© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs219352. doi:10.1242/jcs.219352 RESEARCH ARTICLE VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R–gp130 Ming-Shih Hwang1,*, Michael G. Strainic1,*, Elliot Pohlmann1, Haesuk Kim1, Elzbieta Pluskota2, Diana L. Ramirez-Bergeron3, Edward F. Plow2 and M. Edward Medof1,‡ ABSTRACT signaling thus has broad importance biologically as well as Purified vascular endothelial cell (EC) growth factor receptor-2 clinically. (VEGFR2) auto-phosphorylates upon VEGF-A occupation in vitro, Previous studies reported (Albrecht et al., 2004; Laudes et al., arguing that VEGR2 confers its mitotic and viability signaling in and of 2002) that ECs can synthesize complement component C3, that itself. Herein, we show that, in ECs, VEGFR2 function requires the complement activation fragment C5a can affect EC responses concurrent C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor in vitro, and that the generation of C3a and/or C5a activation (IL-6R)–gp130 co-signaling. C3ar1/C5ar1 or IL-6R blockade totally fragments accompanies numerous disease states (Alawieh et al., abolished VEGFR2 auto-phosphorylation, downstream Src, ERK, 2015; Lillegard et al., 2014; Facciabene et al., 2017; Lechner et al., AKT, mTOR and STAT3 activation, and EC cell cycle entry. VEGF-A 2016; Wu et al., 2017). These findings uniformly were interpreted in augmented production of C3a/C5a/IL-6 and their receptors via a the context of systemic complement activation. We previously two-step p-Tyk2/p-STAT3 process. Co-immunoprecipitation analyses, implicated C3a and C5a receptor (C3ar1 and C5ar1) signaling in the confocal microscopy, ligand pulldown and bioluminescence EC response to vascular wire injury (Sakuma et al., 2010) and, like resonance energy transfer assays all indicated that the four other studies, presumed that the C3a and C5a ligands derived from receptors are physically interactive. Angiogenesis in murine day 5 plasma C3 and C5 proteins. retinas and in adult tissues was accelerated when C3ar1/C5ar1 In prior studies of immune cell activation (Strainic et al., 2008), we signaling was potentiated, but repressed when it was disabled. Thus, found that interacting antigen-presenting dendritic cells (DCs) and C3ar1/C5ar1 and IL-6R–gp130 joint activation is needed to enable cognate T cells locally generate C3a and C5a from endogenously physiological VEGFR2 function. synthesized (as opposed to plasma) complement. The two anaphylatoxins establish autocrine signaling loops with C3ar1 and KEY WORDS: C5ar1, Cellular growth, IL-6R, RTK signaling, VEGF-A, C5ar1 in both partners (Liu et al., 2008; Strainic et al., 2008, 2013). VEGFR2 The joint C3ar1/C5ar1 [G protein-coupled receptor (GPCR)] signal transduction cooperatively promotes DC and T cell proliferation INTRODUCTION (Strainic et al., 2008), as well as viability, both during activation and Growth factors confer their signaling through receptor tyrosine homeostatically (Lalli et al., 2008; Strainic et al., 2008, 2013). Recent kinases (RTKs). Tight regulation of RTK signaling is essential to work by others (Arbore et al., 2016; Liszewski et al., 2013) showed maintain cellular viability homeostatically as well as to enable that C3ar1/C5ar1 signaling operates intracellularly in human CD4+ cellular proliferation physiologically. Important among growth cells and participates in several T cell functions. Our experiments factor-RTK interactions is vascular endothelial growth factor-A traced C3ar1/C5ar1 transduction to activation of phosphoinositide-3 (VEGF-A) ligation of VEGF-A receptor 2 (VEGFR2; also known kinase γ (PI-3Kγ; also known as PIK3R3), AKT (also known as as KDR and Flk-1), a process that controls angiogenesis. VEGFR2 AKT1) and the mammalian target of rapamycin (mTOR) in CD4+ T transduction provides both viability and mitotic signals to vascular cells. Whether and, if so, how this GPCR transduction integrates with endothelial cells (ECs) (Millauer et al., 1993) as well as other cell canonical RTK signaling cascades tied to cellular proliferation and/or types. Virtually all phases of development, growth and tissue repair viability has not been investigated. depend on VEGFR2 signaling. Pathologically, VEGFR2 signaling We hypothesized that the previously reported connections of drives neovascularization that contributes to tumor progression, complement with ECs might be linked to an autocrine C3ar1/C5ar1 atherosclerosis and multiple other disorders (Ferrara et al., 2003). signaling process operating in ECs similarly to that in immune cells, Characterizing the cellular machinery that is integral to VEGFR2 and that autocrine C3ar1/C5ar1 signaling in ECs, if it pertains, might be interconnected with the anti-apoptotic and/or pro-mitotic effects of VEGFR2 signaling. Our analyses not only validated the participation 1Department of Pathology, Case Western Reserve University, Cleveland, OH of EC-produced C3a/C5a in VEGFR2 signaling, but additionally 44106, USA. 2Department of Molecular Cardiology, Lerner Research Institute, uncovered the co-participation of EC-produced IL-6. Mechanistic Cleveland Clinic, Cleveland OH 44195, USA. 3Case Cardiovascular Research Institute and University Hospitals, Case Western Reserve University School of insight came from co-immunoprecipitation (co-IP), C5a and C3a Medicine and University Hospitals, Cleveland, Ohio 44106, USA. ligand pulldown, confocal studies and bioluminescence resonance *These authors contributed equally to this work. energy transfer (BRET) analyses, all of which showed that VEGFR2 is ‡Author for correspondence ([email protected]) associated with C3ar1/C5ar1 and IL-6 receptor (IL-6R) in a signaling platform. Individual blockade of each receptor showed that M.-S.H., 0000-0001-9044-3099; M.G.S., 0000-0003-2438-8540; D.L.R.-B., coordinated operation of all four systems is needed to transmit 0000-0002-0989-3429; E.F.P., 0000-0003-1531-9746; M.E.M., 0000-0001-5239- 2518 VEGFR2 signals into ECs. Taken together, the experiments provide evidence that VEGFR2 Received 23 April 2018; Accepted 20 December 2018 signaling in situ in ECs requires joint C3ar1/C5ar1 and IL-6R– Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs219352. doi:10.1242/jcs.219352 gp130 (also known as IL6ST) signaling to transmit its mitotic and stimulated cells (Fig. 1C)] and both lines expressed C3ar1 and C5ar1 viability activities. The findings thus add to previous concepts of (Fig. 1H), indicative of autocrine C3ar1/C5ar1 signaling operating how VEGFR2 signaling is regulated and how it confers its growth tonically in ECs. Intracellular staining documented intracellular and viability functions under physiological conditions. pools of both C3ar1/C5ar1 and C3a/C5a in unstimulated bEnd.3 cells constitutively, consistent with the tonic C3ar1/C5ar1 signaling RESULTS functioning intracellularly (Fig. 1I). Addition of C3ar1/C5ar1 Autocrine C3ar1/C5ar1 signaling in ECs enables antagonists to bEnd.3 or MS-1 EC cultures induced Fas/FasL VEGF-A-induced growth and sustains viability expression (Fig. 1J), repressed intracellular Bcl-2/Bcl-xL (also As a first test of whether C3ar1/C5ar1 signaling in ECs impacts known as Bcl-2l1) (Fig. 1K), increased Bax/Bim (also known as VEGF-A function, we cultured two murine EC lines (bEnd.3 and Bcl-2l11) mRNAs (Fig. 1K), and rendered each line susceptible to MS-1 cells) with VEGF-A±pharmaceutical C3ar1 and C5ar1 Annexin V staining (Fig. 1L). Studies with primary murine antagonists (C3ar1-A/C5ar1-A). To exclude effects of other factors aortic ECs yielded the same results (Fig. S1D–F). Individual C3ar1 in serum, as generally done in studies of growth factor signaling (Fig. S1G) or C5ar1 (not shown) blockade had partial pro-apoptotic (Cantarella et al., 2002), we used 0.5% bovine serum medium. effects, indicative of C3ar1 and C5ar1 signaling cooperatively Blockade of the receptors or their C3a/C5a ligands [with monoclonal providing viability signals. The pro-apoptotic effects of the joint antibodies (mAbs) specific for C3a/C5a neo-epitopes] abolished C3ar1/C5ar1 blockade closely simulated the reported pro-apoptotic VEGF-A-induced growth of both EC lines (Fig. 1A,B). Added effects of disrupted VEGFR2 signaling (Dias et al., 2002). VEGF-A upregulated C3 and C5 mRNA transcript levels (Fig. S1A) Collectively, the data pointed to a signaling mechanism wherein and increased endogenous C3a and C5a production (Fig. 1C, left and C3ar1/C5ar1 signal transduction is essential for both the anti- right), indicative of the two anaphylatoxins establishing auto- apoptotic and mitotic effects of VEGFR2 signaling. inductive signaling loops. As found for CD4+ T cell proliferation (Strainic et al., 2008, 2013), individual blockade of C3ar1 or C5ar1 VEGF-A-induced C3ar1/C5ar1 signals that promote EC signaling partially inhibited VEGF-A-induced growth (not shown), growth are regulated by DAF whereas blockade of both totally disabled it (Fig. 1C, left and right). The extent of C3ar1/C5ar1 signaling is not only governed by the No effect of the antagonists was observed on IL-2 induction of CTLL level of endogenous complement synthesis but also by the rate of cell growth (Fig. S1B), excluding a non-specific cytotoxic effect. cleavage of the C3a/C5a ligands from the parental C3/C5 proteins, a Studies in human umbilical vein endothelial cells (HUVECs) process which, in part, is regulated by the complement inhibitor validated that the results apply to primary human