Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells Xia V. Yanga, Yajnavalka Banerjeea, Jose´ A. Ferna´ ndeza, Hiroshi Deguchia, Xiao Xua, Laurent O. Mosniera, Rolf. T. Urbanusb, Phillip G. de Grootb, Tara C. White-Adamsc, Owen J. T. McCartyc, and John H. Griffina,1 aDepartment of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037; bLaboratory of Clinical Chemistry and Haematology, University Medical Centre Utrecht, Utrecht, The Netherlands; and cDepartments of Biomedical Engineering and Cell and Developmental Biology, Oregon Health & Science University, Portland, OR 97239 Edited by Charles T. Esmon, Oklahoma Medical Research Foundation, Oklahoma City, OK, and approved November 18, 2008 (received for review August 2, 2008) Binding of activated protein C (APC) to cells triggers multiple ischemic stroke, NMDA excitotoxicity injury, and severe sepsis beneficial cytoprotective activities that suppress apoptosis, inflam- models (11–14) . mation, and endothelial barrier breakdown. One paradigm for Recently, two kinds of findings imply that the APC-EPCR- APC’s signaling emphasizes its binding to endothelial cell protein PAR1 paradigm is not always applicable and that alternative cell C receptor (EPCR) and subsequent protease activated receptor surface receptors might contribute to initiation of cell signaling (PAR)-1 activation. Here we used human monocytic-like U937 cells by APC. First, for a few in vitro assays of APC’s effects on certain to evaluate apolipoprotein E receptor 2 (ApoER2)-dependent sig- cells, it appeared that APC-initiated cell signaling did not naling by APC and found that APC initiated rapid phosphorylation require PAR1 (15, 16) or EPCR (17). Second, in a report of of Tyr-220 in the adaptor protein disabled-1 (Dab1) and of Ser-473 APC-platelet interactions, an important role was posited for in Akt. APC also induced phosphorylation of Ser-9 in glycogen APC binding to apolipoprotein E receptor-2 [ApoER2; aka LDL synthase kinase 3␤ (GSK3␤), which was blocked by the PI3K receptor (LDLR)-related protein 8; LRP8] (18) and to glycop- inhibitor LY294002. Receptor-associated protein (RAP), a general rotein Ib␣ (19). ApoER2 is a member of the LDLR family that antagonist for binding of ligands to LDL receptor family members, includes, among others, the LDLR, the LDLR related protein inhibited APC-induced phosphorylation of Dab1 and GSK3␤, (LRP), ApoER2, and the very low density lipoprotein receptor whereas anti-EPCR or anti-PAR1 blocking antibodies did not. (VLDLR) (20–23). Various LDLR family members provide Knocking down ApoER2 by using siRNA-ablated APC induced Dab1 major endocytotic activities for cells whereas other family mem- phosphorylation, suggesting that RAP-sensitive APC-induced sig- bers also play prominent roles for signal transduction. LDLR naling requires ApoER2. In surface plasmon resonance equilibrium family members can bind a spectrum of ligands on various cells, binding studies, APC bound with high affinity to soluble (s) ApoER2 not limited to lipoproteins, and ligation of many members of this (apparent Kd, Ϸ30 nM) but not to soluble very low density lipopro- receptor family suffices to initiate cell signaling. tein receptor. RAP blocked APC binding to sApoER2 but not to The physiologic neuronal Reelin signaling pathway involves sEPCR. RAP blocked binding of U937 cells to immobilized APC. RAP Reelin ligation of both ApoER2 and VLDLR and subsequent also blocked APC’s ability to inhibit endotoxin-induced tissue phosphorylation of the adaptor protein disabled-1 (Dab1) in- factor pro-coagulant activity of U937 cells. Thus, we propose that volving the Src family kinases Src and Fyn, followed by phos- ligation of ApoER2 by APC signals via Dab1 phosphorylation and phorylation of phosphatidylinositol 3-phosphate kinase (PI3K), subsequent activation of PI3K and Akt and inactivation of GSK3␤, Akt, and glycogen synthase kinase 3␤ (GSK3␤) (20–23). Re- thereby contributing to APC’s beneficial effects on cells. ceptor-associated protein (RAP) (24) is an antagonist for ApoER2 ligand binding that blocks Reelin’s effects on cells. endothelial protein C receptor ͉ reelin ͉ Akt ͉ PI3K ͉ monocyte Remarkably, murine genetic deficiencies of Reelin, ApoER2 plus VLDLR, Dab1, or Src plus Fyn produce mice with indis- ecombinant activated protein C (APC) reduces mortality in tinguishable phenotypes (reeler, scrambler, yotari) (22, 23, 25–29) Rsevere sepsis (1). APC, a well known plasma protein, can involving impaired motor coordination, compromised gait, trem- exert both anticoagulant and cytoprotective activities by dis- ors, and ataxia, plus abnormal patterns of neuronal positioning tinctly different mechanisms (2–4). Because two potent antico- in the brain. Moreover, reeler adult mice exhibit learning defects agulant plasma proteins, antithrombin and tissue factor pathway and have neurons with compromised long-term potentiation inhibitor, failed to reduce mortality in large phase III trials of functioning (30, 31). severe sepsis (5, 6), one may infer that one or more of APC’s Monocytic cell lines, such as the human leukemic monoblast cytoprotective actions contribute to mortality reduction by APC U937 cell line, have been used to study APC’s ability to modulate therapy. APC’s cytoprotective actions include inter alii anti-apo- monocyte apoptosis, phagocytosis, inflammation, and tissue ptotic and anti-inflammatory activities, alterations of gene ex- factor generation (4, 8, 32–34). To evaluate the potential rele- pression, pro-angiogenic activity, and endothelial barrier stabi- vance of ApoER2 for APC cell signaling mechanisms, APC- lization (4, 7, 8). initiated signaling in U937 cells was assayed based on the In vitro studies of the effects of APC on various cells support a paradigm for APC’s cell signaling based on two key receptors, Author contributions: X.V.Y., H.D., and J.H.G. designed research; X.V.Y., Y.B., J.A.F., H.D., endothelial protein C receptor (EPCR) (9) and the G protein- 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 coupled receptor protease activated receptor-1 (PAR1) (10), 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. which mediate downstream cytoprotective effects (4). Central to analyzed data; and X.V.Y. and J.H.G. wrote the paper. this paradigm is the binding of APC’s ␥-carboxyglutamic acid- The authors declare no conflict of interest. rich N-terminal domain to the EPCR with subsequent cleavage This article is a PNAS Direct Submission. of PAR1 by APC’s protease domain, followed by PAR1-induced 1To whom correspondence should be addressed. E-mail: jgriffi[email protected]. direct effects of APC on cells. Strong support for the in vivo This article contains supporting information online at www.pnas.org/cgi/content/full/ relevance of this mechanism for triggering cell signaling by 0807594106/DCSupplemental. pharmacologic doses of APC comes from studies of murine © 2008 by The National Academy of Sciences of the USA 274–279 ͉ PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807594106 Downloaded by guest on September 25, 2021 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 phos- phorylation of Dab1 with consequent PI3K-dependent signaling via phosphorylations of Akt and GSK3␤. 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 GSK3␤. 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 phosphory- lation 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 Fig. 1. APC stimulates RAP-sensitive phosphorylation of Dab-1 and GSK-3␤. ApoER2 by using siRNA ablated APC-induced Dab1 phosphor- (A) Cells were incubated with or without APC (2 ␮g/mL) for 5 or 10 min at 37 °C. ylation (Fig. 2 A and B), indicating that ApoER2 is required for Dab1 was immunoprecipitated from cell lysates and was separated on SDS/ Dab1 phosphorylation under these conditions. Similar to the PAGE and blotted with anti-phosphorylated Tyr (␣-pY) or anti-Dab1 (␣-Dab1) Reelin signaling pathway that causes activation of Akt by phos- as described in Materials and Methods.(B) U937 cells were pre-treated with phorylation of Ser-473 (39, 40) downstream of Dab1 phosphor- control, anti-EPCR mAb RCR252 (15 ␮g/mL), RAP (2 ␮M), or anti-PAR1 anti- ylation, APC caused rapid phosphorylation of Ser-473 in Akt bodies (ATAP2 plus WEDE15, 15 ␮g/mL each) for 15 min, then incubated with ␮ (Fig.