Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas

Abstract

PTEN is a tumor suppressor gene mutated in many human cancers, and its expression is reduced or absent in almost half of hepatoma patients. We used the Cre-loxP system to generate a hepatocyte-specific null mutation of Pten in mice (AlbCrePten(flox/flox) mice). AlbCrePten(flox/flox) mice showed massive hepatomegaly and steatohepatitis with triglyceride accumulation, a phenotype similar to human nonalcoholic steatohepatitis. Adipocyte-specific genes were induced in mutant hepatocytes, implying adipogenic-like transformation of these cells. Genes involved in lipogenesis and beta-oxidation were also induced, possibly as a result of elevated levels of the transactivating factors PPARgamma and SREBP1c. Importantly, the loss of Pten function in the liver led to tumorigenesis, with 47% of AlbCrePten(flox/flox) livers developing liver cell adenomas by 44 weeks of age. By 74-78 weeks of age, 100% of AlbCrePten(flox/flox) livers showed adenomas and 66% had hepatocellular carcinomas. AlbCrePten(flox/flox) mice also showed insulin hypersensitivity. In vitro, AlbCrePten(flox/flox) hepatocytes were hyperproliferative and showed increased hyperoxidation with abnormal activation of protein kinase B and MAPK. Pten is thus an important regulator of lipogenesis, glucose metabolism, hepatocyte homeostasis, and tumorigenesis in the liver.

Figure 1

Generation of hepatocyte-specific Pten-deficient (AlbCrePtenflox/flox) mice. (A) Targeting strategy. Exons of the murine Pten gene are represented by filled boxes, loxP sites by filled arrowheads. The probe used to analyze Southern blots is indicated and has been described previously (14). The floxed (Ptenflox) and deleted (Pten^Ø) alleles are shown. (B) Genomic Southern blot. DNA (20 ∝g) extracted from total liver cells of the indicated genotypes was digested with HindIII. The vast majority of total liver cells from AlbCrePtenflox/flox mice showed deletion of the Pten gene. (C) Western blot analysis of Pten protein expression by total liver cells of the indicated genotype. Actin, loading control. Liver samples were obtained from 8-week-old mice.

Figure 2

Liver anomalies in AlbCrePtenflox/flox mice. (A and B) Macroscopic analyses of livers. Hepatomegaly and pale coloring was observed in 10-week-old (A) and 40-week-old (B) AlbCrePtenflox/flox mice. For all panels: +/+, AlbCrePten+/+; flox/+, AlbCrePtenflox/+, and flox/flox, AlbCrePtenflox/flox mice. (C) Decreased liver weight. The liver weight/body weight ratio was analyzed in +/+, flox/+, and flox/flox mice at 10 weeks (n = 11 mice/group) and 40 weeks (n = 15). For all panels, where appropriate, results are expressed as the mean ± SEM of the indicated number of mice per group. Statistical differences were determined using the Student’s t test: *P < 0.05. (D) Decreased number of liver cells. Absolute numbers of total liver cells were determined in +/+, flox/+, and flox/flox livers at 10 weeks (n = 7 mice/group) and 40 weeks (n = 7).

Figure 3

Steatohepatitis in AlbCrePtenflox/flox mice. (A) Histological analyses of liver sections from +/+ and flox/flox mice sacrificed at 10 weeks of age. The first and second rows show a lower (∞10) and higher (∞400) magnification of H&E-stained livers, respectively. Lipid accumulation was observed around the central vein area (C), whereas the periportal vein area (P) was almost intact. Lipid accumulation was confirmed by Oil-red O staining (third row). Liver fibrosis was not evident as determined by Azan staining (fourth row). Lipid accumulation was also confirmed by electron microscopy (fifth row). (B) Histological analyses of liver sections of +/+, flox/+, and flox/flox mice (without precancerous tumors) sacrificed at 40 weeks of age. The first and second rows show a lower (∞10) and higher (∞400) magnification of H&E-stained liver sections, respectively. The third row (left) shows an accumulation of lipid in the mutant liver that resembles adipocytes in a fat tissue. Additional features of mutant livers were steatohepatitis and Mallory bodies (third row right, arrows), sinusoidal fibrogenic changes (fourth row left), and an accumulation of inflammatory cells (fourth row right). Focal lipid-laden hepatocytes within diffuse mild fatty changes were noted in the livers of some flox/+ mice (fifth row).

Figure 4

Expression of adipogenic and lipogenic genes in the absence of hepatic Pten. (A) RT-PCR analyses of the indicated genes in +/+, flox/+, and flox/flox livers. The relative intensity of each amplified PCR band as measured by NIH Image was as follows: PPARγ +/+, 1; flox/+, 2; flox/flox, 8; PPARα (1, 1, 1); C/EBPα (1, 1, 1); C/EBPβ (1, 1, 0.7); C/EBPδ (1, 1, 1); SREBP1c (1, 1.5, 3); adiponectin (1, 1, 4); adipsin (1, 1, 4); aP2 (1, 1, 4); FAS (1, 1, 3); ACC (1, 1, 2); SCD1 (1, 1, 2); AOX (1, 1, 2); L-PBE (1, 1, 2); PTL (1, 1, 2.5); β-actin (1, 1, 1). Data shown are representative of five independent experiments. (B) Expression of adipocyte-specific and hepatocyte-specific markers in the liver. Immunohistochemical double staining using antibodies specific for adiponectin and albumin revealed the strong coexpression of these molecules in flox/flox liver cells (lower panel) compared with +/+ liver cells (upper panel). Magnification ∞1000.

Figure 5

Liver tumors observed in AlbCrePtenflox/flox mice. (A) Macroscopic view of representative liver adenomas (arrows) observed in 9/19 flox/flox mice of 40_44 weeks of age. (B) H&E-stained section of a liver cell adenoma (T) in (A) flox/flox male mouse, showing its distinct demarcation from the surrounding nontumorous liver tissue. The adenoma hepatocytes contain large quantities of lipid. (C) H&E-stained section of a flox/+ liver (male, 42 weeks). (D) Macroscopic view of a representative mutant liver (male) showing the HCCs observed in 8/12 flox/flox mice at 74_78 weeks of age. (E) H&E-stained section of the flox/flox (male) liver in (D), showing an HCC with a trabecular-like arrangement that disrupts normal liver architecture. (F) Lung of the mouse in D, showing a metastasis (T). Magnifications are ∞40 (B), ∞400 (C), ∞400 (E), and ∞400 (F).

Figure 6

Increased hepatocyte proliferation, phosphorylation of PKB/Akt, Foxo1, and MAPK (ERK1/2), and H2O2 production in AlbCrePtenflox/flox mice. (A) Enhanced hepatocyte proliferation. Left panel: BrdU⁺ hepatocytes (arrows) were counted 4 days after administration of BrdU in drinking water. Right panel: data are expressed as the mean percentage of BrdU⁺ hepatocytes ± SEM for 1 ∞ 10⁴ cells per mouse for four mice per group. (B) No effect on apoptosis. Left panel: TUNEL⁺ hepatocytes (arrows) were counted. Right panel: data are expressed as the mean percentage of TUNEL⁺ hepatocytes ± SEM for 1 ∞ 10⁴ cells per mouse for four mice per group. For A and B, results shown are one experiment representative of 3 trials. (C) Increased phosphorylation of signal transducers. The phosphorylated forms of PKB/Akt, Foxo1, and ERK1/2 in liver lysates of the indicated genotypes were determined by immunoblotting. Total PKB/Akt, Foxo1, and ERK1/2 levels were evaluated as controls. (D) Increased H2O2 production. H2O2 concentrations in liver homogenates from 10-week-old +/+, flox/+, and flox/flox mice were determined by the phenol red method. Results are expressed as the mean H2O2 produced ± SEM for four mice per group and are representative of three trials.