Transforming growth factor-β signaling in hepatocytes promotes hepatic fibrosis and carcinogenesis in mice with hepatocyte-specific deletion of TAK1

Abstract

Background & aims: Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is activated in different cytokine signaling pathways. Deletion of Tak1 from hepatocytes results in spontaneous development of hepatocellular carcinoma (HCC), liver inflammation, and fibrosis. TGF-β activates TAK1 and Smad signaling, which regulate cell death, proliferation, and carcinogenesis. However, it is not clear whether TGF-β signaling in hepatocytes, via TGF-β receptor-2 (Tgfbr2), promotes HCC and liver fibrosis.

Methods: We generated mice with hepatocyte-specific deletion of Tak1 (Tak1ΔHep), as well as Tak1/Tgfbr2DHep and Tak1/Smad4ΔHep mice. Tak1flox/flox, Tgfbr2ΔHep, and Smad4ΔHep mice were used as controls, respectively. We assessed development of liver injury, inflammation, fibrosis, and HCC. Primary hepatocytes isolated from these mice were used to assess TGF-β-mediated signaling.

Results: Levels of TGF-β, TGF-βR2, and phospho-Smad2/3 were increased in HCCs from Tak1ΔHep mice, which developed liver fibrosis and inflammation by 1 month and HCC by 9 months. However, Tak1/Tgfbr2ΔHep mice did not have this phenotype, and their hepatocytes did not undergo spontaneous cell death or compensatory proliferation. Hepatocytes from Tak1ΔHep mice incubated with TGF-β did not activate p38, c-Jun N-terminal kinase, or nuclear factor-κB; conversely, TGF-β-mediated cell death and phosphorylation of Smad2/3 were increased, compared with control hepatocytes. Blocking the Smad pathway inhibited TGF-β-mediated death of Tak1-/- hepatocytes. Accordingly, disruption of Smad4 reduced the spontaneous liver injury, inflammation, fibrosis, and HCC that develops in Tak1ΔHep mice. Levels of the anti-apoptotic protein Bcl-xL, β-catenin, connective tissue growth factor, and vascular endothelial growth factor were increased in HCC from Tak1ΔHep mice, but not in HCCs from Tak1/Tgfbr2ΔHep mice. Injection of N-nitrosodiethylamine induced HCC formation in wild-type mice, but less in Tgfbr2ΔHep mice.

Conclusions: TGF-β promotes development of HCC in Tak1ΔHep mice by inducing hepatocyte apoptosis and compensatory proliferation during early phases of tumorigenesis, and inducing expression of anti-apoptotic, pro-oncogenic, and angiogenic factors during tumor progression.

Figure 1

TGF-β signaling is required for spontaneous hepatocarcinogenesis in Tak1^ΔHep mice. (A) Immunohistochemistry for TGFβR2 and phosphorylated Smad2/3 in liver tissues from 9-month-old WT, and nontumor livers and tumors from 9-month-old Tak1^ΔHep mice is shown. Original magnification ×200 for TGFβR2 staining and 400× for Smad2/3 staining. (B) Western blotting for phosphorylated Smad2 and Smad3 and TAK1 in liver tissues from 9-month-old WT, and nontumor liver tissues and tumors from 9-month-old Tak1^ΔHep mice. β-actin was used as a loading control. (C) Expression of Tgfb1, Tgfbr2, and Tgfbr1 messenger RNA in liver tissues from 9-month-old WT, and nontumor liver tissues and tumors from 9-month-old Tak1^ΔHep mice was measured by quantitative real-time polymerase chain reaction. NT, nontumor liver, T, tumors. (n=6, each samples) (D, left) Immunoblots for TAK1, TGFβR2, phospho-Smad2, phospho-Smad3, Smad4 in the livers of WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice at 1 month of age are shown. β-actin was used as a loading control. (D, right) Representative macroscopic pictures of livers of 9-month-old WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice. (E) The number of tumors per mouse was counted and the maximum diameter of individual tumor nodules was measured (WT, n = 10; Tak1^ΔHep, n = 29; Tak1/Tgfbr2^ΔHep, n = 27; Tgfbr2^ΔHep, n = 10). (F) H&E staining. Data are represented as mean ± standard error of mean.*P < .05; **P < .01.

Figure 2

Ablation of Tgfbr2 in Tak1^ΔHep mice reduces spontaneous liver inflammation. (A) H&E staining in the livers of 1- and 9-month-old WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice. Original magnification 200×. (B) Serum ALT levels were measured in WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice at age 1, 4, and 9 months (n = 8 at the each time point). (C, D) Immunohistochemistry for F4/80 in WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice at 1 month of age. Quantification (C) and representative pictures (D). Original magnification 200×. (E) Hepatic messenger RNA expression of inflammatory genes (Tnf, Il6, Ccl2, and Il1b) in mice at the age of 1 month was determined by quantitative real-time polymerase chain reaction. Data are represented as mean ± standard error of mean. *P < .05; **P < .01.

Figure 3

Loss of Tgfbr2 in Tak1^ΔHep mice suppresses spontaneous liver fibrosis. (A, B) WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice at the age of 1, 4, and 9 months (n = 7 at the each time point) were used for analysis. Fibrillar collagen deposition was determined by Sirius red staining (A) and its quantification are shown in (B). Original magnification 100×. (C, D) Expression of α–smooth muscle actin in 9-month-old mice was determined by immunohistochemistry. Quantification (C) and representative pictures (D). Original magnification 320×. (E) Hepatic messenger RNA expressions of fibrogenic markers, including Col1A1, Acta2, Timp1, and Tgfb1, were determined by quantitative real-time polymerase chain reaction in 1-month-old WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice. Data are reported as mean ± standard error of mean. *P < .05; **P < .01.

Figure 4

Additional deletion of Tgfbr2 in hepatocytes inhibits spontaneous apoptosis and compensatory regeneration in the livers of Tak1^ΔHep mice. (A) Apoptotic hepatocytes were evaluated by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining (A, upper) and proliferating hepatocytes were evaluated by immunohistochemistry for proliferation cell nuclear antigen (PCNA) (A, lower) in the livers of WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice at the 1 month of age (n = 7). Original magnification 320×. (B, C) Quantification for TUNEL staining (B) and staining for PCNA (C). (D) Hepatic messenger RNA expression of apototic genes (Bcl-2, Bax) in the livers of WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice at 1 month of age was determined by quantitative real-time polymerase chain reaction. (E) Immunoblots for caspase 3, cleaved caspase 3, PCNA, and cyclin D1 in the livers of WT, Tak1^ΔHep, Tak1/Tgfbr2^ΔHep, and Tgfbr2^ΔHep mice at 1 month of age. Data are reported as mean± standard error of mean. *P < .05; **P < .01.

Figure 5

Ablation of Tak1 in hepatocytes abolishes NF-κB activation and increases susceptibility to TGF-β–mediated apoptosis. (A) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining (left) and its quantification (right) in the primary hepatocytes from WT and Tak1^ΔHep mice after incubation with TGF-β1 (10 ng/mL) for 24 h. Original magnification ×320. (B) Phospho-p38, phospho-JNK, phospho-Smad2, phospho-Smad3, caspase 3, cleaved caspase 3, TAK1, and β-actin were determined by immunoblot analysis after primary hepatocytes from WT and Tak1^ΔHep mice were incubated with TGF-β1 (10 ng/mL) for the indicated time periods. (C, D) When stimulated with TGF-β (10 ng/mL) for 24 h, NF-κB was activated in the primary hepatocytes from the NF-κB-GFP reporter mice with Tak1 sufficiency and deficiency, and their GFP expression was determined by microscopy (C) and Western blotting (D). Original magnification ×400. (E) Adenoviral-IκB super repressor inhibited NF-κB activation, which, as a result, caused apoptosis in primary hepatocytes from WT when stimulated with TGF-β (10 ng/mL) for 24 h. Apoptosis was determined with TUNEL staining. Data are reported as mean ± standard error of mean. *P < .05; **P < .01.

Figure 6

Inactivation of Smad molecules reduces spontaneous liver injury, inflammation, fibrosis, and HCC in Tak1^ΔHep mice. (A) Apoptosis in the primary hepatocytes from WT, Tak1^ΔHep, Smad4^ΔHep, and Tak1/Smad4^ΔHep mice after being treated with TGF-β (10 ng/mL) for 24 h were analyzed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. The number of TUNEL-positive cells was counted. (B–E) WT, Tak1^ΔHep, and Tak1/Smad4^ΔHep mice at the age of 1 month (n = 8) were analyzed. (B) Apoptotic hepatocytes evaluated by TUNEL staining (left) and its quantification (lower right). Original magnification 200×. Immunoblots for cleaved caspase 3, caspase 3, Smad4, TAK1, and β-actin are shown (upper right). (C) Serum ALT levels. (D) Hepatic messenger RNA expression of inflammatory genes (Tnf, Il1b, and Ccl2) and fibrogenic genes (Col1A1, Acta2, and Tgfb1) determined by quantitative real-time polymerase chain reaction. (E) Fibrillar collagen deposition was determined by Sirius red staining (left) and its quantification (right). Original magnification ×100. (F) The number of tumors per mouse was counted and the maximum diameter of individual tumor nodules was measured. (WT, n = 10; Tak1^ΔHep, n = 42; Tak1/Smad4^ΔHep, n = 15). Data are represented as mean± standard error of mean. *P < .05; **P < .01.

Figure 7

Oncogenic gene expression in HCC from Tak1^ΔHep mice, and TGF-β signaling in DEN-induced murine HCC and in human HCC. (A–D) Liver tissues in 9-month-old WT and Tgfbr2^ΔHep mice, and nontumor liver tissues and tumor tissues in 9-month-old Tak1^ΔHep, Tak1/Tgfbr2^ΔHep were harvested. (n=7, each samples) (A) Hepatic messenger RNA expression of pro-oncogenes (Ctnnb1, Myc, Yap1, and Wisp1) was determined by quantitative real-time polymerase chain reaction (qPCR). (B) Hepatic messenger RNA expression of Bax and Bcl-xl was assessed by qPCR, and protein expression of Bcl-2 and Bcl-xL are shown by immunoblotting. (C, D) Hepatic messenger RNA expressions of Ctgf (C), Vegfa, and Vegfr2 (D, upper) were assessed by qPCR. (D, lower) Immunostaining for VEGFa (upper), VEGFR2, and CD31 (lower) are shown. Original magnification 320×. (E) Diethylnitrosamine was injected (25 mg/kg) in 14-day-old WT and Tgfbr2^ΔHep mice and their livers were harvested at 9 months after DEN injections. Representative macroscopic pictures (left). The number of tumors per mouse was counted and the maximum diameter of individual tumor nodules was measured (right). (WT, n = 16; Tgfbr2^ΔHep, n = 16). NT, nontumor liver, T, tumors. (F) Immunohistochemistry for phospho-Smad2/3 in liver biopsy samples from patients with chronic hepatitis C (n = 4) and in liver tissues from patients with HCC (n = 4). Data are represented as mean ± standard error of mean. *P < .05; **P < .01.