Original Article
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World J Gastroenterol. Dec 21, 2014; 20(47): 17851-17862
Published online Dec 21, 2014. doi: 10.3748/wjg.v20.i47.17851
Senescent human hepatocytes express a unique secretory phenotype and promote macrophage migration
Katharine M Irvine, Richard Skoien, Nilesh J Bokil, Michelle Melino, Gethin P Thomas, Dorothy Loo, Brian Gabrielli, Michelle M Hill, Matthew J Sweet, Andrew D Clouston, Elizabeth E Powell
Katharine M Irvine, Richard Skoien, Michelle Melino, Andrew D Clouston, Elizabeth E Powell, Centre for Liver Disease Research, School of Medicine, The University of Queensland, Translational Research Institute, Brisbane 4102, Australia
Nilesh J Bokil, Matthew J Sweet, Institute for Molecular Bioscience, The University of Queensland, Brisbane 4067, Australia
Gethin P Thomas, Dorothy Loo, Brian Gabrielli, Michelle M Hill, The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane 4102, Australia
Elizabeth E Powell, Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane 4102, Australia
Author contributions: Irvine KM, Skoien R, Sweet MJ, Clouston AD and Powell EE designed the research; Irvine KM, Skoien R, Bokil NJ, Melino M, Thomas GP and Loo D performed the research; Sweet MJ, Gabrielli B and Hill MM contributed new reagents and tools; Irvine KM, Skoein R, Bokil NJ and Hill MM analysed the data; Irvine KM, Skoein R and Powell EE wrote the paper.
Supported by the National Health and Medical Research Council of Australia (NHMRC), APP1044650 and APP1003108; the Queensland Government’s Smart State Health and Medical Research Fund; the Princess Alexandra Hospital Research and Development Foundation and The Australian Liver Foundation; Irvine KM is the recipient of the Australian Liver Foundation Pauline Hall Fellowship; Powell EE is the recipient of an NHMRC Practitioner Fellowship, APP1004242; Sweet MJ is the recipient of an Australian Research Council (ARC) Future Fellowship, FT100100657; and an honorary NHMRC Senior Research Fellowship, APP1003470; Hill MM is the recipient of an ARC Future Fellowship, FT120100251
Correspondence to: Elizabeth E Powell, Professor, Director, Centre for Liver Disease Research, School of Medicine, The University of Queensland, Translational Research Institute, Brisbane, 37 Kent St, Woolloongabba, Brisbane 4102, Australia. e.powell@uq.edu.au
Telephone: +61-7-34438015 Fax: +61-7-34437779
Received: April 30, 2014
Revised: June 13, 2014
Accepted: July 16, 2014
Published online: December 21, 2014
Processing time: 234 Days and 9.4 Hours
Abstract

AIM: To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype (SASP).

METHODS: Hydrogen peroxide treatment was used to induce senescence in the human HepG2 hepatocyte cell line. Senescence was confirmed by cytochemical staining for a panel of markers including Ki67, p21, heterochromatin protein 1β, and senescence-associated-β-galactosidase activity. Senescent hepatocytes were characterised by gene expression arrays and quantitative polymerase chain reaction (qPCR), and conditioned media was used in proteomic analyses, a human chemokine protein array, and cell migration assays to characterise the composition and function of the hepatocyte SASP.

RESULTS: Senescent hepatocytes induced classical markers of senescence (p21, heterochromatin protein 1β, and senescence-associated-β-galactosidase activity); and downregulated the proliferation marker, Ki67. Hepatocyte senescence induced a 4.6-fold increase in total secreted protein (P = 0.06) without major alterations in the protein profile. Senescence-induced genes were identified by microarray (Benjamini Hochberg-corrected P < 0.05); and, consistent with the increase in secreted protein, gene ontology analysis revealed a significant enrichment of secreted proteins among inducible genes. The hepatocyte SASP included characteristic factors such as interleukin (IL)-8 and IL-6, as well as novel components such as SAA4, IL-32 and Fibrinogen, which were validated by qPCR and/or chemokine protein array. Senescent hepatocyte-conditioned medium elicited migration of inflammatory (granulocyte-macrophage colony stimulating factor, GM-CSF-derived), but not non-inflammatory (CSF-1-derived) human macrophages (P = 0.022), which could contribute to a pro-inflammatory microenvironment in vivo, or facilitate the clearance of senescent cells.

CONCLUSION: Our novel model of hepatocyte senescence provides insights into mechanisms by which senescent hepatocytes may promote chronic liver disease pathogenesis.

Keywords: Cell aging; Chemokines; Hepatocytes; Inflammation; Liver diseases; Macrophages

Core tip: Hepatocyte senescence is observed in all chronic liver diseases of hepatocellular origin, even at early stages of disease progression. Although widely studied in cancer biology, the role of cellular senescence in the pathogenesis of inflammatory diseases is not known. We developed a novel model of stress-induced hepatocyte senescence and used it to demonstrate that senescent human hepatocytes adopt a hyper-secretory phenotype, which is likely to condition their microenvironment and contribute to disease pathogenesis. We used microarray and proteomic analysis to characterise senescent hepatocytes and identify candidate mediators; and confirmed the functional relevance of senescence-associated secretory phenotype by demonstrating that conditioned media from senescent hepatocytes elicits inflammatory macrophage migration.