VEGFR2 Survival and Mitotic Signaling Depends on Joint Activation of Associated C3ar1/C5ar1 and IL-6R–Gp130 Ming-Shih Hwang1,*, Michael G

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 ECs. Blockade of DAF (also known as CD55) (Medof et al., 1984). In immune cells C3ar1/C5ar1 abrogated VEGF-A-induced HUVEC growth in non- (Strainic et al., 2008), DAF restrains the generation of C3a/C5a and confluent, i.e. growing (as opposed to contact inhibited) cultures thereby regulates the intensity of C3ar1/C5ar1 signaling. To (Fig. S1C), whereas added C3a or C5a increased C3 and C5 transcript document whether DAF regulation of autocrine C3ar1/C5ar1 levels 200% in 1 h, further supporting their functioning in auto- signaling is involved in controlling the growth of (non-contact- inductive signaling loops. Incubation of non-confluent HUVECs with inhibited) ECs, we prepared aortic ECs from Daf1–/– and C3ar1–/– IL-1/IFN-γ/TNF-α [linked with VEGF-A function and angiogenesis C5ar1–/– mice and compared the growth rates of each to that of (Mohr et al., 2017; van der Wijk et al., 2017; Voronov et al., 2014)] wild-type (WT) aortic ECs (Fig. 2A). The absence of DAF favored upregulated HUVEC C3 mRNA expression levels (900%). As found EC proliferation (1.77±0.06×105 versus 1.30±0.017×105 cells 24 h for bEnd.3 cells (Fig. S1A), adding VEGF-A to primary murine aortic after plating 105 cells), whereas disabled C3ar1/C5ar1 signaling ECs increased complement C3/C5/C5ar1/C5ar1 mRNA expression virtually abolished it (1.07±0.05×105 cells). Culturing 5×104 ECs levels (Fig. 1D) and heightened C3a/C5a production. of each genotype over 72 h documented ∼2-fold faster growth of Importantly, while we detected C3a/C5a in our initial studies with Daf1–/– ECs than WTs (4×106±0.017 versus 2×105±0.15, serum-free cultures, flow cytometric analyses of permeabilized P<0.005), and markedly impaired growth of C3ar1–/–C5ar1–/– HUVECs (not shown) and bEnd.3 murine ECs (Fig. 1E) showed ECs (1×106±0.05 versus 2×106±0.15, P<0.005) (Fig. 2B). upregulation of C3ar1/C5ar1 and C3a/C5a occurring more Silencing of DAF or of C3ar1 or C5ar1 in human aortic prominently intracellularly. Supplementing HUVEC cultures with a endothelial cells (HAECs) had similar effects (Fig. 2C). Silencing pharmaceutical surrogate of mitochondrial changes associated with of IL-6R likewise suppressed growth (Fig. 2C). Quantitative PCR hypoxia, i.e. the mitochondrial uncoupler protonophore carbonyl (qPCR) of perfused Daf1–/–,WTandC3ar1–/–C5ar1–/– aortas showed cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)+iodoacetate upregulation and downregulation of C3/C5 mRNA transcripts, (IAA) increased complement transcripts 400% in 1 h, linking respectively (Fig. 2D), establishing that the in vitro differences in amplification of autocrine C3ar1/C5ar1 signaling with changes growth rates correlated with in vivo differences in aortic complement accompanying hypoxia (not shown). In cell cycle assays, adding C5a production. Addition of VEGF-A to serum-starved aortic C3ar1–/– to (non-confluent) HUVECs promoted cell cycle transition from G0 C5ar1–/– ECs resulted in blunted EC proliferation compared to WT into G2 similarly to adding VEGF-A (Fig. 1F, upper row), whereas ECs (Fig. 2E). Curiously, unlike the virtually abrogating effects of C3ar1/C5ar1 blockade prevented VEGF-A induction of cell cycle disrupting C3ar1/C5ar1 signaling in WT ECs with the C3ar1/C5ar1 progression (Fig. 1F, lower row). Together, the data argued that antagonists or anti-C3a/anti-C5a mAbs (Fig. 1AB), VEGF-A VEGF-A mitotic function in ECs is dependent on intracellular retained some growth-inductive capacity in the genetically deleted C3ar1/C5ar1 co-signaling in ECs. C3ar1–/–C5ar1–/– ECs. To establish whether VEGFR2 viability signaling in ECs is likewise interconnected with autocrine C3ar1/C5ar1 signaling, we Compensatory changes maintaining viability and growth examined unstimulated cultures of murine bEnd.3 and MS-1 EC lines in C3ar1–/–C5ar1–/– ECs under serum-free conditions (again to eliminate effects of serum To determine the mechanism underlying the partial retention of growth mediators). The culture supernatants of both lines contained low of C3ar1–/–C5ar1–/– ECs compared to C3ar1/C5ar1-antagonized levels of C3a and C5a (Fig. 1G) [<10% of levels in VEGF-A- WT ECs, we first compared VEGFR2 expression levels and Journal of Cell Science

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Fig. 1. Autocrine C3ar1/C5ar1 signaling is essential for EC viability. (A,B) bEnd.3 (A) or MS-1 (B) cells were incubated for 3 days with medium alone, VEGF-A (VEGF)±C3ar1-A/C5ar1-A (RA) or anti-C3a/anti-C5a mAbs, and growth was assayed daily. (C) bEnd.3 or MS-1 cells were incubated with VEGF-A±RA, after which culture supernatants were assayed for C3a and C5a by ELISAs. (D) Primary murine aortic ECs were incubated with VEGF-A, after which C3, C5, C3ar1 and C5ar1 mRNA levels were quantified by qPCR. (E) Serum-starved HAECs were stimulated with VEGF-A, and intracellular as well as extracellular expression of C5a and C5ar1 was assessed by flow cytometry. (F) HUVECs were incubated with C5a, VEGF-A or VEGF-A±RA, or medium alone, stained with propidium iodide and assayed by flow cytometry (% G2/M+ is indicated). (G) C3a and C5a in culture supernatants from bEnd.3 and MS-1 cells were quantitated by ELISA. (H) C3ar1 and C5ar1 expression in bEnd.3 and MS-1 cells was assayed by flow cytometry (dashed line, isotype control; solid line, anti-C3ar1/anti-C5ar1 mAb-stained cells). Results are representative of four repeat assays. (I) Intracellular and extracellular expression of C5ar1, C5a, C3ar1 and C3a in bEnd.3 cells as assessed by flow cytometry. (J) bEnd.3 and MS-1 cells were incubated for 24 h with RA, after which Fas and FasL expression was assayed by flow cytometry. Red, specific mAb in treated cells; turquoise, specific Ab in untreated cell; purple, Ig control. (K) bEnd.3 cells were incubated for 8 h with RA or anti-C3a/C5a mAbs, after which Bcl-2, Bcl-xL, Bax, and Bim mRNA expression levels were quantified by qPCR. (L) Annexin V staining in bEnd.3 and MS-1 cells incubated with RA, as above. Error bars show s.d. *P<0.05. endogenous VEGF-A production. Untreated C3ar1–/–C5ar1–/– ECs levels of IL-7 receptor (IL-7R) (Fig. 2J), the latter of which provides showed >600% more tonic VEGF-A production than untreated WT for (homeostatic) growth of immune cells to physiological numbers. ECs (Fig. 2F) and 55% increased surface levels of VEGFR2 Culture supernatants of C3ar1–/–C5ar1–/– ECs showed increased (Fig. 2G). C3ar1–/–C5ar1–/– ECs also expressed ∼130% higher tonic production of IL-6 (Fig. 2K), the significance of which will be levels of β3 integrin (CD61) (see Discussion) than WT ECs described below. Since both IL-6R and IL-7R signaling are linked (Fig. 2H), the activation of which is regulated by VEGFR2 signaling to PI-3Kα (also known as PIK3R1) activation and survival (Byzova et al., 2000) and is implicated in EC viability (Brooks et al., signaling (Carrette and Surh, 2012; Leu et al., 2003; Thang et al., 1994; Scatena et al., 1998). Additionally, C3ar1–/–C5ar1–/– ECs 2010), their increases in conjunction with the increases in VEGFR2 expressed ∼800% higher levels of IL-6R (Fig. 2I) and 400% higher and CD61 signaling could account for the compensated survival of Journal of Cell Science

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Fig. 2. Autocrine C3ar1 and C5ar1 signaling participates in VEGF-A-induced angiogenesis in vivo. (A) Equal numbers of primary murine aortic ECs isolated from the aortic rings of Daf1–/–, WT and C3ar1–/–C5ar1–/– mice were cultured for 2 weeks in basal EC growth medium. All panels at same magnification. (B) Equal numbers of primary ECs of each genotype were cultured for 3 days in EC growth medium (consisting of DMEM/F12 medium containing 20% FBS, 2 mM L-glutamine, 1% non-essential amino acid, 0.09 mg/ml EC growth supplement, 1% antibiotic/antimycotic, 100 units/ml penicillin, 100 g/ml streptomycin, 5 ng/ml VEGF-A and 0.09 mg/ml heparin) and cell counts quantified daily. (C) HAECs were transfected with siRNA targeting DAF, C5ar1+C3ar1 or scrambled control. Forty-eight hours after transfection, HAEC growth was monitored daily. (D) C3 and C5 mRNA transcripts harvested from perfused aortas of each genotype relative to WTs. (E) WT or C3ar1–/–C5ar1–/– ECs were incubated for 3 days with media or VEGF-A and growth was quantified daily. (F) VEGF-A production in supernatants and cell lysates of serum-starved primary ECs. (G) Primary aortic ECs from WT and C3ar1–/–C5ar1–/– mice were assayed for VEGFR2 expression by flow cytometry. (H) Primary aortic WT and C3ar1–/–C5ar1–/– ECs were assayed for β3 integrin (CD61) expression by flow cytometry. (I,J) Primary WT and C3ar1–/–C5ar1–/– mouse aortic ECs were assayed for IL-6R (I) and IL-7R (J) expression by flow cytometry. (K) Primary WT and C3ar1–/–C5ar1–/– mouse aortic ECs were incubated with C5a for 24 h and assayed for IL-6 production by ELISA. Error bars show s.d. *P<0.05, **P<0.005.

C3ar1–/–C5ar1–/– ECs compared to C3ar1/C5ar1-antagonized two-dimensional vascular tube formation assays with HUVECs WT ECs. and VEGF-A±C3ar1/C5ar1 blockade. Culturing HUVECs with EBM-2 basal medium containing supplemental growth factors C3ar1/C5ar1 signaling in ECs is essential for yielded the typical growth pattern of an EC tubal network consisting angiogenesis in vivo of cluster regions with a few branches (Fig. 3A, left). Substitution of To validate that autocrine C3ar1/C5ar1 signaling is interconnected exogenous VEGF-A for the basal growth factor cocktail simulated with VEGFR2 function physiologically, we first performed the increase in branch points observed with the cocktail (Fig. 3A, Journal of Cell Science

4 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs219352. doi:10.1242/jcs.219352 middle), whereas the inclusion of C3ar1-A/C5ar1-A together with Y1059. As a control, we used the Src kinase inhibitor, PP2, which VEGF-A virtually abolished tube formation (Fig. 3A, right). prevents VEGFR2 auto-phosphorylation. PP2 suppressed tonic To examine in vivo angiogenesis, we used three distinct mouse VEGFR2 auto-phosphorylation by 59% (Fig. 4A,B). C3ar1/C5ar1 models. In the first, we implanted RM1 prostate tumors into the antagonism had a comparable suppressive effect (62% reduction). flanks of male WT, Daf1–/– and C3ar1–/–C5ar1–/– mice. These Importantly, while VEGF-A increased auto-phosphorylation 290%, tumors are dependent on tumor-associated angiogenesis for their C3ar1/C5ar1 blockade completely abolished the VEGF-A-induced progression (Huang et al., 2005). Examination of blood vessels at increase (to 15% below that in the medium control), approaching day 10 in stroma for the EC marker CD31 (also known as Pecam1) that of PP2. Conversely, added C5a increased basal auto- showed significantly reduced blood vessel density and vascular area phosphorylation >100%. Replicate analyses gave near identical in C3ar1–/–C5ar1–/– mice compared to WT controls (9772±799 results. versus 15,699±1591 µm2, P=0.004, n=8) (Fig. 3B,C). In contrast, Previous studies of retinal pigment epithelial cells (Fukuoka angiogenesis was significantly enhanced in Daf1–/– recipients of and Medof, 2001; Fukuoka et al., 2003) and of CD4+ T cells RM tumors (23,458±1976 versus 15,699±1591 µm2, P<0.01, n=8). (Strainic et al., 2008) unexpectedly found that added C5a can induce Consistent with the increased blood supply, the tumors in Daf1–/– IL-6 production. Other studies of ECs found that either added mice tended to be larger (359±185.31 versus 227.93±201.28 mg, VEGF-A or added IL-6 can activate STAT3 (Wei et al., 2003). P=0.18) than in WTs, and those in C3ar1–/–C5ar1–/– mice tended to The mechanisms underlying these interconnections were not be smaller (164.33±89.7 versus 227.93±201.28 mg, P=0.29) established. Prompted by these observations, we investigated (Fig. 3D). While the difference in each case compared to WTs did whether VEGFR2 signaling and C3ar1/C5ar1 transduction in ECs not reach statistical significance, the difference in growth might be linked to each other via autocrine IL-6R signaling and between Daf1–/– recipients versus C3ar1–/–C5ar1–/– recipients did activation of STAT3. (359±185.31 versus 164.33±89.7 mg, P=0.025). We incubated cultures of MS-1 ECs with (1) medium alone, In the second model, we placed non-penetrating sutures in the VEGF-A alone, or VEGF-A+anti-IL-6 mAb, or with (2) medium corneas of WT, Daf1–/–, C3ar1–/–C5ar1–/– and Daf1–/–C3ar1–/– alone, IL-6 alone, or IL-6+C3ar1-A/C5ar1-A, after which we C5ar1–/– mice. This assay measures neo-angiogenesis since the assayed MS-1 cell proliferation over 72 h. VEGF-A induction of cornea is normally avascular. Beginning at day 5, we examined growth was abolished by anti-IL-6 mAb treatment (Fig. 4C). IL-6 corneas by fluorescence microscopy after intravenously addition induced EC proliferation comparably to that of VEGF-A administering fluorescent dextran beads. In contrast to the gradual addition (Fig. 4D). However, the IL-6-induced growth, like that of ingrowth of blood vessels at the corneal margins starting at day 9 in VEGF-A, was abolished by C3ar1/C5ar1 blockade (Fig. 4D). WT mice, accelerated and more robust influx occurred in Daf1–/– Incubation of the ECs with either VEGF-A or IL-6 alone induced mice. Virtually none occurred in the corneas from C3ar1–/–C5ar1–/– STAT3 phosphorylation (66% and 121% increases, respectively), or Daf1–/–C3ar1–/–C5ar1–/– mice (Fig. 3E), despite the but the STAT3 phosphorylation in both cases was reversed by compensatory changes (Fig. 2F–K) connected with the genetic C3ar1/C5ar1 antagonism (>70% and virtually 100%, respectively) absence of C3ar1/C5ar1 signaling as opposed to C3ar1/C5ar1- (Fig. 4E). These data argued that STAT3 phosphorylation by either antagonized WTs. Similar results were obtained in the absence of agonist was dependent on concomitant C3ar1/C5ar1 signaling. sutures (M. Kohen and M.E.M., unpublished), excluding suture Consistent with VEGFR2 signaling being interconnected with both responses in the differences. C3ar1/C5ar1 and IL-6R signaling, VEGF-A induction of C3/C5 In the third model, we compared angiogenesis in newborn murine mRNA transcription (Fig. 1D) and of local C3a/C5a generation day 5 retinas. Mice are born blind. They develop retinal vasculature (Fig. 1C) was attenuated by anti-IL-6 mAb (not shown). during the first week of life. Analysis of vascular growth in this Collectively, these findings indicated that VEGF-A interconnects model consequently is a widely accepted system to exclude effects with C3ar1/C5ar1 signaling via the induction of IL-6 and its of most, if not all, exogenous stimuli. We avoided addition of consequent signaling through IL-6R–gp130 to activate STAT3. exogenous VEGF-A, as used by some others, because we wanted to assess endogenous VEGF-A–VEGFR2-dependent growth. We ECs tonically produce VEGF-A, C5a and IL-6, and each measured angiogenesis by distance from the optic nerve to the amplifies the others angiogenic front and by the extent of branching at the angiogenic Prompted by the above interconnection of VEGFR2, C3ar1/C5ar1 front. Newborn day 5 retinas from Daf1–/– mice showed more robust and IL-6R signaling, we assayed culture supernatants of serum- angiogenesis than those from WTs, whereas those from C3ar1–/– starved MS-1 ECs for production of the corresponding VEGF-A, C5ar1–/– mice showed blunted angiogenesis (Fig. 3F–I). Similar C3a/C5a and IL-6 ligands. As found for the tonic production of C3a results were obtained in multiple repeat assays with mice from each and C5a by ECs shown in Fig. 1G, enzyme-linked immunosorbent group. Collectively, these in vivo studies thus argued that joint assays (ELISAs) documented concurrent tonic production of IL-6 C3ar1/C5ar1 and VEGFR2 signaling is integral to angiogenesis and VEGF-A (Fig. 4F). Kinetic analyses showed that, at 4–6h,each physiologically. ligand induced 100–400% increases in the production of the others (Fig. 4F). The increased production of each agonist coincided with VEGFR2 phosphorylation is regulated by C3ar1/C5ar1 an amplified burst of Tyk2 and STAT3 phosphorylation (Fig. 4G), signaling and is tied to IL-6R signaling together with upregulated expression levels of VEGFR2, C5ar1 and We next investigated how C3ar1/C5ar1 transduction C3ar1, measurable prominently intracellularly (Fig. 4H). Blockade mechanistically interconnects with VEGFR2 signaling. We first of C3ar1/C5ar1 or IL-6 signaling abolished both the initial and tested whether adding C5a to primary aortic WT ECs affects amplified activation of Tyk2 and STAT3 (Fig. 4G). Moreover, VEGFR2 auto-phosphorylation and whether canonical VEGFR2 blockade of C3ar1/C5ar1 or IL-6R signaling abolished the auto-phosphorylation is affected by antagonizing C3ar1/C5ar1 upregulated expression of all of the receptors (Fig. 4H). These signaling. To study VEGFR2 auto-phosphorylation, we used an findings pointed to an interdependent signaling mechanism 1094 mAb specific for the VEGFR2 auto-phosphorylation sites p-Y / involving all four ligand-receptor systems. Journal of Cell Science

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Fig. 3. C3ar1/C5ar1 signaling induces VEGFR2 phosphorylation. (A) HUVECs were incubated for 24 h with EBM-2 medium, VEGF-A or VEGF-A+RA. (B–D) Male mice (n=8) were inoculated subcutaneously with RM1 prostate cancer cells. (B) Representative images of tumor sections of WT, Daf1–/– and C3ar1–/– C5ar1–/– mice, following CD31 staining to identify blood vessels (brown). Scale bars: 150 μm. (C) The numbers of CD31-positive areas were quantified in 5–10 independent fields per tumor implant. (D) Tumors were collected 10 days after injection and weighed. (E) Corneal neovascularization assessed by fluorescence microscopy using fluorescein-dextran in WT, Daf1–/–, C3ar1–/– C5ar1–/– and Daf1–/–C3ar1–/–C5ar1–/– (n=3 for each), induced by placing a non-penetrating suture in one cornea of each mouse. Representative data are shown. (F–I) Retinas from day 5 neonatal mice (n=6 WT, 7 Daf1–/– and 5 C3ar1–/–C5ar1–/– mice) were isolated and the retinal vasculature was visualized with isolectin B4. (F–H) Whole mounts of the retinas (F) were analyzed for retinal vessel length (distance to angiogenic from optic nerve) (G) and number of retinal branch points (H). ON, optical nerve. AF, angiogenic front (distance from

ON). (I) Representative images depicting the angiogenic front of retinal growth. Scale bars: 50 μm. Error bars show s.d. *P<0.02; NS, non-significant. Journal of Cell Science

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Fig. 4. VEGF-A induces C3ar1/C5ar1 signaling in ECs via IL-6 and p-STAT3. (A) Serum-starved primary ECs were incubated for 5 min with VEGF-A alone, VEGF-A+RA, RA alone or C5a alone, after which the p-Y1094/Y1059 VEGFR2 auto-phosphorylation sites were assessed by immunoblotting. PP2 was used as a control. (B) Densitometry of bands from three separate experiments from A showed that all changes were significant compared to untreated cells (*P<0.05, **P<0.005). (C) Serum-starved MS-1 cells were cultured for 3 days with medium alone or with VEGF-A±anti-IL-6 neutralizing mAb and growth was quantified daily. (D) Serum-starved MS-1 cells were cultured with IL-6±RA and growth was quantified daily. (E) Serum- starved MS-1 cells were incubated with VEGF-A, VEGF-A±RA, anti-IL-6 mAb or IL-6±RA, after which pY705-STAT3 was assayed by flow cytometry. (F) Following incubation of serum-starved MS-1 cells with medium alone, the cells were alternatively incubated for increasing times with VEGF-A, C5a or VEGF-A, after which endogenously produced IL-6, endogenously produced VEGF-A or endogenously produced C5a in the culture supernatant was assayed by ELISA. (G,H) Serum-starved bEnd.3 cells were incubated for increasing times with VEGF-A±RA or anti-IL-6 mAb, and assayed for pY743-STAT3 and p-Tyk2 expression (G) or VEGFR2, C5ar1 and C3ar1 intracellular expression (H) by flow cytometry. Error bars show s.d.

Downstream RTK signaling depends on joint activation concurrently abolished activation of Src (Fig. 5A), AKT (Fig. 5A) of C3ar1/C5ar1 and IL-6R–gp130 and ERK (Fig. 5A) (as assessed by Luminex). Conversely, addition We next investigated how the signaling via C3ar1/C5ar1 and of C5a or IL-6 induced the phosphorylation of all three signaling IL-6R–gp130 impacts the canonical signaling pathways classically intermediates (Fig. 5B). Consistent with C3ar1/C5ar1 and IL-6R connected with VEGF-A induction of VEGFR2 signaling. We signaling being interconnected with each other and with VEGFR2 assayed phosphorylation of Src, AKT and ERK by Luminex signaling, the activation of Src, AKT and ERK by added C5a was (multiplex) assay, as well as activation of mTOR. We additionally inhibited by anti-IL-6 mAb (Fig. 5C), and that induced by IL-6 was measured activation of STAT3. Blockade of either C3ar1/C5ar1 or inhibited by C3ar1/C5ar1 blockade (Fig. 5D). Identical results were

IL-6R signaling in cultures of VEGF-A-treated bEnd.3 cells obtained with human HeLa cells (Fig. 5E). Total levels of each Journal of Cell Science

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Fig. 5. Signaling studies. (A) bEnd.3 cells were incubated for 5 min with VEGF-A±RA or anti-IL-6 mAb, and cell extracts were assayed for p-Src, p-AKT and p-ERK by Luminex assay. (B) bEnd.3 cells were incubated for 5 min with medium alone, C5a or IL-6, and cell extracts were assayed for p-Src, p-AKT and p-ERK by Luminex assay. (C,D) bEnd.3 cells were incubated with C5a (C) or IL-6±SU5416 (30 μM) (D), after which cell extracts were assayed for Src, AKT and ERK phosphorylation by Luminex assay. (E) HeLa cells were incubated with VEGF-A±RA, after which cell extracts were assayed for p-AKT, p-ERK and p-Src by Luminex assay. (F) bEnd.3 cells were incubated for 1 h with C5a, C3a or VEGF-A, after which cells were lysed and assayed for PLC activity by EnzCheck Direct Phospholipase C Assay Kit. (G–I) bEnd.3 cells were incubated with C5a or IL-6±SU5416, after which cells were assayed for Src (G), AKT (H) and ERK (I) phosphorylation by Luminex assay. (J) bEnd.3 cells were incubated with VEGF-A or VEGF+SU5416, after which culture supernatants were assayed for C5a and IL-6 by ELISA. (K) bEnd.3 cells were incubated with C5a or IL-6±SU5416 and assayed for Bcl-2, Bcl-xL, Bim and Bax mRNA expression. Error bars show s.d. *P<0.05. signaling intermediate were not affected (Fig. S2A). VEGF-A- C5ar1 signaling can occur as a result of C5ar1 coupling to Gαq as well induced activations of mTOR (Fig. S2B) and of STAT3 (Fig. S2C) as Gαi (Amatruda et al., 1993; Buhl et al., 1993; Jiang et al., 1996; were similarly inhibited by C3ar1/C5ar1 blockade. These findings Lee et al., 1995). However, one report (Jiang et al., 1996) implicated indicated that two canonical signaling pathways downstream of the Gβγ subunits as being the predominant driver of PLC activity. VEGFR2 activation are dependent on concomitant C3ar1/C5ar1 and IL-6R–gp130 signaling. C3ar1/C5ar1 and IL-6R–gp130 signaling depend on VEGFR2 Another downstream signaling pathway connected with VEGFR2 signaling signaling or transactivation to it is activation of PLC proteins and A corollary of VEGFR2, C3ar1/C5ar1 and IL-6R–gp130 signaling induction of Ca2+ release. To test whether C3ar1/C5ar1 signaling can being interdependent is that C3ar1/C5ar1 or IL-6R–gp130 signaling activate PLCs, and whether VEGF-A induced activation of PLCs is should be dependent on VEGFR2 signaling. To test this concept, we dependent on C3ar1/C5ar1 signaling, we incubated bEnd.3 cells examined how disrupting VEGFR2 signaling would impact C5a with C5a, C3a or VEGFR2±C3ar1-A/C5ar1-A and examined PLC and IL-6 signaling. activity. Both C5a and C3a activated PLC comparably to VEGF-A, Prior studies found that C5a or IL-6 can induce activation of Src, and C3ar1-A/C5ar1-A inhibited the VEGF-A induction of PLC AKT and ERK (Huang et al., 2014; Rodriguez-Berriguete et al., activity (Fig. 5F). 2010; Wegiel et al., 2008), and attributed the results to C5ar1 or Activation of PLCs is usually mediated by Gαq-coupled GPCRs. IL-6R signaling in and of themselves. To assess whether VEGFR2

Prior studies by others have provided evidence that PLC activation by signaling is required for these processes, we added C5a or IL-6 to Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs219352. doi:10.1242/jcs.219352 cultures of bEnd.3 ECs with or without the VEGFR2 biotin-labeled ligand. Each of the four receptors brought down some pharmaceutical antagonist (SU5416). Quantitation of signaling C5a as early as 5 min, but brought down increasingly more over the intermediates 5 min later showed that the VEGFR2 antagonist next hour (Fig. 6D), with the greatest incorporation occurring at abolished the C5a- or IL-6-induced activation of Src (Fig. 5G), AKT 24 h. This finding that C5a remained associated with all of the (Fig. 5H) and ERK (Fig. 5I). signaling partners at every time point further supported the We next tested whether VERGR2 signaling is needed for proposition that C5ar1 functions in a complex containing endogenous C5a or IL-6 production. Addition of SU5416 to VEGFR2, C5ar1, C3ar1 and IL-6R that exists tonically at low cultures of VEGF-A-treated bEnd.3 cells blocked the production of levels and is upregulated by amplified VEGFR2 signaling. both C5a and IL-6 (Fig. 5J). The same result was found for the As a third way to assess physical proximity/association that avoids reported anti-apoptotic effects of added C5a (Lalli et al., 2008; alterations in the cells by extraction, added mAbs/ligands or IPs, we Strainic et al., 2008) and IL-6 (Leu et al., 2003). While either C5a or performed BRET assays. We co-transfected HEK293 cells with IL-6 upregulated anti-apoptotic Bcl-2/Bcl-xL and downregulated C5ar1 linked to GFP and VEGFR2 linked to luciferase. Addition of pro-apoptotic Bax/Bim (Leu et al., 2003; Spets et al., 2002), the the luciferase substrate followed by luminescence analysis showed a anti-apoptotic effects of both were abolished by blockade of strong BRET signal (Fig. 6F). The reverse experiment with C5ar1- VEGFR2 signaling (Fig. 5K). Luc and VEGFR2-GFP gave the same result (Fig. 6F). An identical These converse findings thus functionally implicate VEGFR2 pattern was seen with C5ar1-GFP and C5aR-Luc (Fig. 6F). signaling as an essential participant in either C3ar1/C5ar1 or Experiments using protease-activated receptor 1 (PAR-1; also IL-6R–gp130 signaling, paralleling the requisite participation of known as F2R), showed no dimerization between PAR-1 and C3ar1/C5ar1 and IL-6R–gp130 signaling in VEGFR2 signaling. VEGFR2 (data not shown). We were unable to test gp130 because of This functional interdependence operating in the opposite technical problems in preparing the gp130 constructs. direction further supports the proposition that VEGFR2 confers As a fourth way to determine whether the four receptors are its downstream signaling interdependently with C3ar1/C5ar1 and co-associated and localize the cellular site of their co-association, IL-6R–gp130 signaling. we examined bEnd.3 cells by confocal microscopy following the addition of AlexaFluor-488 (green)-labeled anti-VEGFR2, VEGFR2, C5ar1 and IL-6R–gp130 are co-associated with AlexaFluor-647 (blue)-labeled anti-C5ar1 and AlexaFluor-546 each other (red)-labeled anti-IL-6R mAbs. Both the patterns of the staining The findings that added C5a caused VEGFR2 phosphorylation in and the overlay were consistent with some plasma membrane co- <5 min (Fig. 4A,B) and that added C5a, VEGF-A or IL-6 each evoked localization but predominantly intracellular co-localization (Fig. 6F, the production of the others (Fig. 4F), prompted the hypothesis that upper row, unstimulated; lower row, VEGF-A activated for 1 h). VEGFR2, C5ar1 and IL-6R–gp130 might co-exist together in a This finding has been observed (Gerhartz et al., 1994) in connection signaling platform in which each is associated with the other directly or with agonist occupation of other receptors but interpreted in the indirectly. To test this, we used four complementary methods that context of internalization following receptor ligation on the cell analyze protein association. First, we performed co-IP experiments. surface. An identical merge was observed for VEGFR2 with C3ar1 Immunoblots (Fig. 6A) of anti-C5ar1 immunoprecipitations (IPs) and IL-6R (Fig. S3A) and for VEGFR2 with C5ar1 and gp-130 showed C3ar1, VEGFR2 and gp130, those of anti-VEGFR2 IPs (not shown). The intracellular staining showed that the receptors showed C3ar1, C5ar1 and gp130, and those of anti-gp130 IPs showed co-localized in endosomes (Fig. 6G), whereas only p-Tyk2 C3ar1, C5ar1 and VEGFR2. Immunoblots of a control IP with anti- (Fig. S3B) and STAT3 (Fig. S3C) were detectable (spotty CD31 mAb showed no associated C3ar1, C5ar1, VEGFR2 or gp130 distribution) in the nucleus (Fig. S3B,C, green). The latter finding (Fig. 6A). Representative primary blots are shown in Fig. S4. To is similar to the spotty co-nuclear translocation of p-Tyk2 together establish whether the receptor associations depended on each other and with p-STAT3 reported in the context of type 1 IFN signaling exclude non-specific pulldowns, we repeated co-IPs in bEnd.3 cells (Ahmed et al., 2013; Ragimbeau et al., 2001). pre-transfected with VEGFR2, C5ar1 or IL-6R small interfering RNA Collectively, the data argue that VEGFR2 functions within a (siRNA). Knockdown of any of one of the three receptors abolished signaling complex containing C3ar1/C5ar1 and IL-6R–gp130, and the co-IP of the others (Fig. S5). that signaling induced by agonist occupation of each of the receptors As a second test of association, we added biotin-labeled C3a or interconnects with joint-signaling of all of the partners. The data C5a to cultures of bEnd.3 cells in the absence or presence of show that this joint signaling is needed for downstream VEGFR2 VEGF-A. After incubation for 30 min, we prepared cell extracts, signaling via all three of its canonical signaling cascades. A performed anti-C3ar1, anti-C5ar1, anti-VEGFR2, anti-gp130 or diagrammatic representation of the signaling complex is shown in anti-CD31 IPs, and probed immunoblots of eluents with Fig. 6H. streptavidin-peroxidase to determine whether immunoprecipitating each interactive partner would pull down the biotin-labeled C3a or DISCUSSION C5a ligands. The mAbs against C3ar1, C5ar1, VEGFR2 and gp130, The expression and biological functions of C3ar1 and C5ar1 for but not that against CD31, brought down some biotin-labeled C3a many years had been thought to be restricted to phagocytic myeloid (Fig. 6B, upper bands) and biotin-labeled C5a (Fig. 6C, upper cells, i.e. neutrophils, monocytes and macrophages, in the context bands) in the absence of added VEGF-A and more in the presence of interacting with plasma C3a and C5a generated on targets of of VEGF-A (Fig. 6B,C, lower bands). The amounts of systemic complement activation for purposes of attracting and immunoprecipitated (IP’d) C3a and IP’d C5a (Fig. 6B,C, lower activating these effector cell types (Owen et al., 2013). While recent bands) were 100–300% greater in cells treated with VEGF-A. These studies of adaptive immune responses have connected C3ar1 and data were consistent with tonic assembly of the signaling complex C5ar1 signaling with T cell activation (Heeger et al., 2005; Lalli and amplification of assembly by VEGF-A. We next examined the et al., 2008; Liszewski et al., 2013; Liu et al., 2008; Strainic et al., kinetics of biotin-labeled C5a incorporation into the interactive 2008, 2013), and one study (Schraufstatter et al., 2002) found that complex by performing sequential IPs after the addition of each exogenously added C5a (presumed to correspond to serum C5a) Journal of Cell Science

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Fig. 6. See next page for legend. can impact EGFR signaling, no study has integrally linked C3ar1 activation of all interactive receptors. Second, in the absence of and C5ar1 signaling with VEGFR2 or other RTK signaling. contact inhibition of ECs (as is usually experimentally modeled in The data herein provide several unexpected insights. First, they confluent HUVEC plates), i.e. conditions of non-contact-inhibited show that VEGFR2 interacts in a heretofore-undescribed signaling EC growth connected with vascular repair; ECs tonically generate platform containing VEGFR2, C5ar1, C3ar1 and IL-6R–gp130. C3a/C5a. Autocrine C3ar1/C5ar1 signaling resulting from this

VEGFR2 growth and viability signaling depends on the joint endogenous C3a/C5a production participates in the mitotic and Journal of Cell Science

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Fig. 6. VEGFR2, gp130, C3ar1 and C5ar1 are co-associated. (A) Anti- delineated below that sustain mTORC1 activation and enable cell C3ar1, anti-C5ar1, anti-VEGFR2, anti-gp130 and anti-CD31 IPs were growth. By analogy to our previous results in T cells (Strainic et al., prepared from detergent extracts of serum-starved bEnd.3 cells and 2008), one of these is that the Gβɣ subunits of the C3ar1/C5ar1 G immunoblots of the IPd proteins were probed for C5ar1, C3ar1, VEGFR2, ɣ gp130 and CD31. (B–D) Following 3 h pre-incubation with cytochalasin D proteins activate PI-3K needed for the generation of (to prevent receptor internalization), serum-starved bEnd.3 cells were phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] upon 308 473 incubated for 30 min with VEGF-A. Biotin-labeled C5a or biotin-labeled-C3a which AKT is phosphorylated at T and S by PDK1 and by was then added for 5 min, after which the cells were extracted and anti-C5ar1, mTORC-2, respectively. Our findings that C3ar1/C5ar1 blockade anti-VEGFR2, anti-gp130 and anti-CD31 IPs prepared. Immunoblots of the prevented p-VEGFR2 phosphorylation indicate that while it is proteins IPd by the different antibodies were probed for biotin-C3a (B) or known that RTK-induced PI-3Kα activation participates in iotin-C5a (C) with streptavidin peroxidase (representative of two repeat experiments). (D) Serum-starved bEnd.3 cells were incubated with VEGF-A, PtdIns(3,4,5)P3 generation, as highlighted in the literature (Fan and after which biotin-labeled C5a was added for 5 min. The cells were extracted, Weiss, 2010; McKinnon et al., 2005), C3ar1/C5ar1 induced anti-C5ar1, anti-VEGFR2, anti-gp130 and anti-CD31 IPs prepared, and activation of PI-3Kγ (which generally has not been concomitantly immunoblots of the IPd proteins were probed for biotin-C5a with streptavidin assessed in studies of RTK activation) is also needed. An implication peroxidase. Densitometry of band intensity for each IP was graphed over time. of this concept is that both PI-3K isoforms function jointly to (E) HEK293 cells were transfected with VEGFR2-Luc donor plasmid and maintain sufficient levels of PtdIns(3,4,5)P3 for optimal AKT increasing (0-5 µg) amounts of acceptor C5ar1-GFP plasmid. A second set of phosphorylation. The second VEGFR2 growth mechanism derives cells was transfected with C5ar1-Luc donor plasmid and increasing (0-5 µg) amounts of acceptor VEGFR2-GFP or C5ar1-GFP plasmid. Immediately from the findings in this study that VEGFR induction of ERK after the addition of 5 μM luciferase substrate BRET signal was collected; activation requires both C3ar1/C5ar1 and IL-6R–gp130 signaling. no interaction between C5ar1 or VEGFR2 and a different GPCR, the This pathway involves activation of Src that, as indicated, is likewise protease-activated receptor 1 (PAR-1) was observed (not shown). Error bars dependent on C3ar1/C5ar1 signaling. While we did not study SHP-2 show s.d. (F) bEnd.3 cells were plated on slides and, following serum (also known as PTPN11), which also participates, SHP-2 activation starvation for 24 h, the cells were stained with differentially tagged antibodies is known to be dependent on IL-6R–gp130 signaling, which the against VEGFR2, C5ar1 and IL-6R, and examined by confocal microscopy (cells were visualized at 63×; 5× digital enlargements of region marked by experiments herein showed is linked with C3ar1/C5ar1 signaling. yellow arrow are below each image). Scale bar: 200 μm. (G) bEnd.3 cells were The findings that C3ar1/C5ar1 and VEGFR2 signaling jointly plated on slides and, following serum starvation for 24 h, the cells were fixed participate in AKT phosphorylation will provide a framework for and permeabilized, then stained for VEGFR2 and EEA1, or C5ar1 and EEA1, quantitative and kinetic analyses of the PI-3Kα and PI-3Kɣ co- and examined by confocal microscopy. Arrows indicate colocalization (cells signaling requirement. The third pathway is activation of PLCs, μ – were visualized at 63×). Scale bars: 200 m. (H) Diagram of the VEGFR2 IL- which past studies have alternatively attributed to activated Gαq/11 6R–C5ar1 signaling complex and integrated signaling cascade. (Zeng et al., 2003) or Gβɣ (Jiang et al., 1996) subunits deriving from C5a ligation of C5ar1. The coupling of C5ar1 to Gαq as well as to survival signaling that has been attributed to VEGFR2 signaling in Gαi might explain past findings by Zeng et al. (2003) that a Gαq and of itself. Independent mouse models of RM prostate cancer antisense oligonucleotide can completely inhibit VEGF-A-induced growth, of ocular angiogenesis, and of angiogenesis in newborn phosphorylation of VEGFR2. mouse day 5 retinas documented the involvement of C3ar1/C5ar1 As highlighted in the Results, it has been reported that IL-6R– signaling in angiogenesis in vivo. Third, the data show that, in gp130 signaling (Wegenka et al., 1994) or C5ar1 signaling (Strainic addition to tonic C3a/C5a generation, non-contact-inhibited ECs et al., 2013; Zhang et al., 2007) in and of themselves can activate constitutively produce IL-6 and VEGF-A. Autocrine signaling, AKT and ERK, but all studies related to this were performed in WT concomitantly through C3ar1/C5ar1, IL-6R–gp130 and VEGFR2, cells containing the full array of other components. The operates homeostatically in ECs. Growth factor occupation of interpretations were that each of the pathways are independent VEGFR2 amplifies these four signaling loops in a two-phase Tyk2- processes, not interdependent or interconnected, and not in any way and STAT3-dependent manner. The auto-inductive VEGFR2, functionally dependent on VEGFR2 signaling. The data in this C5ar1/C3ar1 and IL-6R–gp130 signaling resulting from the study demonstrated that, when C3ar1/C5ar1 signal transduction is ligation of increased amounts of each agonist gives rise to abolished, downstream VEGFR2 and IL-6R signaling is also augmented p-Tyk2-dependent STAT3 phosphorylation. This suppressed. Likewise, when IL-6R signaling is blocked, amplification is a requisite process for VEGFR2 growth signaling downstream C3ar1/C5ar1 as well as VEGFR2 signaling are as exemplified by the upregulated expression of all of the receptors blocked. Furthermore, the VEGFR2 signaling inhibitor SU5416 and the angiogenic response of ECs to VEGF-A. Our experiments blocked VEGF-A induction of C5a and IL-6 production and verify that this VEGFR2 co-signaling is an obligate process in non- consequent C5ar1 and IL-6R–gp130-induced activation of AKT contact-inhibited HUVECs, HAECs and HeLa cells, as in mouse and ERK. It also blocked downstream signaling events when cells cells. Among the implications of this requirement is that in intact were stimulated with either C5a or IL-6. Based on the state of cells (as opposed to the purified state), C3ar1/C5ar1 signaling is knowledge at the time, others reported that Src can be activated by needed to enable VEGFR2 phosphorylation as well as to enable EC IL-6R–gp130 signaling (Wang et al., 2013) in intact cells without cell cycle progression. Lastly, the experiments show that the four appreciating the requirement of the other components, while the receptor systems function in an interactive system that is localized experiments herein show that disruption of C3ar1/C5ar1 or IL-6– predominantly intracellularly. The precise site at which the gp130 signaling abolishes VEGFR2-induced Src activation. complexes form and how they interact will require further study. The same relationship pertains for activation of STAT3. The data C3ar1/C5ar1, IL-6R–gp130 may interact with VEGFR2 directly or herein show that C3ar1/C5ar1 signaling is involved in VEGFR2 and indirectly via an adaptor protein. More studies will be needed to IL-6R–gp130 activation of STAT3. In this vein, it will be of interest characterize the association and determine how association impacts to examine whether other reported signaling interactions between the joint autocrine signaling of each signaling partner. GPCRs and VEGFR2 or other RTKs involve C3ar1/C5ar1 or The linkage of C3ar1/C5ar1 signaling with cell cycle progression IL-6R–gp130, or are transmitted within some other signaling reflects the fact that this signaling is needed for three pathways interconnection. Relevant to the connection of C3ar1/C5ar1 and Journal of Cell Science

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IL-6R–gp130 signaling with STAT3 activation, STAT3 sites are It is important to emphasize that VEGFR2 signaling is complex. present in the promoters of the C3/C5/C3ar1/C5ar1 and Il-6 genes. The interaction of many other proteins, including heparin, Preliminary studies indicate that 473p-STAT3 is needed for neuropilins, semaphorins and integrins, have been described transcription of the complement genes (K. Johnson and M.E.M., (Neufeld et al., 2002; Neufeld et al., 2007; Uniewicz and Fernig, unpublished). More work will be needed, however, to precisely 2008). VEGFR2 interaction with other receptors (Greenberg et al., characterize the interaction between p-STAT3 and the promoters of 2008; Lu et al., 2012), e.g. the ability of VEGFR2 to confer a these genes. negative growth signal (Hatzi et al., 2002), has also been Given the findings herein that disruption of C3ar1/C5ar1 described. It will be informative to study the mechanistic signaling in WT ECs induces EC apoptosis as well as abolishes connection of the findings herein with these previously VEGFR2 signaling, why then is genetic disruption of both GPCRs described processes. not lethal? The experiments on VEGFR2 signaling in C3ar1−/− Finally, in addition to physiological significance, the findings C5ar1−/− ECs shed light on this issue. The experiments herein potentially could have important clinical implications. The found that C3ar1–/–C5ar1–/– ECs possess ∼160% upregulated connection of C5a and IL-6 with VEGFR2 signaling would expression of VEGFR2, tonically produce ∼600% more VEGF-A explain an EC intrinsic link between inflammation and and show 160% upregulated expression of CD61. In addition, they angiogenesis. While the joint activation process delineated herein showed that C3ar1–/–C5ar1–/– ECs possess ∼400% higher IL-7R, opens the possibility that targeting C3ar1/C5ar1 plus IL-6R or show ∼800% upregulated expression of IL-6R and tonically gp130 could help repress cancer-associated angiogenesis and/or produce ∼100% more IL-6, the latter two alterations, like those of inflammation, drugs jointly targeted to more extensive, as yet VEGFR2 and CD61, both associated with viability and growth. unidentified, components in this newly described signaling Such compensatory upregulation of VEGFR2 has been evoked as complex could provide therapeutic possibilities not yet envisioned. the explanation for why angiogenesis is not suppressed in mice deficient in the β3 integrin subunit (Reynolds et al., 2002), even MATERIALS AND METHODS though inhibitors of αVβ3 suppress angiogenesis in vivo (Brooks Reagents and antibodies et al., 1995; Friedlander et al., 1995; West et al., 2012). While we VEGF-A was purchased from ProSpec Bio (Ness Ziona, Israel) or Miltenyi –/– –/– did not study compensations in Il-6 or gp130 mice, others Biotec (San Diego, CA). C3ar1 antagonist (C3ar1-A) and C5ar1 antagonist (Durlacher-Betzer et al., 2018; Kuhn et al., 2014) have. With respect (C5ar1-A) are from Calbiochem (EMD Millipore, Billerica, MA). Anti-C3a to its requirement for cell viability and growth, most tellingly, one and anti-C5a mAbs were purchased from BD Biosciences (San Diego, CA). group studying astrocytes (Quintana et al., 2013; Quintana et al., Anti-C3ar1 and anti-C5ar1 were purchased from Santa Cruz Biotechnology 2008) found that while global IL-6R knockout (which leads to (Santa Cruz, CA). Endothelial cell growth supplement was purchased compensations) had a small effect, conditional knockout in from BD Biosciences. Anti-phospho-VEGFR2 (p-Y1054/p-Y1059) was astrocytes led to their apoptosis. In support of the involvement of purchased from Invitrogen (Camarillo, CA). Anti-p-STAT3 and anti-p-Tyk2 IL-6R in cell growth, other papers have linked IL-6 to intestinal were purchased from Cell Signaling Technology (Danvers, MA). Mouse C3a was purchased from BD Biosciences. Mouse C5a was purchased from epithelial cell and fibroblast growth, VEGF-A secretion from Hycult Biotech (Uden, The Netherlands). Mouse C3a and C5a were prostate cancer cells (Ishii et al., 2018), bone metastases in breast biotinylated using EZ-Link NHS-Biotin kits from Thermo Fisher Scientific cancer (Wakabayashi et al., 2018) and hepatocyte proliferation. (Rockford, IL). AlexaFluor-488, -546, -555 and -647 labeling kits were Additionally, IL-6R has been linked to increased gp130 expression purchased from Life Technologies (Camarillo, CA). For confocal and upregulation of leukemia inhibitory factor (LIF), an alternative microscopy, anti-C5ar1 and anti-C3ar1 were labeled with AlexaFluor- gp-130 signaling system able to compensate for IL-6R deficiency 546, anti-VEGFR2 was labeled with AlexaFluor-488, and anti-IL-6R as (Ezure et al., 2000; Liu et al., 2000). well as anti-gp130 were labeled with AlexaFluor-647. SU5416 was While it is natural to wonder why the findings in this paper have purchased from Tocris (Bristol, UK). not been described until now, most studies that have focused on characterizing VEGFR2 signaling have been performed under Isolation of primary aortic ECs, smooth muscle cells and primary conditions of contact inhibition (in confluent plates). Other studies mouse embryonic fibroblasts ’ have been performed in transformed cell lines that overexpress bEnd.3 cells and MS-1 murine EC lines were cultured in Dulbecco s modified Eagle medium (DMEM) with 10% and 5% fetal bovine serum VEGFR2, or carried out with the use of high concentrations of (FBS), respectively. Primary mouse aortic ECs were isolated from WT exogenously added growth factor to facilitate identification of Daf1–/–, C3ar1–/–C5ar1–/– C57BL/6 mice at ages from 1 to 4 months, by VEGFR2 phosphorylation sites and downstream signaling utilizing a non-mechanical and non-enzymatic method as described intermediates. They thus have been performed under conditions previously (Mahabeleshwar et al., 2006). Outgrowth of ECs from aortic that likely do not require C3ar1/C5ar1 signaling to the same extent, rings occurred over 72 h in the absence of antibiotics. After removing aortic or involve super-physiological VEGFR2 or other RTK signaling rings, the ECs were maintained in DMEM/F12 medium containing 20% such that they overcome C3ar1/C5ar1 and IL-6R–gp130 regulation. FBS, 2 mM L-glutamine, 1% nonessential amino acid, 0.09 mg/ml EC Relevant to the participation of C3ar1/C5ar1 and IL-6R–gp130 in growth supplement, 1% antibiotic/antimycotic, 100 units/ml penicillin, physiological VEGFR2 signaling, it is noteworthy that the results in 100 g/ml streptomycin, 5 ng/ml VEGF-A and 0.09 mg/ml heparin until this study explain puzzling reports of C5a and/or IL-6 linkages with confluent. The medium was changed the following day and the cells were passaged several times. VEGFR2 that have been ascribed to serum C5a and IL-6, respectively (Derer et al., 2012; Karacsonyi et al., 2012; Kato Gene expression and flow cytometry et al., 2012; Melnick et al., 2013; Rada et al., 2011; Schraufstatter RNA was isolated by the TRIzol method (Invitrogen), and reverse et al., 2002; Shure et al., 1992; Song et al., 2011; Suzuki et al., 2011; transcription and qPCR were performed as described (Strainic et al., Wang et al., 2011; Yao et al., 2010). In no cases in these associations 2008, 2013). have molecular mechanisms been delineated, or the involvement of After culturing in 10% FBS-supplemented DMEM, harvested cells were C3ar1/C5ar1 and IL-6R–gp130 in an associated signaling platform stained with goat anti-C3ar1 or anti-C5ar1 mAbs followed by biotin-labeled been proposed. anti-goat and finally with streptavidin-APC. Intracellular staining Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs219352. doi:10.1242/jcs.219352 was performed in formalin-permeabilized cells with subtraction of (BioLegend)+Streptavidin-Tx Red (Life Sciences) and anti-IL-6R–biotin extracellular values. (BioLegend) for 1 h. The slides were washed 3× with 1× PBS and fixed with 2% formaldehyde. Images were collected using a 63× (1.4 NA) oil Luminex (Multiplex) assay immersion objective on an UltraVIEW Vox (Perkin Elmer, Waltham, MA) ECs were stimulated for increasing times with different agonists as indicated spinning disk confocal system attached to a Leica DMI 6000B inverted in the figure legends. After stimulation, cells were assayed for phospho (p)- microscope. Resulting images were subjected to deconvolution using Src, total (t)-Src, p-AKT, t-AKT, p-ERK and t-ERK using Millipore’s Autoquant software embedded in Metamorph imaging software multiplex assay, according to the manufacturer’s instructions. Briefly, cells (Downington, PA). were placed on ice immediately following incubation, centrifuged at 4°C, lysed in the buffer provided by the company, incubated with the capture Co-IP assays beads followed by the detection beads, washed, then assayed on the Bioplex Cells were lysed in 1× Cell Lysis Buffer (10×) (Cell Signaling Technology, 2200 (Bio-Rad, Hercules, CA). #9803) supplemented with 1 mM PMSF and one Complete Mini protease inhibitor tablet (Roche, #11836153001) for 10 min on ice (1 mM Na PLC activity assay orthovanadate was used to inhibit phosphatase activity when lysates were to Lysates from activated cells were assayed for PLC activity by an EnzCheck be assayed for kinase activity). Lysates were then sonicated 3× for 2 min to Direct Phospholipase C Assay kit (Molecular Probes, Eugene, OR), as per break apart nucleic acids, after which the cells were centrifuged for 10 min at the manufacturer’s instructions. Briefly, lysates are incubated with a 12,000 g. Clean supernatants were transferred to new tubes and incubated proprietary substrate, which is cleaved by the PLC to release a dye-labeled with Protein-A/G beads to preclear the lysates and prevent nonspecific diacylglycerol. The reaction production has excitation and emission maxima co-IP. Antibodies against VEGFR2, C5ar1, C3ar1, IL-6R, gp130 or CD31 of 509 nm and 516 nm, respectively, and were measured using a were added, and samples were incubated overnight at 4°C. Protein-A/G SpectraMax M2 microplate reader. beads (Santa Cruz Biotechnology) were used to pull down antibodies and immunoprecipitates were assayed by western blotting, ELISA or protein Quantitation of cell growth activity kits, as described. For studies with bEnd.3 and MS-1 cells, between 5×104 and 50×104 cells were seeded in 6-, 12- or 24-well plates (see individual figure legends for BRET details) and allowed to adhere for 24 h. Following culturing in 0.5% FBS BRET analyses were performed as described (de la Fuente et al., 2012). with DMEM for another 24 h, the cells were treated as described in the Briefly, HEK293 cells (1×105) were transfected with 0.5 μg VEGFR2-Luc figure legends. Growth was quantified manually with Trypan Blue donor plasmid and increasing amounts (0–5 μg) of C5ar1-GFP acceptor exclusion. At least 95% of cells were viable in all experiments. Growth plasmid or C5ar1-Luc donor plasmid and increasing amounts (0–5 μg) of experiments used 30 ng/ml VEGF-A, 10 ng/ml C3ar1-A and C5ar1-A, and C5ar1-GFP or VEGFR2-GFP acceptor plasmids. Emission was detected 1 μg/ml anti-C3a and anti-C5a mAbs. using a Victor 3 plate reader equipped with the appropriate BRET2 filter set (410 nm with 80-nm bandpass and 515 nm with 30-nm bandpass; C3a/C5a/VEGF-A ELISAs and propidium iodide (PI) staining PerkinElmer Life Sciences). Emission at 410 and 515 nm was collected ELISAs were done as previously described (Strainic et al., 2008, 2013). PI immediately after the addition of 5 μm luciferase substrate (coelenterazine staining was performed as per the manufacturer’s instructions using 400a; Biotium Inc., Hayward, CA). BRET signal was calculated by the ratio an Annexin V PI staining kit (BD Biosciences). of emission at 515 nm to emission at 410 nm minus the BRET in the absence of GFP. In BRET studies, specific interactions are detected by a HUVEC tube formation hyperbolic increase in BRET signal (ratio of emission at 510/410 nm) as the Fifty microliters of the growth factor-reduced Matrigel (BD Biosciences) ratio of GFP receptor/RLuc receptor increases, whereas non-specific was allowed to polymerize in a 96-well plate at 37°C for 30 min. Triplicates interactions increase linearly. Data were analyzed with GraphPad Prism 6 of 25,000 HUVECs were plated onto the prepared Matrigel in a volume using best model (hyperbolic versus linear) for each experiment. GFP of 150 µl EBM-2 medium (Lonza) under the conditions described in the expression was determined by excitation at 495 nm and emission at 515 nm. legend of Fig. 3A. After 15 h, images were captured using a light Luciferase expression was determined by adding 5 μm coelenterazine H microscope at high magnification. (Invitrogen) and reading total light emission without a filter. Each BRET experiment was performed in three independent experiments. Tumor angiogenesis and corneal neovascularization models Male mice were injected subcutaneously with 1.5×106 RM1 prostate cancer Newborn retinal angiogenesis model cells. Tumors were collected 10 days after injection and were weighed, Analyses of 5-day-old murine retinal angiogenesis were performed as photographed, snap-frozen in OCT and processed for immunohistochemical described (Pitulescu et al., 2010). Briefly, eye globes from 5-day-old mice staining with biotin-conjugated rat anti-mouse CD31 antibody (BD were harvested and fixed for 1 h at room temperature with 4% Biosciences, San Jose, CA). Stained sections were analyzed using paraformaldehyde in 1× PBS. After fixation, globes were washed with 1× fluorescent or bright-field imaging microscopy (Leica, Germany) and PBS and retinas isolated via dissection. Retinas were blocked with a solution ImagePro Plus Capture and Analysis software version 6.1 (Media of 1% BSA in 0.3% Triton X-100 and 1× PBS overnight at 4°C. Retinas were Cybernetics). CD31-positive areas were quantified in 5–10 independent equilibrated with 1 mM MgCl2, 1 mM CaCl2, 0.1 mM MnCl2,1%(vol/vol) fields per tumor implant. Corneal neovascularization was induced in 6- to Triton X-100 and 1× PBS three times for 20 min each. After equilibration, 8-week-old (C57BL/6) WT, Daf1–/–, C3ar1–/–C5ar1–/– and Daf1–/– C3ar1–/– retinal vasculature was stained with Tomato-Red-labeled isolectin B4 and C5ar1–/– mice (n=5 each group) by placing a non-penetrating suture (0-11 retinas were visualized by fluorescent microscopy at 40×. Resulting images Nylon, Alcon Inc.) in the center of the cornea under a dissecting microscope. were subjected to deconvolution using Autoquant software embedded in On days 7, 9, 11 and 15, corneal vessels were examined after intravenous Metamorph imaging software (Downington, PA). Distances from optic nerve administration of 100 μl of 2.5% fluorescein-dextran (Sigma-Aldrich) by to angiogenic front were measured in Metamorph. fluorescence microscopy. Both protocols were approved by the Institutional Animal Care Centers (IACUC). Statistics Power calculations and animal numbers were determined using the Confocal microscopy information provided by ‘http://statpages.org’ and the therein-linked Russ bEnd.3 cells were grown overnight on ibiTreat eight-chamber μ-Slides Lenth’s power and sample-size calculator obtainable through ‘https:// (ibidi, Martinsried, Germany). Cells were treated with cytochalasin D homepage.divms.uiowa.edu/~rlenth/Power/index.html’. To achieve a true for 3 h after which they were stained with a cocktail containing 2 μg/ml difference between a mean of 0.5 and a power of 0.2, testing the difference of each of anti-C5ar1–AF647 (Abd Serotec), anti-VEGFR2-AF488 between two means via an unpaired, two-tailed Student’s t-test, 10 mice are Journal of Cell Science

13 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs219352. doi:10.1242/jcs.219352 required in each group and there are 10 experimental groups. Statistical surface expression levels on effector mechanisms of EGFR antibodies. significance for all experimental data was determined by Student’s t-test J. Immunol. 189, 5230-5239. (unpaired, two-tailed) performed in Microsoft Excel, SigmaPlot or Dias, S., Shmelkov, S. V., Lam, G. and Rafii, S. (2002). VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and GraphPad Prism 6 with a significance threshold value of P<0.05. Except apoptosis inhibition. Blood 99, 2532-2540. where indicated, all experiments were repeated at least three times. Data are Durlacher-Betzer, K., Hassan, A., Levi, R., Axelrod, J., Silver, J. and Naveh- presented as mean values with s.d. Many, T. (2018). Interleukin-6 contributes to the increase in fibroblast growth factor 23 expression in acute and chronic kidney disease. Kidney Int. 94, 315-325. Acknowledgements Ezure, T., Sakamoto, T., Tsuji, H., Lunz, J. G., III, Murase, N., Fung, J. J. and The authors thank Andrew Esposito and Scott Howell for corneal studies, imaging Demetris, A. J. (2000). The development and compensation of biliary cirrhosis in assistance and confocal analyses, Fengqi An for generation of the knockouts, interleukin-6-deficient mice. Am. J. Pathol. 156, 1627-1639. Sathyamangla v Naga Prasad for discussion and Dawn Smith for help with cells and Facciabene, A., De Sanctis, F., Pierini, S., Reis, E. S., Balint, K., Facciponte, J., culturing. Rueter, J., Kagabu, M., Magotti, P., Lanitis, E. et al. (2017). Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, Competing interests and tumor control during t-cell immunotherapy. Oncoimmunology 6, e1326442. The authors declare no competing or financial interests. Fan, Q. W. and Weiss, W. A. (2010). Targeting the RTK-PI3K-mTOR axis in malignant glioma: overcoming resistance. Curr. Top. Microbiol. Immunol. 347, 279-296. Author contributions Ferrara, N., Gerber, H. P. and LeCouter, J. (2003). The biology of VEGF and its Conceptualization: M.E.M.; Methodology: M.-S.H., M.G.S., E. Pohlmann, receptors. Nat. Med. 9, 669-676. E. Pluskota, D.L.R.-B., E.F.P., M.E.M.; Formal analysis: M.G.S., D.L.R.-B., E.F.P., Friedlander, M., Brooks, P. C., Shaffer, R. W., Kincaid, C. M., Varner, J. A. and M.E.M.; Investigation: M.-S.H., M.G.S., E. Pohlmann, H.K., E. Pluskota, M.E.M.; Cheresh, D. A. (1995). Definition of two angiogenic pathways by distinct alpha v Resources: D.L.R.-B., E.F.P., M.E.M.; Data curation: M.G.S., M.E.M.; Writing - integrins. Science 270, 1500-1502. original draft: M.E.M.; Writing - review & editing: M.G.S., D.L.R.-B., E.F.P., Fukuoka, Y. and Medof, E. M. (2001). C5a receptor-mediated production of IL-8 by M.E.M.; Visualization: M.G.S.; Supervision: M.E.M., M.G.S.; Project administration: the human retinal pigment epithelial cell line, ARPE-19. Curr. Eye Res. 23, 320-325. M.E.M., M.G.S.; Funding acquisition: M.E.M. Fukuoka, Y., Strainic, M. and Medof, M. E. (2003). Differential cytokine expression of human retinal pigment epithelial cells in response to stimulation by C5a. Clin. Funding Exp. Immunol. 131, 248-253. This work was supported by the National Institutes of Health [EY10889 (M.E.M.) Gerhartz, C., Dittrich, E., Stoyan, T., Rose-John, S., Yasukawa, K., Heinrich, HL109561 (M.E.M.), AR067182 (M.E.M.), HL096597 (D.L.R.-B.), HL073311 P. C. and Graeve, L. (1994). Biosynthesis and half-life of the interleukin-6 (E.F.P.) HL096062 (E.F.P.)], National Multiple Sclerosis Society [RG3664-A-1 receptor and its signal transducer gp130. Eur. J. Biochem. 223, 265-274. (M.E.M.)] and the American Heart Association [14POST18580011 (M.G.S.)]. Greenberg, J. I., Shields, D. J., Barillas, S. G., Acevedo, L. M., Murphy, E., Deposited in PMC for release after 12 months. Huang, J., Scheppke, L., Stockmann, C., Johnson, R. S., Angle, N. et al. (2008). A role for VEGF as a negative regulator of pericyte function and vessel Supplementary information maturation. Nature 456, 809-813. Hatzi, E., Murphy, C., Zoephel, A., Rasmussen, H., Morbidelli, L., Ahorn, H., Supplementary information available online at Kunisada, K., Tontsch, U., Klenk, M., Yamauchi-Takihara, K. et al. (2002). N- http://jcs.biologists.org/lookup/doi/10.1242/jcs.219352.supplemental myc oncogene overexpression down-regulates IL-6; evidence that IL-6 inhibits angiogenesis and suppresses neuroblastoma tumor growth. Oncogene 21, References 3552-3561. Ahmed, C. M., Noon-Song, E. N., Kemppainen, K., Pascalli, M. P. and Johnson, Heeger, P. S., Lalli, P. N., Lin, F., Valujskikh, A., Liu, J., Muqim, N., Xu, Y. and H. M. (2013). Type I IFN receptor controls activated TYK2 in the nucleus: Medof, M. E. (2005). Decay-accelerating factor modulates induction of T cell implications for EAE therapy. J. Neuroimmunol. 254, 101-109. immunity. J. Exp. Med. 201, 1523-1530. Alawieh, A., Elvington, A., Zhu, H., Yu, J., Kindy, M. S., Atkinson, C. and Huang, X., Raskovalova, T., Lokshin, A., Krasinskas, A., Devlin, J., Watkins, S., Tomlinson, S. (2015). Modulation of post-stroke degenerative and regenerative Wolf, S. F. and Gorelik, E. (2005). Combined antiangiogenic and immune therapy processes and subacute protection by site-targeted inhibition of the alternative of prostate cancer. Angiogenesis 8, 13-23. pathway of complement. J. Neuroinflammation 12, 247. Huang, Y.-H., Yang, H.-Y., Hsu, Y.-F., Chiu, P.-T., Ou, G. and Hsu, M.-J. (2014). Albrecht, E. A., Chinnaiyan, A. M., Varambally, S., Kumar-Sinha, C., Barrette, Src contributes to IL6-induced vascular endothelial growth factor-C expression in T. R., Sarma, J. V. and Ward, P. A. (2004). C5a-induced gene expression in lymphatic endothelial cells. Angiogenesis 17, 407-418. human umbilical vein endothelial cells. Am. J. Pathol. 164, 849-859. Ishii, K., Sasaki, T., Iguchi, K., Kajiwara, S., Kato, M., Kanda, H., Hirokawa, Y., Amatruda, T. T., III, Gerard, N. P., Gerard, C. and Simon, M. I. (1993). Specific Arima, K., Mizokami, A. and Sugimura, Y. (2018). Interleukin-6 induces VEGF interactions of chemoattractant factor receptors with G-proteins. J. Biol. Chem. secretion from prostate cancer cells in a manner independent of androgen 268, 10139-10144. receptor activation. Prostate 78, 849-856. Arbore, G., West, E. E., Spolski, R., Robertson, A. A., Klos, A., Rheinheimer, C., Jiang, H., Kuang, Y., Wu, Y., Smrcka, A., Simon, M. I. and Wu, D. (1996). Dutow, P., Woodruff, T. M., Yu, Z. X., O’Neill, L. A. et al. (2016). T helper 1 Pertussis toxin-sensitive activation of phospholipase C by the C5a and fMet-Leu- immunity requires complement-driven NLRP3 inflammasome activity in CD4(+) T cells. Science 352, aad1210. Phe receptors. J. Biol. Chem. 271, 13430-13434. Brooks, P. C., Montgomery, A. M., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G. Karacsonyi, C., Lee, J. H., Shanmugam, N. and Kagan, E. (2012). Epidermal and Cheresh, D. A. (1994). Integrin alpha v beta 3 antagonists promote tumor growth factor receptor signaling mediates vesicant-induced airway epithelial regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157-1164. secretion of interleukin-6 and production of mucin. Am. J. Respir. Cell Mol. Biol. Brooks, P. C., Stromblad, S., Klemke, R., Visscher, D., Sarkar, F. H. and 46, 157-164. Cheresh, D. A. (1995). Antiintegrin alpha v beta 3 blocks human breast cancer Kato, K., Otsuka, T., Kondo, A., Matsushima-Nishiwaki, R., Natsume, H., growth and angiogenesis in human skin. J. Clin. Invest. 96, 1815-1822. Kozawa, O. and Tokuda, H. (2012). AMP-activated protein kinase regulates Buhl, A. M., Eisfelder, B. J., Worthen, G. S., Johnson, G. L. and Russell, M. PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts: involvement of (1993). Selective coupling of the human anaphylatoxin C5a receptor and alpha 16 mitogen-activated protein kinases. Life Sci. 90, 71-76. in human kidney 293 cells. FEBS Lett. 323, 132-134. Kuhn, K. A., Manieri, N. A., Liu, T. C. and Stappenbeck, T. S. (2014). IL-6 Byzova, T. V., Goldman, C. K., Pampori, N., Thomas, K. A., Bett, A., Shattil, S. J. stimulates intestinal epithelial proliferation and repair after injury. PLoS ONE 9, and Plow, E. F. (2000). A mechanism for modulation of cellular responses to e114195. VEGF: activation of the integrins. Mol. Cell 6, 851-860. Lalli, P. N., Strainic, M. G., Yang, M., Lin, F., Medof, M. E. and Heeger, P. S. Cantarella, G., Lempereur, L., Presta, M., Ribatti, D., Lombardo, G., Lazarovici, P., (2008). Locally produced C5a binds to T cell-expressed C5aR to enhance effector Zappala, G., Pafumi, C. and Bernardini, R. (2002). Nerve growth factor-endothelial T-cell expansion by limiting antigen-induced apoptosis. Blood 112, 1759-1766. cell interaction leads to angiogenesis in vitro and in vivo. FASEB J. 16, 1307-1309. Laudes, I. J., Chu, J. C., Huber-Lang, M., Guo, R. F., Riedemann, N. C., Sarma, Carrette, F. and Surh, C. D. (2012). IL-7 signaling and CD127 receptor regulation in J. V., Mahdi, F., Murphy, H. S., Speyer, C., Lu, K. T. et al. (2002). Expression and the control of T cell homeostasis. Semin. Immunol. 24, 209-217. function of C5a receptor in mouse microvascular endothelial cells. J. Immunol. de la Fuente, M., Noble, D. N., Verma, S. and Nieman, M. T. (2012). Mapping 169, 5962-5970. human protease-activated receptor 4 (PAR4) homodimer interface to Lechner, J., Chen, M., Hogg, R. E., Toth, L., Silvestri, G., Chakravarthy, U. and transmembrane helix 4. J. Biol. Chem. 287, 10414-10423. Xu, H. (2016). Higher plasma levels of complement C3a, C4a and C5a increase Derer, S., Bauer, P., Lohse, S., Scheel, A. H., Berger, S., Kellner, C., Peipp, M. the risk of subretinal fibrosis in neovascular age-related macular degeneration:

and Valerius, T. (2012). Impact of epidermal growth factor receptor (EGFR) cell Complement activation in AMD. Immun. Ageing 13,4. Journal of Cell Science

14 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs219352. doi:10.1242/jcs.219352

Lee, C. H., Katz, A. and Simon, M. I. (1995). Multiple regions of G alpha 16 (2010). Relationship between IL-6/ERK and NF-kappaB: a study in normal and contribute to the specificity of activation by the C5a receptor. Mol. Pharmacol. 47, pathological human prostate gland. Eur. Cytokine Netw. 21, 241-250. 218-223. Sakuma, M., Morooka, T., Wang, Y., Shi, C., Croce, K., Gao, H., Strainic, M., Leu, C. M., Wong, F. H., Chang, C., Huang, S. F. and Hu, C. P. (2003). Interleukin-6 Medof, M. E. and Simon, D. I. (2010). The intrinsic complement regulator decay- acts as an antiapoptotic factor in human esophageal carcinoma cells through the accelerating factor modulates the biological response to vascular injury. activation of both STAT3 and mitogen-activated protein kinase pathways. Arterioscler. Thromb. Vasc. Biol. 30, 1196-1202. Oncogene 22, 7809-7818. Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F. and Lillegard, K. E., Loeks-Johnson, A. C., Opacich, J. W., Peterson, J. M., Bauer, Giachelli, C. M. (1998). NF-kappaB mediates alphavbeta3 integrin-induced A. J., Elmquist, B. J., Regal, R. R., Gilbert, J. S. and Regal, J. F. (2014). endothelial cell survival. J. Cell Biol. 141, 1083-1093. Differential effects of complement activation products c3a and c5a on Schraufstatter, I. U., Trieu, K., Sikora, L., Sriramarao, P. and DiScipio, R. (2002). cardiovascular function in hypertensive pregnant rats. J. Pharmacol. Exp. Ther. Complement c3a and c5a induce different signal transduction cascades in 351, 344-351. endothelial cells. J. Immunol. 169, 2102-2110. Liszewski, M. K., Kolev, M., Le Friec, G., Leung, M., Bertram, P. G., Fara, A. F., Shure, D., Senior, R. M., Griffin, G. L. and Deuel, T. F. (1992). PDGF AA Subias, M., Pickering, M. C., Drouet, C., Meri, S. et al. (2013). Intracellular homodimers are potent chemoattractants for fibroblasts and neutrophils, and for complement activation sustains T cell homeostasis and mediates effector monocytes activated by lymphocytes or cytokines. Biochem. Biophys. Res. differentiation. Immunity 39, 1143-1157. Commun. 186, 1510-1514. Liu, Z., Sakamoto, T., Yokomuro, S., Ezure, T., Subbotin, V., Murase, N., Song, L., Rawal, B., Nemeth, J. A. and Haura, E. B. (2011). JAK1 activates STAT3 Contrucci, S. and Demetris, A. J. (2000). Acute obstructive cholangiopathy in activity in non-small-cell lung cancer cells and IL-6 neutralizing antibodies can interleukin-6 deficient mice: compensation by leukemia inhibitory factor (LIF) suppress JAK1-STAT3 signaling. Mol. Cancer Ther. 10, 481-494. suggests importance of gp-130 signaling in the ductular reaction. Liver 20, 114-124. Spets, H., Stromberg, T., Georgii-Hemming, P., Siljason, J., Nilsson, K. and Liu, J., Lin, F., Strainic, M. G., An, F., Miller, R. H., Altuntas, C. Z., Heeger, P. S., Jernberg-Wiklund, H. (2002). Expression of the bcl-2 family of pro- and anti- Tuohy, V. K. and Medof, M. E. (2008). IFN-gamma and IL-17 production in apoptotic genes in multiple myeloma and normal plasma cells: regulation during experimental autoimmune encephalomyelitis depends on local APC-T cell interleukin-6(IL-6)-induced growth and survival. Eur. J. Haematol. 69, 76-89. complement production. J. Immunol. 180, 5882-5889. Strainic, M. G., Liu, J., Huang, D., An, F., Lalli, P. N., Muqim, N., Shapiro, V. S., Lu, K. V., Chang, J. P., Parachoniak, C. A., Pandika, M. M., Aghi, M. K., Dubyak, G. R., Heeger, P. S. and Medof, M. E. (2008). Locally produced Meyronet, D., Isachenko, N., Fouse, S. D., Phillips, J. J., Cheresh, D. A. et al. complement fragments C5a and C3a provide both costimulatory and survival (2012). VEGF inhibits tumor cell invasion and mesenchymal transition through a signals to naive CD4+ T cells. Immunity 28, 425-435. MET/VEGFR2 complex. Cancer Cell 22, 21-35. Strainic, M. G., Shevach, E. M., An, F., Lin, F. and Medof, M. E. (2013). Absence of Mahabeleshwar, G. H., Somanath, P. R. and Byzova, T. V. (2006). Methods for signaling into CD4(+) cells via C3aR and C5aR enables autoinductive TGF-beta1 isolation of endothelial and smooth muscle cells and in vitro proliferation assays. signaling and induction of Foxp3(+) regulatory T cells. Nat. Immunol. 14, 162-171. Methods Mol. Med. 129, 197-208. Suzuki, Y., Nakazawa, M., Suzuki, K., Yamazaki, H. and Miyagawa, Y. (2011). McKinnon, R. D., Waldron, S. and Kiel, M. E. (2005). PDGF alpha-receptor signal Expression profiles of cytokines and chemokines in vitreous fluid in diabetic ’ strength controls an RTK rheostat that integrates phosphoinositol 3 -kinase and retinopathy and central retinal vein occlusion. Jpn. J. Ophthalmol. 55, 256-263. phospholipase Cgamma pathways during oligodendrocyte maturation. Thang, P. H., Ruffin, N., Brodin, D., Rethi, B., Cam, P. D., Hien, N. T., Lopalco, L., J. Neurosci. 25, 3499-3508. Vivar, N. and Chiodi, F. (2010). The role of IL-1beta in reduced IL-7 production by Medof, M. E., Kinoshita, T. and Nussenzweig, V. (1984). Inhibition of complement stromal and epithelial cells: a model for impaired T-cell numbers in the gut during activation on the surface of cells after incorporation of decay-accelerating factor HIV-1 infection. J. Intern. Med. 268, 181-193. (DAF) into their membranes. J. Exp. Med. 160, 1558-1578. Uniewicz, K. A. and Fernig, D. G. (2008). Neuropilins: a versatile partner of Melnick, M., Sedghizadeh, P. P., Deluca, K. A. and Jaskoll, T. (2013). extracellular molecules that regulate development and disease. Front. Biosci. 13, Cytomegalovirus-induced salivary gland pathology: resistance to kinase 4339-4360. inhibitors of the upregulated host cell EGFR/ERK pathway is associated with van der Wijk, A. E., Vogels, I. M. C., van Noorden, C. J. F., Klaassen, I. and CMV-dependent stromal overexpression of IL-6 and fibronectin. Herpesviridae 4,1. Schlingemann, R. O. (2017). TNFalpha-Induced Disruption of the Blood-Retinal Millauer, B., Wizigmann-Voos, S., Schnurch, H., Martinez, R., Moller, N. P., Barrier In Vitro Is Regulated by Intracellular 3′,5′-Cyclic Adenosine Risau, W. and Ullrich, A. (1993). High affinity VEGF binding and developmental Monophosphate Levels. Invest. Ophthalmol. Vis. Sci. 58, 3496-3505. expression suggest Flk-1 as a major regulator of vasculogenesis and Voronov, E., Carmi, Y. and Apte, R. N. (2014). The role IL-1 in tumor-mediated angiogenesis. Cell 72, 835-846. angiogenesis. Front. Physiol. 5, 114. Mohr, T., Haudek-Prinz, V., Slany, A., Grillari, J., Micksche, M. and Gerner, C. Wakabayashi, H., Hamaguchi, T., Nagao, N., Kato, S., Iino, T., Nakamura, T. and (2017). Proteome profiling in IL-1beta and VEGF-activated human umbilical vein Sudo, A. (2018). Interleukin-6 receptor inhibitor suppresses bone metastases in a endothelial cells delineates the interlink between inflammation and angiogenesis. PLoS ONE 12, e0179065. breast cancer cell line. Breast Cancer 25, 566-574. Neufeld, G., Kessler, O. and Herzog, Y. (2002). The interaction of Neuropilin-1 and Wang, H. C., Yeh, H. H., Huang, W. L., Lin, C. C., Su, W. P., Chen, H. H., Lai, W. W. Neuropilin-2 with tyrosine-kinase receptors for VEGF. Adv. Exp. Med. Biol. 515, and Su, W. C. (2011). Activation of the signal transducer and activator of 81-90. transcription 3 pathway up-regulates estrogen receptor-beta expression in lung Neufeld, G., Lange, T., Varshavsky, A. and Kessler, O. (2007). Semaphorin adenocarcinoma cells. Mol. Endocrinol. 25, 1145-1158. signaling in vascular and tumor biology. Adv. Exp. Med. Biol. 600, 118-131. Wang, Y., van Boxel-Dezaire, A. H., Cheon, H., Yang, J. and Stark, G. R. (2013). Owen, J. A., Punt, J., Stranford, S. A., Jones, P. P. and Kuby, J. (2013). Kuby STAT3 activation in response to IL-6 is prolonged by the binding of IL-6 receptor to Immunology. W. H. Freeman and Company. EGF receptor. Proc. Natl. Acad. Sci. USA 110, 16975-16980. Pitulescu, M. E., Schmidt, I., Benedito, R. and Adams, R. H. (2010). Inducible Wegenka, U. M., Lutticken, C., Buschmann, J., Yuan, J., Lottspeich, F., Muller- gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in Esterl, W., Schindler, C., Roeb, E., Heinrich, P. C. and Horn, F. (1994). The mice. Nat. Protoc. 5, 1518-1534. interleukin-6-activated acute-phase response factor is antigenically and Quintana, A., Molinero, A., Borup, R., Nielsen, F. C., Campbell, I. L., Penkowa, functionally related to members of the signal transducer and activator of M. and Hidalgo, J. (2008). Effect of astrocyte-targeted production of IL-6 on transcription (STAT) family. Mol. Cell. Biol. 14, 3186-3196. traumatic brain injury and its impact on the cortical transcriptome. Dev. Neurobiol. Wegiel, B., Bjartell, A., Culig, Z. and Persson, J. L. (2008). Interleukin-6 activates 68, 195-208. PI3K/Akt pathway and regulates cyclin A1 to promote prostate cancer cell survival. Quintana, A., Erta, M., Ferrer, B., Comes, G., Giralt, M. and Hidalgo, J. (2013). Int. J. Cancer 122, 1521-1529. Astrocyte-specific deficiency of interleukin-6 and its receptor reveal specific roles Wei, L. H., Kuo, M. L., Chen, C. A., Chou, C. H., Lai, K. B., Lee, C. N. and Hsieh, in survival, body weight and behavior. Brain Behav. Immun. 27, 162-173. C. Y. (2003). Interleukin-6 promotes cervical tumor growth by VEGF-dependent Rada, B., Gardina, P., Myers, T. G. and Leto, T. L. (2011). Reactive oxygen angiogenesis via a STAT3 pathway. Oncogene 22, 1517-1527. species mediate inflammatory cytokine release and EGFR-dependent mucin West, X. Z., Meller, N., Malinin, N. L., Deshmukh, L., Meller, J., Mahabeleshwar, secretion in airway epithelial cells exposed to Pseudomonas pyocyanin. Mucosal G. H., Weber, M. E., Kerr, B. A., Vinogradova, O. and Byzova, T. V. (2012). Immunol. 4, 158-171. Integrin beta3 crosstalk with VEGFR accommodating tyrosine phosphorylation as Ragimbeau, J., Dondi, E., Vasserot, A., Romero, P., Uze, G. and Pellegrini, S. a regulatory switch. PLoS ONE 7, e31071. (2001). The receptor interaction region of Tyk2 contains a motif required for its Wu, L., Tan, X., Liang, L., Yu, H., Wang, C., Zhang, D., Kijlstra, A. and Yang, P. nuclear localization. J. Biol. Chem. 276, 30812-30818. (2017). The Role of Mitochondria-Associated Reactive Oxygen Species in the Reynolds, L. E., Wyder, L., Lively, J. C., Taverna, D., Robinson, S. D., Huang, X., Amyloid beta Induced Production of Angiogenic Factors b y ARPE-19 Cells. Curr. Sheppard, D., Hynes, R. O. and Hodivala-Dilke, K. M. (2002). Enhanced Mol. Med. 17, 140-148. pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 Yao, Z., Fenoglio, S., Gao, D. C., Camiolo, M., Stiles, B., Lindsted, T., integrins. Nat. Med. 8, 27-34. Schlederer, M., Johns, C., Altorki, N., Mittal, V. et al. (2010). TGF-beta IL-6 Rodriguez-Berriguete, G., Prieto, A., Fraile, B., Bouraoui, Y., de Bethencourt, axis mediates selective and adaptive mechanisms of resistance to molecular

F. R., Martinez-Onsurbe, P., Olmedilla, G., Paniagua, R. and Royuela, M. targeted therapy in lung cancer. Proc. Natl. Acad. Sci. USA 107, 15535-15540. Journal of Cell Science

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Zeng, H., Zhao, D., Yang, S., Datta, K. and Mukhopadhyay, D. (2003). Zhang, X., Kimura, Y., Fang, C., Zhou, L., Sfyroera, G., Lambris, J. D., Wetsel, Heterotrimeric G alpha q/G alpha 11 proteins function upstream of vascular R. A., Miwa, T. and Song, W. C. (2007). Regulation of Toll-like endothelial growth factor (VEGF) receptor-2 (KDR) phosphorylation in vascular receptor-mediated inflammatory response by complement in vivo. Blood 110, permeability factor/VEGF signaling. J. Biol. Chem. 278, 20738-20745. 228-236. Journal of Cell Science