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.

Neoplastic Transformation of the Peribiliary Stem Cell Niche in Cholangiocarcinoma Arisen in Primary Sclerosing Cholangitis

“…Interestingly, previous reports have suggested that the loss of primary cilia has a major role in CCA pathogenesis and that its disruption in cholangiocytes is mediated by HDAC6 expression and by autophagy (i.e., ciliophagy) . Primary cilia are emerging as an important structure in influencing the proliferation and differentiation of stem cells by the modulation of signaling pathways . Interestingly, neoplastic PBGs without primary cilia maintained high expression of Gli‐1, suggesting a noncanonical activation of the hedgehog pathway .…”

“…(38) Primary cilia are emerging as an important structure in influencing the proliferation and differentiation of stem cells by the modulation of signaling pathways. (37,39) Interestingly, neoplastic PBGs without primary cilia maintained high expression of Gli-1, suggesting a noncanonical activation of the hedgehog pathway. (39,40) Taken together, our data clearly indicate that PSC carcinogenesis is characterized by the loss of epithelial cell polarization and the acquisition of EMT traits in PBGs and associated BTSCs.…”

The hedgehog pathway in nonalcoholic fatty liver disease

“…However, some peri-portal hepatocytes exhibit nuclear Gli-2 in fatty livers, consistent with induction of Hh signaling (Fleig et al, 2007; Syn et al, 2009; Swiderska-Syn et al, 2013). It is possible that Gli-2 expression in those hepatocytes might have resulted from non-canonical (i.e., Smo- independent) activation of Gli-2 (Grzelak et al, 2014; Grzelak et al, 2017) by factors other than Hh ligands, such as TGF-β (Dennler et al, 2007; Johnson et al, 2011). Alternatively, the Gli-2-positive hepatocytes might be the immature progeny of Hh-responsive progenitor cells that still retain PC that permit ligand-depended canonical activation of the Hh pathway (Ochoa et al, 2010).…”

“…Further, Gli-expressing hepatocytes have been shown to release other Hh-inducible factors, such as osteopontin, that promote inflammation and fibrogenesis (Kwon et al, 2016). Hepatic progenitor cells strongly react to canonical Hh pathway activation with increased proliferation and viability but decreased hepatocytic differentiation (Sicklick et al, 2006; Fleig et al, 2007; Syn et al, 2009; Grzelak et al, 2017; Jung et al, 2010). Hh-responsive progenitor cells also release many factors that can promote the wound-healing response, such as platelet-derived growth factor (PDGF) and osteopontin which activate hepatic stellate cells (Omenetti et al, 2008; Syn et al, 2011), and proinflammatory chemokines (for example, CXCL16 and osteopontin) (Omenetti et al, 2009) which recruit immune cells to the liver.…”

“…In all models, hepatic Hh pathway activation has been observed consistently. Similarly, in human NASH the Hh pathway is activated and the level of pathway activity correlates with the severity of liver damage (as assessed by numbers of ballooned hepatocytes, levels of portal inflammation, markers of liver repair (aka accumulation of progenitor cells and activated myofibroblasts), and fibrosis severity) (Guy et al, 2012; Grzelak et al, 2017; Syn et al, 2009; Machado et al, 2015). …”