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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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HSCs upregulated non-canonical effectors upon Wnt stimulus.(A) Western blot for total (T) and phosphorylated (P) forms of JNK and CamKii in two preparations of rat aHSCs treated for 24 hours with either control (-), Wnt5a or Wnt10b conditioned media. (B) Western blot for Dvl2 in two preparations of rat HSCs at 0,4,6,8 and 10 days in culture. (C) Western Blot for Dvl2 in LX-2 cells overexpressing Wnt3a, Wnt5a or Wnt10b. (D) Diagram illustrating points in Wnt pathway for which inhibitors were chosen: Wnt secretion (IWP2) and Dvl-PDZ domain function (Dvl-PDZ). (E) Western Blot for Dvl2 in rat HSCs treated with 1–20 μM of IWP2 (F) qRT-PCR for profibrotic markers in vehicle control (DMSO) and 20μM IWP2 treated rat aHSCs, (n = 4). (G)qRT-PCR for NFAT3, Sox9 and Axin2 in vehicle control (DMSO) and 5μM PDZ-Inhibitor treated rat aHSCs, (n = 3) (H) qRT-PCR for profibrotic markers in vehicle control and 5μM PDZ-Inhibitor treated rat aHSCs (n = 3). qRT-PCR results expressed as fold change normalised to control ± SEM *p<0.05 (Student’s t-test).
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pone.0142794.g006: HSCs upregulated non-canonical effectors upon Wnt stimulus.(A) Western blot for total (T) and phosphorylated (P) forms of JNK and CamKii in two preparations of rat aHSCs treated for 24 hours with either control (-), Wnt5a or Wnt10b conditioned media. (B) Western blot for Dvl2 in two preparations of rat HSCs at 0,4,6,8 and 10 days in culture. (C) Western Blot for Dvl2 in LX-2 cells overexpressing Wnt3a, Wnt5a or Wnt10b. (D) Diagram illustrating points in Wnt pathway for which inhibitors were chosen: Wnt secretion (IWP2) and Dvl-PDZ domain function (Dvl-PDZ). (E) Western Blot for Dvl2 in rat HSCs treated with 1–20 μM of IWP2 (F) qRT-PCR for profibrotic markers in vehicle control (DMSO) and 20μM IWP2 treated rat aHSCs, (n = 4). (G)qRT-PCR for NFAT3, Sox9 and Axin2 in vehicle control (DMSO) and 5μM PDZ-Inhibitor treated rat aHSCs, (n = 3) (H) qRT-PCR for profibrotic markers in vehicle control and 5μM PDZ-Inhibitor treated rat aHSCs (n = 3). qRT-PCR results expressed as fold change normalised to control ± SEM *p<0.05 (Student’s t-test).

Mentions: Having failed to find evidence for β-catenin-dependent canonical Wnt activity in HSC we turned our attention to non-canonical Wnt signalling which is mediated via JNK- and CamKii/NFAT-dependent pathways[23,24]. Exposure of primary rat HSC to Wnt ligands Wnt5a and Wnt10b led to a substantial induction of active phosphorylated JNK (p-JNK) and enhanced expression of CamKii (Fig 6A). Dishevelled proteins (Dvl1-3) function as key membrane-proximal intracellular mediators of canonical and non-canonical Wnt signalling [25]. Phosphorylation of Dvl2 has been functionally associated with activation of non-canonical Wnt signal transduction and can be assessed by Dvl2 electrophoretic mobility shift[26]. Immunoblotting for Dvl2 with protein extracts from primary rat HSC representing a time course for culture-induced activation not only showed activation-dependent induction of Dvl2 expression (appearing at culture day 3) but also revealed a progressive electromobility shift as a function of days in culture, with day 7 aHSC predominantly expressing the low mobility hyperphosphorylated modified Dvl2 (Fig 6B). Of note, treatment of protein extracts with alkaline phosphatase selectively depleted this lower mobility form of Dvl2 confirming that the electrophoretic shift is due to phosphorylation (S4 Fig) Over-expression of Wnt3a, Wnt5a or Wnt10b into LX-2 cells induced an Dvl2 electrophoretic mobility shift confirming the ability of HSC to support Wnt signalling via Dvl2 activation (Fig 6C). Moreover, media collected from Wnt5a transfected LX-2 (S5 Fig) was able to activate low-mobility forms of Dvl2 in primary rat HSC (S6A Fig) and stimulated a modest increase in expression of α-SMA and transforming growth factor beta (TGFβ1) transcripts (S6B Fig). To determine the functional relevance of Wnt signal transduction in HSC the effects of Wnt inhibitors that have distinct molecular targets (as illustrated in Fig 6D) was examined in primary rat aHSC. IWP2 is an antagonist of Porcupine (Porc), which is responsible for the palmitoylation and subsequent secretion of Wnt ligands[27]. Dvl-PDZ targets the central PDZ domain of the Dvl proteins preventing their interaction with Fzd receptors[28]. ICG001 is an inhibitor of β-catenin interaction with its transcriptional co-activator CBP and as such is an inhibitor of canonical Wnt signalling[29]. Treatment of rat aHSC with IWP2 dose-dependently suppressed p-Dvl2 (top band on Western blot, Fig 6E) and suppressed α-SMA and collagen IA1 mRNA expression (Fig 6F). Dvl-PDZ, which is expected to suppress all downstream Wnt signalling, lowered expression of common Wnt target genes Sox9 and Axin2 (Fig 6G) and (as seen with IWP2) also significantly inhibited expression of α-SMA and collagen IA1 transcripts (Fig 6H). By contrast ICG001 was without effect on fibrogenic gene expression (S7A Fig) but did appear to cause rounding up of cells and apoptosis as determined by acridine orange staining (S7B and S7C Fig). We conclude from the effects of IWP2 and Dvl-PDZ that non-canonical Wnt signalling contributes to regulation of fibrogenic gene expression in aHSC.


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)

HSCs upregulated non-canonical effectors upon Wnt stimulus.(A) Western blot for total (T) and phosphorylated (P) forms of JNK and CamKii in two preparations of rat aHSCs treated for 24 hours with either control (-), Wnt5a or Wnt10b conditioned media. (B) Western blot for Dvl2 in two preparations of rat HSCs at 0,4,6,8 and 10 days in culture. (C) Western Blot for Dvl2 in LX-2 cells overexpressing Wnt3a, Wnt5a or Wnt10b. (D) Diagram illustrating points in Wnt pathway for which inhibitors were chosen: Wnt secretion (IWP2) and Dvl-PDZ domain function (Dvl-PDZ). (E) Western Blot for Dvl2 in rat HSCs treated with 1–20 μM of IWP2 (F) qRT-PCR for profibrotic markers in vehicle control (DMSO) and 20μM IWP2 treated rat aHSCs, (n = 4). (G)qRT-PCR for NFAT3, Sox9 and Axin2 in vehicle control (DMSO) and 5μM PDZ-Inhibitor treated rat aHSCs, (n = 3) (H) qRT-PCR for profibrotic markers in vehicle control and 5μM PDZ-Inhibitor treated rat aHSCs (n = 3). qRT-PCR results expressed as fold change normalised to control ± SEM *p<0.05 (Student’s t-test).
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pone.0142794.g006: HSCs upregulated non-canonical effectors upon Wnt stimulus.(A) Western blot for total (T) and phosphorylated (P) forms of JNK and CamKii in two preparations of rat aHSCs treated for 24 hours with either control (-), Wnt5a or Wnt10b conditioned media. (B) Western blot for Dvl2 in two preparations of rat HSCs at 0,4,6,8 and 10 days in culture. (C) Western Blot for Dvl2 in LX-2 cells overexpressing Wnt3a, Wnt5a or Wnt10b. (D) Diagram illustrating points in Wnt pathway for which inhibitors were chosen: Wnt secretion (IWP2) and Dvl-PDZ domain function (Dvl-PDZ). (E) Western Blot for Dvl2 in rat HSCs treated with 1–20 μM of IWP2 (F) qRT-PCR for profibrotic markers in vehicle control (DMSO) and 20μM IWP2 treated rat aHSCs, (n = 4). (G)qRT-PCR for NFAT3, Sox9 and Axin2 in vehicle control (DMSO) and 5μM PDZ-Inhibitor treated rat aHSCs, (n = 3) (H) qRT-PCR for profibrotic markers in vehicle control and 5μM PDZ-Inhibitor treated rat aHSCs (n = 3). qRT-PCR results expressed as fold change normalised to control ± SEM *p<0.05 (Student’s t-test).
Mentions: Having failed to find evidence for β-catenin-dependent canonical Wnt activity in HSC we turned our attention to non-canonical Wnt signalling which is mediated via JNK- and CamKii/NFAT-dependent pathways[23,24]. Exposure of primary rat HSC to Wnt ligands Wnt5a and Wnt10b led to a substantial induction of active phosphorylated JNK (p-JNK) and enhanced expression of CamKii (Fig 6A). Dishevelled proteins (Dvl1-3) function as key membrane-proximal intracellular mediators of canonical and non-canonical Wnt signalling [25]. Phosphorylation of Dvl2 has been functionally associated with activation of non-canonical Wnt signal transduction and can be assessed by Dvl2 electrophoretic mobility shift[26]. Immunoblotting for Dvl2 with protein extracts from primary rat HSC representing a time course for culture-induced activation not only showed activation-dependent induction of Dvl2 expression (appearing at culture day 3) but also revealed a progressive electromobility shift as a function of days in culture, with day 7 aHSC predominantly expressing the low mobility hyperphosphorylated modified Dvl2 (Fig 6B). Of note, treatment of protein extracts with alkaline phosphatase selectively depleted this lower mobility form of Dvl2 confirming that the electrophoretic shift is due to phosphorylation (S4 Fig) Over-expression of Wnt3a, Wnt5a or Wnt10b into LX-2 cells induced an Dvl2 electrophoretic mobility shift confirming the ability of HSC to support Wnt signalling via Dvl2 activation (Fig 6C). Moreover, media collected from Wnt5a transfected LX-2 (S5 Fig) was able to activate low-mobility forms of Dvl2 in primary rat HSC (S6A Fig) and stimulated a modest increase in expression of α-SMA and transforming growth factor beta (TGFβ1) transcripts (S6B Fig). To determine the functional relevance of Wnt signal transduction in HSC the effects of Wnt inhibitors that have distinct molecular targets (as illustrated in Fig 6D) was examined in primary rat aHSC. IWP2 is an antagonist of Porcupine (Porc), which is responsible for the palmitoylation and subsequent secretion of Wnt ligands[27]. Dvl-PDZ targets the central PDZ domain of the Dvl proteins preventing their interaction with Fzd receptors[28]. ICG001 is an inhibitor of β-catenin interaction with its transcriptional co-activator CBP and as such is an inhibitor of canonical Wnt signalling[29]. Treatment of rat aHSC with IWP2 dose-dependently suppressed p-Dvl2 (top band on Western blot, Fig 6E) and suppressed α-SMA and collagen IA1 mRNA expression (Fig 6F). Dvl-PDZ, which is expected to suppress all downstream Wnt signalling, lowered expression of common Wnt target genes Sox9 and Axin2 (Fig 6G) and (as seen with IWP2) also significantly inhibited expression of α-SMA and collagen IA1 transcripts (Fig 6H). By contrast ICG001 was without effect on fibrogenic gene expression (S7A Fig) but did appear to cause rounding up of cells and apoptosis as determined by acridine orange staining (S7B and S7C Fig). We conclude from the effects of IWP2 and Dvl-PDZ that non-canonical Wnt signalling contributes to regulation of fibrogenic gene expression in aHSC.

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