Reply to: “The many faces of Hedgehog signalling in the liver: Recent progress reveals striking cellular diversity and the importance of microenvironments”
Authors: Candice Alexandra Grzelak, Nicholas David Sigglekow, D. Neil Watkins, Geoffrey William McCaughan
Journal: Journal of Hepatology (2014)

The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injuryJournal of HepatologyVol. 60Issue 1PreviewIn 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. Full-Text PDF The many faces of Hedgehog signalling in the liver: Recent progress reveals striking cellular diversity and the importance of microenvironmentsJournal of HepatologyVol. 61Issue 6PreviewWe have read with great interest the work of Grzelak and colleagues. The authors report the occurrence of primary cilia-positive cell populations and have characterised detailed features of Hedgehog (Hh) signalling during chronic liver injury, induced by thioacetamide (TAA) [1]. They describe an attractive scenario for driving fibrosis and repair in response to TAA treatment based on local niches and two signalling routes: (i) the canonical pathway involving primary cilia and SMO and (ii) an apparently SMO- and primary cilium-independent pathway. Full-Text PDF Open Access We thank Matz-Soja and colleagues for their interest and critique of our article [[1]Grzelak C.A. Martelotto L.G. Sigglekow N.D. Patkunanathan B. Ajami K. Calabro S.R. et al.The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury.J Hepatol. 2014; 60: 143-151Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. We agree with Matz-Soja et al. that generalisations regarding the contribution of Smoothened (SMO)-dependent and SMO-independent GLI-mediated signals during liver injury processes should be made with caution. We concur that more work is required to delineate how, within various liver cell niches, Hedgehog (Hh) pathway signalling components elicit canonical or ‘non-canonical’ Hh signalling responses during liver injury.The focus of our article was to highlight the assumption that GLI2 expression alone as an accurate indicator of canonical Hh pathway activity in the liver, may be an oversimplification. Data from our in vivo model suggest that canonical SMO-dependent GLI-mediated signals can occur within primary cilia (Pc) positive/GLI2 positive liver progenitor cell (LPC) populations, while in Pc negative/GLI2 positive populations GLI-mediated signals may be driven in a SMO-independent manner.Hh signalling pathway components coordinate diverse responses (both GLI-dependent and GLI-independent), categorised into five specific pathways (Table 1), recently reviewed [2Aberger F. Ruiz I.A.A. Context-dependent signal integration by the GLI code: the oncogenic load, pathways, modifiers and implications for cancer therapy.Semin Cell Dev Biol. 2014; 33C: 93-104Crossref Scopus (120) Google Scholar, 3Teperino R. Aberger F. Esterbauer H. Riobo N. Pospisilik J.A. Canonical and non-canonical Hedgehog signalling and the control of metabolism.Semin Cell Dev Biol. 2014; 33C: 81-92Crossref Scopus (89) Google Scholar]. Table 1 illustrates the complexity of Hh pathway signalling; a comprehensive understanding of Hh signalling processes within the liver would require that all five aspects be addressed. In our paper, we specifically studied canonical Hh (SMO-dependent GLI-mediated) signalling in vivo. Our results also point towards a role for SMO-independent GLI-mediated signalling involvement in chronic liver injury. We acknowledge that we have not incorporated type I (PTCH1) or type II (SMO-dependent GLI-independent) non-canonical Hh signals within our working model (see Table 1), which does not suggest the irrelevance of these in chronic liver injury. Indeed, Matz-Soja recently showed the importance of SMO-dependent GLI-independent signals within hepatocytes [[4]Matz-Soja M. Aleithe S. Marbach E. Bottger J. Arnold K. Schmidt-Heck W. et al.Hepatic Hedgehog signaling contributes to the regulation of IGF1 and IGFBP1 serum levels.Cell Commun Signal. 2014; 12: 11Crossref PubMed Scopus (37) Google Scholar]. However, within our current understanding GLI is either activated via Pc/SMO, or through multiple SMO-independent mechanisms such as PI3K/AKT, S6K, RAS, TGFβ/SMAD, MEK/ERK, RTK, and EWS/FLI1 (reviewed in [[3]Teperino R. Aberger F. Esterbauer H. Riobo N. Pospisilik J.A. Canonical and non-canonical Hedgehog signalling and the control of metabolism.Semin Cell Dev Biol. 2014; 33C: 81-92Crossref Scopus (89) Google Scholar]). Currently, there is no evidence of Hh/PTCH1/SMO/GLI ‘canonical’ signalling occurring independently of the Pc.Table 1Summary of known canonical and non-canonical Hedgehog signalling cascades. Open table in a new tab We are perplexed by a few conclusions drawn by Matz-Soja et al. from the current literature. Matz-Soja et al. wrote, ‘besides canonical (cilia- and SMO-dependent) signalling in endothelial cells, cholangiocytes, activated hepatic stellate cells (HSC) and progenitor cells, there is obviously cilia-independent but SMO-dependent signalling in mature healthy hepatocytes, Kupffer cells and, most probably, quiescent HSCs.’ The conclusion that canonical Hh signalling occurs in liver endothelial cells (ECs) and activated HSCs has been drawn prematurely, as the presence of Pc (required for canonical Hh signalling) was not evaluated in the study referenced [[5]Michelotti G.A. Xie G. Swiderska M. Choi S.S. Karaca G. Kruger L. et al.Smoothened is a master regulator of adult liver repair.J Clin Invest. 2013; 123: 2380-2394PubMed Google Scholar] or in previous studies.The presence of Pc on HSCs is debatable. Confusingly, Matz-Soja et al. inferred that activated but not quiescent HSCs express Pc. Our data from TAA-induced chronic liver injury demonstrate that vimentin positive HSCs are Pc negative, suggesting that activated HSCs do not express a Pc, at least in mice. Further, a recent publication suggested myofibroblasts disassemble Pc during activation in vitro [[6]Rozycki M. Lodyga M. Lam J. Miranda M.Z. Fatyol K. Speight P. et al.The fate of the primary cilium during myofibroblast transition.Mol Biol Cell. 2014; 25: 643-657Crossref PubMed Scopus (24) Google Scholar]. In human liver however, a minority of HSCs can assemble Pc under certain circumstances (http://www.bowserlab.org/primarycilia/cilialist.html) and we have observed Pc on a minority of vimentin positive HSCs in human alcoholic liver disease (unpublished data). Other electron microscopy (EM) studies have also reported <5% HSCs express a Pc in human cirrhotic injury [[7]Tobe K. Tsuchiya T. Itoshima T. Nagashima H. Kobayashi T. Electron microscopy of fat-storing cells in liver diseases with special reference to cilia and cytoplasmic cholesterol crystals.Arch Histol Jpn. 1985; 48: 435-441Crossref PubMed Scopus (9) Google Scholar].The concern over the use of pan Hh antibodies is unwarranted, as the pan Hh antibody used detects both Sonic Hh (SHH) and Indian Hh (IHH). Ligand detection was corroborated separately with in situ Shh mRNA and IHH ligand protein detection. IHH positive ballooned hepatocytes were denoted as ‘damaged’ on pathologist recommendations, due to altered cell morphology and significantly increased AST/ALT serum levels in 20 week TAA mice, in keeping with previous studies that identify damaged hepatocytes as a source of ligand [[8]Rangwala F. Guy C.D. Lu J. Suzuki A. Burchette J.L. Abdelmalek M.F. et al.Increased production of sonic hedgehog by ballooned hepatocytes.J Pathol. 2011; 224: 401-410Crossref PubMed Scopus (122) Google Scholar].We note that Fig. 1 of the ‘Letter to the Editor’ inaccurately references our data with regard to LPC’s ‘known’ involvement in epithelial-to-mesenchymal transition (EMT). In actuality, we observed and described paracrine epithelial-epithelial (hepatocyte-LPC) canonical Hh signalling.In summary, future research in this field should be focused towards dissecting out the contribution of all five potential Hh signalling responses. This includes identification of Hh ligand, Pc, SMO and GLI protein source(s) in vivo, coincident expression of Hh co-receptors (CDON, BOC, and GAS1), and the Hh pathway activation state (ratio of GLI-A:GLI-R, and GLI1 Hh target gene expression) itself. These in vivo analyses combined with functional analysis of cellular proliferation, viability, migration, apoptosis, cell cycle progression, calcium signalling and metabolism will allow future studies to properly delineate what combination of Hh signal(s) occur within various liver cell niches. This will aid in addressing many of the current unknowns within the field. We thank the authors for their interest and comments and hope we have adequately addressed their concerns.Conflict of interestThe authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. We thank Matz-Soja and colleagues for their interest and critique of our article [[1]Grzelak C.A. Martelotto L.G. Sigglekow N.D. Patkunanathan B. Ajami K. Calabro S.R. et al.The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury.J Hepatol. 2014; 60: 143-151Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. We agree with Matz-Soja et al. that generalisations regarding the contribution of Smoothened (SMO)-dependent and SMO-independent GLI-mediated signals during liver injury processes should be made with caution. We concur that more work is required to delineate how, within various liver cell niches, Hedgehog (Hh) pathway signalling components elicit canonical or ‘non-canonical’ Hh signalling responses during liver injury. The focus of our article was to highlight the assumption that GLI2 expression alone as an accurate indicator of canonical Hh pathway activity in the liver, may be an oversimplification. Data from our in vivo model suggest that canonical SMO-dependent GLI-mediated signals can occur within primary cilia (Pc) positive/GLI2 positive liver progenitor cell (LPC) populations, while in Pc negative/GLI2 positive populations GLI-mediated signals may be driven in a SMO-independent manner. Hh signalling pathway components coordinate diverse responses (both GLI-dependent and GLI-independent), categorised into five specific pathways (Table 1), recently reviewed [2Aberger F. Ruiz I.A.A. Context-dependent signal integration by the GLI code: the oncogenic load, pathways, modifiers and implications for cancer therapy.Semin Cell Dev Biol. 2014; 33C: 93-104Crossref Scopus (120) Google Scholar, 3Teperino R. Aberger F. Esterbauer H. Riobo N. Pospisilik J.A. Canonical and non-canonical Hedgehog signalling and the control of metabolism.Semin Cell Dev Biol. 2014; 33C: 81-92Crossref Scopus (89) Google Scholar]. Table 1 illustrates the complexity of Hh pathway signalling; a comprehensive understanding of Hh signalling processes within the liver would require that all five aspects be addressed. In our paper, we specifically studied canonical Hh (SMO-dependent GLI-mediated) signalling in vivo. Our results also point towards a role for SMO-independent GLI-mediated signalling involvement in chronic liver injury. We acknowledge that we have not incorporated type I (PTCH1) or type II (SMO-dependent GLI-independent) non-canonical Hh signals within our working model (see Table 1), which does not suggest the irrelevance of these in chronic liver injury. Indeed, Matz-Soja recently showed the importance of SMO-dependent GLI-independent signals within hepatocytes [[4]Matz-Soja M. Aleithe S. Marbach E. Bottger J. Arnold K. Schmidt-Heck W. et al.Hepatic Hedgehog signaling contributes to the regulation of IGF1 and IGFBP1 serum levels.Cell Commun Signal. 2014; 12: 11Crossref PubMed Scopus (37) Google Scholar]. However, within our current understanding GLI is either activated via Pc/SMO, or through multiple SMO-independent mechanisms such as PI3K/AKT, S6K, RAS, TGFβ/SMAD, MEK/ERK, RTK, and EWS/FLI1 (reviewed in [[3]Teperino R. Aberger F. Esterbauer H. Riobo N. Pospisilik J.A. Canonical and non-canonical Hedgehog signalling and the control of metabolism.Semin Cell Dev Biol. 2014; 33C: 81-92Crossref Scopus (89) Google Scholar]). Currently, there is no evidence of Hh/PTCH1/SMO/GLI ‘canonical’ signalling occurring independently of the Pc. We are perplexed by a few conclusions drawn by Matz-Soja et al. from the current literature. Matz-Soja et al. wrote, ‘besides canonical (cilia- and SMO-dependent) signalling in endothelial cells, cholangiocytes, activated hepatic stellate cells (HSC) and progenitor cells, there is obviously cilia-independent but SMO-dependent signalling in mature healthy hepatocytes, Kupffer cells and, most probably, quiescent HSCs.’ The conclusion that canonical Hh signalling occurs in liver endothelial cells (ECs) and activated HSCs has been drawn prematurely, as the presence of Pc (required for canonical Hh signalling) was not evaluated in the study referenced [[5]Michelotti G.A. Xie G. Swiderska M. Choi S.S. Karaca G. Kruger L. et al.Smoothened is a master regulator of adult liver repair.J Clin Invest. 2013; 123: 2380-2394PubMed Google Scholar] or in previous studies. The presence of Pc on HSCs is debatable. Confusingly, Matz-Soja et al. inferred that activated but not quiescent HSCs express Pc. Our data from TAA-induced chronic liver injury demonstrate that vimentin positive HSCs are Pc negative, suggesting that activated HSCs do not express a Pc, at least in mice. Further, a recent publication suggested myofibroblasts disassemble Pc during activation in vitro [[6]Rozycki M. Lodyga M. Lam J. Miranda M.Z. Fatyol K. Speight P. et al.The fate of the primary cilium during myofibroblast transition.Mol Biol Cell. 2014; 25: 643-657Crossref PubMed Scopus (24) Google Scholar]. In human liver however, a minority of HSCs can assemble Pc under certain circumstances (http://www.bowserlab.org/primarycilia/cilialist.html) and we have observed Pc on a minority of vimentin positive HSCs in human alcoholic liver disease (unpublished data). Other electron microscopy (EM) studies have also reported <5% HSCs express a Pc in human cirrhotic injury [[7]Tobe K. Tsuchiya T. Itoshima T. Nagashima H. Kobayashi T. Electron microscopy of fat-storing cells in liver diseases with special reference to cilia and cytoplasmic cholesterol crystals.Arch Histol Jpn. 1985; 48: 435-441Crossref PubMed Scopus (9) Google Scholar]. The concern over the use of pan Hh antibodies is unwarranted, as the pan Hh antibody used detects both Sonic Hh (SHH) and Indian Hh (IHH). Ligand detection was corroborated separately with in situ Shh mRNA and IHH ligand protein detection. IHH positive ballooned hepatocytes were denoted as ‘damaged’ on pathologist recommendations, due to altered cell morphology and significantly increased AST/ALT serum levels in 20 week TAA mice, in keeping with previous studies that identify damaged hepatocytes as a source of ligand [[8]Rangwala F. Guy C.D. Lu J. Suzuki A. Burchette J.L. Abdelmalek M.F. et al.Increased production of sonic hedgehog by ballooned hepatocytes.J Pathol. 2011; 224: 401-410Crossref PubMed Scopus (122) Google Scholar]. We note that Fig. 1 of the ‘Letter to the Editor’ inaccurately references our data with regard to LPC’s ‘known’ involvement in epithelial-to-mesenchymal transition (EMT). In actuality, we observed and described paracrine epithelial-epithelial (hepatocyte-LPC) canonical Hh signalling. In summary, future research in this field should be focused towards dissecting out the contribution of all five potential Hh signalling responses. This includes identification of Hh ligand, Pc, SMO and GLI protein source(s) in vivo, coincident expression of Hh co-receptors (CDON, BOC, and GAS1), and the Hh pathway activation state (ratio of GLI-A:GLI-R, and GLI1 Hh target gene expression) itself. These in vivo analyses combined with functional analysis of cellular proliferation, viability, migration, apoptosis, cell cycle progression, calcium signalling and metabolism will allow future studies to properly delineate what combination of Hh signal(s) occur within various liver cell niches. This will aid in addressing many of the current unknowns within the field. We thank the authors for their interest and comments and hope we have adequately addressed their concerns. Conflict of interestThe authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.