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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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Extracellular interactors of Wnt are upregulated upon HSC transdifferentiation.(A) Products of RT-PCR for sFRP1, sFRP4 and Dkk3 in rat qHSCs and aHSCs. visualised on agarose gels using UV. (B) Western blot for sFRP4 in culture activated rat HSCs. (C) qRT-PCR for Wnt target genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4) (D) qRT-PCR for fibrogenic genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4) (E) qRT-PCR for adipogenic genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4). Results expressed as fold change normalised to control ± SEM; (n = 4). *p<0.05, (Student’s T-test).
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pone.0142794.g003: Extracellular interactors of Wnt are upregulated upon HSC transdifferentiation.(A) Products of RT-PCR for sFRP1, sFRP4 and Dkk3 in rat qHSCs and aHSCs. visualised on agarose gels using UV. (B) Western blot for sFRP4 in culture activated rat HSCs. (C) qRT-PCR for Wnt target genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4) (D) qRT-PCR for fibrogenic genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4) (E) qRT-PCR for adipogenic genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4). Results expressed as fold change normalised to control ± SEM; (n = 4). *p<0.05, (Student’s T-test).

Mentions: The interaction of Wnt ligands with their cell surface receptors is under tight extracellular regulation by soluble proteins that compete for their binding; these include members of the secreted frizzled-related proteins (sFRP1-5) and the dickkopf (Dkk1-4) family. HSC activation was accompanied by induction of sFRP1, sFRP4 and Dkk3 mRNAs which was detected by both semi-quantitative (Fig 3A) and quantitative (S1B Fig) assays. By contrast only low levels of sFRP2 (S1B Fig) and other Wnt antagonists (Dkk1, Dkk2, Dkk4, sFRP5 –not shown) were found in HSC with no obvious differences in expression between quiescent and activated cells. Western blot confirmed induction of sFRP4 protein with HSC activation and maintenance of its expression in fully activated myofibroblasts (Fig 3B). Of note, canonical Wnt signalling has been reported to be critical for adipogenic differentiation and to be strongly suppressed by sFRP4 [16]. Hence, culture-induced sFRP4 (and SFRP1 and Dkk3) combined with reduced surface expression of several Wnt receptors may be important for the switch from the adipogenic phenotype of qHSC to the myofibroblastic phenotype of aHSC. Treatment of LX2 cells with recombinant sFRP1 decreased expression of the fibrogenic gene TGF1β, albeit not significantly. Treatment of LX2 cells with recombinant sFRP5 demonstrated a greater effect, significantly reducing expression of Wnt targets (cMyc, CyclinD) (Fig 3C) and fibrogenic genes (Col1A1, αSMA, PDGFα)(Fig 3D). Additionally we found a repressive effect on the expression of the adipogenesis-associated genes CEBPα and CD36, however the impact of sFRP5 on the adipogenic phenotype was limited as we found no effect on LRXα, PPARγ or adipsin (Fig 3E).


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)

Extracellular interactors of Wnt are upregulated upon HSC transdifferentiation.(A) Products of RT-PCR for sFRP1, sFRP4 and Dkk3 in rat qHSCs and aHSCs. visualised on agarose gels using UV. (B) Western blot for sFRP4 in culture activated rat HSCs. (C) qRT-PCR for Wnt target genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4) (D) qRT-PCR for fibrogenic genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4) (E) qRT-PCR for adipogenic genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4). Results expressed as fold change normalised to control ± SEM; (n = 4). *p<0.05, (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.g003: Extracellular interactors of Wnt are upregulated upon HSC transdifferentiation.(A) Products of RT-PCR for sFRP1, sFRP4 and Dkk3 in rat qHSCs and aHSCs. visualised on agarose gels using UV. (B) Western blot for sFRP4 in culture activated rat HSCs. (C) qRT-PCR for Wnt target genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4) (D) qRT-PCR for fibrogenic genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4) (E) qRT-PCR for adipogenic genes in LX-2 cells treated with 5nM recombinant sFRP1 or sFRP5 (n = 4). Results expressed as fold change normalised to control ± SEM; (n = 4). *p<0.05, (Student’s T-test).
Mentions: The interaction of Wnt ligands with their cell surface receptors is under tight extracellular regulation by soluble proteins that compete for their binding; these include members of the secreted frizzled-related proteins (sFRP1-5) and the dickkopf (Dkk1-4) family. HSC activation was accompanied by induction of sFRP1, sFRP4 and Dkk3 mRNAs which was detected by both semi-quantitative (Fig 3A) and quantitative (S1B Fig) assays. By contrast only low levels of sFRP2 (S1B Fig) and other Wnt antagonists (Dkk1, Dkk2, Dkk4, sFRP5 –not shown) were found in HSC with no obvious differences in expression between quiescent and activated cells. Western blot confirmed induction of sFRP4 protein with HSC activation and maintenance of its expression in fully activated myofibroblasts (Fig 3B). Of note, canonical Wnt signalling has been reported to be critical for adipogenic differentiation and to be strongly suppressed by sFRP4 [16]. Hence, culture-induced sFRP4 (and SFRP1 and Dkk3) combined with reduced surface expression of several Wnt receptors may be important for the switch from the adipogenic phenotype of qHSC to the myofibroblastic phenotype of aHSC. Treatment of LX2 cells with recombinant sFRP1 decreased expression of the fibrogenic gene TGF1β, albeit not significantly. Treatment of LX2 cells with recombinant sFRP5 demonstrated a greater effect, significantly reducing expression of Wnt targets (cMyc, CyclinD) (Fig 3C) and fibrogenic genes (Col1A1, αSMA, PDGFα)(Fig 3D). Additionally we found a repressive effect on the expression of the adipogenesis-associated genes CEBPα and CD36, however the impact of sFRP5 on the adipogenic phenotype was limited as we found no effect on LRXα, PPARγ or adipsin (Fig 3E).

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