Proc Natl Acad Sci U S A 99(26): 16881-16886

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

Department of Molecular Genetics, University of Illinois College of Medicine, 900 South Ashland Avenue, Chicago, IL 60607

^{To whom correspondence should be addressed at: Department of Molecular Genetics (M/C 669), University of Illinois College of Medicine, 900 South Ashland Avenue, Room 2220 MBRB, Chicago, IL 60607-7170. E-mail:}ude.ciu@atsocbor.

Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved October 22, 2002

Received 2002 Sep 19

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}^{(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.}

Keywords: knock-out mouse, Cdc25A, Cdc25B, cyclin-dependent kinase inhibitor p21^Cip1

Abstract

Cell division is a tightly regulated process, especially at the initiation of DNA replication (S-phase) and at the entry into mitosis (M-phase). Progression through the cell cycle is regulated by temporal activation of multiple cyclin-dependent kinases (Cdk). In addition to assembly with a cyclin-regulatory subunit, Cdk activity requires dephosphorylation of the Cdk catalytic subunit by the Cdc25A, Cdc25B, or Cdc25C phosphatase protein (1–3) and is negatively regulated by Cdk inhibitor p21^{(p21), p27}^{, and p16}⁽4). Cdk2 activity in complex with cyclin E and cyclin A is critical for S-phase progression because cyclin E/A-Cdk2 cooperates with cyclin D-Cdk4/6 to phosphorylate the retinoblastoma (RB) protein, which releases bound E2F transcription factor and allows it to stimulate expression of proliferation-specific target genes (5, 6). Likewise, phosphorylation of critical target proteins by the active cyclin B-Cdk1 complex mediates progression into mitosis (7).

The Forkhead Box (Fox) transcription factors are an extensive family of transcription factors, consisting of more than 50 mammalian proteins (8) that share homology in the winged helix DNA-binding domain (9). Its members play important roles in regulating expression of genes involved in cellular proliferation (10), differentiation (11–13), apoptosis (14), transformation (15), longevity (16), and metabolic homeostasis (17). The mammalian liver is one of the few adult organs capable of completely regenerating itself in response to injury, and which is mediated by the release of growth factors and cytokines that stimulate reentry of terminally differentiated hepatocytes into the cell cycle (18–21). In regenerating liver, increased hepatic expression of FoxM1B levels occurs at the G₁/S transition of the cell cycle; its levels remain elevated throughout the period of proliferation, suggesting that the delayed early FoxM1B transcription factor plays a role in cell cycle progression (22). Premature expression of FoxM1B (HFH-11B) levels in regenerating liver of transgenic (TG) mice accelerated the onset of hepatocyte DNA replication and mitosis through stimulating earlier expression of cell cycle genes (10, 23). Analysis of cDNA microarrays shows that diminished proliferation exhibited by human fibroblasts from either the elderly or genetically aged patients with Hutchinson-Gilford progeria compared with proliferating young human fibroblasts is associated with reduced expression of FoxM1B and its cell cycle target genes (24). Recent liver regeneration studies indicate that maintaining hepatocyte expression of FoxM1B in 12-month old (old-aged) TG mice is sufficient to increase hepatocyte DNA replication and mitosis and reestablish expression of cell cycle regulatory genes to levels found in young regenerating mouse liver (25, 26). These results suggest the hypothesis that Foxm1b controls the transcriptional network of genes essential for cell cycle progression.

In this study, we performed partial hepatectomy (PHx) operations to induce hepatic regeneration, and we demonstrated that albumin-promoter-driven Cre recombinase (Alb-Cre)-mediated hepatocyte-specific deletion of the Foxm1b Floxed (fl/fl) allele resulted in significant reduction in hepatocyte DNA replication and inhibition of mitosis. Diminished hepatocyte proliferation in regenerating Alb-Cre Foxm1b^{liver was associated with altered expression of proteins that limit Cdk1 and Cdk2 activity required for normal cell cycle progression into DNA replication and mitosis.}

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Acknowledgments

We thank H. Ye for isolation of the Foxm1b genomic clone and R. Franks for injection of Foxm1b-targeted ES cells into mouse blastocysts. We also thank P. Raychaudhuri, K. Krupczak-Hollis, V. Kalinichenko, Y. Zhou, M. Major, and F. Rausa for critically reviewing the manuscript. This work was supported by National Institutes of Health Grant DK 54687 from National Institute of Diabetes and Digestive and Kidney Diseases (to R.H.C.).

Acknowledgments

Abbreviations

Cdk, cyclin-dependent kinase
RB, retinoblastoma
Foxm1b, Forkhead Box m1b
PHx, partial hepatectomy
Alb-Cre, albumin-promoter-driven Cre recombinase
fl, Floxed
neo, neomycin
ES, embryonic stem
BrdUrd, 5 bromo-2′-deoxyuridine
p21, p21^Cip1
RPA, RNase protection assays
CMV, cytomegalovirus

Abbreviations

Notes

This paper was submitted directly (Track II) to the PNAS office.

Notes

This paper was submitted directly (Track II) to the PNAS office.

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