The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury

Abstract

Background & aims: 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.

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.