The metazoan liver exhibits a remarkable capacity to regenerate lost liver mass without leaving a scar following partial hepatectomy (PH). Whilst previous studies have identified components of several different signaling pathways that are essential for activation of hepatocyte proliferation during liver regeneration, the mechanisms that enable such regeneration to occur without accompanying scar formation remain poorly understood. Here we use the adult zebrafish liver, which can regenerate within two weeks following PH, as a new genetic model to address this important question. We focus on the role of Digestive-organ-expansion-factor (Def), a nucleolar protein which has recently been shown to complex with calpain3 (Capn3) to mediate p53 degradation specifically in the nucleolus, in liver regeneration. Firstly, we show that Def expression is up-regulated in the wild-type liver following amputation, and that the defhi429/+ heteroozygous mutant (def+/？) suffers from haploinsufficiency of Def in the liver. We then show that the expression of pro-inflammatory cytokines is up-regulated in the def+/？ liver, which leads to distortion of the migration and the clearance of leukocytes after PH. Transforming growth factor β (TGFβ) signalling is thus activated in the wound epidermis in def+/？ due to a prolonged inflammatory response, which leads to fibrosis at the amputation site. Fibrotic scar formation in def+/？ is blocked by the over-expression of Def, by the loss-of-function of p53, and by treatment with anti-inflammation drug dexamethasone or TGFβ-signalling inhibitor SB431542. We finally show that the Def- p53 pathway suppresses fibrotic scar formation, at least in part, through the regulation of the expression of the pro-inflammatory factor, high-mobility group box 1. We conclude that the novel Def- p53 nucleolar pathway functions specifically to prevent a scar formation at the amputation site in a normal amputated liver.
References
[1]
Taub R (2004) Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 5: 836–847. doi: 10.1038/nrm1489
Michalopoulos GK (2010) Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas. Am J Pathol 176: 2–13.
[4]
Costa RH, Kalinichenko VV, Holterman AX, Wang X (2003) Transcription factors in liver development, differentiation, and regeneration. Hepatology 38: 1331–1347. doi: 10.1016/j.hep.2003.09.034
[5]
Schachtrup C, Le MN, Passino MA, Akassoglou K (2011) Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair. Cell Cycle 10: 1764–1771. doi: 10.4161/cc.10.11.15828
[6]
Tao T, Peng J (2009) Liver development in zebrafish (Danio rerio). J Genet Genomics 36: 325–334. doi: 10.1016/s1673-8527(08)60121-6
[7]
Curado S, Stainier DY (2010) deLiver'in regeneration: injury response and development. Semin Liver Dis 30: 288–295. doi: 10.1055/s-0030-1255357
[8]
Shin D, Weidinger G, Moon RT, Stainier DY (2012) Intrinsic and extrinsic modifiers of the regulative capacity of the developing liver. Mech Dev 128: 525–535. doi: 10.1016/j.mod.2012.01.005
[9]
Kan NG, Junghans D, Izpisua Belmonte JC (2009) Compensatory growth mechanisms regulated by BMP and FGF signaling mediate liver regeneration in zebrafish after partial hepatectomy. FASEB J 23: 3516–3525. doi: 10.1096/fj.09-131730
[10]
Sadler KC, Krahn KN, Gaur NA, Ukomadu C (2007) Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1. Proc Natl Acad Sci U S A 104: 1570–1575. doi: 10.1073/pnas.0610774104
[11]
Charette JM, Baserga SJ (2010) The DEAD-box RNA helicase-like Utp25 is an SSU processome component. RNA 16: 2156–2169. doi: 10.1261/rna.2359810
[12]
Harscoet E, Dubreucq B, Palauqui JC, Lepiniec L (2010) NOF1 encodes an Arabidopsis protein involved in the control of rRNA expression. PLoS One 5: e12829. doi: 10.1371/journal.pone.0012829
[13]
Chen J, Ruan H, Ng SM, Gao C, Soo HM, et al. (2005) Loss of function of def selectively up-regulates Delta113p53 expression to arrest expansion growth of digestive organs in zebrafish. Genes Dev 19: 2900–2911. doi: 10.1101/gad.1366405
[14]
Tao T, Shi H, Guan Y, Huang D, Chen Y, et al. (2013) Def defines a conserved nucleolar pathway that leads p53 to proteasome-independent degradation. Cell Res 23: 620–634. doi: 10.1038/cr.2013.16
[15]
Korzh S, Pan X, Garcia-Lecea M, Winata CL, Pan X, et al. (2008) Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish. BMC Dev Biol 8: 84. doi: 10.1186/1471-213x-8-84
[16]
Stadelmann WK, Digenis AG, Tobin GR (1998) Physiology and healing dynamics of chronic cutaneous wounds. Am J Surg 176: 26S–38S. doi: 10.1016/s0002-9610(98)00183-4
[17]
Yang SL, Aw SS, Chang C, Korzh S, Korzh V, et al. (2011) Depletion of Bhmt elevates sonic hedgehog transcript level and increases beta-cell number in zebrafish. Endocrinology 152: 4706–4717. doi: 10.1210/en.2011-1306
[18]
Dong PD, Munson CA, Norton W, Crosnier C, Pan X, et al. (2007) Fgf10 regulates hepatopancreatic ductal system patterning and differentiation. Nat Genet 39: 397–402. doi: 10.1038/ng1961
[19]
Masuzaki R, Zhao SR, Csizmadia E, Yannas I, Karp SJ (2013) Scar formation and lack of regeneration in adult and neonatal liver after stromal injury. Wound Repair Regen 21: 122–130. doi: 10.1111/j.1524-475x.2012.00868.x
[20]
Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31: 11–24. doi: 10.1016/0092-8674(82)90400-7
[21]
Bataller R, Gabele E, Parsons CJ, Morris T, Yang L, et al. (2005) Systemic infusion of angiotensin II exacerbates liver fibrosis in bile duct-ligated rats. Hepatology 41: 1046–1055. doi: 10.1002/hep.20665
[22]
Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M, et al. (1999) Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad 3. J Invest Dermatol 112: 49–57. doi: 10.1046/j.1523-1747.1999.00477.x
[23]
Gressner AM, Weiskirchen R, Breitkopf K, Dooley S (2002) Roles of TGF-beta in hepatic fibrosis. Front Biosci 7: d793–d807. doi: 10.2741/gressner
[24]
Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, et al. (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62: 65–74. doi: 10.1124/mol.62.1.65
[25]
Ho DM, Chan J, Bayliss P, Whitman M (2006) Inhibitor-resistant type I receptors reveal specific requirements for TGF-beta signaling in vivo. Dev Biol 295: 730–742. doi: 10.1016/j.ydbio.2006.03.050
[26]
Jaquet V, Scapozza L, Clark RA, Krause KH, Lambeth JD (2009) Small-molecule NOX inhibitors: ROS-generating NADPH oxidases as therapeutic targets. Antioxid Redox Signal 11: 2535–2552. doi: 10.1089/ars.2009.2585
[27]
Bhattacharyya S, Brown DE, Brewer JA, Vogt SK, Muglia LJ (2007) Macrophage glucocorticoid receptors regulate Toll-like receptor 4-mediated inflammatory responses by selective inhibition of p38 MAP kinase. Blood 109: 4313–4319. doi: 10.1182/blood-2006-10-048215
[28]
Harada A, Sekido N, Akahoshi T, Wada T, Mukaida N, et al. (1994) Essential involvement of interleukin-8 (IL-8) in acute inflammation. J Leukoc Biol 56: 559–564. doi: 10.1016/1043-4666(94)90129-5
[29]
Lalani I, Bhol K, Ahmed AR (1997) Interleukin-10: biology, role in inflammation and autoimmunity. Ann Allergy Asthma Immunol 79: 469–483. doi: 10.1016/s1081-1206(10)63052-9
[30]
Radtke S, Wuller S, Yang XP, Lippok BE, Mutze B, et al. (2010) Cross-regulation of cytokine signalling: pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation. J Cell Sci 123: 947–959. doi: 10.1242/jcs.065326
[31]
Li L, Yan B, Shi YQ, Zhang WQ, Wen ZL (2012) Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. J Biol Chem 287: 25353–25360. doi: 10.1074/jbc.m112.349126
[32]
Tao T, Shi H, Huang D, Peng J (2013) Def functions as a cell autonomous factor in organogenesis of digestive organs in zebrafish. PLoS One 8: e58858. doi: 10.1371/journal.pone.0058858
[33]
Tsung A, Sahai R, Tanaka H, Nakao A, Fink MP, et al. (2005) The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J Exp Med 201: 1135–1143. doi: 10.1084/jem.20042614
[34]
Scaffidi P, Misteli T, Bianchi ME (2002) Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418: 191–195. doi: 10.1038/nature00858
[35]
Yan HX, Wu HP, Zhang HL, Ashton C, Tong C, et al. (2013) p53 promotes inflammation-associated hepatocarcinogenesis by inducing HMGB1 release. J Hepatol 59: 762–768. doi: 10.1016/j.jhep.2013.05.029
[36]
DiPietro LA, Burdick M, Low QE, Kunkel SL, Strieter RM (1998) MIP-1alpha as a critical macrophage chemoattractant in murine wound repair. J Clin Invest 101: 1693–1698. doi: 10.1172/jci1020
[37]
Wetzler C, Kampfer H, Stallmeyer B, Pfeilschifter J, Frank S (2000) Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair. J Invest Dermatol 115: 245–253. doi: 10.1046/j.1523-1747.2000.00029.x
[38]
Her GM, Chiang CC, Chen WY, Wu JL (2003) In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio). FEBS Lett 538: 125–133. doi: 10.1016/s0014-5793(03)00157-1
[39]
Wan H, Korzh S, Li Z, Mudumana SP, Korzh V, et al. (2006) Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter. Exp Cell Res 312: 1526–1539. doi: 10.1016/j.yexcr.2006.01.016
[40]
Zhang Y, Bai XT, Zhu KY, Jin Y, Deng M, et al. (2008) In vivo interstitial migration of primitive macrophages mediated by JNK-matrix metalloproteinase 13 signaling in response to acute injury. J Immunol 181: 2155–2164. doi: 10.4049/jimmunol.181.3.2155
[41]
Berghmans S, Murphey RD, Wienholds E, Neuberg D, Kutok JL, et al. (2005) tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. Proc Natl Acad Sci U S A 102: 407–412. doi: 10.1073/pnas.0406252102
[42]
Huang H, Ruan H, Aw MY, Hussain A, Guo L, et al. (2008) Mypt1-mediated spatial positioning of Bmp2-producing cells is essential for liver organogenesis. Development 135: 3209–3218. doi: 10.1242/dev.024406
