title: Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease.
abstract: Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (>99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMO-dependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness.
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.
authors.forename: Candice Alexandra
authors.identifier: http://orcid.org/0000-0001-8645-4297
authors.initials: CA
authors.lastname: Grzelak
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.
authors.forename: Nicholas David
authors.initials: ND
authors.lastname: Sigglekow
authors.affiliation: School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Bentley, Western Australia, Australia.
authors.forename: Janina Elke Eleonore
authors.initials: JE
authors.lastname: Tirnitz-Parker
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.
authors.forename: Elizabeth Jane
authors.initials: EJ
authors.lastname: Hamson
authors.affiliation: Ageing and Alzheimers Institute, Centre for Education and Research on Ageing, University of Sydney, Concord Hospital, Sydney, New South Wales, Australia.|ANZAC Research Institute, University of Sydney, Concord Hospital, Sydney, New South Wales, Australia.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Warren
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.|A.W. Morrow Gastroenterology and Liver Centre, R.P.A.H., Camperdown, New South Wales, Australia.
authors.forename: Bharvi
authors.initials: B
authors.lastname: Maneck
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.
authors.forename: Jinbiao
authors.initials: J
authors.lastname: Chen
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.
authors.forename: Bramilla
authors.initials: B
authors.lastname: Patkunanathan
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.
authors.forename: Jade
authors.initials: J
authors.lastname: Boland
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.
authors.forename: Robert
authors.initials: R
authors.lastname: Cheng
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.|A.W. Morrow Gastroenterology and Liver Centre, R.P.A.H., Camperdown, New South Wales, Australia.|Faculty of Medicine, University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Nicholas Adam
authors.initials: NA
authors.lastname: Shackel
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.
authors.forename: Devanshi
authors.initials: D
authors.lastname: Seth
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.|A.W. Morrow Gastroenterology and Liver Centre, R.P.A.H., Camperdown, New South Wales, Australia.
authors.forename: David Geoffrey
authors.initials: DG
authors.lastname: Bowen
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
authors.forename: Luciano Gastón
authors.initials: LG
authors.lastname: Martelotto
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute, Darlinghurst, New South Wales, Australia.
authors.forename: D Neil
authors.initials: DN
authors.lastname: Watkins
authors.affiliation: Liver Injury and Cancer, Centenary Institute, Camperdown, New South Wales, Australia.|A.W. Morrow Gastroenterology and Liver Centre, R.P.A.H., Camperdown, New South Wales, Australia.|Faculty of Medicine, University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Geoffrey William
authors.initials: GW
authors.lastname: McCaughan
mesh_terms: D000328:Adult; D000368:Aged; D002923:Cilia / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism; D005260:Female; D053823:Hedgehog Proteins / Q000235:genetics* / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism; D008107:Liver Diseases / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D000075746:Zinc Finger Protein Gli2
chemical_list: C082593:GLI2 protein, human; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; C509239:SHH protein, human; D000075746:Zinc Finger Protein Gli2