Impaired postnatal hepatocyte proliferation and liver regeneration in mice lacking c-jun in the liver

Abstract

Mice lacking the AP-1 transcription factor c-jun die at mid-gestation showing heart defects and impaired hepatogenesis. To inactivate c-jun in hepatocytes, mice carrying a floxed c-jun allele were generated. Perinatal liver-specific c-jun deletion caused reduced hepatocyte proliferation and decreased body size. After partial hepatectomy, half of the mutants died and liver regeneration was impaired. This phenotype was not present in mice lacking the N-terminal phosphorylation sites of c-Jun. The failure to regenerate was accompanied by increased cell death and lipid accumulation in hepatocytes. Moreover, cyclin-dependent kinases and several cell cycle regulators were affected, resulting in inefficient G(1)-S phase progression. These studies identify c-Jun as a critical regulator of hepatocyte proliferation and survival during liver development and regeneration.

Fig. 1. Generation of mice harbouring a floxed c-jun allele. (A) Schematic representation of the targeting strategy employed to generate a floxed allele of c-jun. The c-jun open reading frame is represented by a rectangle, thin lines represent untranslated regions of the c-jun locus. The neomycine resistance gene (NeoR), the thymidine kinase gene (tk) and the diphtheria toxin alpha gene (DTα) are indicated; loxP sites are shown as triangles. X, XbaI; Xh, XhoI; P, PstI; E, EcoRI; H, HindIII. (B) Southern blot analysis of genomic DNA from the parental ES cell line E14.1 (+/+) and from three targeted (targ/+) ES cell clones. (C) After transient tranfection of cre, ES cell clones were selected for the loss of the NeoR and tk genes. Southern blot analysis of genomic DNA from the parental ES cell line E14.1 (+/+) and from three floxed (f/+) ES cell clones.

Fig. 2. Analysis of mice lacking c-jun in the liver. (A) Photograph of 8-week-old female c-jun^Δli and littermate control mice. (B) Southern blot of genomic DNA isolated from various organs of a c-jun^f/f Alfp-cre mouse (c-jun^Δli). Br, brain; Li, liver; Pa, pancreas; Sp, spleen; Ki, kidney. (C) PCR analysis of genomic DNA isolated from various organs of a c-jun^Δli mouse. T, tail; Sp, spleen; Ki, kidney; Li, liver; Th, thymus; Lu, lung; M, skeletal muscle; Br, brain, He, Heart; Sk, skin; Pa, pancreas; Bl, bladder; Mg, mammary gland; Ut, uterus; Bo, bone; f/f, c-jun^f/f control; Li*, liver of c-jun^f/f Mx-cre (c-jun^Δli*) mouse; +/+, wild-type control. (D) Postnatal growth rate of one litter of c-jun^f/f (black squares) and c-jun^Δli mice (white triangles). The result is representative of six litters analysed from three independent breeding cages. (E) Quantification of BrdU-immunostaining of c-jun^f/f (black bar) and c-jun^Δli livers (white bar) 7 and 14 days after birth.

Fig. 3. c-jun is required for liver regeneration. Histological analysis of wild-type (A, B), c-jun^Δli (Alfp-cre; C, D, I), c-jun^Δli* (Mx-cre; E, F, J) and junAA/AA (G, H) livers before (A, C, E, G), 36 h after (B, D, F, H) and 72 h after (I, J) hepatectomy. In (D), (F), (I) and (J), arrowheads indicate areas of necrotic tissue; some fat-containing vacuoles are indicated by arrows (magnification ×20).

Fig. 4. Increased cell death and decreased hepatocyte proliferation after hepatectomy in the absence of c-jun. (A) Southern blot analysis of genomic DNA isolated from various organs of one pIpC-treated c-jun^f/+ Mx-cre+ mouse. Ki, kidney; Sp, spleen; Li, liver; Th, thymus; Te, testes; BM, bone marrow; Br, brain. (B) Southern blot analysis of genomic DNA isolated from tail (Tl) and liver (Li) of pIpC-treated (+) and untreated (–) c-jun^f/– heterozygous mice before and after hepatectomy. c-jun^– is the null allele (Hilberg et al., 1993). (C, G) Electronmicroscope image of a control (C) and a c-jun^Δli mutant liver (G) 48 h after hepatectomy. The hepatocyte nucleus is indicated by ‘N’ and fat-containing vacuoles by arrows. (D, H) Oil red O-staining of a control (D) and a c-jun^Δli* mutant (H) liver 48 h after hepatectomy. Neutral lipids are visualized as red droplets in (H). (E, I) TUNEL-staining of a control (E) and a c-jun^Δli* mutant liver (I) 48 h after hepatectomy. Some TUNEL-positive cells are indicated by arrows. (F, J) BrdU-immunostaining of c-jun^f/f (F) and c-jun^Δli* (J) livers 48 h after hepatectomy. Arrows and arrowheads point to some BrdU-positive non- parenchymal cells and hepatocytes, respectively (J). (K, L) Quantification of BrdU-positive hepatocytes (K) and non-parenchymal cells (L) of c-jun^f/f (black lines) and c-jun^Δli* (red lines) mice after hepatectomy. Magnification: (C, G), ×3150; (D, H), ×20; (E, F, I, J), ×40.

Fig. 5. Molecular analysis of regenerating livers lacking c-jun. (A) AP-1, STAT and NF-κB DNA-binding in nuclear extracts from c-jun^f/f, c-jun^Δli and c-jun^Δli* livers; numbers below the AP-1 electrophoretic mobility-shift assay of c-jun^f/f and c-jun^Δli livers represent the fold induction of DNA-binding activity relative to the respective 0 time point; the asterisk indicates binding of p50 homodimers. (B) Immuno precipitated cyclin D1-, cyclin E- or cyclin B1-associated kinase activity in c-jun^f/f and c-jun^Δli* livers isolated at the indicated times after hepatectomy. Numbers below the cyclin D1-kinase activity represent the fold induction relative to the respective 0 time point. The presence of cyclin D1, cyclin E and cyclin B1 in the immunoprecipitates was confirmed by western blot analysis.

Fig. 6. Protein and RNA expression in regenerating livers lacking c-jun. (A, B) Expression of various proteins in c-jun^f/f, c-jun^Δli* (A) and c-jun^Δli (B) livers isolated at the indicated time points after hepatectomy. β-actin was used as a loading control. (C) Induction of c-jun, c-fos and fox mRNA (loading control), p21, PCNA and cyclinD1 in regenerating c-jun^f/f and c-jun-mutant livers at the indicated time points after hepatectomy.