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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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Expression of TCF/LEF transcription factors appears reduced in LX-2 cells.(A) Western Blot for TCF1, TCF3, TCF4 and LEF1in separate samples of LX-2 and HEK293 cells (B) TOPFLASH assay in HEK293 cells after 24hours incubation with conditioned medium from either HEK293 or LX-2 cells, (n = 4). (C) TOPFLASH assay in LX-2 cells overexpressing LEF1 and Ser37-βcatenin (n = 3) (D) TOPFLASH assay in HEK293 cells overexpressing Ser37-βCatenin or Ser37-βCatenin and sFRP4, (n = 3). Luciferase results represented as fold change of Firefly to Renilla *p<0.05 (Student’s t-test).
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pone.0142794.g005: Expression of TCF/LEF transcription factors appears reduced in LX-2 cells.(A) Western Blot for TCF1, TCF3, TCF4 and LEF1in separate samples of LX-2 and HEK293 cells (B) TOPFLASH assay in HEK293 cells after 24hours incubation with conditioned medium from either HEK293 or LX-2 cells, (n = 4). (C) TOPFLASH assay in LX-2 cells overexpressing LEF1 and Ser37-βcatenin (n = 3) (D) TOPFLASH assay in HEK293 cells overexpressing Ser37-βCatenin or Ser37-βCatenin and sFRP4, (n = 3). Luciferase results represented as fold change of Firefly to Renilla *p<0.05 (Student’s t-test).

Mentions: To determine the ability of aHSC to respond to canonical Wnt signalling, human LX-2 were co-transfected with the canonical Wnt luciferase reporter Topflash and expression vectors for Wnt3a and Wnt10b. To control these assays similar transfections were carried out in HEK293 cells which are known to support β-catenin-dependent Wnt signalling[17]. As anticipated co-transfection of the canonical Wnts activated the Topflash reporter in HEK293 cells, however no reporter activity was detected in LX-2 cells transfected with either Wnt3a or Wnt10b (Fig 4A). In addition, transfection of Wnt10b failed to induce expression of the Wnt target gene Axin2 in LX-2 which by contrast was up-regulated 3-fold by Wnt10b transfection in HEK293 cells (Fig 4B). Over-expression of a constitutive active mutant β-catenin (S2 Fig) induced Topflash activity by 40-fold in HEK293 cells (Fig 4C) and enhanced the expression of endogenous Axin2, cMyc and Cyclin D (Fig 4D). However, neither the reporter nor endogenous Axin2, cMyc and Cyclin D were stimulated in LX-2 cells, this confirming that the human HSC cell line does not support canonical Wnt signalling (Fig 4C and 4D). Expression of constitutive active β-catenin induced Topflash in primary rat dermal fibroblasts but was inactive in primary rat aHSC (Fig 4E). These data were indicative of defective β-catenin-dependent Wnt signalling in HSC. To investigate this further, rat HSC protein extracts were probed by immunoblot for expression of active (de-phosphorylated) and total β-catenin (Fig 4F) Freshly isolated qHSC lacked expression of active β-catenin, whereas HSC cultured for 7 days expressed abundant levels of active β-catenin such that pharmacological activation with a GSK3β inhibitor (CT99021) made little difference to the amount of active protein detected. Furthermore, active β-catenin was readily detected in the HSC nucleus, this ruling out a defect in nuclear translocation (S3 Fig). Once in the nucleus, β-catenin interacts with a variety of different proteins that impact on its stability and its ability to stimulate target gene transcription[18,19]. Scaffold proteins (Pyg1, Pyg2, BCL9, BCL92) that tether β-catenin to its transcription factor partners the TCF/LEF proteins, were all expressed at the mRNA level in LX-2 and HEK293 (Fig 4G) as was the nuclear repressor of β-catenin Chibby, which in primary rat HSC was found to undergo a dramatic diminution in expression with culture-induced activation (Fig 4H). The transcriptional activation of β-catenin-target genes is dependent on its interaction with TCF/LEF transcription factors [20]. Comparison of the expression of TCF/LEF proteins between HEK293 and LX-2 cells indicated a very low level of TCF1 and LEF1 in the HSC line and decreased levels of TCF4, by contrast TCF3 was expressed to a similar level between HEK293 and LX-2 (Fig 5A). These data were of particular interest since TCF1, TCF4 and LEF1 are generally considered to be activators of β-catenin-dependent transcription whereas TCF3 may have a more repressive function[21,22]. However, over-expression of LEF1 did not enable LX-2 to support transcriptional activity of co-transfected constitutive active β-catenin (Fig 5B). This result indicates that aHSC may establish multiple regulatory checkpoints to ensure blockade of canonical Wnt signalling. Indeed, exposure of HEK293 cells to LX-2-conditioned media is able to partly inhibit constitutive active β-catenin signalling (Fig 5C), which may be due to the secretion of inhibitory sFRPs by HSC, this idea being confirmed by demonstrating the ability of overexpressed sFRP4 to inhibit Topflash reporter in HEK293 transfected with constitutive active β-catenin (Fig 5D).


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)

Expression of TCF/LEF transcription factors appears reduced in LX-2 cells.(A) Western Blot for TCF1, TCF3, TCF4 and LEF1in separate samples of LX-2 and HEK293 cells (B) TOPFLASH assay in HEK293 cells after 24hours incubation with conditioned medium from either HEK293 or LX-2 cells, (n = 4). (C) TOPFLASH assay in LX-2 cells overexpressing LEF1 and Ser37-βcatenin (n = 3) (D) TOPFLASH assay in HEK293 cells overexpressing Ser37-βCatenin or Ser37-βCatenin and sFRP4, (n = 3). Luciferase results represented as fold change of Firefly to Renilla *p<0.05 (Student’s t-test).
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Related In: Results  -  Collection

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pone.0142794.g005: Expression of TCF/LEF transcription factors appears reduced in LX-2 cells.(A) Western Blot for TCF1, TCF3, TCF4 and LEF1in separate samples of LX-2 and HEK293 cells (B) TOPFLASH assay in HEK293 cells after 24hours incubation with conditioned medium from either HEK293 or LX-2 cells, (n = 4). (C) TOPFLASH assay in LX-2 cells overexpressing LEF1 and Ser37-βcatenin (n = 3) (D) TOPFLASH assay in HEK293 cells overexpressing Ser37-βCatenin or Ser37-βCatenin and sFRP4, (n = 3). Luciferase results represented as fold change of Firefly to Renilla *p<0.05 (Student’s t-test).
Mentions: To determine the ability of aHSC to respond to canonical Wnt signalling, human LX-2 were co-transfected with the canonical Wnt luciferase reporter Topflash and expression vectors for Wnt3a and Wnt10b. To control these assays similar transfections were carried out in HEK293 cells which are known to support β-catenin-dependent Wnt signalling[17]. As anticipated co-transfection of the canonical Wnts activated the Topflash reporter in HEK293 cells, however no reporter activity was detected in LX-2 cells transfected with either Wnt3a or Wnt10b (Fig 4A). In addition, transfection of Wnt10b failed to induce expression of the Wnt target gene Axin2 in LX-2 which by contrast was up-regulated 3-fold by Wnt10b transfection in HEK293 cells (Fig 4B). Over-expression of a constitutive active mutant β-catenin (S2 Fig) induced Topflash activity by 40-fold in HEK293 cells (Fig 4C) and enhanced the expression of endogenous Axin2, cMyc and Cyclin D (Fig 4D). However, neither the reporter nor endogenous Axin2, cMyc and Cyclin D were stimulated in LX-2 cells, this confirming that the human HSC cell line does not support canonical Wnt signalling (Fig 4C and 4D). Expression of constitutive active β-catenin induced Topflash in primary rat dermal fibroblasts but was inactive in primary rat aHSC (Fig 4E). These data were indicative of defective β-catenin-dependent Wnt signalling in HSC. To investigate this further, rat HSC protein extracts were probed by immunoblot for expression of active (de-phosphorylated) and total β-catenin (Fig 4F) Freshly isolated qHSC lacked expression of active β-catenin, whereas HSC cultured for 7 days expressed abundant levels of active β-catenin such that pharmacological activation with a GSK3β inhibitor (CT99021) made little difference to the amount of active protein detected. Furthermore, active β-catenin was readily detected in the HSC nucleus, this ruling out a defect in nuclear translocation (S3 Fig). Once in the nucleus, β-catenin interacts with a variety of different proteins that impact on its stability and its ability to stimulate target gene transcription[18,19]. Scaffold proteins (Pyg1, Pyg2, BCL9, BCL92) that tether β-catenin to its transcription factor partners the TCF/LEF proteins, were all expressed at the mRNA level in LX-2 and HEK293 (Fig 4G) as was the nuclear repressor of β-catenin Chibby, which in primary rat HSC was found to undergo a dramatic diminution in expression with culture-induced activation (Fig 4H). The transcriptional activation of β-catenin-target genes is dependent on its interaction with TCF/LEF transcription factors [20]. Comparison of the expression of TCF/LEF proteins between HEK293 and LX-2 cells indicated a very low level of TCF1 and LEF1 in the HSC line and decreased levels of TCF4, by contrast TCF3 was expressed to a similar level between HEK293 and LX-2 (Fig 5A). These data were of particular interest since TCF1, TCF4 and LEF1 are generally considered to be activators of β-catenin-dependent transcription whereas TCF3 may have a more repressive function[21,22]. However, over-expression of LEF1 did not enable LX-2 to support transcriptional activity of co-transfected constitutive active β-catenin (Fig 5B). This result indicates that aHSC may establish multiple regulatory checkpoints to ensure blockade of canonical Wnt signalling. Indeed, exposure of HEK293 cells to LX-2-conditioned media is able to partly inhibit constitutive active β-catenin signalling (Fig 5C), which may be due to the secretion of inhibitory sFRPs by HSC, this idea being confirmed by demonstrating the ability of overexpressed sFRP4 to inhibit Topflash reporter in HEK293 transfected with constitutive active β-catenin (Fig 5D).

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