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. 2013 Feb;57(2):656-66.
doi: 10.1002/hep.26019.

Deficiency of G-protein-coupled Bile Acid Receptor Gpbar1 (TGR5) Enhances Chemically Induced Liver Carcinogenesis

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Deficiency of G-protein-coupled Bile Acid Receptor Gpbar1 (TGR5) Enhances Chemically Induced Liver Carcinogenesis

Wei-Dong Chen et al. Hepatology. .
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Abstract

Gpbar1 (TGR5), a membrane-bound bile acid receptor, is well known for its roles in regulation of energy homeostasis and glucose metabolism. TGR5 activation also inhibits nuclear factor κB (NF-κB)-mediated inflammation. Here we show that TGR5 deficiency enhances chemically induced liver carcinogenesis, and that TGR5 is a negative regulator of signal transducer and activator of transcription 3 (STAT3) signaling. Mice lacking TGR5 were much more susceptible to diethylnitrosamine (DEN)-induced acute liver injury and liver carcinogenesis than wildtype (WT) mice. Consistent with the increasing incidence of liver cancer in TGR5(-/-) mice, hepatocyte death, compensatory proliferation, and gene expression of certain inflammatory cytokines and matrix metalloproteinases were more sensitive to DEN induction in the absence of TGR5 signaling. In vitro, TGR5 activation greatly inhibited proliferation and migration of human liver cancer cells. We then found that TGR5 activation strongly suppressed STAT3 signaling in vitro and in vivo. Furthermore, we observed that TGR5 antagonizes the STAT3 pathway through suppressing STAT3 phosphorylation, its transcription activity, and DNA binding activity, which suggests that TGR5 antagonizes liver tumorigenesis at least in part by inhibiting STAT3 signaling.

Conclusion: These findings identify TGR5 as a novel liver tumor suppressor that may serve as an attractive therapeutic tool for human liver cancer.

Figures

Figure 1
Figure 1
TGR5−/− mouse livers are more sensitive to DEN-induced acute liver injury. (A) Levels of inflammatory serum marker, ALT in serum from WT and TGR5−/− (TGR5KO) mice that were treated with either vehicle (0.9% NaCl) or DEN at 100 mg/kg body weight (n = 4–5). *P < 0.05, **P < 0.005. (B) Induction of proinflammatory gene expression in response to DEN was significantly greater in TGR5−/− mice than WT mice (n = 4–5). *P < 0.05 versus WT mice at the same time point. (C) Representative Ki67 staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of Ki67-positive cells per field. Arrows indicate Ki67 positive cells. Twenty microscopic fields were counted. *P < 0.05 versus WT groups (n = 4–5).
Figure 1
Figure 1
TGR5−/− mouse livers are more sensitive to DEN-induced acute liver injury. (A) Levels of inflammatory serum marker, ALT in serum from WT and TGR5−/− (TGR5KO) mice that were treated with either vehicle (0.9% NaCl) or DEN at 100 mg/kg body weight (n = 4–5). *P < 0.05, **P < 0.005. (B) Induction of proinflammatory gene expression in response to DEN was significantly greater in TGR5−/− mice than WT mice (n = 4–5). *P < 0.05 versus WT mice at the same time point. (C) Representative Ki67 staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of Ki67-positive cells per field. Arrows indicate Ki67 positive cells. Twenty microscopic fields were counted. *P < 0.05 versus WT groups (n = 4–5).
Figure 1
Figure 1
TGR5−/− mouse livers are more sensitive to DEN-induced acute liver injury. (A) Levels of inflammatory serum marker, ALT in serum from WT and TGR5−/− (TGR5KO) mice that were treated with either vehicle (0.9% NaCl) or DEN at 100 mg/kg body weight (n = 4–5). *P < 0.05, **P < 0.005. (B) Induction of proinflammatory gene expression in response to DEN was significantly greater in TGR5−/− mice than WT mice (n = 4–5). *P < 0.05 versus WT mice at the same time point. (C) Representative Ki67 staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of Ki67-positive cells per field. Arrows indicate Ki67 positive cells. Twenty microscopic fields were counted. *P < 0.05 versus WT groups (n = 4–5).
Figure 2
Figure 2
TGR5 deficiency promotes DEN-induced liver cancer. (A) TGR5−/− mice developed tumors after 8 months of DEN treatment on the 26 day after birth (n=24). Arrows indicate tumors. (B) Representative H&E staining of liver sections and ALT levels of WT and TGR5−/− mice after 8 months of DEN treatment (n=24). NT, non-tumor; T, tumor. *P < 0.05. Con, control; DEN, DEN-treated groups. Black arrows, bile ducts; White arrow, inflammatory cells; Arrowheads, focal necrosis. (C) Representative PCNA staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of PCNA positive cells per field. The number of cells in at least 20 microscopic fields was counted. Arrows indicate PCNA positive cells. *P < 0.05. (D) Statistical analysis of the number of Ki67 positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (E) Representative TUNEL staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of TUNEL positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (F) Gene expression of MMP2 and MMP12 in WT and TGR5−/− livers after DEN administration. *P < 0.05 and **P < 0.05 versus the control groups.
Figure 2
Figure 2
TGR5 deficiency promotes DEN-induced liver cancer. (A) TGR5−/− mice developed tumors after 8 months of DEN treatment on the 26 day after birth (n=24). Arrows indicate tumors. (B) Representative H&E staining of liver sections and ALT levels of WT and TGR5−/− mice after 8 months of DEN treatment (n=24). NT, non-tumor; T, tumor. *P < 0.05. Con, control; DEN, DEN-treated groups. Black arrows, bile ducts; White arrow, inflammatory cells; Arrowheads, focal necrosis. (C) Representative PCNA staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of PCNA positive cells per field. The number of cells in at least 20 microscopic fields was counted. Arrows indicate PCNA positive cells. *P < 0.05. (D) Statistical analysis of the number of Ki67 positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (E) Representative TUNEL staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of TUNEL positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (F) Gene expression of MMP2 and MMP12 in WT and TGR5−/− livers after DEN administration. *P < 0.05 and **P < 0.05 versus the control groups.
Figure 2
Figure 2
TGR5 deficiency promotes DEN-induced liver cancer. (A) TGR5−/− mice developed tumors after 8 months of DEN treatment on the 26 day after birth (n=24). Arrows indicate tumors. (B) Representative H&E staining of liver sections and ALT levels of WT and TGR5−/− mice after 8 months of DEN treatment (n=24). NT, non-tumor; T, tumor. *P < 0.05. Con, control; DEN, DEN-treated groups. Black arrows, bile ducts; White arrow, inflammatory cells; Arrowheads, focal necrosis. (C) Representative PCNA staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of PCNA positive cells per field. The number of cells in at least 20 microscopic fields was counted. Arrows indicate PCNA positive cells. *P < 0.05. (D) Statistical analysis of the number of Ki67 positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (E) Representative TUNEL staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of TUNEL positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (F) Gene expression of MMP2 and MMP12 in WT and TGR5−/− livers after DEN administration. *P < 0.05 and **P < 0.05 versus the control groups.
Figure 2
Figure 2
TGR5 deficiency promotes DEN-induced liver cancer. (A) TGR5−/− mice developed tumors after 8 months of DEN treatment on the 26 day after birth (n=24). Arrows indicate tumors. (B) Representative H&E staining of liver sections and ALT levels of WT and TGR5−/− mice after 8 months of DEN treatment (n=24). NT, non-tumor; T, tumor. *P < 0.05. Con, control; DEN, DEN-treated groups. Black arrows, bile ducts; White arrow, inflammatory cells; Arrowheads, focal necrosis. (C) Representative PCNA staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of PCNA positive cells per field. The number of cells in at least 20 microscopic fields was counted. Arrows indicate PCNA positive cells. *P < 0.05. (D) Statistical analysis of the number of Ki67 positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (E) Representative TUNEL staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of TUNEL positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (F) Gene expression of MMP2 and MMP12 in WT and TGR5−/− livers after DEN administration. *P < 0.05 and **P < 0.05 versus the control groups.
Figure 2
Figure 2
TGR5 deficiency promotes DEN-induced liver cancer. (A) TGR5−/− mice developed tumors after 8 months of DEN treatment on the 26 day after birth (n=24). Arrows indicate tumors. (B) Representative H&E staining of liver sections and ALT levels of WT and TGR5−/− mice after 8 months of DEN treatment (n=24). NT, non-tumor; T, tumor. *P < 0.05. Con, control; DEN, DEN-treated groups. Black arrows, bile ducts; White arrow, inflammatory cells; Arrowheads, focal necrosis. (C) Representative PCNA staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of PCNA positive cells per field. The number of cells in at least 20 microscopic fields was counted. Arrows indicate PCNA positive cells. *P < 0.05. (D) Statistical analysis of the number of Ki67 positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (E) Representative TUNEL staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of TUNEL positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (F) Gene expression of MMP2 and MMP12 in WT and TGR5−/− livers after DEN administration. *P < 0.05 and **P < 0.05 versus the control groups.
Figure 2
Figure 2
TGR5 deficiency promotes DEN-induced liver cancer. (A) TGR5−/− mice developed tumors after 8 months of DEN treatment on the 26 day after birth (n=24). Arrows indicate tumors. (B) Representative H&E staining of liver sections and ALT levels of WT and TGR5−/− mice after 8 months of DEN treatment (n=24). NT, non-tumor; T, tumor. *P < 0.05. Con, control; DEN, DEN-treated groups. Black arrows, bile ducts; White arrow, inflammatory cells; Arrowheads, focal necrosis. (C) Representative PCNA staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of PCNA positive cells per field. The number of cells in at least 20 microscopic fields was counted. Arrows indicate PCNA positive cells. *P < 0.05. (D) Statistical analysis of the number of Ki67 positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (E) Representative TUNEL staining of sections from WT and TGR5−/− livers (magnification, ×200) and statistical analysis of the number of TUNEL positive cells per field. The number of cells in at least 20 microscopic fields was counted. *P < 0.05. (F) Gene expression of MMP2 and MMP12 in WT and TGR5−/− livers after DEN administration. *P < 0.05 and **P < 0.05 versus the control groups.
Figure 3
Figure 3
TGR5 activation impairs proliferation and migration of human liver cancer cells. (A) TGR5 activation by its ligand inhibited proliferation of HepG2 cells. Proliferation of cells was analyzed using MTT assay. TGR5 plasmid was transfected into HepG2 cells and then the ligand was added into the culture after 24 hours. After 1, 2 and 3 days of treatment, MTT assay was performed to determine cell proliferation. *P<0.05 versus the control groups (n=3). Con, control. (B) TGR5-transfected cells with ligand treatment exhibited a lower scratch closure rate than the controls in in vitro scratch assay (n=3). Wound healing was recorded 2.5 days after transfection. (C) Transwell cell migration assay confirmed that TGR5 activation inhibited HepG2 cell migration (n=3). *P < 0.05 versus the control groups. (D) TGR5 activation suppressed gene expression of MMP7 and MMP9 in HepG2 cells. *P < 0.05 versus the control groups (n=3). The mRNA levels were measured 2 days after transfection. The n=3 refers to 3 independent experiments, each performed in triplicate.
Figure 3
Figure 3
TGR5 activation impairs proliferation and migration of human liver cancer cells. (A) TGR5 activation by its ligand inhibited proliferation of HepG2 cells. Proliferation of cells was analyzed using MTT assay. TGR5 plasmid was transfected into HepG2 cells and then the ligand was added into the culture after 24 hours. After 1, 2 and 3 days of treatment, MTT assay was performed to determine cell proliferation. *P<0.05 versus the control groups (n=3). Con, control. (B) TGR5-transfected cells with ligand treatment exhibited a lower scratch closure rate than the controls in in vitro scratch assay (n=3). Wound healing was recorded 2.5 days after transfection. (C) Transwell cell migration assay confirmed that TGR5 activation inhibited HepG2 cell migration (n=3). *P < 0.05 versus the control groups. (D) TGR5 activation suppressed gene expression of MMP7 and MMP9 in HepG2 cells. *P < 0.05 versus the control groups (n=3). The mRNA levels were measured 2 days after transfection. The n=3 refers to 3 independent experiments, each performed in triplicate.
Figure 4
Figure 4
TGR5 inhibits STAT3 phosphorylation in vitro and in vivo. (A) Immunoblot analysis for phosphorylated STAT3 (p-STAT3) and total STAT3 (T-STAT3) in total protein pools from WT and TGR5−/− mouse livers after DEN treatment for 8 months (n=24). Western blot was performed in triplicate. *P < 0.05. (B) TGR5 overexpression with ligand treatment suppressed IL-6-induced p-STAT3 in HepG2 cells. Cells were treated with ligand for 18 hours and then were treated with IL-6 for 2 hours. (n=3) The data of relative protein levels in (A) and (B) are expressed as fold change over the ratio of p-STAT3 to T-STAT3 in the control group (lane 1). (C) Immunoblot analysis for p-STAT3 and T-STAT3 in total protein pools from WT and TGR5−/− mouse livers that were treated with or without LPS (three independent experiments, 5–6 mice per group). Mice were fed a diet containing 10 mg of 23(S)-mCDCA/kg diet or standard rodent chow for 3 days. After fasted overnight, mice were treated with LPS (20 mg/kg) or PBS for 6 hours.
Figure 4
Figure 4
TGR5 inhibits STAT3 phosphorylation in vitro and in vivo. (A) Immunoblot analysis for phosphorylated STAT3 (p-STAT3) and total STAT3 (T-STAT3) in total protein pools from WT and TGR5−/− mouse livers after DEN treatment for 8 months (n=24). Western blot was performed in triplicate. *P < 0.05. (B) TGR5 overexpression with ligand treatment suppressed IL-6-induced p-STAT3 in HepG2 cells. Cells were treated with ligand for 18 hours and then were treated with IL-6 for 2 hours. (n=3) The data of relative protein levels in (A) and (B) are expressed as fold change over the ratio of p-STAT3 to T-STAT3 in the control group (lane 1). (C) Immunoblot analysis for p-STAT3 and T-STAT3 in total protein pools from WT and TGR5−/− mouse livers that were treated with or without LPS (three independent experiments, 5–6 mice per group). Mice were fed a diet containing 10 mg of 23(S)-mCDCA/kg diet or standard rodent chow for 3 days. After fasted overnight, mice were treated with LPS (20 mg/kg) or PBS for 6 hours.
Figure 4
Figure 4
TGR5 inhibits STAT3 phosphorylation in vitro and in vivo. (A) Immunoblot analysis for phosphorylated STAT3 (p-STAT3) and total STAT3 (T-STAT3) in total protein pools from WT and TGR5−/− mouse livers after DEN treatment for 8 months (n=24). Western blot was performed in triplicate. *P < 0.05. (B) TGR5 overexpression with ligand treatment suppressed IL-6-induced p-STAT3 in HepG2 cells. Cells were treated with ligand for 18 hours and then were treated with IL-6 for 2 hours. (n=3) The data of relative protein levels in (A) and (B) are expressed as fold change over the ratio of p-STAT3 to T-STAT3 in the control group (lane 1). (C) Immunoblot analysis for p-STAT3 and T-STAT3 in total protein pools from WT and TGR5−/− mouse livers that were treated with or without LPS (three independent experiments, 5–6 mice per group). Mice were fed a diet containing 10 mg of 23(S)-mCDCA/kg diet or standard rodent chow for 3 days. After fasted overnight, mice were treated with LPS (20 mg/kg) or PBS for 6 hours.
Figure 5
Figure 5
Activation of TGR5 antagonizes STAT3 transactivity and its DNA binding activity. (A) TGR5 suppressed STAT3 transactivity induced by IL-6 in a TGR5 dose-dependent manner. HepG2 cells were cotransfected with the STAT3 reporter plasmid, phRL-TK, and increasing amounts of TGR5 expression plasmid at 1:10, 2:10, 3:10 or 5:10 ratios with the STAT3 reporter plasmid. The same dose of TGR5 plasmid was used in the third and fourth columns. After transfection, cells were treated with 23(S)-mCDCA (10 μM) or vehicle (DMSO) for 18 hours and then treated with IL-6 (20ng/mL) for 6 hours. *P < 0.005 versus the IL-6 treated groups. RLU, relative luciferase units. (B) EMSA showed that activation of TGR5 suppressed LPS-induced STAT3 DNA-binding activity (n=5–6).
Figure 5
Figure 5
Activation of TGR5 antagonizes STAT3 transactivity and its DNA binding activity. (A) TGR5 suppressed STAT3 transactivity induced by IL-6 in a TGR5 dose-dependent manner. HepG2 cells were cotransfected with the STAT3 reporter plasmid, phRL-TK, and increasing amounts of TGR5 expression plasmid at 1:10, 2:10, 3:10 or 5:10 ratios with the STAT3 reporter plasmid. The same dose of TGR5 plasmid was used in the third and fourth columns. After transfection, cells were treated with 23(S)-mCDCA (10 μM) or vehicle (DMSO) for 18 hours and then treated with IL-6 (20ng/mL) for 6 hours. *P < 0.005 versus the IL-6 treated groups. RLU, relative luciferase units. (B) EMSA showed that activation of TGR5 suppressed LPS-induced STAT3 DNA-binding activity (n=5–6).

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