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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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Wnt5a stimulus influences HSC survival and expression of profibrotic markers in Kuppfer cells.(A) Rat aHSCs were treated with control or Wnt5a conditioned medium and visualised by bright field microscopy. (B) Proliferation was assessed by MTT assay (C) Acridine Orange staining was used to quantify apoptotic response upon serum withdrawal (D) Migratory potential was assessed by scratch wound assay (E) Apoptotic response in LX-2 cells overexpressing Wnt 5a orWnt10b was also assessed by acridine orange staining in standard culture conditions (10% FCS) or serum free conditions. Data presented as number of apoptotic cells as percentage of total cell number. (F) qRT-PCR for profibrotic markers in control or Wnt5a conditioned medium treated rat Kuppfer cells, (n = 3) (G) Western Blot for TGFB1 expression in control or Wnt5a conditioned medium treated Kuppfer cells. qRT-PCR results are expressed as fold change normalised to control ± SEM *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).
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pone.0142794.g007: Wnt5a stimulus influences HSC survival and expression of profibrotic markers in Kuppfer cells.(A) Rat aHSCs were treated with control or Wnt5a conditioned medium and visualised by bright field microscopy. (B) Proliferation was assessed by MTT assay (C) Acridine Orange staining was used to quantify apoptotic response upon serum withdrawal (D) Migratory potential was assessed by scratch wound assay (E) Apoptotic response in LX-2 cells overexpressing Wnt 5a orWnt10b was also assessed by acridine orange staining in standard culture conditions (10% FCS) or serum free conditions. Data presented as number of apoptotic cells as percentage of total cell number. (F) qRT-PCR for profibrotic markers in control or Wnt5a conditioned medium treated rat Kuppfer cells, (n = 3) (G) Western Blot for TGFB1 expression in control or Wnt5a conditioned medium treated Kuppfer cells. qRT-PCR results are expressed as fold change normalised to control ± SEM *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).

Mentions: Our observation that HSC secrete and respond to Wnt5a by activating Dvl2 and downstream non-canonical Wnt signalling encouraged us to further examine the functional contribution of this Wnt ligand to fibrogenesis. As before, primary rat aHSC were exposed to conditioned media from cultures of Wnt5a over-expressing LX-2 cells prior to determining effects on morphology (Fig 7A), proliferation by MTT assay (Fig 7B) and expression of PCNA and c-Myc (S8 Fig), apoptosis by acridine orange staining (Fig 7C and S9 Fig) and wound-induced migration by scratch wound assay (Fig 7D). The only phenotypic characteristic that was responsive to Wnt5a was HSC apoptosis where a modest protection to serum-starvation-induced cell death was measured (Fig 7C). We were also able to show protective effects of Wnt5a in transfected LX-2, whereas by contrast over-expression of Wnt10b appeared to stimulate apoptosis (Fig 7E). Finally, we were interested to determine if HSC-derived Wnt5a is able to exert paracrine effects on Kupffer cells (KC). To this end, primary rat KC were exposed to either control LX-2 conditioned media or to media conditioned by LX-2 transfected with Wnt5a. Measurement of transcripts for secreted inflammatory and fibrogenic mediators was performed and indicated that paracrine Wnt5a can stimulate KC expression of pro-fibrogenic TGFβ1 and monocyte chemoattractant protein 1 (MCP1) (Fig 7F). Western blot confirmed that Wnt5a induced TGFβ1 at the protein level (Fig 7G). We conclude that Wnt5a can exert autocrine control over HSC survival and stimulate the expression of soluble profibrogenic mediators by KC, this suggests that Wnt5a contributes to the persistence of fibrogenesis in the diseased liver.


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)

Wnt5a stimulus influences HSC survival and expression of profibrotic markers in Kuppfer cells.(A) Rat aHSCs were treated with control or Wnt5a conditioned medium and visualised by bright field microscopy. (B) Proliferation was assessed by MTT assay (C) Acridine Orange staining was used to quantify apoptotic response upon serum withdrawal (D) Migratory potential was assessed by scratch wound assay (E) Apoptotic response in LX-2 cells overexpressing Wnt 5a orWnt10b was also assessed by acridine orange staining in standard culture conditions (10% FCS) or serum free conditions. Data presented as number of apoptotic cells as percentage of total cell number. (F) qRT-PCR for profibrotic markers in control or Wnt5a conditioned medium treated rat Kuppfer cells, (n = 3) (G) Western Blot for TGFB1 expression in control or Wnt5a conditioned medium treated Kuppfer cells. qRT-PCR results are expressed as fold change normalised to control ± SEM *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142794.g007: Wnt5a stimulus influences HSC survival and expression of profibrotic markers in Kuppfer cells.(A) Rat aHSCs were treated with control or Wnt5a conditioned medium and visualised by bright field microscopy. (B) Proliferation was assessed by MTT assay (C) Acridine Orange staining was used to quantify apoptotic response upon serum withdrawal (D) Migratory potential was assessed by scratch wound assay (E) Apoptotic response in LX-2 cells overexpressing Wnt 5a orWnt10b was also assessed by acridine orange staining in standard culture conditions (10% FCS) or serum free conditions. Data presented as number of apoptotic cells as percentage of total cell number. (F) qRT-PCR for profibrotic markers in control or Wnt5a conditioned medium treated rat Kuppfer cells, (n = 3) (G) Western Blot for TGFB1 expression in control or Wnt5a conditioned medium treated Kuppfer cells. qRT-PCR results are expressed as fold change normalised to control ± SEM *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).
Mentions: Our observation that HSC secrete and respond to Wnt5a by activating Dvl2 and downstream non-canonical Wnt signalling encouraged us to further examine the functional contribution of this Wnt ligand to fibrogenesis. As before, primary rat aHSC were exposed to conditioned media from cultures of Wnt5a over-expressing LX-2 cells prior to determining effects on morphology (Fig 7A), proliferation by MTT assay (Fig 7B) and expression of PCNA and c-Myc (S8 Fig), apoptosis by acridine orange staining (Fig 7C and S9 Fig) and wound-induced migration by scratch wound assay (Fig 7D). The only phenotypic characteristic that was responsive to Wnt5a was HSC apoptosis where a modest protection to serum-starvation-induced cell death was measured (Fig 7C). We were also able to show protective effects of Wnt5a in transfected LX-2, whereas by contrast over-expression of Wnt10b appeared to stimulate apoptosis (Fig 7E). Finally, we were interested to determine if HSC-derived Wnt5a is able to exert paracrine effects on Kupffer cells (KC). To this end, primary rat KC were exposed to either control LX-2 conditioned media or to media conditioned by LX-2 transfected with Wnt5a. Measurement of transcripts for secreted inflammatory and fibrogenic mediators was performed and indicated that paracrine Wnt5a can stimulate KC expression of pro-fibrogenic TGFβ1 and monocyte chemoattractant protein 1 (MCP1) (Fig 7F). Western blot confirmed that Wnt5a induced TGFβ1 at the protein level (Fig 7G). We conclude that Wnt5a can exert autocrine control over HSC survival and stimulate the expression of soluble profibrogenic mediators by KC, this suggests that Wnt5a contributes to the persistence of fibrogenesis in the diseased liver.

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