title: The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury.
abstract: OBJECTIVE
  In vertebrates, canonical Hedgehog (Hh) pathway activation requires Smoothened (SMO) translocation to the primary cilium (Pc), followed by a GLI-mediated transcriptional response. In addition, a similar gene regulation occurs in response to growth factors/cytokines, although independently of SMO signalling. The Hh pathway plays a critical role in liver fibrosis/regeneration, however, the mechanism of activation in chronic liver injury is poorly understood. This study aimed to characterise Hh pathway activation upon thioacetamide (TAA)-induced chronic liver injury in vivo by defining Hh-responsive cells, namely cells harbouring Pc and Pc-localised SMO.
  
  
  METHODS
  C57BL/6 mice (wild-type or Ptc1(+/-)) were TAA-treated. Liver injury and Hh ligand/pathway mRNA and protein expression were assessed in vivo. SMO/GLI manipulation and SMO-dependent/independent activation of GLI-mediated transcriptional response in Pc-positive (Pc(+)) cells were studied in vitro.
  
  
  RESULTS
  In vivo, Hh activation was progressively induced following TAA. At the epithelial-mesenchymal interface, injured hepatocytes produced Hh ligands. Progenitors, myofibroblasts, leukocytes and hepatocytes were GLI2(+). Pc(+) cells increased following TAA, but only EpCAM(+)/GLI2(+) progenitors were Pc(+)/SMO(+). In vitro, SMO knockdown/hGli3-R overexpression reduced proliferation/viability in Pc(+) progenitors, whilst increased proliferation occurred with hGli1 overexpression. HGF induced GLI transcriptional activity independently of Pc/SMO. Ptc1(+/-) mice exhibited increased progenitor, myofibroblast and fibrosis responses.
  
  
  CONCLUSIONS
  In chronic liver injury, Pc(+) progenitors receive Hh ligand signals and process it through Pc/SMO-dependent activation of GLI-mediated transcriptional response. Pc/SMO-independent GLI activation likely occurs in Pc(-)/GLI2(+) cells. Increased fibrosis in Hh gain-of-function mice likely occurs by primary progenitor expansion/proliferation and secondary fibrotic myofibroblast expansion, in close contact with progenitors.
authors: Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia.
authors: Candice Alexandra
authors: CA
authors: Grzelak
authors: Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia; Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors: Luciano Gastón
authors: LG
authors: Martelotto
authors: Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia.
authors: Nicholas David
authors: ND
authors: Sigglekow
authors: Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia.
authors: Bramilla
authors: B
authors: Patkunanathan
authors: Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia.
authors: Katerina
authors: K
authors: Ajami
authors: Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia.
authors: Sarah Ruth
authors: SR
authors: Calabro
authors: School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, Australia.
authors: Benjamin James
authors: BJ
authors: Dwyer
authors: School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, Australia.
authors: Janina Elke Eleonore
authors: JE
authors: Tirnitz-Parker
authors: Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia.
authors: D Neil
authors: DN
authors: Watkins
authors: Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia.
authors: Fiona Jane
authors: FJ
authors: Warner
authors: Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia; A. W. Morrow Gastroenterology and Liver Centre, R.P.A.H., Camperdown, NSW, Australia; Faculty of Medicine, University of Sydney, Sydney, NSW, Australia.
authors: Nicholas Adam
authors: NA
authors: Shackel
authors: Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia; A. W. Morrow Gastroenterology and Liver Centre, R.P.A.H., Camperdown, NSW, Australia; Faculty of Medicine, University of Sydney, Sydney, NSW, Australia. Electronic address: g.mccaughan@centenary.org.au.
authors: Geoffrey William
authors: GW
authors: McCaughan
mesh_terms: D000818:Animals; D056486:Chemical and Drug Induced Liver Injury / Q000473:pathology*; D002908:Chronic Disease; D002923:Cilia / Q000502:physiology*; D058750:Epithelial-Mesenchymal Transition; D053823:Hedgehog Proteins / Q000502:physiology*; D051741:Kruppel-Like Transcription Factors / Q000032:analysis / Q000502:physiology; D008099:Liver / Q000473:pathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins / Q000032:analysis; D043562:Receptors, G-Protein-Coupled / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D000072086:Smoothened Receptor; D013853:Thioacetamide; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2
chemical_list: C082593:GLI2 protein, human; C498520:Gli1 protein, mouse; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D043562:Receptors, G-Protein-Coupled; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D013853:Thioacetamide
keywords: ALD; ALT; CK; DHH; Desert Hedgehog; EGF; EMT; EpCAM; GLI; GLI cleaved repressor; GLI full-length activator; GLI-A; GLI-Kruppel family of transcription factors; GLI-R; HGF; HSC; Hedgehog; Hedgehog signalling pathway; Hh; IHH; Indian Hedgehog; LPC; Liver progenitor cells; MCDE; N-Hh; N-terminal Hedgehog signalling peptide; NT2; Non-canonical cell signalling; PTCH1; Patched 1; Pc; Primary cilia; Ptc1(+/−); Ptc1-lacZ reporter; SHH; SMO; Smoothened; Sonic Hedgehog; TAA; Thioacetamide; alanine aminotransferase; alcoholic liver disease; cytokeratin; epidermal growth factor; epithelial cell adhesion molecule; epithelial-to-mesenchymal transition; hepatic stellate cell; hepatocyte growth factor; liver progenitor cell; methionine choline-deficient diet+ethionine; non-targeting control; primary cilia; thioacetamide; α-SMA; α-smooth muscle actin