title: Senescent human hepatocytes express a unique secretory phenotype and promote macrophage migration.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  Our novel model of hepatocyte senescence provides insights into mechanisms by which senescent hepatocytes may promote chronic liver disease pathogenesis.
authors.affiliation: 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.
authors.forename: Katharine M
authors.initials: KM
authors.lastname: Irvine
authors.affiliation: 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.
authors.forename: Richard
authors.initials: R
authors.lastname: Skoien
authors.affiliation: 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.
authors.forename: Nilesh J
authors.initials: NJ
authors.lastname: Bokil
authors.affiliation: 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.
authors.forename: Michelle
authors.initials: M
authors.lastname: Melino
authors.affiliation: 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.
authors.forename: Gethin P
authors.initials: GP
authors.lastname: Thomas
authors.affiliation: 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.
authors.forename: Dorothy
authors.initials: D
authors.lastname: Loo
authors.affiliation: 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.
authors.forename: Brian
authors.initials: B
authors.lastname: Gabrielli
authors.affiliation: 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.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Hill
authors.affiliation: 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.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Sweet
authors.affiliation: 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.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Clouston
authors.affiliation: 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.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Powell
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D059447:Cell Cycle Checkpoints; D016922:Cellular Senescence / Q000187:drug effects; D002633:Chemotaxis; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018836:Inflammation Mediators / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D018384:Oxidative Stress; D019899:Paracrine Communication; D010641:Phenotype; D059467:Transcriptome
chemical_list: D015415:Biomarkers; D017077:Culture Media, Conditioned; D016207:Cytokines; D018836:Inflammation Mediators; D006861:Hydrogen Peroxide
keywords: Cell aging; Chemokines; Hepatocytes; Inflammation; Liver diseases; Macrophages