Abstract
Receptor-interacting protein kinase 3 (RIPK3) is a multifunctional intracellular protein that was first recognized as an important component of the necroptosis programmed cell death pathway. RIPK3 is also highly expressed in non-necroptotic murine embryonic endothelial cells (ECs) during vascular development, indicating its potential contribution to angiogenesis. To test this hypothesis, we generated mice lacking endothelial RIPK3 and found non-lethal embryonic and perinatal angiogenesis defects in multiple vascular beds. Our in vitro data indicate that RIPK3 supports angiogenesis by regulating growth factor receptor degradation in ECs. We found that RIPK3 interacted with the membrane trafficking protein myoferlin to sustain expression of vascular endothelial growth factor receptor 2 (VEGFR2) in cultured ECs following vascular endothelial growth factor A (VEGFA) stimulation. Restoration of myoferlin, which was diminished after RIPK3 knockdown, rescued decreased VEGFR2 expression and vascular sprouting in RIPK3-deficient ECs after VEGFA treatment. In addition, we found that RIPK3 modulated expression of genes involved in endothelial identity by inhibiting ERK signaling independently of growth factor receptor turnover. Altogether, our data reveal unexpected non-necroptotic roles for RIPK3 in ECs and evidence that RIPK3 promotes developmental angiogenesis in vivo.
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Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, Chan FK (2009) Phosphorylation-driven assembly of the RIP1–RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137(6):1112–1123. https://doi.org/10.1016/j.cell.2009.05.037
He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X (2009) Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 137(6):1100–1111. https://doi.org/10.1016/j.cell.2009.05.021
Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G (2010) Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol 11(10):700–714. https://doi.org/10.1038/nrm2970
Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, Dong MQ, Han J (2009) RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325(5938):332–336. https://doi.org/10.1126/science.1172308
Samson AL, Zhang Y, Geoghegan ND, Gavin XJ, Davies KA, Mlodzianoski MJ, Whitehead LW, Frank D, Garnish SE, Fitzgibbon C, Hempel A, Young SN, Jacobsen AV, Cawthorne W, Petrie EJ, Faux MC, Shield-Artin K, Lalaoui N, Hildebrand JM, Silke J, Rogers KL, Lessene G, Hawkins ED, Murphy JM (2020) MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis. Nat Commun 11(1):3151. https://doi.org/10.1038/s41467-020-16887-1
Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I, Hulpiau P, Weber K, Sehon CA, Marquis RW, Bertin J, Gough PJ, Savvides S, Martinou JC, Bertrand MJ, Vandenabeele P (2014) MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep 7(4):971–981. https://doi.org/10.1016/j.celrep.2014.04.026
Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148(1–2):213–227. https://doi.org/10.1016/j.cell.2011.11.031
Yang Z, Wang Y, Zhang Y, He X, Zhong CQ, Ni H, Chen X, Liang Y, Wu J, Zhao S, Zhou D, Han J (2018) RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis. Nat Cell Biol 20(2):186–197. https://doi.org/10.1038/s41556-017-0022-y
Newton K (2015) RIPK1 and RIPK3: critical regulators of inflammation and cell death. Trends Cell Biol 25(6):347–353. https://doi.org/10.1016/j.tcb.2015.01.001
Lawlor KE, Khan N, Mildenhall A, Gerlic M, Croker BA, D’Cruz AA, Hall C, Kaur Spall S, Anderton H, Masters SL, Rashidi M, Wicks IP, Alexander WS, Mitsuuchi Y, Benetatos CA, Condon SM, Wong WW, Silke J, Vaux DL, Vince JE (2015) RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun 6:6282. https://doi.org/10.1038/ncomms7282
Varfolomeev EE, Schuchmann M, Luria V, Chiannilkulchai N, Beckmann JS, Mett IL, Rebrikov D, Brodianski VM, Kemper OC, Kollet O, Lapidot T, Soffer D, Sobe T, Avraham KB, Goncharov T, Holtmann H, Lonai P, Wallach D (1998) Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 9(2):267–276. https://doi.org/10.1016/s1074-7613(00)80609-3
Yeh WC, Itie A, Elia AJ, Ng M, Shu HB, Wakeham A, Mirtsos C, Suzuki N, Bonnard M, Goeddel DV, Mak TW (2000) Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development. Immunity 12(6):633–642. https://doi.org/10.1016/s1074-7613(00)80214-9
Yeh WC, de la Pompa JL, McCurrach ME, Shu HB, Elia AJ, Shahinian A, Ng M, Wakeham A, Khoo W, Mitchell K, El-Deiry WS, Lowe SW, Goeddel DV, Mak TW (1998) FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science 279(5358):1954–1958. https://doi.org/10.1126/science.279.5358.1954
Kaiser WJ, Upton JW, Long AB, Livingston-Rosanoff D, Daley-Bauer LP, Hakem R, Caspary T, Mocarski ES (2011) RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 471(7338):368–372. https://doi.org/10.1038/nature09857
Dillon CP, Oberst A, Weinlich R, Janke LJ, Kang TB, Ben-Moshe T, Mak TW, Wallach D, Green DR (2012) Survival function of the FADD-CASPASE-8-cFLIP(L) complex. Cell Rep 1(5):401–407. https://doi.org/10.1016/j.celrep.2012.03.010
Dillon CP, Tummers B, Baran K, Green DR (2016) Developmental checkpoints guarded by regulated necrosis. Cell Mol Life Sci 73(11–12):2125–2136. https://doi.org/10.1007/s00018-016-2188-z
Colijn S, Gao S, Ingram KG, Menendez M, Muthukumar V, Silasi-Mansat R, Chmielewska JJ, Hinsdale M, Lupu F, Griffin CT (2020) The NuRD chromatin-remodeling complex enzyme CHD4 prevents hypoxia-induced endothelial Ripk3 transcription and murine embryonic vascular rupture. Cell Death Differ 27(2):618–631. https://doi.org/10.1038/s41418-019-0376-8
Tisch N, Freire-Valls A, Yerbes R, Paredes I, La Porta S, Wang X, Martin-Perez R, Castro L, Wong WW, Coultas L, Strilic B, Grone HJ, Hielscher T, Mogler C, Adams RH, Heiduschka P, Claesson-Welsh L, Mazzone M, Lopez-Rivas A, Schmidt T, Augustin HG, Ruiz de Almodovar C (2019) Caspase-8 modulates physiological and pathological angiogenesis during retina development. J Clin Invest 129(12):5092–5107. https://doi.org/10.1172/JCI122767
Strilic B, Yang L, Albarran-Juarez J, Wachsmuth L, Han K, Muller UC, Pasparakis M, Offermanns S (2016) Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis. Nature 536(7615):215–218. https://doi.org/10.1038/nature19076
Hanggi K, Vasilikos L, Valls AF, Yerbes R, Knop J, Spilgies LM, Rieck K, Misra T, Bertin J, Gough PJ, Schmidt T, de Almodovar CR, Wong WW (2017) RIPK1/RIPK3 promotes vascular permeability to allow tumor cell extravasation independent of its necroptotic function. Cell Death Dis 8(2):e2588. https://doi.org/10.1038/cddis.2017.20
Reventun P, Sanchez-Esteban S, Cook A, Cuadrado I, Roza C, Moreno-Gomez-Toledano R, Munoz C, Zaragoza C, Bosch RJ, Saura M (2020) Bisphenol A induces coronary endothelial cell necroptosis by activating RIP3/CamKII dependent pathway. Sci Rep 10(1):4190. https://doi.org/10.1038/s41598-020-61014-1
Yang L, Joseph S, Sun T, Hoffmann J, Thevissen S, Offermanns S, Strilic B (2019) TAK1 regulates endothelial cell necroptosis and tumor metastasis. Cell Death Differ 26(10):1987–1997. https://doi.org/10.1038/s41418-018-0271-8
Newton K, Dugger DL, Wickliffe KE, Kapoor N, de Almagro MC, Vucic D, Komuves L, Ferrando RE, French DM, Webster J, Roose-Girma M, Warming S, Dixit VM (2014) Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis. Science 343(6177):1357–1360. https://doi.org/10.1126/science.1249361
Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M (2001) Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol 230(2):230–242. https://doi.org/10.1006/dbio.2000.0106
Wang Y, Nakayama M, Pitulescu ME, Schmidt TS, Bochenek ML, Sakakibara A, Adams S, Davy A, Deutsch U, Luthi U, Barberis A, Benjamin LE, Makinen T, Nobes CD, Adams RH (2010) Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465(7297):483–486. https://doi.org/10.1038/nature09002
Simons M, Gordon E, Claesson-Welsh L (2016) Mechanisms and regulation of endothelial VEGF receptor signalling. Nat Rev Mol Cell Biol 17(10):611–625. https://doi.org/10.1038/nrm.2016.87
Redpath GM, Sophocleous RA, Turnbull L, Whitchurch CB, Cooper ST (2016) Ferlins show tissue-specific expression and segregate as plasma membrane/late endosomal or trans-Golgi/recycling ferlins. Traffic 17(3):245–266. https://doi.org/10.1111/tra.12370
Bernatchez PN, Acevedo L, Fernandez-Hernando C, Murata T, Chalouni C, Kim J, Erdjument-Bromage H, Shah V, Gratton JP, McNally EM, Tempst P, Sessa WC (2007) Myoferlin regulates vascular endothelial growth factor receptor-2 stability and function. J Biol Chem 282(42):30745–30753. https://doi.org/10.1074/jbc.M704798200
Bernatchez PN, Sharma A, Kodaman P, Sessa WC (2009) Myoferlin is critical for endocytosis in endothelial cells. Am J Physiol Cell Physiol 297(3):C484–C492. https://doi.org/10.1152/ajpcell.00498.2008
Sharma A, Yu C, Leung C, Trane A, Lau M, Utokaparch S, Shaheen F, Sheibani N, Bernatchez P (2010) A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis. Arterioscler Thromb Vasc Biol 30(11):2196–2204. https://doi.org/10.1161/ATVBAHA.110.208108
Yu C, Sharma A, Trane A, Utokaparch S, Leung C, Bernatchez P (2011) Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo. Vascul Pharmacol 55(1–3):26–33. https://doi.org/10.1016/j.vph.2011.04.001
Koch S, Claesson-Welsh L (2012) Signal transduction by vascular endothelial growth factor receptors. Cold Spring Harb Perspect Med 2(7):a006502. https://doi.org/10.1101/cshperspect.a006502
Simons M (2012) An inside view: VEGF receptor trafficking and signaling. Physiology (Bethesda) 27(4):213–222. https://doi.org/10.1152/physiol.00016.2012
Kovalenko D, Yang X, Nadeau RJ, Harkins LK, Friesel R (2003) Sef inhibits fibroblast growth factor signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent ERK activation. J Biol Chem 278(16):14087–14091. https://doi.org/10.1074/jbc.C200606200
Hata A, Chen YG (2016) TGF-beta signaling from receptors to Smads. Cold Spring Harb Perspect Biol 8(9):022061. https://doi.org/10.1101/cshperspect.a022061
Deng Y, Larrivee B, Zhuang ZW, Atri D, Moraes F, Prahst C, Eichmann A, Simons M (2013) Endothelial RAF1/ERK activation regulates arterial morphogenesis. Blood 121(19):3988–3996. https://doi.org/10.1182/blood-2012-12-474601
Deng Y, Atri D, Eichmann A, Simons M (2013) Endothelial ERK signaling controls lymphatic fate specification. J Clin Invest 123(3):1202–1215. https://doi.org/10.1172/JCI63034
Eichmann A, Simons M (2012) VEGF signaling inside vascular endothelial cells and beyond. Curr Opin Cell Biol 24(2):188–193. https://doi.org/10.1016/j.ceb.2012.02.002
Yoon S, Kovalenko A, Bogdanov K, Wallach D (2017) MLKL, the protein that mediates necroptosis, also regulates endosomal trafficking and extracellular vesicle generation. Immunity 47(1):51-65 e57. https://doi.org/10.1016/j.immuni.2017.06.001
Douanne T, Andre-Gregoire G, Trillet K, Thys A, Papin A, Feyeux M, Hulin P, Chiron D, Gavard J, Bidere N (2019) Pannexin-1 limits the production of proinflammatory cytokines during necroptosis. EMBO Rep 20(10):e47840. doi:https://doi.org/10.15252/embr.201947840
Rasheed A, Robichaud S, Nguyen MA, Geoffrion M, Wyatt H, Cottee ML, Dennison T, Pietrangelo A, Lee R, Lagace TA, Ouimet M, Rayner KJ (2020) Loss of MLKL (mixed lineage kinase domain-like protein) decreases necrotic core but increases macrophage lipid accumulation in atherosclerosis. Arterioscler Thromb Vasc Biol 40(5):1155–1167. https://doi.org/10.1161/ATVBAHA.119.313640
Bruno J, Brumfield A, Chaudhary N, Iaea D, McGraw TE (2016) SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes. J Cell Biol 214(1):61–76. doi:https://doi.org/10.1083/jcb.201509052
Colijn S, Muthukumar V, Xie J, Gao S, Griffin CT (2020) Cell-specific and athero-protective roles for RIPK3 in a murine model of atherosclerosis. Dis Model Mech 13(1):dmm041962. https://doi.org/10.1242/dmm.041962
Fantin A, Vieira JM, Plein A, Maden CH, Ruhrberg C (2013) The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis. Nat Protoc 8(2):418–429. https://doi.org/10.1038/nprot.2013.015
Montoya-Zegarra JA, Russo E, Runge P, Jadhav M, Willrodt AH, Stoma S, Norrelykke SF, Detmar M, Halin C (2019) AutoTube: a novel software for the automated morphometric analysis of vascular networks in tissues. Angiogenesis 22(2):223–236. https://doi.org/10.1007/s10456-018-9652-3
Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M (2014) iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159(4):896–910. https://doi.org/10.1016/j.cell.2014.10.010
Tual-Chalot S, Allinson KR, Fruttiger M, Arthur HM (2013) Whole mount immunofluorescent staining of the neonatal mouse retina to investigate angiogenesis in vivo. J Vis Exp 77:e50546. https://doi.org/10.3791/50546
Nakatsu MN, Davis J, Hughes CC (2007) Optimized fibrin gel bead assay for the study of angiogenesis. J Vis Exp (3):186. https://doi.org/10.3791/186
Acknowledgements
We thank Jun Xie for assistance with mouse colony maintenance, Kate Wheeler for technical help with this project, and Griffin lab members for helpful discussions. We also thank Steve Hartson (Oklahoma State University Protein Resource Core Facility) for assistance with LC-MS/MS. This work was supported by NIH grants R35HLI44605 (to C.T.G.) and P30GM114731 and by an AHA Predoctoral Fellowship #19PRE34380708 (to S.G.).
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Conceptualization, S.G and C.T.G.; Investigation, S.G.; Writing, S.G. and C.T.G.; Funding acquisition, C.T.G. and S.G.
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Gao, S., Griffin, C.T. RIPK3 modulates growth factor receptor expression in endothelial cells to support angiogenesis. Angiogenesis 24, 519–531 (2021). https://doi.org/10.1007/s10456-020-09763-5
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DOI: https://doi.org/10.1007/s10456-020-09763-5