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Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells, Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells.

Corbett L, Mann J, Mann DA - PLoS ONE (2015)

Bottom Line: We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK.Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively.We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways.

View Article: PubMed Central - PubMed

Affiliation: Fibrosis Research Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom.

ABSTRACT
The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis.

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Possible Points of Canonical Wnt Inhibition.This study has highlighted six possible points of canonical Wnt inhibition occurring in HSCs. (1) Predominantly non-canonical ligand expression. Non canonical signalling is inhibitory to canonical signalling (2) Decreased Fzd receptor expression, reducing response to canonical ligands secreted by other cells (3) Increased sFRP expression (4) Increased expression of other morphogens such as Notch, able to directly inhibit βCatenin. In the nucleus, HSCs express higher levels of the repressive TCF3 in comparision to the other TCF/LEF family members associated with transcriptional activation(5) (6) Transdifferentiation is associated with upregulation of SOX9, direct inhibitor of βCatenin activity.
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pone.0142794.g008: Possible Points of Canonical Wnt Inhibition.This study has highlighted six possible points of canonical Wnt inhibition occurring in HSCs. (1) Predominantly non-canonical ligand expression. Non canonical signalling is inhibitory to canonical signalling (2) Decreased Fzd receptor expression, reducing response to canonical ligands secreted by other cells (3) Increased sFRP expression (4) Increased expression of other morphogens such as Notch, able to directly inhibit βCatenin. In the nucleus, HSCs express higher levels of the repressive TCF3 in comparision to the other TCF/LEF family members associated with transcriptional activation(5) (6) Transdifferentiation is associated with upregulation of SOX9, direct inhibitor of βCatenin activity.

Mentions: The main conclusion from our data is that at least in vitro there is little concrete evidence in favour of a role for canonical β-catenin-dependent Wnt signalling in aHSC. Rather on the contrary, the aHSC is lacking a number of factors for this pathway to be active (Fig 8). These deficiencies include low-level autocrine production of canonical Wnt ligands, a global down-regulation of Fzd receptor genes, abundant expression of repressive sFRP proteins including sFRP4, a well-established suppressor of β-catenin-dependent signalling[16], and low-level expression of the transcriptional mediators TCF1, TCF4 and LEF1. Despite repeated attempts in LX-2 and with numerous independent cultures of primary HSC we were unable to obtain measurable levels of β-catenin-dependent Topflash activity. Surprisingly this was also the case when co-transfecting with a constitutive active β-catenin that bypasses the need for upstream signalling events. The precise deficiency in aHSC that prevents β-catenin-dependent Wnt signalling is not yet clear but may as we suggest be due to a combined low-level expression of numerous key regulatory components. Alternatively, we have shown that as HSC transdifferentiate they acquire abundant expression of hyper-phosphorylated Dvl2, which in this form negatively regulates canonical Wnt[30]. Conceptually it is not unreasonable for canonical Wnt signalling to be suppressed in aHSC given that Wnt-activated β-catenin is required for adipogenesis[31]. Highly active canonical Wnt signalling may be incompatible with HSC transdifferention and as previously reported by Kordes et al [13] is more expected to be a feature of the adipogeneic phenotype of the qHSC. Despite these observations we were able to confirm the expression of β-catenin in aHSC and its localisation to the nucleus. A likely explanation for this apparent paradox is that β-catenin might function as a signalling molecule outside of the context of the canonical Wnt pathway and in this way be required for aHSC phenotype and/or function. As an example, interactions between β-catenin and members of the TGF-β1 regulated Smad proteins have been observed indicating coupling of Wnt and Smad components that may expand the mechanisms by which these proteins regulate gene transcription[32,33]. In order to properly define functions for β-catenin in HSC and their fibrogenic activities it will be necessary to genetically manipulate the β-catenin gene in vivo and we await these future experiments with much interest.


Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells, Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells.

Corbett L, Mann J, Mann DA - PLoS ONE (2015)

Possible Points of Canonical Wnt Inhibition.This study has highlighted six possible points of canonical Wnt inhibition occurring in HSCs. (1) Predominantly non-canonical ligand expression. Non canonical signalling is inhibitory to canonical signalling (2) Decreased Fzd receptor expression, reducing response to canonical ligands secreted by other cells (3) Increased sFRP expression (4) Increased expression of other morphogens such as Notch, able to directly inhibit βCatenin. In the nucleus, HSCs express higher levels of the repressive TCF3 in comparision to the other TCF/LEF family members associated with transcriptional activation(5) (6) Transdifferentiation is associated with upregulation of SOX9, direct inhibitor of βCatenin activity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643911&req=5

pone.0142794.g008: Possible Points of Canonical Wnt Inhibition.This study has highlighted six possible points of canonical Wnt inhibition occurring in HSCs. (1) Predominantly non-canonical ligand expression. Non canonical signalling is inhibitory to canonical signalling (2) Decreased Fzd receptor expression, reducing response to canonical ligands secreted by other cells (3) Increased sFRP expression (4) Increased expression of other morphogens such as Notch, able to directly inhibit βCatenin. In the nucleus, HSCs express higher levels of the repressive TCF3 in comparision to the other TCF/LEF family members associated with transcriptional activation(5) (6) Transdifferentiation is associated with upregulation of SOX9, direct inhibitor of βCatenin activity.
Mentions: The main conclusion from our data is that at least in vitro there is little concrete evidence in favour of a role for canonical β-catenin-dependent Wnt signalling in aHSC. Rather on the contrary, the aHSC is lacking a number of factors for this pathway to be active (Fig 8). These deficiencies include low-level autocrine production of canonical Wnt ligands, a global down-regulation of Fzd receptor genes, abundant expression of repressive sFRP proteins including sFRP4, a well-established suppressor of β-catenin-dependent signalling[16], and low-level expression of the transcriptional mediators TCF1, TCF4 and LEF1. Despite repeated attempts in LX-2 and with numerous independent cultures of primary HSC we were unable to obtain measurable levels of β-catenin-dependent Topflash activity. Surprisingly this was also the case when co-transfecting with a constitutive active β-catenin that bypasses the need for upstream signalling events. The precise deficiency in aHSC that prevents β-catenin-dependent Wnt signalling is not yet clear but may as we suggest be due to a combined low-level expression of numerous key regulatory components. Alternatively, we have shown that as HSC transdifferentiate they acquire abundant expression of hyper-phosphorylated Dvl2, which in this form negatively regulates canonical Wnt[30]. Conceptually it is not unreasonable for canonical Wnt signalling to be suppressed in aHSC given that Wnt-activated β-catenin is required for adipogenesis[31]. Highly active canonical Wnt signalling may be incompatible with HSC transdifferention and as previously reported by Kordes et al [13] is more expected to be a feature of the adipogeneic phenotype of the qHSC. Despite these observations we were able to confirm the expression of β-catenin in aHSC and its localisation to the nucleus. A likely explanation for this apparent paradox is that β-catenin might function as a signalling molecule outside of the context of the canonical Wnt pathway and in this way be required for aHSC phenotype and/or function. As an example, interactions between β-catenin and members of the TGF-β1 regulated Smad proteins have been observed indicating coupling of Wnt and Smad components that may expand the mechanisms by which these proteins regulate gene transcription[32,33]. In order to properly define functions for β-catenin in HSC and their fibrogenic activities it will be necessary to genetically manipulate the β-catenin gene in vivo and we await these future experiments with much interest.

Bottom Line: We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK.Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively.We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways.

View Article: PubMed Central - PubMed

Affiliation: Fibrosis Research Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom.

ABSTRACT
The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis.

Show MeSH
Related in: MedlinePlus