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Farnesoid X receptor associates with β-catenin and inhibits its activity in hepatocellular carcinoma.

Liu X, Zhang X, Ji L, Gu J, Zhou M, Chen S - Oncotarget (2015)

Bottom Line: Activation of FXR attenuated the DNA-binding activity of β-Catenin/TCF4, and subsequently, its targeting gene-cyclin D1 expression.Importantly, FXR expression was markedly reduced in human HCC, an event which correlated with aberrant activation of β-Catenin.These data identified FXR as a negative regulator of HCC development through direct suppression of Wnt/β-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical college, Fudan University, Shanghai, China.

ABSTRACT
The association between the temporal activation of Wnt/β-catenin pathway and the spontaneous hepatocellular carcinoma (HCC) development in Farnesoid X receptor (FXR) knockout mice is not well understood. We found that Huh7 cells depleted with FXR by RNAi showed enhanced cell growth, migration and invasion in vitro and accelerated tumor xenografts formation in nude mice. And these phenotypes were attenuated by simultaneous knockdown of β-catenin with RNAi. Furthermore, we identified that FXR could bind with β-Catenin through AF1 domain, and disrupt the assembly of the core β-Catenin/TCF4 complex. Activation of FXR attenuated the DNA-binding activity of β-Catenin/TCF4, and subsequently, its targeting gene-cyclin D1 expression. Importantly, FXR expression was markedly reduced in human HCC, an event which correlated with aberrant activation of β-Catenin. These data identified FXR as a negative regulator of HCC development through direct suppression of Wnt/β-catenin pathway.

No MeSH data available.


Related in: MedlinePlus

FXR attenuates β-Catenin/TCF4 complex mediated cyclin D1 transcription(A) FXR did not bind with TCF4. Co-immunoprecipitation was carried out with anti-Myc agarose in lysates from HEK293T cells co-transfected with Myc-FXR and HA-TCF4. TCF4 was not detected using antibodies against HA. (B) FXR agonist GW4064 attenuated β-Catenin/TCF4 complex formation. Co-immunoprecipitation was carried out using anti-TCF4 antibody, followed by IgG agarose incubation in lysates from Huh7 cells treated with 1 μM or 4 μM GW4064 for 24 hours. Bound β-Catenin was detected by Western blot. (C) Down-regulation of FXR enhanced β-Catenin/TCF4 complex formation. Immunoprecipitation was carried out using anti-TCF4 antibody, followed by IgG agarose incubation in lysates from Huh7 cells transfected with FXR siRNA (#1 or #2) or control siRNA for 24 hours. Bound β-Catenin was detected by Western blot. (D) FXR agonist GW4064 impaired the binding of TCF4 with Cyclin D1 promoter. Wild type Cyclin D1 promoter (Cyclin D1-WT; 0.1 μg), or a mutated Cyclin D1 loss of TCF binding site (CyclinD1-mTCF; 0.1μg) and pRL-TK plasmid were transfected into HEK293 cells for 24 hours. Luciferase activity was measured using cell lysates 24 hours after cells were treated with FXR agonists GW4064 (1 or 4 μM). GW4064 impaired the binding of TCF4 with Cyclin D1-WT but not CyclinD1-mTCF. (E) Down-regulation of FXR promoted the binding of TCF4 with Cyclin D1 promoter. FXR siRNA #2 or control siRNA were transfected into HEK293T cells in combination with Cyclin D1-WT or CyclinD1-mTCF. Luciferase activity was measured 24 hour after transfection. FXR siRNA #2 promoted the binding of TCF4 with Cyclin D1-WT but not CyclinD1-mTCF. (F) FXR agonist GW4064 reduced the association between β-Catenin/TCF4 complex and the TCF binding sites from Cyclin D1 promoter. ChIP analysis was carried out using antibodies against TCF4, β-Catenin, or normal IgG in lysates from Huh7 cells. DNA fragment precipitates were amplified by PCR (35 or 38 cycles). (G) Down-regulation of FXR promoted the association between β-Catenin/TCF4 complex and the TCF binding sites from Cyclin D1 promoter. ChIP analysis was carried out using antibodies against TCF4 or β-Catenin, or normal IgG in lysates of Huh7 cells transfected with FXR siRNA #2 or control siRNA. DNA fragment precipitates were amplified by PCR (35 or 38 cycles). (H) Model of mechanism for the FXR mediated repression of the canonical Wnt/β-Catenin signaling. Upon activation, FXR, through its interaction with β-Catenin, prevents β-Catenin/TCF4 complex formation, subsequently represses transcription of its downstream target genes, such as cyclin D1. Error bars represent ± SEM of three independent samples. *, p < 0.01; **, p < 0.001.
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Figure 4: FXR attenuates β-Catenin/TCF4 complex mediated cyclin D1 transcription(A) FXR did not bind with TCF4. Co-immunoprecipitation was carried out with anti-Myc agarose in lysates from HEK293T cells co-transfected with Myc-FXR and HA-TCF4. TCF4 was not detected using antibodies against HA. (B) FXR agonist GW4064 attenuated β-Catenin/TCF4 complex formation. Co-immunoprecipitation was carried out using anti-TCF4 antibody, followed by IgG agarose incubation in lysates from Huh7 cells treated with 1 μM or 4 μM GW4064 for 24 hours. Bound β-Catenin was detected by Western blot. (C) Down-regulation of FXR enhanced β-Catenin/TCF4 complex formation. Immunoprecipitation was carried out using anti-TCF4 antibody, followed by IgG agarose incubation in lysates from Huh7 cells transfected with FXR siRNA (#1 or #2) or control siRNA for 24 hours. Bound β-Catenin was detected by Western blot. (D) FXR agonist GW4064 impaired the binding of TCF4 with Cyclin D1 promoter. Wild type Cyclin D1 promoter (Cyclin D1-WT; 0.1 μg), or a mutated Cyclin D1 loss of TCF binding site (CyclinD1-mTCF; 0.1μg) and pRL-TK plasmid were transfected into HEK293 cells for 24 hours. Luciferase activity was measured using cell lysates 24 hours after cells were treated with FXR agonists GW4064 (1 or 4 μM). GW4064 impaired the binding of TCF4 with Cyclin D1-WT but not CyclinD1-mTCF. (E) Down-regulation of FXR promoted the binding of TCF4 with Cyclin D1 promoter. FXR siRNA #2 or control siRNA were transfected into HEK293T cells in combination with Cyclin D1-WT or CyclinD1-mTCF. Luciferase activity was measured 24 hour after transfection. FXR siRNA #2 promoted the binding of TCF4 with Cyclin D1-WT but not CyclinD1-mTCF. (F) FXR agonist GW4064 reduced the association between β-Catenin/TCF4 complex and the TCF binding sites from Cyclin D1 promoter. ChIP analysis was carried out using antibodies against TCF4, β-Catenin, or normal IgG in lysates from Huh7 cells. DNA fragment precipitates were amplified by PCR (35 or 38 cycles). (G) Down-regulation of FXR promoted the association between β-Catenin/TCF4 complex and the TCF binding sites from Cyclin D1 promoter. ChIP analysis was carried out using antibodies against TCF4 or β-Catenin, or normal IgG in lysates of Huh7 cells transfected with FXR siRNA #2 or control siRNA. DNA fragment precipitates were amplified by PCR (35 or 38 cycles). (H) Model of mechanism for the FXR mediated repression of the canonical Wnt/β-Catenin signaling. Upon activation, FXR, through its interaction with β-Catenin, prevents β-Catenin/TCF4 complex formation, subsequently represses transcription of its downstream target genes, such as cyclin D1. Error bars represent ± SEM of three independent samples. *, p < 0.01; **, p < 0.001.

Mentions: Next, we investigated whether FXR also interacts with the other core transcription factor, TCF4. HEK293 cells were transiently transfected with Myc-tagged FXR and HA-tagged TCF4. The interaction between FXR and TCF4 was tested with co-immunoprecipitation assay. We found that HA-TCF4 was not presented in the cell lysate immunoprecipitated with Myc agarose (Figure 4A). β-Catenin and TCF4 protein level were unaffected by FXR specific agonist GW4064 in Huh7 cells (Figure 4B, input). We then investigated whether FXR regulates the stability of β-Catenin-TCF4 complex. We found that, upon FXR activation in GW4064 treated Huh7 cells, the binding between β-Catenin and TCF4 was decreased (Figure 4B). In contrast, when FXR expression was depleted by two independent siRNA in Huh7 cells, the interaction between β-Catenin and TCF4 was enhanced (Figure 4C).


Farnesoid X receptor associates with β-catenin and inhibits its activity in hepatocellular carcinoma.

Liu X, Zhang X, Ji L, Gu J, Zhou M, Chen S - Oncotarget (2015)

FXR attenuates β-Catenin/TCF4 complex mediated cyclin D1 transcription(A) FXR did not bind with TCF4. Co-immunoprecipitation was carried out with anti-Myc agarose in lysates from HEK293T cells co-transfected with Myc-FXR and HA-TCF4. TCF4 was not detected using antibodies against HA. (B) FXR agonist GW4064 attenuated β-Catenin/TCF4 complex formation. Co-immunoprecipitation was carried out using anti-TCF4 antibody, followed by IgG agarose incubation in lysates from Huh7 cells treated with 1 μM or 4 μM GW4064 for 24 hours. Bound β-Catenin was detected by Western blot. (C) Down-regulation of FXR enhanced β-Catenin/TCF4 complex formation. Immunoprecipitation was carried out using anti-TCF4 antibody, followed by IgG agarose incubation in lysates from Huh7 cells transfected with FXR siRNA (#1 or #2) or control siRNA for 24 hours. Bound β-Catenin was detected by Western blot. (D) FXR agonist GW4064 impaired the binding of TCF4 with Cyclin D1 promoter. Wild type Cyclin D1 promoter (Cyclin D1-WT; 0.1 μg), or a mutated Cyclin D1 loss of TCF binding site (CyclinD1-mTCF; 0.1μg) and pRL-TK plasmid were transfected into HEK293 cells for 24 hours. Luciferase activity was measured using cell lysates 24 hours after cells were treated with FXR agonists GW4064 (1 or 4 μM). GW4064 impaired the binding of TCF4 with Cyclin D1-WT but not CyclinD1-mTCF. (E) Down-regulation of FXR promoted the binding of TCF4 with Cyclin D1 promoter. FXR siRNA #2 or control siRNA were transfected into HEK293T cells in combination with Cyclin D1-WT or CyclinD1-mTCF. Luciferase activity was measured 24 hour after transfection. FXR siRNA #2 promoted the binding of TCF4 with Cyclin D1-WT but not CyclinD1-mTCF. (F) FXR agonist GW4064 reduced the association between β-Catenin/TCF4 complex and the TCF binding sites from Cyclin D1 promoter. ChIP analysis was carried out using antibodies against TCF4, β-Catenin, or normal IgG in lysates from Huh7 cells. DNA fragment precipitates were amplified by PCR (35 or 38 cycles). (G) Down-regulation of FXR promoted the association between β-Catenin/TCF4 complex and the TCF binding sites from Cyclin D1 promoter. ChIP analysis was carried out using antibodies against TCF4 or β-Catenin, or normal IgG in lysates of Huh7 cells transfected with FXR siRNA #2 or control siRNA. DNA fragment precipitates were amplified by PCR (35 or 38 cycles). (H) Model of mechanism for the FXR mediated repression of the canonical Wnt/β-Catenin signaling. Upon activation, FXR, through its interaction with β-Catenin, prevents β-Catenin/TCF4 complex formation, subsequently represses transcription of its downstream target genes, such as cyclin D1. Error bars represent ± SEM of three independent samples. *, p < 0.01; **, p < 0.001.
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Figure 4: FXR attenuates β-Catenin/TCF4 complex mediated cyclin D1 transcription(A) FXR did not bind with TCF4. Co-immunoprecipitation was carried out with anti-Myc agarose in lysates from HEK293T cells co-transfected with Myc-FXR and HA-TCF4. TCF4 was not detected using antibodies against HA. (B) FXR agonist GW4064 attenuated β-Catenin/TCF4 complex formation. Co-immunoprecipitation was carried out using anti-TCF4 antibody, followed by IgG agarose incubation in lysates from Huh7 cells treated with 1 μM or 4 μM GW4064 for 24 hours. Bound β-Catenin was detected by Western blot. (C) Down-regulation of FXR enhanced β-Catenin/TCF4 complex formation. Immunoprecipitation was carried out using anti-TCF4 antibody, followed by IgG agarose incubation in lysates from Huh7 cells transfected with FXR siRNA (#1 or #2) or control siRNA for 24 hours. Bound β-Catenin was detected by Western blot. (D) FXR agonist GW4064 impaired the binding of TCF4 with Cyclin D1 promoter. Wild type Cyclin D1 promoter (Cyclin D1-WT; 0.1 μg), or a mutated Cyclin D1 loss of TCF binding site (CyclinD1-mTCF; 0.1μg) and pRL-TK plasmid were transfected into HEK293 cells for 24 hours. Luciferase activity was measured using cell lysates 24 hours after cells were treated with FXR agonists GW4064 (1 or 4 μM). GW4064 impaired the binding of TCF4 with Cyclin D1-WT but not CyclinD1-mTCF. (E) Down-regulation of FXR promoted the binding of TCF4 with Cyclin D1 promoter. FXR siRNA #2 or control siRNA were transfected into HEK293T cells in combination with Cyclin D1-WT or CyclinD1-mTCF. Luciferase activity was measured 24 hour after transfection. FXR siRNA #2 promoted the binding of TCF4 with Cyclin D1-WT but not CyclinD1-mTCF. (F) FXR agonist GW4064 reduced the association between β-Catenin/TCF4 complex and the TCF binding sites from Cyclin D1 promoter. ChIP analysis was carried out using antibodies against TCF4, β-Catenin, or normal IgG in lysates from Huh7 cells. DNA fragment precipitates were amplified by PCR (35 or 38 cycles). (G) Down-regulation of FXR promoted the association between β-Catenin/TCF4 complex and the TCF binding sites from Cyclin D1 promoter. ChIP analysis was carried out using antibodies against TCF4 or β-Catenin, or normal IgG in lysates of Huh7 cells transfected with FXR siRNA #2 or control siRNA. DNA fragment precipitates were amplified by PCR (35 or 38 cycles). (H) Model of mechanism for the FXR mediated repression of the canonical Wnt/β-Catenin signaling. Upon activation, FXR, through its interaction with β-Catenin, prevents β-Catenin/TCF4 complex formation, subsequently represses transcription of its downstream target genes, such as cyclin D1. Error bars represent ± SEM of three independent samples. *, p < 0.01; **, p < 0.001.
Mentions: Next, we investigated whether FXR also interacts with the other core transcription factor, TCF4. HEK293 cells were transiently transfected with Myc-tagged FXR and HA-tagged TCF4. The interaction between FXR and TCF4 was tested with co-immunoprecipitation assay. We found that HA-TCF4 was not presented in the cell lysate immunoprecipitated with Myc agarose (Figure 4A). β-Catenin and TCF4 protein level were unaffected by FXR specific agonist GW4064 in Huh7 cells (Figure 4B, input). We then investigated whether FXR regulates the stability of β-Catenin-TCF4 complex. We found that, upon FXR activation in GW4064 treated Huh7 cells, the binding between β-Catenin and TCF4 was decreased (Figure 4B). In contrast, when FXR expression was depleted by two independent siRNA in Huh7 cells, the interaction between β-Catenin and TCF4 was enhanced (Figure 4C).

Bottom Line: Activation of FXR attenuated the DNA-binding activity of β-Catenin/TCF4, and subsequently, its targeting gene-cyclin D1 expression.Importantly, FXR expression was markedly reduced in human HCC, an event which correlated with aberrant activation of β-Catenin.These data identified FXR as a negative regulator of HCC development through direct suppression of Wnt/β-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical college, Fudan University, Shanghai, China.

ABSTRACT
The association between the temporal activation of Wnt/β-catenin pathway and the spontaneous hepatocellular carcinoma (HCC) development in Farnesoid X receptor (FXR) knockout mice is not well understood. We found that Huh7 cells depleted with FXR by RNAi showed enhanced cell growth, migration and invasion in vitro and accelerated tumor xenografts formation in nude mice. And these phenotypes were attenuated by simultaneous knockdown of β-catenin with RNAi. Furthermore, we identified that FXR could bind with β-Catenin through AF1 domain, and disrupt the assembly of the core β-Catenin/TCF4 complex. Activation of FXR attenuated the DNA-binding activity of β-Catenin/TCF4, and subsequently, its targeting gene-cyclin D1 expression. Importantly, FXR expression was markedly reduced in human HCC, an event which correlated with aberrant activation of β-Catenin. These data identified FXR as a negative regulator of HCC development through direct suppression of Wnt/β-catenin pathway.

No MeSH data available.


Related in: MedlinePlus