The Forkhead Box m1b transcription factor is essential for hepatocyte DNA replication and mitosis during mouse liver regeneration

Abstract

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA-binding domain and whose members play essential roles in cellular proliferation, differentiation, transformation, longevity, and metabolic homeostasis. Liver regeneration studies with transgenic mice demonstrated that FoxM1B regulates the onset of hepatocyte DNA replication and mitosis by stimulating expression of cell cycle genes. Here, we demonstrate that albumin-promoter-driven Cre recombinase-mediated hepatocyte-specific deletion of the Foxm1b Floxed (fl) targeted allele resulted in significant reduction in hepatocyte DNA replication and inhibition of mitosis after partial hepatectomy. Reduced DNA replication in regenerating Foxm1b(-/-) hepatocytes was associated with sustained increase in nuclear staining of the cyclin-dependent kinase (Cdk) inhibitor p21(Cip1) (p21) protein between 24 and 40 h after partial hepatectomy. Furthermore, increased nuclear p21 levels and reduced expression of Cdc25A phosphatase coincided with decreases in Cdk2 activation and hepatocyte progression into S-phase. Moreover, the significant reduction in hepatocyte mitosis was associated with diminished mRNA levels and nuclear expression of Cdc25B phosphatase and delayed accumulation of cyclin B1 protein, which is required for Cdk1 activation and entry into mitosis. Cotransfection studies demonstrate that FoxM1B protein directly activated transcription of the Cdc25B promoter region. Our present study shows that the mammalian Foxm1b transcription factor regulates expression of cell cycle proteins essential for hepatocyte entry into DNA replication and mitosis.

Fig 1.

fl Foxm1b-targeting vector, targeted Foxm1b allele, and deleted Foxm1b allele. (A) Diagrammatical representation of the mouse Foxm1b locus with seven exons (exons A1 and A2 are found in the Foxm1a splicing isoform, and exon A1 is found in the Foxm1c isoform). Also shown is the Foxm1b fl-targeting vector, the Foxm1b fl-targeted allele, and the deleted Foxm1b allele after introduction of the albumin promoter-driven Cre recombinase transgene (Alb-Cre). Hatched boxes indicate the position of the 5′ and 3′ Southern blot hybridization probes and the exons encoding the winged helix DNA-binding domain. Dotted lines indicate restriction enzymes used to introduce the KpnI-containing LoxP sequence (used for 5′ end screening), the LoxP PGK promoter-driven neomycin (neo) gene LoxP positive-selection cassette, and the PGK HSV-TK gene (used for negative selection for nonhomologous recombination). Insertion of the LoxP sequences disrupted the BglII restriction sites (BglII), and deletion by the Alb-Cre recombinase protein removes Foxm1b sequences flanking the two farthest LoxP sites, leaving one LoxP sequence. (B and C) Southern blot of genomic DNA from Foxm1b fl-targeted ES cells showing bands specific to WT and Foxm1b fl loci (pseudogene band indicated by *). (D) Southern blot of liver genomic DNA from Foxm1b fl/fl mice and the Alb-Cre recombinase transgene-mediated hepatocyte deletion (−/−) of the Foxm1b fl/fl allele creating the larger molecular weight DNA fragment. (E) PCR analysis of mouse tail genomic DNA with primers flanking the third intron LoxP sequence resulting in a larger PCR DNA product with the Foxm1b fl-targeted locus.

Fig 2.

Liver regeneration studies demonstrate that Foxm1b-deficient (−/−) hepatocytes lack induction of the Foxm1b mRNA and protein. (A) RPA demonstrate significant reduction in expression of Foxm1b mRNA. Total RNA was prepared from regenerating livers from either fl/fl mice or Alb-Cre Foxm1b^−/− mice at indicated hours after PHx (numbers above panels) and was used for RPA to demonstrate that regenerating Foxm1b^−/− hepatocytes failed to induce high levels of Foxm1b mRNA. Immunohistochemical staining of regenerating liver sections (40 h after PHx) with Foxm1b antibody demonstrates that regenerating Alb-Cre Foxm1b^−/− hepatocytes (C) display undetectable nuclear staining of Foxm1b protein compared with regenerating Foxm1b fl/fl hepatocytes (B).

Fig 3.

Regenerating Alb-Cre Foxm1b^−/− mouse liver displays significant decreases in hepatocyte DNA replication and mitosis. (A) Graphic representation of diminished BrdUrd incorporation during mouse liver regeneration in Foxm1b-deficient hepatocytes. Graphically presented is the BrdUrd incorporation (DNA replication) detected by immunohistochemical staining (10, 25) at the indicated hours after PHx with either 8-week-old Alb-Cre Foxm1b^−/− mice or Foxm1b fl/fl littermates. The mean of the number of BrdUrd-positive hepatocyte nuclei per 1,000 hepatocytes ± SE was calculated for each time point (three mice per time point). (B) Graphic representation of diminished hepatocyte mitosis in regenerating livers of Foxm1b-deficient vs. fl Foxm1b mouse liver between 32 and 52 h after PHx. Hepatocyte mitosis is expressed as the mean of the number of mitotic figures found per 1,000 hepatocytes ± SD using three mice per time point (10, 25).

Fig 4.

Regenerating Alb-Cre Foxm1b^−/− liver display increased p21 protein levels and nuclear staining. (A) Western blot analysis with p21 antibody reveals increased p21 protein levels in regenerating Alb-Cre Foxm1b^−/− liver. Total protein extracts were isolated from regenerating liver of Foxm1b fl/fl and Alb-Cre Foxm1b^−/− mice and analyzed for protein expression of p21 by Western blot analysis. Shown below the panels is the fold increase in expression levels with respect to regenerating Foxm1b fl/fl liver at the 24-h time point. Immunohistochemical staining of regenerating liver sections at the indicated time points after PHx with p21 antibody demonstrates increased hepatocyte nuclear staining of p21 protein in Alb-Cre Foxm1b^−/− liver (F–I) compared with Foxm1b fl/fl littermates (B–E). (B–I, ×100 magnification.)

Fig 5.

Regenerating Alb-Cre Foxm1b^−/− liver exhibit diminished Cdc25A protein levels and Cdk2 activity. (A) Western blot analysis with Cdc25A antibody reveals diminished Cdc25A phosphatase protein levels in regenerating Alb-Cre Foxm1b^−/− liver. (B) Diminished Cdk2 kinase activity in regenerating Alb-Cre Foxm1b^−/− liver. Total protein extracts were isolated from regenerating liver, immunoprecipitated with Cdk2 antibody, and used for Cdk2 kinase assays with RB protein substrate. Position of the phosphorylated and hyperphosphorylated (*) RB protein is indicated on panels. (C) Western blot analysis with cyclin B1 antibody reveals delayed cyclin B1 protein levels in regenerating Alb-Cre Foxm1b^−/− liver. A nonspecific band reacting with the cyclin B1 antibody is labeled by NS. Shown below the panels is the fold increase in expression levels with respect to regenerating Foxm1b fl/fl liver at the 24- or 32-h time points.

Fig 6.

Regenerating Alb-Cre Foxm1b^−/− liver exhibits diminished expression and nuclear staining of cdc25B phosphatase protein. (A) RPA demonstrate that regenerating Alb-Cre Foxm1b^−/− liver exhibited reduced Cdc25B mRNA levels and slight decreases in expression of cyclin A2, cyclin B1, and Cdk1. (B and C) Undetectable hepatocyte nuclear Cdc25B protein staining in regenerating liver of Alb-Cre Foxm1b^−/− mice. Immunohistochemical staining of regenerating liver sections (40 h after PHx) with Cdc25B antibody demonstrates that regenerating Alb-Cre Foxm1b^−/− hepatocytes display undetectable Cdc25B nuclear staining (C) compared with regenerating Foxm1b fl/fl hepatocytes (B). (D) Increased phosphorylation of Cdk1 protein in regenerating Alb-Cre Foxm1b^−/− liver. Western blot analysis with Cdk-1 phospho-tyrosine-15 antibody reveals increased Cdk-1 phosphorylation in regenerating Alb-Cre Foxm1b^−/− liver resulting from diminished cdc25B phosphatase levels. (E) Diminished Cdk1-dependent phosphorylation of histone H1 protein in regenerating Alb-Cre Foxm1b^−/− liver. The Cdk1 protein was immunoprecipitated from regenerating liver extracts and used for kinase assays with H1 protein phosphorylation substrate. Shown below the panels is the fold increase in expression levels with respect to regenerating liver at 24 h after PHx and (E) with respect to Foxm1b fl/fl 32-h time point. (F) Foxm1b directly activates transcription of the Cdc25B promoter in cotransfection assays. The osteosarcoma U2OS cell line was cotransfected with the mouse −200-bp Cdc25B promoter luciferase plasmid and CMV expression vector containing either human FoxM1B full-length cDNA (1–748) or transcriptionally inactive FoxM1B (1–688) deletion as described (25). Graphic presentation of normalized fold induction of Cdc25B promoter expression in response to FoxM1B cDNA cotransfection, with CMV-empty vector control set at 1.0. Two transfection experiments were performed in duplicate and used to determine mean fold induction ± SD. (G). Diagram depicting FoxM1B regulation of cell cycle genes. Blue arrows represent positive regulation and black lines represent negative regulation.