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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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Activated HSCs express Wnt4, Wnt5a and Wnt6.(A) Products of RT-PCR for Wnt4, Wnt5a, Wnt6, Wnt7b, Wnt9b and Wnt3a in day 7 rat aHSCs, liver and brain (+ve) visualised on agarose gels using UV. Water only (-ve) serves as negative control. β-Actin serves as a loading control (B) qRT-PCR for Wnt4 and Wnt5a in culture activated rat HSCs (quiescent (qHSC) or day 7 activated HSC (aHSC) (n = 5). (C) qRT-PCR for Wnt4 and Wnt5a in rat HSCs after 0,4,6,8 and 10 days in culture, (n = 3) (D) Western Blot demonstrates increased Wnt5a expression in rat HSCs activated after 7 days in culture. Wnt3a protein expression is absent in both quiescent (qHSC) and activated (aHSC) cells. αSMA and TGFβ 1 serve as markers of HSC activation. Huh7 protein lysate serves as a positive control (E) Western Blot for Wnt5a protein in whole cell rat aHSC (Day 7) lysate and concentrated conditioned media (F) qRT-PCR for Wnt4 and Wnt5a in HSCs isolated from mouse livers injured by carbon tetrachloride injection (CCl4) or Bile Duct Ligation (BDL) (n = 3) (G) Products of RT-PCR for Wnt3a, Wnt10b and Wnt5a in 3 preparations of human HSCs visualised on agarose gels using UV. Wnt overexpressing LX-2 cells were used as a positive control (+ve). qRT-PCR results expressed as fold change normalised to control ± SEM. *p<0.05 (Student’s T-test).
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pone.0142794.g001: Activated HSCs express Wnt4, Wnt5a and Wnt6.(A) Products of RT-PCR for Wnt4, Wnt5a, Wnt6, Wnt7b, Wnt9b and Wnt3a in day 7 rat aHSCs, liver and brain (+ve) visualised on agarose gels using UV. Water only (-ve) serves as negative control. β-Actin serves as a loading control (B) qRT-PCR for Wnt4 and Wnt5a in culture activated rat HSCs (quiescent (qHSC) or day 7 activated HSC (aHSC) (n = 5). (C) qRT-PCR for Wnt4 and Wnt5a in rat HSCs after 0,4,6,8 and 10 days in culture, (n = 3) (D) Western Blot demonstrates increased Wnt5a expression in rat HSCs activated after 7 days in culture. Wnt3a protein expression is absent in both quiescent (qHSC) and activated (aHSC) cells. αSMA and TGFβ 1 serve as markers of HSC activation. Huh7 protein lysate serves as a positive control (E) Western Blot for Wnt5a protein in whole cell rat aHSC (Day 7) lysate and concentrated conditioned media (F) qRT-PCR for Wnt4 and Wnt5a in HSCs isolated from mouse livers injured by carbon tetrachloride injection (CCl4) or Bile Duct Ligation (BDL) (n = 3) (G) Products of RT-PCR for Wnt3a, Wnt10b and Wnt5a in 3 preparations of human HSCs visualised on agarose gels using UV. Wnt overexpressing LX-2 cells were used as a positive control (+ve). qRT-PCR results expressed as fold change normalised to control ± SEM. *p<0.05 (Student’s T-test).

Mentions: We began by determining the expression of Wnt ligands at the transcript level in rat aHSC (Fig 1A). We observed expression of Wnt 4, 5a and 6 while Wnts 7b, 9b or 3a could not be detected. Comparison between freshly isolated qHSC and 7-day cultured aHSC demonstrated activation-associated enhancement of Wnt4 and Wnt5a indicating that these ligands may be more highly expressed in fibrogenic aHSC (Fig 1B). A more detailed time course of rat HSC activation revealed that Wnt4 and Wnt5a transcripts progressively accumulate as the cell adopts its activated phenotype with maximal expression observed between days 6 and 10 when the cells have matured to a myofibroblastic state (Fig 1C) Western blotting for alpha smooth muscle actin (α-SMA) and TGFβ confirmed that 7-day cultured rat HSC were in an activated state while blotting for Wnt5a confirmed induction of this Wnt ligand at the protein level (Fig 1D). By comparison we did not detect expression of Wnt3a protein in either qHSC or aHSC. We were also able to identify expression of Wnt5a in the aHSC conditioned media indicative of active secretion of Wnt5a protein (Fig 1E). To corroborate these data, Wnt4 and Wnt5a mRNA were induced in ex-vivo mouse HSC isolated from either CCL4- or BDL-injured livers (Fig 1F). Finally, expression of Wnt5a, Wnt 3a and Wnt10b was determined in human HSCs. In contrast to previous studies we failed to detect significant levels of canonical Wnt3a and only low levels of Wnt10b whereas Wnt5a was readily detectable in human HSC. (Fig 1G)


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)

Activated HSCs express Wnt4, Wnt5a and Wnt6.(A) Products of RT-PCR for Wnt4, Wnt5a, Wnt6, Wnt7b, Wnt9b and Wnt3a in day 7 rat aHSCs, liver and brain (+ve) visualised on agarose gels using UV. Water only (-ve) serves as negative control. β-Actin serves as a loading control (B) qRT-PCR for Wnt4 and Wnt5a in culture activated rat HSCs (quiescent (qHSC) or day 7 activated HSC (aHSC) (n = 5). (C) qRT-PCR for Wnt4 and Wnt5a in rat HSCs after 0,4,6,8 and 10 days in culture, (n = 3) (D) Western Blot demonstrates increased Wnt5a expression in rat HSCs activated after 7 days in culture. Wnt3a protein expression is absent in both quiescent (qHSC) and activated (aHSC) cells. αSMA and TGFβ 1 serve as markers of HSC activation. Huh7 protein lysate serves as a positive control (E) Western Blot for Wnt5a protein in whole cell rat aHSC (Day 7) lysate and concentrated conditioned media (F) qRT-PCR for Wnt4 and Wnt5a in HSCs isolated from mouse livers injured by carbon tetrachloride injection (CCl4) or Bile Duct Ligation (BDL) (n = 3) (G) Products of RT-PCR for Wnt3a, Wnt10b and Wnt5a in 3 preparations of human HSCs visualised on agarose gels using UV. Wnt overexpressing LX-2 cells were used as a positive control (+ve). qRT-PCR results expressed as fold change normalised to control ± SEM. *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.g001: Activated HSCs express Wnt4, Wnt5a and Wnt6.(A) Products of RT-PCR for Wnt4, Wnt5a, Wnt6, Wnt7b, Wnt9b and Wnt3a in day 7 rat aHSCs, liver and brain (+ve) visualised on agarose gels using UV. Water only (-ve) serves as negative control. β-Actin serves as a loading control (B) qRT-PCR for Wnt4 and Wnt5a in culture activated rat HSCs (quiescent (qHSC) or day 7 activated HSC (aHSC) (n = 5). (C) qRT-PCR for Wnt4 and Wnt5a in rat HSCs after 0,4,6,8 and 10 days in culture, (n = 3) (D) Western Blot demonstrates increased Wnt5a expression in rat HSCs activated after 7 days in culture. Wnt3a protein expression is absent in both quiescent (qHSC) and activated (aHSC) cells. αSMA and TGFβ 1 serve as markers of HSC activation. Huh7 protein lysate serves as a positive control (E) Western Blot for Wnt5a protein in whole cell rat aHSC (Day 7) lysate and concentrated conditioned media (F) qRT-PCR for Wnt4 and Wnt5a in HSCs isolated from mouse livers injured by carbon tetrachloride injection (CCl4) or Bile Duct Ligation (BDL) (n = 3) (G) Products of RT-PCR for Wnt3a, Wnt10b and Wnt5a in 3 preparations of human HSCs visualised on agarose gels using UV. Wnt overexpressing LX-2 cells were used as a positive control (+ve). qRT-PCR results expressed as fold change normalised to control ± SEM. *p<0.05 (Student’s T-test).
Mentions: We began by determining the expression of Wnt ligands at the transcript level in rat aHSC (Fig 1A). We observed expression of Wnt 4, 5a and 6 while Wnts 7b, 9b or 3a could not be detected. Comparison between freshly isolated qHSC and 7-day cultured aHSC demonstrated activation-associated enhancement of Wnt4 and Wnt5a indicating that these ligands may be more highly expressed in fibrogenic aHSC (Fig 1B). A more detailed time course of rat HSC activation revealed that Wnt4 and Wnt5a transcripts progressively accumulate as the cell adopts its activated phenotype with maximal expression observed between days 6 and 10 when the cells have matured to a myofibroblastic state (Fig 1C) Western blotting for alpha smooth muscle actin (α-SMA) and TGFβ confirmed that 7-day cultured rat HSC were in an activated state while blotting for Wnt5a confirmed induction of this Wnt ligand at the protein level (Fig 1D). By comparison we did not detect expression of Wnt3a protein in either qHSC or aHSC. We were also able to identify expression of Wnt5a in the aHSC conditioned media indicative of active secretion of Wnt5a protein (Fig 1E). To corroborate these data, Wnt4 and Wnt5a mRNA were induced in ex-vivo mouse HSC isolated from either CCL4- or BDL-injured livers (Fig 1F). Finally, expression of Wnt5a, Wnt 3a and Wnt10b was determined in human HSCs. In contrast to previous studies we failed to detect significant levels of canonical Wnt3a and only low levels of Wnt10b whereas Wnt5a was readily detectable in human HSC. (Fig 1G)

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