The Forkhead Box m1b transcription factor is
essential for hepatocyte DNA replication and
mitosis during mouse liver regeneration
Xinhe Wang, Hiroaki Kiyokawa, Margaret B. Dennewitz, and Robert H. Costa*
Department of Molecular Genetics, University of Illinois College of Medicine, 900 South Ashland Avenue, Chicago, IL 60607
Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved October 22, 2002 (received for review September 19, 2002)
The Forkhead Box (Fox) proteins are an extensive family of tran-
scription factors that shares homology in the winged helix DNA-
binding domain and whose members play essential roles in cellular
proliferation, differentiation, transformation, longevity, and met-
abolic 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 re-
duction in hepatocyte DNA replication and inhibition of mitosis
after partial hepatectomy. Reduced DNA replication in regenerat-
ing Foxm1b?/?hepatocytes was associated with sustained in-
crease in nuclear staining of the cyclin-dependent kinase (Cdk)
inhibitor p21Cip1(p21) protein between 24 and 40 h after partial
hepatectomy. Furthermore, increased nuclear p21 levels and re-
in Cdk2 activation and hepatocyte progression into S-phase. More-
over, the significant reduction in hepatocyte mitosis was associ-
ated with diminished mRNA levels and nuclear expression of
Cdc25B phosphatase and delayed accumulation of cyclin B1 pro-
tein, which is required for Cdk1 activation and entry into mitosis.
Cotransfection studies demonstrate that FoxM1B protein directly
study shows that the mammalian Foxm1b transcription factor
regulates expression of cell cycle proteins essential for hepatocyte
entry into DNA replication and mitosis.
knock-out mouse ? Cdc25A ? Cdc25B ? cyclin-dependent kinase inhibitor
mitosis (M-phase). Progression through the cell cycle is regu-
lated by temporal activation of multiple cyclin-dependent ki-
nases (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 phospha-
tase protein (1–3) and is negatively regulated by Cdk inhibitor
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 ex-
pression 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 mam-
malian liver is one of the few adult organs capable of completely
ell division is a tightly regulated process, especially at the
initiation of DNA replication (S-phase) and at the entry into
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
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 dimin-
ished 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
In this study, we performed partial hepatectomy (PHx) oper-
ations 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 al-
tered expression of proteins that limit Cdk1 and Cdk2 activity
required for normal cell cycle progression into DNA replication
Materials and Methods
Generation of Mice with fl Foxm1b-Targeted Allele and Hepatocyte-
Specific Deletion. Mouse Foxm1b genomic DNA was isolated
from mouse 129SvJ genomic library (Stratagene) and charac-
terization of the intron and exon boundaries was determined as
described (22). We constructed a triple-LoxP Foxm1b-targeting
vector to generate an ‘‘fl’’ mouse Foxm1b-targeted locus con-
the winged helix DNA-binding domain and LoxP PGK-1 neo-
exon 7 (Fig. 1A). The PGK-1 promoter-driven herpes simplex
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Cdk, cyclin-dependent kinase; RB, retinoblastoma; Foxm1b, Forkhead Box
m1b; PHx, partial hepatectomy; Alb-Cre, albumin-promoter-driven Cre recombinase; fl,
RPA, RNase protection assays; CMV, cytomegalovirus.
*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: firstname.lastname@example.org.
www.pnas.org?cgi?doi?10.1073?pnas.252570299 PNAS ?
December 24, 2002 ?
vol. 99 ?
no. 26 ?
virus-thymidine kinase (HSV-TK) gene was placed outside of
the Foxm1b gene homology region for negative selection of
nonhomologous recombination during targeted embryonic stem
(ES) cell selection by using ganciclovir. We used this Foxm1b
Genome Systems, St. Louis), which were propagated on mouse
embryo fibroblasts feeder cells (Genome Systems) and selected
for neo (G418) and HSV-TK (ganciclovir) resistance, as de-
scribed (27). ES cells with the appropriate Foxm1b fl-targeted
locus were identified by Southern blot analysis and were used to
generate chimeric mice by injecting them into mouse blastocysts.
Mice containing the Foxm1b fl targeted allele were determined
by Southern blot analysis with 5? and 3? probes (Fig. 1 A–C) and
PCR amplification with primers flanking the LoxP sequence
located in the third exon (Fig. 1E). These Foxm1b primers
included the sense 5?-taggagatacactgttatat-3? and the antisense
5?-tgtgggaaaatgcttacaaaag-3?. The chimeric mice were bred with
C57?B6 WT mice to produce Foxm1b fl?? mice, which were
backcrossed to generate viable Foxm1b fl?fl mice. Liver regen-
eration studies used Foxm1b fl?fl mice that were bred into the
C57?B6 background for four generations. Hepatocyte-specific
deletion of the Foxm1b fl?fl allele was accomplished through
breeding with the Alb-Cre recombinase C57B6 transgenic mice
(The Jackson Laboratory; ref. 28). Hepatocyte-specific deletion
of the Foxm1b fl?fl allele was verified by using liver genomic
DNA for Southern blot analysis (Fig. 1D).
Partial Hepatectomy Surgery, Immunohistochemical Staining, West-
ern Blot Analysis, and RNase Protection Assays (RPA).Eight-weekold
Alb-Cre Foxm1b?/?mice or Foxm1b fl?fl littermates were
subjected to PHx operations to induce liver regeneration (10,
25). Three mice at each time point were killed by using CO2
asphyxiation at the following intervals after PHx: 24, 28, 32, 36,
40, 44, 48, and 52 h and 7 days. The regenerating livers were
(10), (ii) to isolate total protein extract, and (iii) for paraffin
embedding (29). DNA replication was monitored by immuno-
histochemical detection of 5-bromo-2?-deoxyuridine (BrdUrd,
Sigma) incorporation as described (10, 25). By using three
regenerating livers per time point, we counted the number of
BrdUrd-positive nuclei per 1,000 hepatocytes to calculate the
mean number of BrdUrd-positive cells (? SE), as described (10,
25). Three regenerating liver sections at 32, 36, 40, 44, 48, and
52 h after PHx were stained with hematoxylin and eosin and
examined for hepatocyte mitotic figures (mitosis). Hepatocyte
mitosis is expressed as the mean of the number of mitotic figures
per 1,000 hepatocytes ? SD, as described (10, 25).
Antibodies specific to FoxM1B (10, 22), p21 (Calbiochem), or
Cdc25B (Santa Cruz Biotechnology) proteins were used for
immunohistochemical detection of paraffin-embedded 5-?m
sections of regenerating liver by using methods described (10, 22,
26). For Western blot analysis, 50 ?g of total liver protein (29)
was separated on SDS?PAGE and transferred to Protran mem-
brane (Schleicher & Schuell). The signals from primary p21
(Calbiochem; 1:750), Cdc25A (Santa Cruz Biotechnology,
1:200), cyclin B1 (Santa Cruz Biotechnology, 1:200) and Cdk1
phosphotyrosine-15-specific (Cell Signaling, Berkeley, CA;
1:1,000) antibodies were amplified by biotin-conjugated anti-
rabbit IgG (Bio-Rad) and detected with Enhanced Chemilumi-
nescence Plus (Amersham Pharmacia). Total RNA was pre-
pared from mouse liver at indicated hours after PHx by using
RNA-STAT-60 (Tel-Test, Friendswood, TX) and was used for
cell cycle regulatory genes or the p21 gene, and expression levels
were calculated as described (23, 25, 26).
Immunoprecipitation Kinase Assays. Either the Cdk1 (2 ?g, Lab-
vision) or Cdk2 antibody (1 ?g, Santa Cruz Biotechnology) and
immunoprecipitate these kinases from 200 ?g of total protein
extracts from regenerating liver. The immunoprecipitated Cdk1
and Cdk2 proteins were used for kinase assays with [32P]?-ATP
and either the Cdk2 substrate Rb protein (Santa Cruz Biotech-
nology) or the Cdk1 substrate histone H1 protein (Santa Cruz
Biotechnology), as described (25, 27). One half of the kinase
reaction was separated by SDS?PAGE and exposed to a phos-
phorimager screen, scanned with the Storm 860 phosphorim-
ager, and phosphorylated bands were quantitated with the
IMAGEQUANT program (Amersham Pharmacia).
U2OS Cell Cotransfection Assays. U2OS cells were transfected with
50 ng of either CMV (cytomegalovirus)-FoxM1B (1–748)
cDNA, CMV-FoxM1B (1–688) cDNA, or CMV-empty expres-
sion vectors and 1500 ng of the ?200-bp mouse Cdc25B-
promoter luciferase plasmid using Fugene6 (Roche Molecular
Biochemicals). Protein extracts were prepared from transfected
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 in-
troduce the KpnI-containing LoxP sequence (used for 5? end screening), the
LoxP PGK promoter-driven neomycin (neo) gene LoxP positive-selection cas-
sette, and the PGK HSV-TK gene (used for negative selection for nonhomolo-
gous 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 (pseudo-
gene band indicated by*). (D) Southern blot of liver genomic DNA from
Foxm1b fl?fl mice and the Alb-Cre recombinase transgene-mediated hepato-
cyte 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.
fl Foxm1b-targeting vector, targeted Foxm1b allele, and deleted
www.pnas.org?cgi?doi?10.1073?pnas.252570299 Wang et al.
U2OS cells at 24 h after DNA transfection, and the Dual-
Luciferase Assay System (Promega) was used to measure lucif-
erase enzyme activity, as described (25). Results are presented
as mean fold induction of Cdc25B promoter activity ? SD from
two separate experiments in duplicate, with the CMV-empty
value set at 1.0.
Alb-Cre Recombinase Mediates Hepatocyte-Specific Deletion of the
Mouse Foxm1b fl?fl-Targeted Allele. To generate mice with a
hepatocyte-specific deletion of the mouse Foxm1b gene, we used
a triple-LoxP Foxm1b fl-targeting vector (Fig. 1A) for electro-
poration of mouse ES cells. We selected G418 (neo) and
ganciclovir (HSV-TK)-resistant ES cells containing a Foxm1b
fl-targeted allele (27) and used them to generate Foxm1b fl?fl
mice (see Materials and Methods for details). Albumin promoter-
driven Alb-Cre mediates hepatocyte-specific deletion of the
mouse Foxm1b fl?fl-targeted allele, removing the entire winged
helix DNA-binding domain and the C-terminal transcriptional-
activation domain (22), thereby preventing expression of func-
tional Foxm1b protein (Fig. 1A). Mouse ES cells containing
homologous recombination with the Foxm1b fl-targeting vector
were identified by Southern blot analysis. By using the 5?
is detected by a smaller molecular weight DNA fragment be-
cause of the additional KpnI site engineered at the end of the
LoxP sequence (Fig. 1B). By using the 3? genomic probe and
BglII?XbaI digestion (Fig. 1A), the Foxm1b fl allele is detected
by a larger band because of the additional 1.6-kb PGK-neo
positive selection cassette (Fig. 1C). After mouse blastocyst
with germ line transmission and bred them to generate viable
Foxm1b fl?fl C57?B6 mice (Fig. 1D). PCR amplification of
mouse tail genomic DNA with primers that flanked the third
intron LoxP site detected the larger molecular weight product
derived from the Foxm1b fl allele (Fig. 1E). Breeding the
Alb-Cre recombinase transgene into the Foxm1b fl?fl mouse
genetic background allowed hepatocyte-specific deletion of the
Foxm1b locus within 6 weeks after birth (28), as detected by a
larger molecular weight band in Southern blots of BglII?XbaI-
digested liver genomic DNA (Fig. 1D, band indicated by ???).
Foxm1b Is Required for Normal Hepatocyte DNA Replication and
Mitosis in Regenerating Liver. Adult Alb-Cre Foxm1b?/?livers
were histologically normal before the PHx and exhibited no
difference in liver weight?body weight ratio compared with
Foxm1b fl?fl controls. Eight-week old Alb-Cre Foxm1b?/?mice
or Foxm1b fl?fl littermates were subjected to PHx, and their
regenerating livers were harvested at different intervals between
24 and 52 h and 7 days after surgery. Regenerating liver from
Foxm1b fl?fl littermates displayed normal induction of Foxm1b
mRNA and abundant hepatocyte nuclear staining of Foxm1b
protein at 40 h after PHx (Fig. 2 A and B). Confirming
hepatocyte-specific deletion of the Foxm1b fl?fl allele, we found
that regenerating Alb-Cre Foxm1b?/?liver failed to induce high
levels of Foxm1b mRNA and displayed no hepatocyte nuclear
staining of Foxm1b protein in any of the micrographs examined
(Fig. 2 B and C and data not shown). The Foxm1b fl?fl mice
exhibited a bifunctional S-phase peak in hepatocyte DNA rep-
lication, as evidenced by BrdUrd incorporation between 32 and
44 h after PHx (Fig. 3A). In contrast, regenerating Alb-Cre
Foxm1b?/?liver displayed a significant reduction in hepatocyte
DNA replication, but BrdUrd incorporation was still detectable
during these time points (Fig. 3A). A more significant reduction
in hepatocyte mitosis was observed in regenerating Alb-Cre
Foxm1b?/?liver, as evidenced by the paucity of mitotic figures
between 36 to 52 h after PHx compared with regenerating
Foxm1b fl?fl liver (Fig. 3B). This diminished hepatocyte mitosis
in regenerating Alb-Cre Foxm1b?/?liver caused hepatocyte
hypertrophy and compensatory increase in liver size at 7 days
after PHx, but they did not exhibit any increase in apoptosis, as
measured by terminal deoxynucleotidyltransferase-mediated
dUTP assay (see Supporting Text and Fig. 7, which are published
These studies suggest that Foxm1b is critical for mediating
normal levels of hepatocyte DNA replication and is essential for
progression into mitosis.
Regenerating Alb-Cre Foxm1b?/?Liver Displays Increased Levels of
p21 and Diminished Cdc25A Protein Expression Leading to Decreased
Cdk2 Activity. Previous CCl4 liver injury studies demonstrated
that increased hepatocyte levels of FoxM1B mediated reduced
expression of Cdk inhibitor p21 protein before S-phase (23).
Because Alb-Cre Foxm1b?/?liver displayed minimal changes in
hepatocytes lack induction of the Foxm1b mRNA and protein. (A) RPA dem-
onstrate significant reduction in expression of Foxm1b mRNA. Total RNA was
mice at indicated hours after PHx (numbers above panels) and was used for
RPA to demonstrate that regenerating Foxm1b?/?hepatocytes failed to in-
duce high levels of Foxm1b mRNA. Immunohistochemical staining of regen-
erating liver sections (40 h after PHx) with Foxm1b antibody demonstrates
that regenerating Alb-Cre Foxm1b?/?hepatocytes (C) display undetectable
decreases in hepatocyte DNA replication and mitosis. (A) Graphic represen-
Foxm1b-deficient hepatocytes. Graphically presented is the BrdUrd incorpo-
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
the mean of the number of mitotic figures found per 1,000 hepatocytes ? SD
using three mice per time point (10, 25).
Regenerating Alb-Cre Foxm1b?/?mouse liver displays significant
Wang et al.
December 24, 2002 ?
vol. 99 ?
no. 26 ?
either cyclin D or cyclin E mRNA levels after PHx (data not
shown), we next examined whether regenerating Alb-Cre
Foxm1b?/?liver exhibited altered expression of the p21 protein.
Western blot analysis of protein extracts from regenerating
Alb-Cre Foxm1b?/?livers showed that they displayed increased
p21 protein levels compared with the Foxm1b fl?fl control liver
(Fig. 4A). Furthermore, whereas regenerating Foxm1b fl?fl
hepatocytes exhibited only a transient increase in nuclear p21
staining at 32 h after PHx (Fig. 4 B–E), Foxm1b?/?hepatocytes
displayed a sustained increase in nuclear p21 protein levels
between 24 to 40 h after PHx (Fig. 4 F–I). Moreover, the
hepatocyte levels of nuclear p21 protein were not uniform, a
finding consistent with detectable levels of hepatocyte DNA
replication in regenerating Alb-Cre Foxm1b?/?liver. Because
regenerating p21?/?liver displayed increased Cdc25A expres-
sion and earlier nuclear localization of Cdc25A (30), we exam-
ined whether increased p21 levels resulted in diminished protein
expression of Cdc25A phosphatase required for Cdk2 activity
(5). Western blot analysis demonstrated that regenerating Alb-
Cre Foxm1b?/?livers displayed reduced Cdc25A protein levels
before S-phase that initiated at 36 h after PHx (Fig. 5A). Thus,
both decreased Cdc25A protein expression and increased nu-
clear expression of p21 protein are consistent with reduced Cdk2
kinase activity necessary for S-phase progression (5). Regener-
ating liver protein extracts prepared from either Foxm1b fl?fl or
Alb-Cre Foxm1b?/?mice were, therefore, immunoprecipitated
RB protein substrate. These experiments demonstrated that
regenerating Alb-Cre Foxm1b?/?liver displayed little hyper-
phosphorylation of the RB protein (Fig. 5B, indicated by*),
thereby demonstrating reduced Cdk2 kinase activity compared
with that of regenerating Foxm1b fl?fl liver. Furthermore, active
cyclin A2-Cdk2 kinase complex is required to phosphorylate the
Cdh1 subunit of the ubiquitin–ligase anaphase-promoting com-
plex (APC), which prevents APC-mediated degradation of
cyclin B at the end of S-phase and thus allows cyclin B accu-
mulation to promote entry into mitosis (5). Consistent with this
concept, Western blot analysis with the cyclin B1 antibody
showed delayed S-phase accumulation of cyclin B1 protein
between 32 and 40 h after PHx in liver extracts prepared from
Alb-Cre Foxm1b?/?mice compared with Foxm1b fl?fl litter-
mates (Fig. 5C). Collectively, diminished DNA replication in
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 expres-
sion 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.)
Regenerating Alb-Cre Foxm1b?/?liver display increased p21 protein
protein levels and Cdk2 activity. (A) Western blot analysis with Cdc25A anti-
body 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 regener-
assays with RB protein substrate. Position of the phosphorylated and hyper-
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.
Regenerating Alb-Cre Foxm1b?/?liver exhibit diminished Cdc25A
www.pnas.org?cgi?doi?10.1073?pnas.252570299Wang et al.
Foxm1b?/?liver is associated with increased Cdk inhibitor p21
protein and decreased Cdc25A phosphatase levels, resulting in
a reduction of cyclin A?E-Cdk2 kinase activity required for
Regenerating Alb-Cre Foxm1b?/?Liver Displays Reduced Cdc25B
Phosphatase Levels Leading to Diminished Cyclin B-Cdk1 Activity. To
identify mitotic regulatory genes whose expression is diminished
in regenerating liver of Alb-Cre Foxm1b?/?mice, RPA were
performed (Fig. 6A). Regenerating Alb-Cre Foxm1b?/?livers
displayed significant reduction in induced levels of Cdc25B
mRNA compared with regenerating Foxm1b fl?fl liver controls,
whereas only slight decreases were found in regenerating hepatic
levels of Cdk1, cyclin A2, and cyclin B1 mRNAs (Fig. 6A).
Whereas Foxm1b fl?fl hepatocytes displayed abundant Cdc25B
nuclear staining at 40 h after PHx (Fig. 6B), undetectable levels
of nuclear Cdc25B protein was found in regenerating Foxm1b?/?
hepatocytes (Fig. 6C). The Cdc25B phosphatase promotes M-
phase progression by dephosphorylation of the Cdk1 tyrosine-15
and threonine-14 residues, in effect providing a mitotic check-
point by regulating activity of the Cdk1?cyclin B kinase (1–3).
Western blot analysis with Cdk-1-specific phosphotyrosine-15
antibody demonstrated increased Cdk-1 phosphorylation in re-
generating liver extracts from Alb-Cre Foxm1b?/?mice (Fig.
6D), a finding consistent with diminished levels of the Cdc25B
phosphatase protein (1–3). In support of diminished Cdk1
kinase activity, immunoprecipitation-kinase assays demon-
strated that regenerating liver protein extracts from Alb-Cre
Foxm1b?/?mice displayed reduced Cdk-1-dependent phosphor-
ylation of the histone H1 substrate (Fig. 6E). These studies
demonstrate that Foxm1b regulates nuclear expression of the
Cdc25B phosphatase protein, an essential activator of M-phase
promoting Cdk1-cyclin B kinase.
Cotransfection assays in osteosarcoma U2OS cells with the
?200-bp mouse Cdc25B promoter luciferase plasmids and CMV
human FoxM1B (1–748) cDNA expression vector demonstrated
that FoxM1B protein stimulated expression of the Cdc25B
promoter (Fig. 6F). In contrast, no transcriptional activation of
the Cdc25B promoter was found with a C-terminal mutant
FoxM1B (1–688) that deletes sequences critical for transcrip-
tional activation (Fig. 6F). These results demonstrate that
Foxm1b regulates transcription of Cdc25B phosphatase gene,
whose expression is essential for activation of Cdk1-cyclin B
kinase and M-phase progression.
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 (10, 22,
23, 26). In this study, we used Alb-Cre recombinase to generate
a hepatocyte-specific deletion of the Foxm1b gene and demon-
strated that Foxm1b is required for normal levels of hepatocyte
DNA replication and is essential for mitosis in regenerating liver.
We found no significant increase in hepatocyte apoptosis in
regenerating Alb-Cre Foxm1b?/?liver (data not shown), sug-
gesting that Foxm1b is required for hepatocyte proliferation but
not survival. Reduced DNA replication in regenerating
Foxm1b?/?hepatocytes coincided with sustained increase in
nuclear staining of the Cdk inhibitor p21 protein between 24 and
40 h after PHx. This increase in nuclear p21 levels and a
reduction in Cdc25A phosphatase expression resulted in de-
creased activation of Cdk2 kinase (Fig. 6G). Cyclin E?A-Cdk2
complex cooperates with cyclin D-Cdk4?6 complex to phosphor-
ylate the RB protein, which releases bound E2F transcription
factor and allows it to stimulate expression of target genes
essential for hepatocyte S-phase progression (5, 6). Further-
more, a significant reduction in hepatocyte mitosis was associ-
ated with reduced mRNA and nuclear protein levels of the
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 regener-
ating liver of Alb-Cre Foxm1b?/?mice. Immunohistochemical staining of
regenerating liver sections (40 h after PHx) with Cdc25B antibody demon-
strates that regenerating Alb-Cre Foxm1b?/?hepatocytes display undetect-
able Cdc25B nuclear staining (C) compared with regenerating Foxm1b fl?fl
hepatocytes (B). (D) Increased phosphorylation of Cdk1 protein in regenerat-
ing Alb-Cre Foxm1b?/?liver. Western blot analysis with Cdk-1 phospho-
(E) Diminished Cdk1-dependent phosphorylation of histone H1 protein in
regenerating Alb-Cre Foxm1b?/?liver. The Cdk1 protein was immunoprecipi-
tated from regenerating liver extracts and used for kinase assays with H1
protein phosphorylation substrate. Shown below the panels is the fold in-
crease 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 normal-
ized fold induction of Cdc25B promoter expression in response to FoxM1B
cDNA cotransfection, with CMV-empty vector control set at 1.0. Two trans-
fection 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 repre-
sent negative regulation.
Wang et al.
December 24, 2002 ?
vol. 99 ?
no. 26 ?
Cdc25B phosphatase and delayed S-phase accumulation of Download full-text
cyclin B1 (Fig. 6G), both of which are necessary to stimulate
Cdk1 activity that mediates phosphorylation of critical target
proteins required for hepatocyte entry into mitosis (7). We
demonstrated that the Cdc25B promoter region is a direct target
for FoxM1B transcriptional activation in cotransfection assays.
Our present study shows that the mammalian Foxm1b transcrip-
tion factor is regulating expression of cell cycle proteins that
stimulate Cdk2 and Cdk1 activity, which are essential for entry
into DNA replication and mitosis, respectively. Consistent with
these findings, elevated FoxM1B levels are found in numerous
carcinomas (33), suggesting that FoxM1B is required for cellular
proliferation in human cancers.
Foxm1b deficiency caused inhibition of regenerating hepatocyte
mitosis, which was associated with undetectable nuclear levels of
Cdc25B phosphatase, but exhibited normal levels of Cdc25C (data
not shown) and correlated with decreased M-phase-promoting
Cdk1 activity. Activation of Cdk1 kinase is controlled by multiple
B proteins and dephosphorylation of the Y15 and T14 residues of
the Cdk1 protein by the Cdc25B and Cdc25C phosphatases (1–3).
To prevent premature activation of Cdk1 kinase activity, these
Cdk1 residues are phosphorylated by the Myt1 and Wee1 kinases,
whose activities are inhibited by phosphorylation via the mitogen-
activated protein kinases (RSK) pathway (1, 34). Stimulation of the
Cdc25C phosphatase activity is mediated by Cdk1?cyclin B phos-
phorylation at mitosis, which in turn, dephosphorylates and further
activates Cdk1 kinase mediating entry into mitosis (35, 36). In
contrast, Cdc25B phosphatase activity appears at late S-phase and
peaks during G2phase; it may be involved in the initiation of the
G2?M transition by activating the Cdk1?cyclin B kinase (37).
Consistent with this hypothesis, microinjection of Cdc25B-specific
antibody inhibits progression of Hs68 cells into mitosis (37). Fur-
thermore, microinjection of the Cdc25B protein, but not the
mitosis (38). Our current liver regeneration studies with Alb-Cre
Foxm1b?/?mice are consistent with the hypothesis that deficiency
in nuclear expression of Cdc25B protein is sufficient to inhibit
hepatocyte mitosis (Fig. 6G).
Maintaining hepatocyte expression of FoxM1B in regenerat-
ing liver of old-aged TG CD-1 mice is sufficient to increase
hepatocyte proliferation and expression of cyclin F, cyclin B1,
cyclin B2, Cdc25B, and p55Cdc, all of which are required for
mitosis (25). Despite the ability of FoxM1B to restore expression
of these mitotic regulatory genes in regenerating old-aged mouse
liver, our current studies show that FoxM1B is required only for
Cdc25B expression in 2-month-old regenerating liver, suggesting
that in young mice, redundant regulatory pathways exist for the
other mitotic regulatory genes. Taken together, our studies
demonstrate that Foxm1b regulates transcription and nuclear
expression levels of the Cdc25B phosphatase, which is essential
for hepatocyte entry into mitosis.
Reduced hepatocyte DNA replication in regenerating Alb-
Cre Foxm1b?/?liver was associated with a transcriptional-
independent stimulation of p21 protein expression (data not
shown) and increased p21 nuclear protein, indicating that
FoxM1B regulates p21 protein levels rather than influencing its
promoter activity. One possible explanation for increased nu-
clear levels of p21 protein is that one of the Foxm1b target genes
is involved in mediating p21 protein degradation. Elevated
transgenic hepatic levels of p21 protein caused significant de-
creases in regenerating hepatocyte DNA replication (39), a
finding consistent with diminished regenerating hepatocyte
DNA replication in Alb-Cre Foxm1b?/?mice. However, regen-
erating Alb-Cre Foxm1b?/?liver exhibited detectable hepato-
cyte DNA replication. One explanation for this phenotype is that
hepatocyte nuclear levels of p21 protein was not uniformly
increased in regenerating Alb-Cre Foxm1b?/?liver, suggesting
to inhibit DNA replication. Collectively, these results suggest
Foxm1b stimulated hepatocyte DNA replication through limiting
p21 protein levels before S-phase and that Foxm1b mediated
is essential for hepatocyte progression into mitosis.
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.).
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