Abstract
Successful wound closure is mainly the result of two cellular processes: migration and proliferation. Apoptosis has also been suggested to play a role in the mechanisms of wound healing. The fast calcium wave (FCW), triggered immediately after a wound is produced, has been proposed to be involved in determining healing responses in epithelia. We have explored the effects of the reversible inhibition of FCW on the apoptotic and proliferative responses of healing bovine corneal endothelial (BCE) cells in culture. The most important findings of this study are that caspase-dependent apoptosis occurs during the healing process, that the amount of apoptosis has a linear dependence on the migrated distance, and that FCW inhibition greatly increases the apoptotic index. We have further been able to establish that FCW plays a role in the control of cell proliferation during BCE wound healing. These results indicate that one of the main roles of the wave is to inhibit an excessive apoptotic response of the healing migrating cells. This property might represent a basic mechanism to allow sufficient migration and proliferation of the healing cells to assure proper restitution of the injured tissue.
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Abe M, Yokoyama Y, Ishikawa O (2012) A possible mechanism of basic fibroblast growth factor-promoted scarless wound healing: the induction of myofibroblast apoptosis. Eur J Dermatol 22:46–53
Akasaka Y, Ono I, Kamiya T, Ishikawa Y, Kinoshita T, Ishiguro S, Yokoo T, Imaizumi R, Inomata N, Fujita K, Akishima-Fukasawa Y, Uzuki M, Ito K, Ishii T (2010) The mechanisms underlying fibroblast apoptosis regulated by growth factors during wound healing. J Pathol 221:285–299
Appleby PA, Shabir S, Southgate J, Walker D (2015) Sources of variability in cytosolic calcium transients triggered by stimulation of homogeneous uro-epithelial cell monolayers. J R Soc Interface 12:20141403
Bement WM, Forscher P, Mooseker MS (1993) A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance. J Cell Biol 121:565–578
Berra-Romani R, Raqeeb A, Avelino-Cruz JE, Moccia F, Oldani A, Speroni F, Taglietti V, Tanzi F (2008) Ca2+ signaling in injured in situ endothelium of rat aorta. Cell Calcium 44:298–309
Carter R, Sykes V, Lanning D (2009) Scarless fetal mouse wound healing may initiate apoptosis through caspase 7 and cleavage of PARP. J Surg Res 156:74–79
Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE (1997) Geometric control of cell life and death. Science 276:1425–1428
Chifflet S, Hernandez JA, Grasso S, Cirillo A (2003) Nonspecific depolarization of the plasma membrane potential induces cytoskeletal modifications of bovine corneal endothelial cells in culture. Exp Cell Res 282:1–13
Chifflet S, Justet C, Hernandez JA, Nin V, Escande C, Benech JC (2012) Early and late calcium waves during wound healing in corneal endothelial cells. Wound Repair Regen 20:28–37
Citi S (1992) Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. J Cell Biol 117:169–178
Clause KC, Barker TH (2013) Extracellular matrix signaling in morphogenesis and repair. Curr Opin Biotechnol 24:830–833
Cole BK, Curto M, Chan AW, McClatchey AI (2008) Localization to the cortical cytoskeleton is necessary for Nf2/merlin-dependent epidermal growth factor receptor silencing. Mol Cell Biol 28:1274–1284
Cordeiro JV, Jacinto A (2013) The role of transcription-independent damage signals in the initiation of epithelial wound healing. Nat Rev Mol Cell Biol 14:249–262
Crosby LM, Luellen C, Zhang Z, Tague LL, Sinclair SE, Waters CM (2011) Balance of life and death in alveolar epithelial type II cells: proliferation, apoptosis, and the effects of cyclic stretch on wound healing. Am J Physiol Lung Cell Mol Physiol 301:L536–L546
D’Hondt C, Ponsaerts R, Srinivas SP, Vereecke J, Himpens B (2007) Thrombin inhibits intercellular calcium wave propagation in corneal endothelial cells by modulation of hemichannels and gap junctions. Invest Ophthalmol Vis Sci 48:120–133
D’Hondt C, Ponsaerts R, De Smedt H, Vinken M, De Vuyst E, De Bock M, Wang N, Rogiers V, Leybaert L, Himpens B, Bultynck G (2011) Pannexin channels in ATP release and beyond: an unexpected rendezvous at the endoplasmic reticulum. Cell Signal 23:305–316
Denis C, Deiteren K, Mortier A, Tounsi A, Fransen E, Proost P, Renauld JC, Lambeir AM (2012) C-terminal clipping of chemokine CCL1/I-309 enhances CCR8-mediated intracellular calcium release and anti-apoptotic activity. PLoS One 7:e34199
Eagle H, Levine EM (1967) Growth regulatory effects of cellular interaction. Nature 213:1102–1106
Folkman J, Moscona A (1978) Role of cell shape in growth control. Nature 273:345–349
Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson SA, Abrams JM, Adam D, Alnemri ES, Altucci L, Andrews D, Annicchiarico-Petruzzelli M, Baehrecke EH, Bazan NG, Bertrand MJ, Bianchi K, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Campanella M, Candi E, Cecconi F, Chan FK, Chandel NS, Cheng EH, Chipuk JE, Cidlowski JA, Ciechanover A, Dawson TM, Dawson VL, De Laurenzi V, De Maria R, Debatin KM, Di Daniele N, Dixit VM, Dynlacht BD, El-Deiry WS, Fimia GM, Flavell RA, Fulda S, Garrido C, Gougeon ML, Green DR, Gronemeyer H, Hajnoczky G, Hardwick JM, Hengartner MO, Ichijo H, Joseph B, Jost PJ, Kaufmann T, Kepp O, Klionsky DJ, Knight RA, Kumar S, Lemasters JJ, Levine B, Linkermann A, Lipton SA, Lockshin RA, Lopez-Otin C, Lugli E, Madeo F, Malorni W, Marine JC, Martin SJ, Martinou JC, Medema JP, Meier P, Melino S, Mizushima N, Moll U, Munoz-Pinedo C, Nunez G, Oberst A, Panaretakis T, Penninger JM, Peter ME, Piacentini M, Pinton P, Prehn JH, Puthalakath H, Rabinovich GA, Ravichandran KS, Rizzuto R, Rodrigues CM, Rubinsztein DC, Rudel T, Shi Y, Simon HU, Stockwell BR, Szabadkai G, Tait SW, Tang HL, Tavernarakis N, Tsujimoto Y, Vanden Berghe T, Vandenabeele P, Villunger A, Wagner EF, Walczak H, White E, Wood WG, Yuan J, Zakeri Z, Zhivotovsky B, Melino G, Kroemer G (2015) Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ 22:58–73
Gomes P, Srinivas SP, Van Driessche W, Vereecke J, Himpens B (2005a) ATP release through connexin hemichannels in corneal endothelial cells. Invest Ophthalmol Vis Sci 46:1208–1218
Gomes P, Srinivas SP, Vereecke J, Himpens B (2005b) ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 46:104–113
Grasso S, Hernandez JA, Chifflet S (2007) Roles of wound geometry, wound size, and extracellular matrix in the healing response of bovine corneal endothelial cells in culture. Am J Physiol Cell Physiol 293:C1327–C1337
Guadamillas MC, Cerezo A, Del Pozo MA (2011) Overcoming anoikis—pathways to anchorage-independent growth in cancer. J Cell Sci 124:3189–3197
Harris RA, Hanrahan JW (1994) Effects of EGTA on calcium signaling in airway epithelial cells. Am J Physiol 267:C1426–C1434
Henson JH, Nazarian R, Schulberg KL, Trabosh VA, Kolnik SE, Burns AR, McPartland KJ (2002) Wound closure in the lamellipodia of single cells: mediation by actin polymerization in the absence of an actomyosin purse string. Mol Biol Cell 13:1001–1014
Hofer AM (2005) Another dimension to calcium signaling: a look at extracellular calcium. J Cell Sci 118:855–862
Jiffar T, Kurinna S, Suck G, Carlson-Bremer D, Ricciardi MR, Konopleva M, Andreeff M, Ruvolo PP (2004) PKC alpha mediates chemoresistance in acute lymphoblastic leukemia through effects on Bcl2 phosphorylation. Leukemia 18:505–512
Justet C, Evans F, Vasilskis E, Hernandez JA, Chifflet S (2013) ENaC contribution to epithelial wound healing is independent of the healing mode and of any increased expression in the channel. Cell Tissue Res 353:53–64
Kato K, Tanaka T, Sadik G, Baba M, Maruyama D, Yanagida K, Kodama T, Morihara T, Tagami S, Okochi M, Kudo T, Takeda M (2011) Protein kinase C stabilizes X-linked inhibitor of apoptosis protein (XIAP) through phosphorylation at Ser(87) to suppress apoptotic cell death. Psychogeriatrics 11:90–97
Kim NG, Koh E, Chen X, Gumbiner BM (2011) E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components. Proc Natl Acad Sci U S A 108:11930–11935
Kimura C, Oike M, Koyama T, Ito Y (2001) Alterations of Ca2+ mobilizing properties in migrating endothelial cells. Am J Physiol Heart Circ Physiol 281:H745–H754
Klepeis VE, Cornell-Bell A, Trinkaus-Randall V (2001) Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells. J Cell Sci 114:4185–4195
Klepeis VE, Weinger I, Kaczmarek E, Trinkaus-Randall V (2004) P2Y receptors play a critical role in epithelial cell communication and migration. J Cell Biochem 93:1115–1133
Kofron M, Heasman J, Lang SA, Wylie CC (2002) Plakoglobin is required for maintenance of the cortical actin skeleton in early Xenopus embryos and for cdc42-mediated wound healing. J Cell Biol 158:695–708
Korkiamaki T, Yla-Outinen H, Leinonen P, Koivunen J, Peltonen J (2005) The effect of extracellular calcium concentration on calcium-mediated cell signaling in NF1 tumor suppressor-deficient keratinocytes. Arch Dermatol Res 296:465–472
Leiper LJ, Walczysko P, Kucerova R, Ou J, Shanley LJ, Lawson D, Forrester JV, McCaig CD, Zhao M, Collinson JM (2006) The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6+/- mouse model of epithelial wound-healing delay. BMC Biol 4:27
Levine EM, Becker Y, Boone CW, Eagle H (1965) Contact inhibition, macromolecular synthesis, and polyribosomes in cultured human diploid fibroblasts. Proc Natl Acad Sci U S A 53:350–356
Li F, Huang Q, Chen J, Peng Y, Roop DR, Bedford JS, Li CY (2010) Apoptotic cells activate the “phoenix rising” pathway to promote wound healing and tissue regeneration. Sci Signal 3:ra13
Li W, Xiong Y, Shang C, Twu KY, Hang CT, Yang J, Han P, Lin CY, Lin CJ, Tsai FC, Stankunas K, Meyer T, Bernstein D, Pan M, Chang CP (2013) Brg1 governs distinct pathways to direct multiple aspects of mammalian neural crest cell development. Proc Natl Acad Sci U S A 110:1738–1743
Lotz MM, Rabinovitz I, Mercurio AM (2000) Intestinal restitution: progression of actin cytoskeleton rearrangements and integrin function in a model of epithelial wound healing. Am J Pathol 156:985–996
Lytton J, Westlin M, Hanley MR (1991) Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J Biol Chem 266:17067–17071
McClatchey AI, Yap AS (2012) Contact inhibition (of proliferation) redux. Curr Opin Cell Biol 24:685–694
Narciso C, Wu Q, Brodskiy P, Garston G, Baker R, Fletcher A, Zartman J (2015) Patterning of wound-induced intercellular Ca(2+) flashes in a developing epithelium. Phys Biol 12:056005
Omelchenko T, Vasiliev JM, Gelfand IM, Feder HH, Bonder EM (2003) Rho-dependent formation of epithelial “leader” cells during wound healing. Proc Natl Acad Sci U S A 100:10788–10793
Orrenius S, Gogvadze V, Zhivotovsky B (2015) Calcium and mitochondria in the regulation of cell death. Biochem Biophys Res Commun 460:72–81
Petroll WM, Ma L, Jester JV, Cavanagh HD, Bean J (2001) Organization of junctional proteins in proliferating cat corneal endothelium during wound healing. Cornea 20:73–80
Poujade M, Grasland-Mongrain E, Hertzog A, Jouanneau J, Chavrier P, Ladoux B, Buguin A, Silberzan P (2007) Collective migration of an epithelial monolayer in response to a model wound. Proc Natl Acad Sci U S A 104:15988–15993
Qian X, Karpova T, Sheppard AM, McNally J, Lowy DR (2004) E-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases. EMBO J 23:1739–1748
Quist AP, Rhee SK, Lin H, Lal R (2000) Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol 148:1063–1074
Razzell W, Evans IR, Martin P, Wood W (2013) Calcium flashes orchestrate the wound inflammatory response through DUOX activation and hydrogen peroxide release. Curr Biol 23:424–429
Rorth P (2009) Collective cell migration. Annu Rev Cell Dev Biol 25:407–429
Sammak PJ, Hinman LE, Tran PO, Sjaastad MD, Machen TE (1997) How do injured cells communicate with the surviving cell monolayer? J Cell Sci 110:465–475
Sato A, Scholl AM, Kuhn EN, Stadt HA, Decker JR, Pegram K, Hutson MR, Kirby ML (2011) FGF8 signaling is chemotactic for cardiac neural crest cells. Dev Biol 354:18–30
Schlegelmilch K, Mohseni M, Kirak O, Pruszak J, Rodriguez JR, Zhou D, Kreger BT, Vasioukhin V, Avruch J, Brummelkamp TR, Camargo FD (2011) Yap1 acts downstream of alpha-catenin to control epidermal proliferation. Cell 144:782–795
Shabir S, Southgate J (2008) Calcium signalling in wound-responsive normal human urothelial cell monolayers. Cell Calcium 44:453–464
Sherwood CL, Lantz RC, Burgess JL, Boitano S (2011) Arsenic alters ATP-dependent Ca(2)+ signaling in human airway epithelial cell wound response. Toxicol Sci 121:191–206
Shi RX, Ong CN, Shen HM (2005) Protein kinase C inhibition and X-linked inhibitor of apoptosis protein degradation contribute to the sensitization effect of luteolin on tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cancer cells. Cancer Res 65:7815–7823
Sonnemann KJ, Bement WM (2011) Wound repair: toward understanding and integration of single-cell and multicellular wound responses. Annu Rev Cell Dev Biol 27:237–263
St Croix B, Sheehan C, Rak JW, Florenes VA, Slingerland JM, Kerbel RS (1998) E-Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27 (KIP1). J Cell Biol 142:557–571
Sung YJ, Sung Z, Ho CL, Lin MT, Wang JS, Yang SC, Chen YJ, Lin CH (2003) Intercellular calcium waves mediate preferential cell growth toward the wound edge in polarized hepatic cells. Exp Cell Res 287:209–218
Tan JQ, Zhang HH, Lei ZJ, Ren P, Deng C, Li XY, Chen SZ (2013) The roles of autophagy and apoptosis in burn wound progression in rats. Burns J Int S Burn Inj 39:1551–1556
Tran PO, Tran QH, Hinman LE, Sammak PJ (1998) Co-ordination between localized wound-induced Ca2+ signals and pre-wound serum signals is required for proliferation after mechanical injury. Cell Prolif 31:155–170
Tran PO, Hinman LE, Unger GM, Sammak PJ (1999) A wound-induced [Ca2+]i increase and its transcriptional activation of immediate early genes is important in the regulation of motility. Exp Cell Res 246:319–326
Waters CM, Sporn PH, Liu M, Fredberg JJ (2002) Cellular biomechanics in the lung. Am J Physiol Lung Cell Mol Physiol 283:L503–L509
Yamaguchi M (2013) The anti-apoptotic effect of regucalcin is mediated through multisignaling pathways. Apoptosis 18:1145–1153
Acknowledgments
We thank Frigorífico Las Piedras, Frigorífico Lorsinal S.A., and the Ecole Nationale Vétérinaire d’Alfort for supplying us with fresh bovine eyes. We are also grateful to the anonymous reviewers for their useful criticisms and suggestions.
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This study was supported by Cooperation Program ECOS Sud France-Uruguay Grant U08H01 (to A. Torriglia and S. Chifflet); Comisión Sectorial de Investigación Científica (CSIC), Universidad de la República, Uruguay (Proyecto Grupos I+D 2014 to S. Chifflet and Magister and Doctoral Fellowships to C. Justet); and Programa de Desarrollo de las Ciencias Básicas (PEDECIBA), Uruguay (to S. Chifflet and J. A. Hernandez).
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Justet, C., Hernández, J.A., Torriglia, A. et al. Fast calcium wave inhibits excessive apoptosis during epithelial wound healing. Cell Tissue Res 365, 343–356 (2016). https://doi.org/10.1007/s00441-016-2388-8
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DOI: https://doi.org/10.1007/s00441-016-2388-8