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Key Event Title
Induction, persistent proliferation/sustained proliferation
|Level of Biological Organization|
Key Event Components
|cellular response to oxidative stress||increased|
|macrophage activation involved in immune response||macrophage||increased|
|hypoxia||hypoxia-inducible factor 1-alpha||decreased|
|hypoxia||von Hippel-Lindau disease tumor suppressor||decreased|
Key Event Overview
AOPs Including This Key Event
|All life stages||High|
Key Event Description
Cellular proliferation rates increase in response to cell death in the liver in order to replace the dying cells. Cellular proliferation refers to the production of new cells to maintain a balance of cell division and cell loss. This key event describes conditions under which this process is persistent or sustained because of chronic exposure. This process is analogous to liver regenerative proliferation (e.g., regerating liver cells following partial hepatectomy), which is often used as a model.
The liver has two modes of regenerating lost cells: (1) via cellular hypertrophy and division of existing cells; or (2) via proliferation of a population of facultative stem cells, called biliary epithelial cells (BECs), located at the Canals of Hering (in zone 1 where canaliculi join and drain into the main bile duct). Facultative stem cells are functional, differentiated cells that will dedifferentiate in response to tissue damage, thereby becoming a population of progenitor cells that can redifferentiate to replace multiple lost cell types.In a process known as ductal expansion, BECs dedifferentiate into oval cells, which then redifferentiate into hepatocytes or BECs in order to regenerate damaged liver tissue. Liver regeneration has been reviewed (Mao, et al. 2014, Stanger 2015, Yanger and Stanger 2011).
At the molecular level, two dimeric transcription factors, AP-1 (particularly the c-Jun monomer) and NF-kappaB, are key players during liver cellular proliferation and regeneration. While neither is expressed in normal liver tissue, they are upregulated during normal hepatic regeneration, and are required for regeneration (Alcorn, et al. 1990, Cressman, et al. 1994, FitzGerald, et al. 1995). Indeed, rodents lacking AP-1 or NF-kappaB display impaired liver regeneration, often leading to death (Behrens, et al. 2002, Schrum, et al. 2000). Both NF-kappaB and c-Jun (AP-1) are required for embryonic liver development, and a loss of either one is embryonic lethal due to widespread cell death and liver degeneration (Behrens, et al. 2002, Eferl, et al. 1999, Jochum, et al. 2001, Li, et al. 1999, Rudolph, et al. 2000).
A causal network for sustained proliferation and regenerative proliferation in liver can also occur via WNT signaling and the following pathways: the network begins with oxidative stress or other mechanisms causing liver tissue injury which in turn causes (2) activation of macrophages and wound repair (Boulter et al., 2012), (3) increased hypoxia through diminished blood supply or activity of reactive oxygen species (Ju et al., 2016, Gonzalez et al., 2018) and (4) increased expression of Wnt ligands (Okabe et al., 2016). The activation of macrophages causes (5) activation of Wnt proteins and Wnt signaling (Boulter et al., 2012, Vanella and Wynn 2017). The activation and/or increased expression of Wnt signaling ligands causes (6) binding of the Wnt ligand to the co-receptors Frizzled (FZD family) and (7) Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) which then (8) recruit and phosphorylate Dishevelled (DVL1) and the scaffold protein Axin (Takigawa and Brown 2008).
The phosphorylation and recruitment of Axin (AXIN1, AXIN2), (33) inhibits formation of the beta-catenin destruction complex, composed of AXIN1 or AXIN2, adenomatosis polyposis coli (APC), beta-catenin (CTNNB1) and glycogen synthase kinase 3 (GSK3), which (10) targets beta-catenin for degradation. Inhibiting formation of the destruction complex increases the amount of available beta-catenin to (11) interact and complex with the transcription factor 7 and lymphoid enhancer-binding factor (TCF/LEF) family of transcription factors (TF7, TCF7L1, TCF7L2, LEF1; Takigawa and Brown 2008). The TCF/LEF:beta-catenin complex then (12) activates transcription of MYC proto-oncogene (MYC) and (13) cyclin D1 or CCND1 (Schuijers et al 2014;Katoh 2017). Activation of Wnt signaling (14) inhibits GSK3 phosphorylation activity which then (15) represses forkhead box M1 (FOXM1) activity, (34) causes increased turnover of CCND1 and (35) increases proteolysis of MYC (Katoh 2017; Gregory et al., 2003).
FOXM1 activates (16) transcription of MYC and (17) transcription of MAPK8, the mitogen-activated protein kinase (also known as JNK1; Wierstra and Alves 2007; Wang et al., 2008). Transcriptional activation of MAPK8 then leads to (36) transcriptional activation of CCND1 (Wang et al., 2008). Transcriptional activation of MYC causes (18) transcription of cyclin-dependent kinase 4 (CDK4) which leads to (19, 20) formation of a CDK4 and CCND1 complex (Wang et al 2011). The cyclin-CDK complex then (21) inhibits activity of the retinoblastoma (RB1) transcriptional corepressor 1 which (22) negatively regulates the cell cycle (Burkhart and Sage 2008). Dysregulation of G1/S transition by inhibition of RB1 and/or FOXM1 (23) leads to cell proliferation (Wierstra and Alves 2007; Burkhart and Sage 2008).
MYC can also be activated via hypoxia signaling where an increase in hypoxia (24) decreases the activity of oxygen sensor hypoxia-inducible factor 1 alpha inhibitor (HIF1AN) thereby reducing the ability of HIF1AN to (25) hydroxylate and inhibit hypoxia-inducible factor 1 alpha (HIF1A) activity (Whyte et al., 2012; Mahon et al., 2001). Hypoxia also can (26) inhibit activity of the von Hippel-Lindau (VHL) tumor suppressor protein which has been shown to (27) hydroxylate HIF1A in an O2 dependent manner marking HIF1A for degradation and inactivation in addition to inhibiting expression of HIF1A (Mahon et al., 2001). In stem cells, activated HIF1A (28) increases expression of TCF/LEF leading to increased expression of genes including MYC (Whyte et al., 2012; Tiburcio et al., 2014).
The long WNT Signaling Pathway Activating noncoding RNA (WSPAR) is often highly expressed in hepatocellular carcinoma cells and has been found to (29) activate expression of members of the TCF/LEF family (Zhan et al 2017). TCF/LEF transcription factors (30) increase transcription of AXIN2 and increase destruction of beta-catenin in a Wnt signaling negative feedback loop (Jho et al., 2002). TCF/LEF transcription factors form a negative feedback loop that inhibits Wnt signaling by (31) activating transcription of the dickkopf Wnt signaling pathway inhibitor 1 (DKK1) which then (32) binds to the LRP co-receptor (Takigawa and Brown 2008). Finally, cellular G1/S transition can also be dysregulated by (35) phosphorylation of RB1 by the 26S proteasome non-ATPase regulatory subunit 10 (PSMD10) which results in an increase in proteosomal degradation of RB1 (Higashitsuji et al., 2005).
How It Is Measured or Detected
- Proliferation. In vivo or in vitro cellular proliferation can be measured following a multiday 5-bromo-2'-deoxyuridine (BrdU) exposure and quantification of BrdU incorporation in DNA by immunohistochemistry. Alternatively, cells or tissue sections may be stained for Ki-67 or proliferating cell nuclear antigen (PCNA) for a snapshot of cellular proliferation. Use of BrdU, Ki-67, and PCNA in risk assessment has been described in detail (Wood, et al. 2015). A variety of commercial kits exist for this assay.
- Regeneration. Liver regeneration can be observed following partial hepatectomy. Method for 2/3 partial hepatectomy have been described (Mitchell and Willenbring 2008, Mitchell and Willenbring 2014)
- Gene expression analysis can be conducted to demonstrate increased expression of AP-1 or NF-kappaB monomers, or decreased expression of negative regulators, which can be used as an indicator that there is increased cellular proliferation in the liver.
- A BrdU-ELISA incorporation in vivo test method is also described in OECD TG 442B (The Local Lymph Node Assay, which is for skin sensitization).
Domain of Applicability
This key event has been well studied in mice, rats (Taub 2004), and zebrafish (Cox and Goessling 2015, Goessling and Sadler 2015), which are all systems that are thought to work in a similar way to human liver cell proliferation and regeneration (Kwon, et al. 2015).
Alcorn, J.A., Feitelberg, S.P., Brenner, D.A., 1990. Transient induction of c-jun during hepatic regeneration. Hepatology 11, 909-915.
Behrens, A., Sibilia, M., David, J.P., Möhle-Steinlein, U., Tronche, F., Schütz, G., Wagner, E.F., 2002. Impaired postnatal hepatocyte proliferation and liver regeneration in mice lacking c-jun in the liver. EMBO J. 21, 1782-1790.
Boulter L, Govaere O, Bird TG, Radulescu S, Ramachandran P, Pellicoro A, Ridgway RA, et al. Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease. Nat Med. 2012 Mar 4;18(4):572-9. doi: 10.1038/nm.2667.
Burkhart DL, Sage J. Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nat Rev Cancer. 2008 Sep;8(9):671-82. doi: 10.1038/nrc2399.
Cox, A.G., Goessling, W., 2015. The lure of zebrafish in liver research: regulation of hepatic growth in development and regeneration. Curr. Opin. Genet. Dev. 32, 153-161.
Cressman, D.E., Greenbaum, L.E., Haber, B.A., Taub, R., 1994. Rapid activation of post-hepatectomy factor/nuclear factor κB in hepatocytes, a primary response in the regenerating liver. J. Biol. Chem. 269, 30429-30435.
Eferl, R., Sibilia, M., Hilberg, F., Fuchsbichler, A., Kufferath, I., Guertl, B., Zenz, R., Wagner, E.F., Zatloukal, K., 1999. Functions of c-Jun in liver and heart development. J. Cell Biol. 145, 1049-1061.
FitzGerald, M.J., Webber, E.M., Donovan, J.R., Fausto, N., 1995. Rapid DNA binding by nuclear factor κB in hepatocytes at the start of liver regeneration. Cell Growth and Differentiation 6, 417-427.
Goessling, W., Sadler, K.C., 2015. Zebrafish: an important tool for liver disease research. Gastroenterology 149, 1361-1377.
Gregory MA, Qi Y, Hann SR. Phosphorylation by glycogen synthase kinase- controls c-myc proteolysis and subnuclear localization. J Biol Chem. 2003 Dec 19;278(51):51606-12. Epub 2003 Oct 16. PubMed PMID: 14563837.
Higashitsuji H, Liu Y, Mayer RJ, Fujita J. The oncoprotein gankyrin negatively regulates both p53 and RB by enhancing proteasomal degradation. Cell Cycle. 2005 Oct;4(10):1335-7. Epub 2005 Oct 17. PubMed PMID: 16177571. Jho EH, Zhang T, Domon C, Joo CK, Freund JN, Costantini F. Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol Cell Biol. 2002 Feb;22(4):1172-83 Katoh M. Canonical and non-canonical WNT signaling in cancer stem cells and their niches: Cellular heterogeneity, omics reprogramming, targeted therapy and tumor plasticity (Review). Int J Oncol. 2017 Nov;51(5):1357-1369. doi: 10.3892/ijo.2017.4129. Jochum, W., Passegué, E., Wagner, E.F., 2001. AP-1 in mouse development and tumorigenesis. Oncogene 20, 2401-2412.
Kwon, Y.J., Lee, K.G., Choi, D., 2015. Clinical implications of advances in liver regeneration. Clin. Mol. Hepatol. 21, 7-13.
Li, Q., Van Antwerp, D., Mercurio, F., Lee, K.F., Verma, I.M., 1999. Severe liver degeneration in mice lacking the IkappaB kinase 2 gene. Science 284, 321-325.
Mao, S.A., Glorioso, J.M., Nyberg, S.L., 2014. Liver regeneration. Transl. Res. 163, 352-362.
Mitchell, C., Willenbring, H., 2008. A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nat. Protoc. 3, 1167-1170.
Mitchell, C., Willenbring, H., 2014. Addendum: A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nat. Protoc. 9, 10.1038/nprot.2014.122.
OECD (2018), Test No. 442B: Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA or –FCM, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264090996-en.
Rudolph, D., Yeh, W.-., Wakeham, A., Rudolph, B., Nallainathan, D., Potter, J., Elia, A.J., Mak, T.W., 2000. Severe liver degeneration and lack of NF-κB activation in NEMO/IKK γ- deficient mice. Genes and Development 14, 854-862.
Schrum, L.W., Black, D., Iimuro, Y., Rippe, R.A., Brenner, D.A., Behrns, K.E., 2000. c-Jun does not mediate hepatocyte apoptosis following NFκB inhibition and partial hepatectomy. J. Surg. Res. 88, 142-149.
Schuijers J, Mokry M, Hatzis P, Cuppen E, Clevers H. Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF. EMBO J. 2014 Jan 13;33(2):146-56. doi: 10.1002/embj.201385358.
Stanger, B.Z., 2015. Cellular homeostasis and repair in the mammalian liver. Annu. Rev. Physiol. 77, 179-200.
Takigawa Y, Brown AM. Wnt signaling in liver cancer. Curr Drug Targets. 2008; 9:1013-24 Tiburcio PD, Choi H, Huang LE. Complex role of HIF in cancer: the known, the unknown, and the unexpected. Hypoxia (Auckl). 2014 Jun 18;2:59-70.
Vannella KM, Wynn TA. Mechanisms of Organ Injury and Repair by Macrophages. Annu Rev Physiol. 2017 Feb 10;79:593-617. doi:10.1146/annurev-physiol-022516-034356.
Wang C, Lisanti MP, Liao DJ. Reviewing once more the c-myc and Ras collaboration: converging at the cyclin D1-CDK4 complex and challenging basic concepts of cancer biology. Cell Cycle. 2011 Jan 1;10(1):57-67.
Wang IC, Chen YJ, Hughes DE, Ackerson T, Major ML, Kalinichenko VV, Costa RH, Raychaudhuri P, Tyner AL, Lau LF. FoxM1 regulates transcription of JNK1 to promote the G1/S transition and tumor cell invasiveness. J Biol Chem. 2008 Jul 25;283(30):20770-8. doi: 10.1074/jbc.M709892200.
Whyte JL, Smith AA, Helms JA. Wnt signaling and injury repair. Cold Spring Harb Perspect Biol. 2012 Aug 1;4(8):a008078. doi: 10.1101/cshperspect.a008078. Wierstra I, Alves J. FOXM1, a typical proliferation-associated transcription factor. Biol Chem. 2007 Dec;388(12):1257-74.
Wierstra I, Alves J. FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha. Biochem Biophys Res Commun. 2006 Sep15;348(1):99-108.
Wood, C.E., Hukkanen, R.R., Sura, R., Jacobson-Kram, D., Nolte, T., Odin, M., Cohen, S.M., 2015. Scientific and Regulatory Policy Committee (SRPC) Review: Interpretation and Use of Cell Proliferation Data in Cancer Risk Assessment. Toxicol. Pathol. 43, 760-775.
Yanger, K., Stanger, B.Z., 2011. Facultative stem cells in liver and pancreas: fact and fancy. Dev. Dyn. 240, 521-529.
Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:146-1473