor 2 (ApoER2) for APC’s anti-apoptotic activity and APC’s stabilization of endothelial barriers. Results show that these two distinct cytoprotective actions of APC require ApoER2. APCinduced Akt activation required ApoER2 as well as PAR1. Moreover, APC induced phosphorylation of endothelial disabled-1 and Akt via Src-PI3K pathways and ApoER2dependent activation of Rac1 to achieve endothelial barrier stabilization. Thus, ApoER2 contributes cooperatively with EPCR and PAR1 to APC-initiated endothelial antiapoptotic and barrier protective signaling, indicating that multiple endothelial receptors can contribute to initiation of APC’s signaling.
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Figure 1. APC requires ApoER2 in addition to EPCR and PAR1 for its anti-apoptotic activity
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(A) Endothelial cells transfected with control siRNA or siRNA specific for EPCR, PAR1 or ApoER2 were exposed to pro-apoptotic staurosporine followed by detected of apoptosis by the APOPercentage assay. *p < 0.0001 (two-tailed t test). (B) Endothelial cells transfected with control siRNA or siRNA specific for EPCR, PAR1 or ApoER2 were exposed to TNFα and apoptosis was detected by TUNEL assay. *p < 0.0002 (two-tailed t test). (C) APC (20 nM) was preincubated with sEPCR (1 μM), sE86A-EPCR (1 μM), sApoER2 (1 μM) or control buffer for 30 min before addition to cells; then staurosporine-induced apoptosis assays were done. *p < 0.02. (D) APC (20 nM) was preincubated with sApoER2 (1 μM), sVLDLR (1 μM) or control buffer for 30 min before addition to cells and then TNFαinduced apoptosis assays were made. *p < 0.001. (E) Representative fluorescent microscopy images are shown for assay mixtures from TNFα-induced apoptosis assays as described in panel D.
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Figure 2. APC-induced signaling in endothelial cells involves phosphorylation of Akt and Dab1
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Phosphorylation of S473-Akt or Y232-Dab1 induced by APC (70 nM) was determined using immunoblots (see Methods). For some studies, cells were pretreated with siRNA against ApoER2 or control siRNA. (A) Time course for Akt phosphorylation at S473 where total Akt antigen levels were used as a loading control (Molecular Weight 60kDa). (B) Time course for Dab1 phosphorylation at Y232 in the presence of Src-kinase inhibitor PP2 (10 μM) or control non-inhibitory PP3 (10 μM) (Molecular Weight 80kDa). (C, D, and E) Effects of various indicated inhibitors on APC-induced Ser473-Akt phosphorylation. Inhibitors were added to cells 20 min before addition of APC (70 nM) and included: (C) the Src kinase inhibitor PP2 (10 μM) or control PP3 (10 μM); (D) the PI3K inhibitor LY294002 (10 μM) or control LY303511 (10 μM); and (E) the PAR1 antagonist SCH79797 (1 μM) or vehicle control containing DMSO. Error bars represent SEM for at least 3 separate experiments. Significance was analyzed using two way ANOVA. * = p < 0.05, **=p
Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2017 March 01. Published in final edited form as: Arterioscler Thromb Vasc Biol. 2016 March ; 36(3): 518–524. doi:10.1161/ATVBAHA.115.306795.
Apolipoprotein E Receptor 2 mediates Activated Protein Cinduced endothelial Akt activation and endothelial barrier stabilization
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Ranjeet K. Sinha, Xia V. Yang*, José A. Fernández, Xiao Xu, Laurent O. Mosnier, and John H. Griffin Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037 USA
Abstract 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.
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Approach and Results—We used siRNA and inhibitors to probe requirements for specific receptors for APC’s anti-apoptotic activity and for phosphorylation of disabled-1 (Dab1) by Src family kinases and of Akt. When siRNA for ApoER2 or endothelial cell protein C receptor or protease activated receptor 1 was employed, APC’s anti-apoptotic activity was ablated, indicating that each of these receptors was required. In EA.hy926 cells, APC induced a 2 to 3-fold increase phosphorylation of Ser473-Akt and Tyr232-Dab1, a phosphorylation known to trigger Dab1mediated signaling in other cell types. Ser473-Akt phosphorylation was inhibited by ApoER2 siRNA or by inhibitors of Src (PP2), PI3K (LY303511), and protease activated receptor 1 (SCH79797). ApoER2 siRNA 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.
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Conclusion—ApoER2 contributes cooperatively with endothelial cell protein C receptor and protease activated receptor 1 to APC-initiated endothelial anti-apoptotic and barrier protective signaling.
Corresponding author: John H. Griffin, Phone (858) 784 8220, [email protected]. *Present address: Regulus Therapeutics. 3545 John Jay Hopkins Ct., Suite 210. San Diego, CA 92121-1121 Disclosure of Conflicts of Interest None.
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Introduction The normal blood serine protease, activated protein C (APC), provides multiple beneficial physiologic and pharmacologic effects, including antithrombotic, cytoprotective, and regenerative actions1–5 and proteolytic detoxification of extracellular histones.6 Multiple receptors on various cell types have been implicated as required for APC’s cytoprotective cell signaling with most reports emphasizing a role for the APC-EPCR-PAR1 signaling paradigm in which biased signaling by cleavage of PAR1 at Arg46 may be central.7, 8 Although direct molecular interactions of purified APC have been well documented for EPCR, PAR1, PAR3, and apolipoprotein E receptor 2 (ApoER2) (also known as LRP8),1–4, 6, 7, 9, 10 the multiplicity of cell types and biologic activities linked to APC’s actions leaves many unanswered questions about mechanisms for APC’s beneficial cell signaling activities.
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One major pharmacologic activity of APC-induced signaling involves its ability to reduce vascular leakage induced by edemagenic mediators (e.g., thrombin, VEGF, P. aeruginosaderived toxins, etc.) that involves increased endothelial Rac1 activation and decreased RhoA activation in vitro and in vivo.1, 5, 7–9, 11–15 On monocytic-like U937 cells, ApoER2 promotes APC-induced activation of this receptor’s adaptor protein, disabled-1 (Dab1), and of Akt signaling, linked to APC anti-inflammatory actions.10 ApoER2 is central to the reelin pathway that mediates reelin-induced signaling and neuronal pattern development.16–22 Reelin binds ApoER2 and triggers phosphorylation of Dab1 involving the Src family kinases (SFK), Src and Fyn. This ligation of ApoER2 causes activation of phosphatidylinositol-3 phosphate kinase (PI3K) and phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β) in neurons. Engagement of endothelial ApoER2 by apoE results in suppression of metastatic endothelial recruitment by melanoma cells, implying that ligation of endothelial ApoER2 by one of its ligands, apo E, triggers beneficial signaling.23 Here we used the human EA.hy926 transformed endothelial cell line to assess APC’s ability to initiate reelin-like signaling and cytoprotection via ApoER2. The results show that APC induces phosphorylation of endothelial Dab-1 and that ApoER2 is required for APC-induced phosphorylation and activation of the PI3K-Akt pathway, activation of endothelial Rac1, and vasculoprotection against thrombin-induced vascular leakage and cytotoxicity.
Materials and Methods Materials and Methods are available in the online-only Data Supplement
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Results ApoER2 mediates protective activity of APC against apoptosis Endothelial cells and other cells contain alternatively spliced ApoER2 variants.10, 21, 24 When the EA.hy926 cells were analyzed for ApoER2 variants by PCR and sequencing (see Methods, Fig. SI), we identified 4 isoforms that are schematically indicated in Fig. SIC with deletions of exons (i) 5, (ii) 5 and 15, (iii) 5, 15 and 18, and (iv) 5 and 18. The N-terminal ligand binding region includes LDL receptor class A (LA) modules, each containing ~ 40
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residues. Given the absence of exon 5 from all endothelial ApoER2 variants, these data indicate that endothelial ApoER2 generally lacks LA domains 4–6 (Fig. SIC).
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Studies of human umbilical vein endothelial cells and EA.hy926 cells showed that APC similarly reduced staurosporine-induced apoptosis for each cell line,25 and we first showed that this endothelial cell cytoprotection required EPCR and PAR1 for its anti-apoptotic activity.26 To determine if ApoER2 is also required, siRNA was used to knock down each of these three receptors in EA.hy926 cells prior to apoptosis assays. When two different proapoptotic stimuli, staurosporine and TNFα, were used and apoptosis was monitored using Apopercentage dye uptake (Fig. 1A) or TUNEL staining (Fig. 1B), respectively, the results showed that knocking down any one of these three receptors ablated APC’s anti-apoptotic activity (Figs. 1A, 1B) (p < 0.0002). Because soluble ectodomains of EPCR and ApoER2 bind APC,10 they were studied as inhibitors of APC-initiated anti-apoptotic activity in each apoptosis model. When sApoER2 or sEPCR was preincubated with APC prior to the apoptosis assays, each one caused loss of APC’s anti-apoptotic activity (Figs. 1C, 1D). Predictably, the negative controls, sE86A-EPCR and soluble very low density lipoprotein receptor (VLDLR) which do not bind APC27, 28 did not cause loss of activity. When cells subjected to TNFα-induced apoptosis were stained with TUNEL or Hoechst dye or their combination, the cell images show that APC’s anti-apoptotic activity was inhibited by its prior incubation with sApoER2 but not with sVLDLR (Fig. 1E). These data support the hypothesis that each one of EPCR, PAR1 and ApoER2 was required for APC’s antiapoptotic activity under the conditions employed. APC requires ApoER2, PAR1, Src kinases and PI3-kinase to induce Akt phosphorylation in endothelial cells
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To determine requirements for ApoER2 for APC’s activation of Akt in endothelial cells,7 cells were treated with ApoER2 siRNA or control siRNA prior to APC. Studies to quantify mRNA levels showed that ApoER2 mRNA was knocked down by > 80% (Fig. SID), and Western blots showed similar reduction of ApoER2 antigen while EPCR antigen levels remained normal (Fig. SID). The ability of APC to cause phosphorylation of Ser473 in Akt was significantly inhibited by knocking down ApoER2 (Fig. 2A), indicating that this phosphorylation required ApoER2. Addition of APC to endothelial cells caused phosphorylation of Tyr232 in Dab1 that was sustained over 5 to 60 min, and this phosphorylation was significantly inhibited by the Src inhibitor PP2 but not by the control compound, PP3. When the requirements for SFK, PAR1, and PI3K for APC-induced Akt phosphorylation were probed using their inhibitors (PP2, SCH79797, and LY294002, or respective control compounds), the results showed that each of these proteins was required for the typical pattern of APC-induced Akt phosphorylation over the 90 min time course (Figs. 2C–E and Fig. SII A–C). Inhibition of either PAR1 or PI3K completely ablated Akt phosphorylation over 90 min (Fig. 2D–E and Fig. SII B–C). Interestingly, although PP2 treatment of endothelial cells completely ablated Dab-1 phosphorylation at up to 90 min (Fig. 2B), it did not reduce the level of Akt phosphorylation seen at 90 min, although it retarded this reaction over 5 to 60 min (Fig. 2C). This suggests that some pathways of APCinitiated signaling which are less efficient without ApoER2-mediated contributions can
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eventually augment Akt activation. In summary, these results show that ApoER2 is required for APC-initiated signaling in endothelial cells. ApoER2 is required for endothelial barrier stabilization by APC
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The vasculoprotective actions of APC for protecting against thrombin-induced vascular leakage on endothelial cells were described for human pulmonary artery endothelial cells using Transendothelial Electric Resistance (TER) assays11 and for EA.hy.926 human endothelial cells based on fluorescent dye leakage assays.12 In both those studies, a requirement for the S1P receptor, S1P1, was shown using siRNA, and a requirement for PI3K was also demonstrated.11 Additional data showed that EPCR and PAR1 were required for APC’s vasculoprotective effects.1 Therefore, here we used both TER and dye (FITCdextran) leakage bioassays to probe the requirement for ApoER2. Endothelial cells were pretreated with either ApoER2 siRNA or control siRNA and then exposed to either thrombin alone or thrombin plus APC. First, without APC addition, data showed that the extensive amount of thrombin-induced endothelial barrier leakage was the same for cells exposed to ApoER2 siRNA or control siRNA (Fig, 3A). Second, APC treatment completely ablated thrombin-induced leakage in controls, whereas ApoER2 siRNA treatment significantly diminished by > 50% the ability of APC to prevent thrombin-induced endothelial barrier leakage (Fig. 3A). When TER assays were used for defining thrombin-induced barrier disruption, the conclusions were the same as for FITC-Dextran leakage studies (Figs.3B, 3C). For cells treated with control siRNA, APC reduced by 50 % thrombin-induced barrier disruption (Fig. 3B) whereas APC had no protective effects when ApoER2 was knocked down (Fig. 3C). Rac1 activation mediates APC’s prevention of thrombin-induced vascular leakage.8, 11 When the effects of knocking down ApoER2 on Rac1 activation was studied, ApoER2 was absolutely essential for the observed APC-induced Rac1 activation (Fig. 3D). Thus, ApoER2 is required for APC’s vasculoprotective activity against thrombin-induced endothelial barrier disruption. APC and reelin share an LA1 domain binding site in ApoER2
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Endothelial ApoER2, similar to other LDL receptor family members, binds its ligands via LA domains.18, 21, 22, 29 A binding site for reelin on ApoER2’s LA1 domain was delineated using mutagenesis, protein structural information, and binding studies.29 We constructed and purified recombinant wild type and variant sApoER2 species comprising LA domains 1, 2, 3 and 7 plus EGF domains A and B with the LA1 domain mutations, K49P/D50S, W64A, or E68G (numbering according to Pro-protein sequence). The mutated 49, 50, and 68 residues involved replacement of ApoER2 residues with those of the highly homologous VLDLR. Binding studies using these purified sApoER2 preparations showed that data for each sApoER2 construct fit a binding isotherm for a 1:1 stoichiometry (Fig. 4A). Mutations of K49P and D50S or of W64A decreased the binding affinity by 75 to 92 % compared to control wild type sApoER2 (Fig. 4A) whereas the E68G mutation in ApoER2 caused no significant change in binding affinity (Fig. 4A). Thus, LA1 domain residues 49, 50 and 64 are essential for normal binding of APC to sApoER2.
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Discussion While neuronal ApoER2 is widely recognized in the neuroscience literature for neuronal and brain cell signaling and biologic outcomes,21, 30–33 there is a dearth of data for biological roles for endothelial ApoER2.23, 24, 34–37 To explore the importance of endothelial ApoER2 for APC’s cytoprotective actions, we used EA.hy926 cells as an endothelial cell surrogate38, 39 to study some of APC’s various activities. Extensive data in this report show that ApoER2 is required for APC’s cytoprotective and signaling activities in this cell line.
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In two distinct apoptosis assays that employed either staurosporine or TNFα, data here show that ApoER2 was required for APC’s anti-apoptotic activity in endothelial cells. Activation of the “Akt cell survival pathway” can provide an important mechanism to reduce apoptosis. APC-induced signaling caused phosphorylation of both Dab1, a known mediator of ApoER2-dependent signaling, and of Akt. The Src inhibitor, PP2, prevented phosphorylation of Dab1 and significantly delayed the phosphorylation of Akt, thereby implicating the known Dab1 signaling partner Src as mediating APC’s signaling. APC-induced Akt phosphorylation was blocked by a PI3K inhibitor and a PAR1 antagonist, indicating that this kinase and this receptor were required for Akt activation. These requirements are partially reminiscent of reelin-initiated activation of Akt downstream of Dab1 phosphorylation that involves Src and PI3K in neurons.17–19, 21, 22 Moreover, Akt phosphorylation in our studies was significantly inhibited by knocking down ApoER2. These data indicate that ApoER2 contributes to APC-induced cytoprotective signaling under the conditions of this study.
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An important protective effect of APC for stressed endothelial cells is its ability to stabilize endothelial barrier function that involves Rac1 activation which is known to require EPCR and PAR1.7, 8, 11, 12, 40 When TER was used to monitor the time course for thrombininduced barrier disruption in the absence or presence of APC, the data showed that knocking down of ApoER2 ablated the ability of APC to prevent endothelial barrier disruption. Thus, ApoER2 is required for APC’s vasculoprotective activity against thrombin-induced endothelial barrier disruption. This discovery is consistent with reports that, in neurons, reelin activates Rac1 via Dab-1, SFKs and PI3K. 20
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Studies of the binding of purified sApoER2 molecular variants to APC here indicated that LA1 domain residues 49, 50 and 64 are essential for normal binding of APC to sApoER2. A model of this LA1 domain face containing these residues shows that the surface which putatively binds APC (black oval in Fig. 4B) overlaps the domain surface previously identified as binding reelin (yellow oval in Fig. 4B).29 It remains to be defined how ligation of this LA1 domain surface mechanistically contributes to intracellular signaling mediated by ApoER2. APC’s cell signaling activities generally require EPCR and APC’s proteolytic GPCR target, namely PAR1, as recently reviewed.5, 41–43 However, other receptors such as PAR3, S1P1, Mac-1, EGFR, Tie-2 and/or other receptors may also contribute, directly or indirectly, to APC’s beneficial signaling by transactivation or crosstalk. Previously we showed that ApoER2 contributes to APC’s actions in U937 cells.10 Here we demonstrate that ApoER2 is required for APC’s antiapoptotic activity and endothelial barrier stabilization. Moreover, we
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found that both ApoER2 and PAR1 were necessary for the normal pattern of APC-induced Akt activation in EA.hy926 cells. These requirements are schematically depicted in Fig. 5 to indicate the complexity and diversity for receptors that bind APC and directly initiate APCinduced signaling. Localization of EPCR, PAR1 and ApoER2 to caveolae44–46 enables localization of the depicted signal initiations that must be integrated for APC’s benefits. Specifically, ligation of ApoER2 promotes intracellular interactions between Dab1 that is bound to ApoER2 and Src kinases resulting in phosphorylation of Dab1. In turn, phosphorylated Dab1 may serve as a scaffold that promotes additional protein – protein interactions that ultimately enhance Akt phosphorylation and activation (Fig. 5). In summary, the message of this paper is that ApoER2, which directly binds APC via its LA1 domain, should be considered as a potential mediator of APC’s actions on endothelial and possibly other cells not yet studied.
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Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments Ranjeet K Sinha, Xia V. Yang Jose A. Fernandez and Xiao Xu performed experiments. Ranjeet K. Sinha, Laurent Mosnier, Xia Yang and John H. Griffin designed the study. Ranjeet K Sinha, Jose A. Fernandez and John H. Griffin wrote the paper. Sources of Funding This work was supported in part by the National Heart, Lung, and Blood Institute (grants HL087618 and HL104165 (L.O.M.) and grants HL031950 and HL052246 (J.H.G.).
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Abbreviations
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APC
activated protein C
ApoER2
apolipoprotein E receptor 2
EPCR
endothelial protein C receptor
PAR
protease activated receptor
LDL
low density lipoprotein
MoAb
monoclonal antibody
PI3K
phosphatidylinositol-3 kinase
Dab-1
disabled-1
s
soluble
S1P1
sphingosine 1-phosphate receptor 1
Rac1
ras-related C3 botulinum toxin substrate 1
SFK
Src family kinase
GPCR
G protein coupled receptor
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Significance
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Activated protein C (APC) provides multiple beneficial physiologic and pharmacologic effects via its antithrombotic and cell signaling actions. Multiple receptors have been implicated to mediate APC’s cytoprotective cell signaling. Many but not all reports emphasize a role for the APC-Endothelial Cell Protein C Receptor (EPCR)-protease activated receptor-1 (PAR1) signaling paradigm. Here we used the human EA.hy926 endothelial cell line to assess the requirement for Apolipoprotein E Receptor 2 (ApoER2) for APC’s anti-apoptotic activity and APC’s stabilization of endothelial barriers. Results show that these two distinct cytoprotective actions of APC require ApoER2. APCinduced Akt activation required ApoER2 as well as PAR1. Moreover, APC induced phosphorylation of endothelial disabled-1 and Akt via Src-PI3K pathways and ApoER2dependent activation of Rac1 to achieve endothelial barrier stabilization. Thus, ApoER2 contributes cooperatively with EPCR and PAR1 to APC-initiated endothelial antiapoptotic and barrier protective signaling, indicating that multiple endothelial receptors can contribute to initiation of APC’s signaling.
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Figure 1. APC requires ApoER2 in addition to EPCR and PAR1 for its anti-apoptotic activity
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(A) Endothelial cells transfected with control siRNA or siRNA specific for EPCR, PAR1 or ApoER2 were exposed to pro-apoptotic staurosporine followed by detected of apoptosis by the APOPercentage assay. *p < 0.0001 (two-tailed t test). (B) Endothelial cells transfected with control siRNA or siRNA specific for EPCR, PAR1 or ApoER2 were exposed to TNFα and apoptosis was detected by TUNEL assay. *p < 0.0002 (two-tailed t test). (C) APC (20 nM) was preincubated with sEPCR (1 μM), sE86A-EPCR (1 μM), sApoER2 (1 μM) or control buffer for 30 min before addition to cells; then staurosporine-induced apoptosis assays were done. *p < 0.02. (D) APC (20 nM) was preincubated with sApoER2 (1 μM), sVLDLR (1 μM) or control buffer for 30 min before addition to cells and then TNFαinduced apoptosis assays were made. *p < 0.001. (E) Representative fluorescent microscopy images are shown for assay mixtures from TNFα-induced apoptosis assays as described in panel D.
Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2017 March 01.
Sinha et al.
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Figure 2. APC-induced signaling in endothelial cells involves phosphorylation of Akt and Dab1
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Phosphorylation of S473-Akt or Y232-Dab1 induced by APC (70 nM) was determined using immunoblots (see Methods). For some studies, cells were pretreated with siRNA against ApoER2 or control siRNA. (A) Time course for Akt phosphorylation at S473 where total Akt antigen levels were used as a loading control (Molecular Weight 60kDa). (B) Time course for Dab1 phosphorylation at Y232 in the presence of Src-kinase inhibitor PP2 (10 μM) or control non-inhibitory PP3 (10 μM) (Molecular Weight 80kDa). (C, D, and E) Effects of various indicated inhibitors on APC-induced Ser473-Akt phosphorylation. Inhibitors were added to cells 20 min before addition of APC (70 nM) and included: (C) the Src kinase inhibitor PP2 (10 μM) or control PP3 (10 μM); (D) the PI3K inhibitor LY294002 (10 μM) or control LY303511 (10 μM); and (E) the PAR1 antagonist SCH79797 (1 μM) or vehicle control containing DMSO. Error bars represent SEM for at least 3 separate experiments. Significance was analyzed using two way ANOVA. * = p < 0.05, **=p
