Carcinogenesis vol.24 no.5 pp.835±841, 2003
DOI: 10.1093/carcin/bgg027
Induction of DNA synthesis in primary mouse hepatocytes is associated with
nuclear pro-transforming growth factor a and erbb-1 and is independent of c-jun
Elisabeth Schausberger1, Robert Eferl2,
Wolfram Parzefall1, Monica Chabikovsky1, Paul Breit1,
Erwin F.Wagner2, Rolf Schulte-Hermann1 and
Bettina Grasl-Kraupp1,3
1
Institut fur Krebsforschung, University of Vienna, Borschkegasse 8a, A-1090
Vienna, Austria and 2Research Institute of Molecular Pathology, Dr.
Bohrgasse 7, A-1030 Vienna, Austria
3
To whom correspondence should be addressed
Email: [email protected]
For growth stimulation of liver cells by hepatocyte growth
factor (HGF) or transforming growth factor a (TGFa) via
receptor tyrosine kinases, c-fos/c-jun has been considered a
point of intersection for cross-talk between the different
signal transduction pathways. Recent evidence strongly
implicates translocation of pro-TGFa into the nucleus as
an important step preceding the initiation of hepatic DNA
synthesis. We asked whether an active c-jun is required for
the nuclear translocation of pro-TGFa and its stimulatory
effect on DNA synthesis. For this purpose we used mice
with c-jun inactivated post partum in hepatocytes by the
Cre-loxP recombination system (c-junDliver). Nuclear fractions from control and c-junDliver mouse livers contained
TGFa as pro-form of 17 kDa and the epidermal growth
factor receptor (erbb-1) with molecular weights of 170 and
150 kDa (truncated form). Hepatocytes were isolated by
collagenase perfusion and cultivated. A lack of c-jun did
not alter the apoptotic activity but significantly suppressed
DNA synthesis in the cultured hepatocytes. In control and
c-junDliver cells DNA synthesis was almost always associated with nuclear presence of pro-TGF. 76.5 6.8% of
hepatocytes with pro-TGF positive nuclei and only 4.52 1.31% of hepatocytes with negative nuclei exhibited DNA
replication. About 85% of the pro-TGF positive nuclei
also contained erbb-1. Treatment of cultures with mature
TGFa or HGF elevated the frequency of pro-TGF positive nuclei replicating DNA; HGF and TGFa-induced
nuclear pro-TGF and DNA synthesis significantly more
in c-junDliver than in control hepatocytes. These results
suggest that (i) a lack of c-jun suppresses basal rates of
DNA replication in hepatocytes; (ii) c-jun deficient hepatocytes show a pronounced growth response towards HGF or
TGFa; (iii) nuclear translocation of pro-TGF together
with erbb-1 and its association with DNA synthesis are
independent of c-jun.
Abbreviations: HGF, hepatocyte growth factor; LI, labelling index; PB,
phenobarbital; pro-TGFa ‡, positive for pro-TGFa; pro-TGFa ÿ, negative for
pro-TGFa; TGFa, transforming growth factor a; TGF-b1, transforming growth
factor b1.
#
Oxford University Press. All rights reserved
Introduction
Primary cultures of hepatocytes, kept under serum-free conditions, are a good in vitro model to dissect the different growth
signals and downstream signalling pathways that lead to a
mitogenic response within the liver. In cultured rat hepatocytes
TGFa stimulates DNA synthesis alone, or synergistically with
other factors (1±4). The hepatocyte growth factor/scatter factor
(HGF/SF) is a further mitogen for rodent and human hepatocytes (5±9). TGFa and HGF bind on the cell surface to erbb- or
Met-receptors, respectively (10). Ligand binding to these tyrosine kinase receptors triggers autophosphorylation, upon
which the receptors associate with various transducers containing SH2 -domains, such as phosphoinositide 3-kinase, the phospholipase Cg, Grb2, ras GAP, p59FYN , Shc and others (3,9,11).
This triggers the activation of protein kinase B and C, the
RAF-MEK-MAPK or the JNK cascade leading to transcriptional changes by phosphorylation of nuclear proteins, including the jun family of transcription factors. Jun-proteins (c-jun,
junB, junD) are key players in the growth-signalling cascade.
They form the transcription factor AP-1, along with fos proteins (c-foc, fosB, fra1, fra2) as well as CREB/ATF and maf
proteins (12). The c-jun proto-oncogene itself is one of the
earliest genes that is transcribed after stimulation of cells with
growth factors and activation of the MAPK and JNK pathways. When cultured rat hepatocytes are incubated with EGF,
TGFa or HGF, there is an early peak of c-jun and c-fos
synthesis, followed by increased AP-1 transcriptional activity
and further potentiation of the activation domain of the c-jun
protein (9,11). Likewise, growth stimulatory xenobiotics, such
as the tumour promoter phenobarbital (PB), induce c-jun
expression in the intact liver (13). Thus, c-fos/c-jun may be
the point of intersection for cross-talk between the different
signal transduction pathways in the liver. C-jun deficient cells
suffer from growth arrest or at least a delayed cell cycle.
Accordingly, c-jun has been considered essential for liver
cell growth. The Cre-loxP recombination system has recently
been used to bypass the embryonic lethality of c-jun knockout
mice and to study c-jun functions in hepatocytes (14±17).
When c-jun was inactivated in hepatocytes during postnatal
life, mice stayed viable and showed no overt liver abnormalities, but they displayed impaired liver regeneration in
response to partial hepatectomy (17).
We have shown recently that hepatocytes in the intact liver
and in primary culture synthesize pro-forms of TGFa, that
translocate to the nucleus in G1 where they appear to serve in
a novel function for mitogenic signalling (4). Nuclear location
of pro-TGFa was induced by various different growth stimuli
in cultured hepatocytes and in the intact liver. It seems to
operate in addition to the well-known signal pathway via
erbb-1 receptor binding of mature TGFa on the cell surface.
The erbb-1 belongs to the family of four erbb receptors, each
of which is able to form heterodimers with other family members and to bind several ligands including TGFa, EGF,
835
E.Schausberger et al.
amphiregulin, b-cellulin and others (18,19). Like pro-TGFa
erbb-1 was found in the nucleus of proliferating cells; it
contains a transactivating domain and associates with the
promoter region of the cyclin D1 gene (20). Upon binding
of heregulin b1 erbb-3 was found to shuttle between nuclear
and non-nuclear compartments of mammary epithelial cells
(21). Erbb-4, a further member of the erbb-receptor family,
may be cleaved by secretase upon activation, which releases
the cytoplasmic domain of the receptor for translocation into
the nucleus. The C-terminus of this erbb-4 fragment has transcriptional activity (22). Multiple modes have been suggested
by which erbb receptors may escape from the cell membrane
and travel to the nucleus (23). Taken together these recent
findings indicate that TGFa and/or erbb-receptors mayÐat
least under certain conditionsÐbypass cytoplasmic phosphorylation cascades, widely thought to be essential in transducing
mitogenic stimuli. Because TGFa is up-regulated in different
types of human malignancies, such as hepatocellular carcinoma, novel therapeutic approaches concentrate on the possible benefit of blocking TGFa-evoked signal transduction, e.g.
by blockade of the receptor or ligand receptor interactions on
the cell surface (24±28). However, human hepatocellular adenoma and carcinoma often express pro-TGFa in their nuclei
(E.Schausberger et al., in preparation). To explore effective
strategies for tumour therapy, it is of great importance to focus
further investigations on the novel pathway of mitogenic signalling by TGFa gene products.
In the present study we investigated whether the high association between nuclear pro-TGFa and DNA replication in
hepatocytes depends on an active c-jun. For this purpose, the
inducible cre-loxP recombination system was used to delete cjun in mouse hepatocytes during postnatal life; the growth
potential of these hepatocytes was tested in an ex vivo system.
We found that c-junDliver mouse hepatocytes show a lower
basal rate of DNA replication in primary culture but are stimulated to DNA synthesis by HGF and mature TGFa to a
much higher extent than control hepatocytes. DNA synthesis
was detected almost exclusively in mouse hepatocyte nuclei
expressing nuclear pro-TGFa and erbb-1. Thus, the nuclear
pro-TGFa system together with nuclear erbb-1 seems to be
involved in DNA replication independent of c-jun. This is
further evidence for novel intracellular mitogenic signal transduction pathways that act in addition to `classical' phosphorylation cascades.
Materials and methods
Animals and treatment
The mice used in this study were recombinant inbred strains between 129 C57/B6 genetic background and were bred at the Research Institute of
Molecular Pathology (Vienna, Austria). Animals were kept under standardized
SPF-conditions. The inducible Mx-cre approach was applied to delete floxed
c-jun alleles in the liver, according to published procedures (15,17). For all
experiments, sex-matched littermates, 3 months old, were used. Two weeks
before collagenase perfusion the group of control animals without the Mx-cre
transgene (c-junf=f ) and the c-junDliver animals with the Mx-cre transgene
(Mx-cre c-junf=f ) were treated with a single injection of 400 mg pI±pC to delete
the floxed c-jun alleles. All experiments were performed according to the
Austrian guidelines for animal care and protection.
Southern blot
Southern blotting was performed according to standard protocols (29). Ten
microgram of genomic DNA was digested with XbaI yielding a 6.9 kb fragment for the floxed c-jun allele and a 3.3 kb fragment for the deleted c-jun
allele. For detection of the bands a 600 bp BamHI fragment from the c-jun
promoter was used as probe.
836
Cell fractionation, one- and two-dimensional gel electrophoresis and
immunoblotting
Nuclei were isolated from the cytoplasmic/membrane fraction applying 2.0 M
sucrose for purification (30). This was followed by nuclear matrix preparation,
as has been described in detail (31). Protein was measured by the Bio-Rad
Assay using BSA as a standard. Twenty micrograms of protein of isolated
nuclei or of the cytoplasmic/membrane fraction were dissolved in 2 SDS
buffer and were applied to one-dimensional SDS±polyacrylamide gels (stacking gel: 5%; resolving gel: 6%). Forty micrograms of the matrix preparations
were dissolved and subjected to high-resolution two-dimensional gel electrophoresis. The separated proteins were transferred and detected, as described
(4,32). The mouse monoclonal antisera against mature TGFa (Oncogene
Science, Uniondale, NY; Ab-1, clone 134A-2B3; 1:300), against the Cterminal amino acids 144±160 of pro-form of wild-type TGFa (InnoGenex,
San Ramon, CA; 1:300), against the extracellular domain of erbb-1 (1:500,
Genosys Biotechnologies, Pampisford, UK), or against residues 985±996 of
the intracellular domain of human erbb-1 (1:500; clone F4; Sigma, St Louis,
MO) were applied as primary antibodies.
Studies on primary hepatocyte cultures
Hepatocyte preparation. Male mice were anaesthetized with isoflurane in a
carrier gas of dinitrogen oxide and oxygen until exsanguination was terminated.
Liver perfusion according to Seglen (33) with modifications as described
(34,35) was done through the portal vein with calcium-free buffer at a constant
flow rate of 4.5 ml/min. After the liver was freed of blood the processus
papilliformis caudatus was ligated, cut off and fixed in Carnoy's solution for
histology (see below). After 10 min of pre-perfusion the perfusion buffer was
switched to collagenase buffer (0.2 mg/ml collagenase Sigma Type IV) at
decreasing flow rates from 4.5 to 2.5 ml/min. Parenchymal cells were isolated
from the initial cell suspension by four cycles of low-speed centrifugation
(30 g, 5 min). Viability of the cell isolates was 80 7% by Trypan Blue exclusion test with an average cell yield of 32 17 million cells per mouse liver.
Culture and treatment. Conditions and treatments of cultures have been
published in detail (35). If not stated differently treatment commenced 4 h
after plating (time point 0 in the experimental protocol) and was renewed with
every medium change. Phenobarbital-Na (PB, Fluka, Buchs, Switzerland) was
dissolved in 0.9% of NaCl and was added to the medium for a final concentration of 1 mM. HGF (Sigma), dissolved in PBS as a stock of 20 ng/ml, was
applied at a final concentration of 10 ng/ml medium. Recombinant human
mature TGFa (UBI, Lake Placid, NY) was prepared as a stock of 10 mg/10 ml
in 10 mM acetic acid and added as final concentrations of 20 and 50 ng/ml.
Histology
Liver tissue of untreated C57/B6 mice, fixed in Carnoy's solution, was processed as described (36,37); three serial sections, 2 mm thick, were cut; one of
the sections was stained with haematoxylin and eosin, the second one for proTGFa and the third one for erbb-1 (4).
Immunostaining for pro-TGFa or erbb-1 of tissue sections and culture plates
For pre-treatment and staining of tissue sections or culture plates see (4,35).
Primary antibodies applied were: mouse monoclonal IgGs against either
recombinant mature TGFa (originally clone 213-4.4, Oncogene Science) or
the C-terminus of human pro-TGFa (Innogenex); rabbit polyclonal IgG against
mature TGFa (Santa Cruz, Santa Cruz, CA 1:50); mouse monoclonal IgG
against extracellular domain of erbb-1 (Genosys Biotechnologies); a sheep
polyclonal IgG against the intracellular domain of human erbb-1 (Fitzgerald
Industries International, Concord, MA).
Double-immunostaining for TGFa and erbb-1; confocal laser scanning
microscopy. The procedure followed published descriptions with the following modifications (4,35): primary antisera (anti-TGFa from Oncogene
Science; anti-erbb-1 from Fitzgerald, both 1:50) were applied together overnight at 4 C; secondary antisera, i.e. Cy2-labelled donkey-anti-sheep (1:100;
Dianova, Hamburg, FRG) and biotinylated rabbit-anti-mouse (Dako, Glostrup,
Denmark; 1:300), were used together for 60 min at room temperature, which
was followed by Cy3-labelled streptavidin (1:1000 in TBS, 60 min room
temperature, Dianova). Stained culture plates were examined by confocal
laser scanning microscopy (TCSNT/SP series, Leica-Microsystems,
Wetzlar, FRG).
Determination of DNA replication and apoptosis in cultures
Newly synthesized DNA was detected with [3 H]thymidine (sp. act. 60±80
Ci/mmol; added at 0.5 mCi/ml to the medium 24 h before harvesting; ARC,
St Louis, MO) and subsequent autoradiography (4,35). Plates were stained for
5 min with 8 mg/ml Hoechst 33258 (Riedel de Haen, Seelze, FRG) and
mounted. To determine DNA synthesis a total of 1000 TGFa ÿ and 300±400
pro-TGFa ‡ hepatocyte nuclei were evaluated per plate. For the evaluation of
Induction of DNA synthesis in primary mouse hepatocytes
apoptosis at least 1000 hepatocyte nuclei per plate were scored for apoptotic
morphology (condensed and fragmented nuclei) as described (38). The percentage of labelled hepatocyte nuclei (LI%) and the incidences of apoptotic
bodies were calculated.
Statistics
For in vitro studies, four hepatocyte cultures per animal, treatment and time
point were run and harvested in parallel. If not stated differently, the mean and
SD of three equally treated cultures from at least four different unfloxed and
floxed donor mice are given. The significance of differences of means was
tested by the non-parametric Wilcoxon's test.
Results
c-jun-is deleted by Cre-lox P recombination
The inducible Mx-cre approach was applied to delete the
floxed c-jun alleles in the liver (15,17). The level of c-jun
deletion in the mice was checked by Southern blot analysis
and nearly 100% deletion of the c-jun alleles was found 2
weeks after injection of pI±pC (Figure 1).
of the acinus (Figure 4A and B). This nuclear location of proTGFa could also be seen with an antiserum raised against the
C-terminus of pro-TGFa (Figure 4E). Nuclear matrix preparations from control and c-junDliver mouse livers were subjected
to separation by two-dimensional gel electrophoresis and to
immunoblotting. TGFa was detectable as pro-form at 17 kDa
and an isoelectric point of 7.5 (Figure 5). The mature form of
TGFa (5.6 kDa) was not found within the nucleus. The
detected spots correspond to those found in rat and human
liver, in which MALDI-TOF analysis verified spot 1 and 2 to
be pro-TGFa wild-type with the characteristic valines at
position 159 and 160 (4). The other two spots could not yet
Basal and induced DNA synthesis in cultured hepatocytes
from control and c-jun DDliver mice
Hepatocytes were isolated from control and c-junDliver mice by
collagenase perfusion and were cultivated. DNA synthesis of
c-junDliver hepatocytes was reduced to 30% of DNA synthesis
of the control cells (Figure 2A). The incidence of apoptoses
was not significantly different in both groups (control: 0.35 0.18; c-junDliver: 0.23 0.11; n ˆ 4). Thus, a lack of c-jun
suppresses DNA replication in adult hepatocytes without
significant effect on the apoptotic activity.
Next we asked whether cultured primary hepatocytes from
control and c-junDliver mice show a different response towards
various growth stimuli. We chose three compounds with presumably different modes of action (39). A relatively high
concentration of PB in the medium for 48 h did not alter
DNA replication in the cultured cells (Figure 3). However,
the two growth factors, HGF and TGFa raised the labelling
index (Figure 3). The relative growth response was more
pronounced in hepatocytes with an inactive c-jun than in control cells. This suggests that the response of hepatocytes
towards TGFa and HGF is not impaired by an inactive c-jun.
We therefore investigated whether the growth response in cjun deficient hepatocytes is triggered by other signalling
mechanisms, such as represented by the nuclear occurrence
of pro-TGFa.
The pro-form of wild-type TGFa and erbb-1 co-localize in
hepatocyte nuclei
In immunostained liver sections of control mice pro-TGFa
was detectable in the cytoplasm, cell membranes and most
prominent in almost all hepatocyte nuclei located in zone III
Fig. 1. Detection of Cre-mediated deletion of c-jun in mouse liver. Southern
blot analysis of DNA from liver of groups of control (c-junf=f ) (‡pI±pC)
transgenic mice (lanes 1 and 2) in comparison to c-junDliver (Mx-cre c-junf=f )
(‡pI±pC) (lanes 3 and 4).The positions of the fragments derived from c-junflox
(Flox) and c-junDliver (D) alleles are indicated.
Fig. 2. DNA synthesis in untreated control (Co) and c-junDliver mouse
hepatocytes associated with nuclear occurrence of pro-TGFa. (A) Per cent of
all hepatocyte nuclei in S-phase; (B) total column gives per cent of all
pro-TGFa ‡ hepatocyte nuclei; the dark portion of the column gives per cent of
pro-TGFa ‡ nuclei in S-phase; (C) total column gives per cent
of all pro-TGFa-neg hepatocyte nuclei; the dark portion of the column
gives per cent of pro-TGFa ÿ nuclei in S-phase. Means SD are given from
four independent experiments after 48 h in culture. Statistics by Wilcoxon's
test: a P 5 0.05.
837
E.Schausberger et al.
be identified unequivocally. Pro-TGFa protein appeared less
abundant in nuclear matrix preparations from c-junDliver than
from control animals (Figure 5). In silver-stained two-dimensional gels of the nuclear matrix proteins, the pattern and
intensity of spots of the wild-type and c-junDliver mice did not
reveal significant differences (not shown).
Immunostains with two different antibodies against the
extracellular (not shown) or intracellular domain (Figure 4C
and D) of erbb-1 showed that the distribution of erbb-1 in the
liver was similar to that of pro-TGFa, i.e. in the cell membranes and nuclei of hepatocytes in zone III of the acinus. In
immunoblots of control and c-junDliver mouse livers erbb-1
appeared predominantly in the cytoplasmic/membrane fraction
and faintly in the nuclear fraction at 170 and 150 kDa (truncated form). These results were obtained with antisera raised
against the extracellular (Figure 6) or the intracellular (not
shown) domain of erbb-1. The cytoplasmic/membrane and
the nuclear fraction of c-junDliver samples appear to contain
less erbb-1 protein than controls (Figure 6).
Fig. 3. Effect of PB, TGFa and HGF on the percentage of DNA synthesis
[LI(%)] in control and c-junDliver mouse hepatocytes. Total column gives per
cent of all hepatocyte nuclei in S-phase (total LI; ¤); the dark portion of the
column (¥) gives per cent of hepatocyte nuclei showing pro-TGFa and
S-phase. Number on the top of the columns indicates fold increases of LI over
control. Abbreviations: C, control hepatocytes, DL, c-junDliver hepatocytes;
TGFa 20, 20 ng of TGFa/ml medium; TGFa 50, 50 ng of TGFa/ml
medium; HGF, 10 ng of HGF/ml medium. Means SD are given from
four independent experiments after 48 h in culture. Statistics by
Wilcoxon's test: a P 5 0.05.
In immunostained primary hepatocyte cultures from control
and c-junDliver mice, confocal laser scanning microscopy
detected pro-TGFa in the nuclei and erbb-1 in the cell membranes and nuclei. Pro-TGFa and erbb-1 often co-localized in
the nucleus (Figure 4F±H); 84.2 7.9% of the pro-TGFa ‡
nuclei were positive for erbb-1 and 62.6 10.6 of erbb-1
positive nuclei also expressed nuclear pro-TGFa.
Basal and induced DNA replication in cultured mouse
hepatocytes is highly associated with nuclear pro-TGFa
In untreated primary cultures the incidence of pro-TGFa ‡
nuclei was ~3-fold higher in controls (23.6 10.7%) than in
c-junDliver hepatocytes (7.9 4.8%); 83.5 9.8% of the mouse
hepatocytes with pro-TGFa ‡ nuclei and only 4.1 3.1% with
negative nuclei replicated DNA, which was observed in both
control and c-junDliver hepatocytes (Figures 2B and C and 4E).
As 84.2% of the pro-TGFa ‡ nuclei also expressed erbb-1, at
least 67.7% of the pro-TGFa ‡ nuclei in DNA synthesis should
be positive for erbb-1. In conclusion, DNA replication in
pro-TGFa ÿ nuclei was a rare event and nuclear pro-TGFa
together with nuclear erbb-1 were strongly associated with
DNA synthesis. Similar findings have been reported for primary rat hepatocyte cultures (4).
We asked whether nuclear pro-TGFa is involved in the
growth response of control and c-junDliver hepatocytes in primary culture. Forty-eight hours after addition of HGF the
percentage of cultured hepatocytes with pro-TGFa ‡ nuclei
was raised 2-fold, paralleled by a doubled frequency of nuclei
in S-phase (Figure 3). The induction of both, DNA synthesis
and pro-TGFa ‡ within the same nuclei, correlated highly
(Pearson's test: r2 ˆ 0.7127; significant at P 5 0.0001).
Treatment of cultures with mature TGFa dose-dependently
induced a similar effect. Thus, the induction of DNA synthesis
by HGF and TGFa was highly associated with de novo occurrence of nuclear pro-TGFa; this mechanism was active in both
control and c-junDliver hepatocytes.
Discussion
The present study suggests that an active c-jun supports DNA
replication in hepatocytes and that the high association
between nuclear pro-TGFa and replicative DNA synthesis
Fig. 4. Pro-TGFa and erbb-1 are localized to hepatocyte nuclei in vivo and in culture. (A) Mouse liver with nuclei positive for pro-TGFa. The central vein is
indicated with an arrow. (B) Detail of (A). In (C) and (D) sections were stained with an antiserum raised against the intracellular domain of erbb-1; erbb-1 (C) is
localized in hepatocyte nuclei of zones II and III of the liver; the central vein is indicated by an arrow; (D) is a detail of (C). Untreated primary culture of control
mouse hepatocytes at 48 h: (E) plate was stained with antiserum against the C-terminus of pro-TGFa; pro-TGFa occurs in hepatocyte nuclei with incorporated
[3 H]thymidine; in (F±H) confocal laser scanning microscopy for erbb-1 (F), pro-TGFa (G), and in (H) overlap of (F) and (G). Magnifications 25 (A), 225 (B and
D), 100 (C), 200 (E±H).
838
Induction of DNA synthesis in primary mouse hepatocytes
Fig. 5. Detection of pro-TGFa as wild-type form by two-dimensional
immunoblots of nuclear matrix proteins of control and c-junDliver mouse liver.
The gel covers a range of pI 3±8 and Mr 6.5±40 kDa. Corresponding spots
(encircled) were analysed from human liver by MALDI-TOF; spots 1 and 2
were identified as wild-type pro-TGFa (4).
Fig. 6. Detection of erbb-1 in one-dimensional immunoblots of nuclear
proteins from mouse liver. Nuclear fraction (N) and cytoplasmic membrane
fraction (C) of control (1) and c-junDliver (2) mouse liver.
does not depend on a functional c-jun, which is supported by
the following findings.
Suppressed basal DNA replication after inactivation of c-jun
C-junDliver hepatocytes showed lower basal DNA synthesis
than control cells. Thus, an inactive c-jun suppresses DNA
replication under the present experimental conditions. This
agrees with previous data showing that c-jun directly regulates
the cyclin D1 and PCNA promoters in fibroblasts. Furthermore, c-jun was shown to control p21 protein levels by inhibiting p53 binding to the p21 promoter. Elevated p21 levels could
account for the delayed cell cycle progression of hepatocytes
lacking c-jun, as shown by impaired liver regeneration in mice
with transgenic overexpression of p21 (40). In hepatocytes
lacking c-jun, mRNA and protein levels of p21 accumulate
and again impair regenerative liver growth (17). Therefore, the
diminished progression through the cell cycle in c-junDliver
hepatocytes might be due to inefficient activation of cyclindependent kinases, which are essential for a proliferative
response.
Mitogen-induced DNA synthesis occurs in the absence of c-jun
The effects of PB or TGFa on DNA replication of cultured
mouse hepatocytes have not yet been studied. In the present
work, treatment of mouse hepatocytes in vitro with 1 mM PB
did not alter DNA synthesis. The findings obtained are consistent with previous ones in cultured primary rat hepatocytes
in which slight stimulation of DNA replication occurred at
1.5 mM and pronounced mitoinhibition at 3 mM (35). Also, in
the intact liver of rats and mice PB affects DNA replication
only marginally and exerts its effect on growth of liver
mostly via suppression of apoptosis (39). The present study
shows that TGFa stimulates DNA synthesis dose-dependently
in cultured mouse hepatocytes. HGF is a well-characterized,
potent mitogen for hepatocytes in different mammalian
species; in vivo infusion of recombinant HGF into untreated
mice results in low level of cell proliferation (8). Recombinant
HGF induced DNA synthesis in primary cultures of rat, mouse
and human hepatocytes (5±7,9). Also, under the conditions of
the present study HGF raised significantly the replication of
DNA in cultured mouse hepatocytes.
Although basal DNA replication was suppressed in
c-junDliver hepatocytes, stimulation was not compromised at
all in the present study. In contrast, the relative induction of
DNA replication was more pronounced in c-junDliver than in
control hepatocytes. It is presently unclear whether the inactivated c-jun is functionally replaced by other jun proteins (jun-B,
jun-D) under our experimental conditions. c-Jun and jun-B
have antagonistic functions in a variety of biological processes
such as cell proliferation, e.g. in fibroblasts c-jun activates and
jun-B suppresses cell proliferation (41). Recent unexpected
data show that jun-B, when introduced by a knock-in strategy
at a supraphysiological gene dosage, can functionally replace
c-jun in jun-null mice, that otherwise would die between day
12.5 and 14.5 due to defects of the heart and the liver (42). As
the mice still die postnatally, c-jun and jun-B may be functionally interchangeable during development but may have different functions after birth. This suggests that in the present study
the induction of DNA synthesis in c-jun deficient hepatocytes,
that have been isolated postpartally, may not involve jun-B.
Pro-TGFa occurs in the nucleus of mouse hepatocytes and is
associated with basal and induced DNA synthesis
Analogous to findings in rat and human liver pro-forms of
wild-type TGFa are present in the nuclei of both, control and
c-junDliver mice. We have shown recently that a subfraction of
the isolated primary hepatocytes synthesize pro-TGFa. Immediately after seeding of the cells pro-TGFa is distributed
evenly throughout the cytoplasm and soon thereafter starts to
translocate to the nucleus; 16 h later hepatocytes show intranuclear accumulation of pro-TGFa without cytoplasmic residues and after a further lag phase of ~22 h almost all of the
positive nuclei replicate DNA (4). Addition of mature TGFa to
the medium increased DNA synthesis exclusively in proTGFa negative hepatocytes. This stimulatory effect was abrogated by neutralizing antibodies or by the erbb-1-specific
tyrosine kinase inhibitor tyrphostin A25. These interventions
did not affect the impact of nuclear pro-TGFa on DNA replication (4). The blockade of pro-TGF by RNA interference is
currently under investigation.
Like in vitro, the start of liver growth in the intact animal
induces de-novo synthesis and nuclear import of pro-TGFa in
hepatocytes scattered throughout the liver lobule. This again is
followed by DNA replication in the fraction of pro-TGFa
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E.Schausberger et al.
positive nuclei. In the resting liver, however, the constitutive
occurrence of nuclear pro-TGFa in hepatocytes of zone III
seems not to be linked to cell replication (4). Thus, under
conditions of induced hepatocellular growth the nuclear occurrence of newly synthesized pro-TGFa is highly associated with
replicative DNA synthesis and may be part of a novel mitogenic-signalling pathway, that acts in addition to the pathway
initiated by binding of TGFa to erbb-receptors at the cell
membrane (4).
One advantage of a direct action of pro-TGFa in the nucleus
becomes evident in culture, where cells often lack intercellular
contacts, cytokines or hormones present in the whole body.
Then, the novel pathway may circumvent the secretion and
possible loss of mature TGFa to the outside of the cell and may
confer autonomy and an inherent growth advantage. The present work shows that mouse hepatocytes utilize this pathway in
primary culture in a way similar to rat hepatocytes. A further
advantage of a direct action of a growth factor in the nucleus
may be to guarantee replication of the cell in case of functional
loss of components of other signal transduction pathways; this
may be used by hepatocytes lacking an active c-jun. A conceivable third advantage is to maintain specificity, which may
be compromised by the degeneracy of signalling pathways,
shared by many different cell surface receptors.
The growth stimulatory effects of TGFa or HGF were associated with induction of nuclear pro-TGFa in cultured mouse
hepatocytes of both controls and and c-junDliver mice. In vivo
various hepatomitogenic signals led to pronounced induction
of nuclear occurrence of pro-TGFa. In vitro the number of
pro-TGFa ‡ hepatocyte nuclei was elevated by treatment with
hepatomitogenic steroid hormones or prostaglandins (4). A
further interesting finding is, that treatment with mature
TGFa induces the nuclear pro-TGFa system. A similar result
was recently obtained in cultured primary rat hepatocytes
treated with EGF (E.Schausberger, manuscript in preparation).
This suggests that two signal pathways, i.e. the `classical'
signal transduction via membrane receptor binding of mature
TGFa and the pathway involving nuclear pro-TGFa are
interdependent.
Possible interaction of pro-TGFa and erbb-1
How pro-TGFa is translocated to the nucleus and its targets
therein are still unclear; the present work indicates that c-jun
may not be involved. However, we found that ~85% of the
pro-TGFa ‡ hepatocyte nuclei also were positive for erbb-1. It
is currently under investigation whether the large TGFa precursor may attach within the binding site of any of the erbbreceptors suited for the small molecule of mature TGFa and
whether pro-TGFa may be co-targeted to the nucleus as a
receptor-bound ligand. Recently it was demonstrated that the
EGF/erbb-1 complex translocates to the nucleus in tissues with
a high proliferative status (20). In the pre-S phase of liver
regeneration 125 I-labelled EGF accumulated in the nucleus of
hepatocytes (43). EGF binding sites and second messenger
systems associated with erbb-1 signalling were isolated from
matrix of rat liver nuclei (44±47). It was also reported that
erbb-1 directly activates the signal transducers and activators
of transcription (STAT) proteins independent of jak-1 (48).
Moreover, evidence was provided that nuclear erbb-1 acts as a
transcription factor or co-activator of cyclin D1 (20). In a
further study erbb-4 was found to be cleaved upon activation;
the released cytoplasmic domain of the receptor translocates
into the nucleus and exerts transcriptional activity (22). The
840
recent findings together with the present study imply that
growth factors/erbb-receptors may bypass the protein phosphorylation cascades and c-jun for the transduction of mitogenic stimuli (23).
In conclusion, the present work shows that the nuclear
occurrence of pro-TGFa is associated with DNA replication
of hepatocytes, which is independent of the function of c-jun.
Further research is necessary to elucidate the mechanisms that
regulate the different intracellular routes of pro-TGFa and that
link nuclear pro-TGFa to DNA replication.
Acknowledgements
The excellent technical assistance of K.Bukowska, M.Kafer, and H.Koudelka
is gratefully acknowledged. This study was also supported by `Herzfeldersche
Familienstiftung'.
References
1. Mead,J.E. and Fausto,N. (1989) Transforming growth factor a may be a
physiological regulator of liver regeneration by means of an autocrine
mechanism. Proc. Natl Acad. Sci. USA, 86, 1558±1562.
2. Michalopoulos,G. and DeFrances,M. (1997) Liver regeneration Science,
276, 61±66.
3. Moriuchi,A., Hirono,S., Ido,A., Ochiai,T., Nakama,T., Uto,H., Hori,T.,
Hayashi,K. and Tsubouchi,H. (2001) Additive and inhibitory effects of
simultaneous treatment with growth factors on DNA synthesis through
MAPK pathway and G1 cyclins in rat hepatocytes. Biochem. Biophys. Res.
Commun., 280, 368±373.
4. Grasl-Kraupp,B., Schausberger,E., Hufnagl,K., Gerner,C., L
ow-Baselli,A.,
Rossmanith,W., Parzefall,W. and Schulte-Hermann.,R. (2002) A novel
mechanism for mitogenic signaling via pro-transforming growth factor a
within hepatocyte nuclei. Hepatology, 35, 1372±1380.
5. Nakamura,T., Teramoto,H. and Ichihara,A. (1986) Purification and characterization of a growth factor from rat platelets for mature parenchymal
hepatocytes in primary culture. Proc. Natl Acad. Sci. USA, 83, 6489±6493.
6. Yee,C.J.,
DeFrances,M.C.,
Bell,A.,
Bowen,W.,
Petersen,B.,
Michalopoulos,G.K. and Zarnegar,R. (1993) Expression and characterization of biologically active human hepatocyte growth factor (HGF) by
insect cells infected with HGF-recombinant baculovirus. Biochemistry, 32,
7922±7931.
7. Petersen,B., Yee,C.J., Bowen,W., Zarnegar,R. and Michalopoulos,G.K.
(1994) Distinct morphological and mito-inhibitory effects induced by
TGF-b1, HGF and EGF on mouse, rat and human hepatocytes. Cell Biol.
Toxicol., 10, 219±230.
8. Roos,F., Ryan,A.M., Chamow,S.M., Bennett,G.L. and Schwall,R.H.
(1995) Induction of liver growth in normal mice by infusion of hepatocyte
growth factor/scatter factor. Am. J. Physiol., 268, G380±G386.
9. Gomez-Lechon,M.J., Guillen,I., Ponsoda,X., Fabra,R., Trullenque,R.,
Nakamura,T. and Castell,J.V. (1996) Cell cycle progression proteins
(cyclins), oncogene expression and signal transduction during the
proliferative response of human hepatocytes to hepatocyte growth factor.
Hepatology, 23, 1012±1019.
10. Naldini,L., Vigna,E., Narsimhan,R.P., Gaudino,G., Zarnegar,R.,
Michalopoulos,G.K. and Comoglio,P.M. (1991) Hepatocyte growth factor
(HGF) stimulates the tyrosine kinase activity of the receptor encoded by
the proto-oncogene c-MET. Oncogene, 6, 501±504.
11. Brenner,D.A. (1998) Signal transduction during liver regeneration.
J. Gastroenterol. Hepatol., 13(suppl.), 93±95.
12. Wagner,E.F. (2001) AP-1. Oncogene, 20, 2334±2335.
13. Holden,P.R., Odum,J., Soames,A.R., Foster,J.R., Elcombe,C.R. and
Tugwood,J.D. (1998) Immediate-early gene expression during regenerative and mitogen-induced liver growth in the rat. J. Biochem. Mol.
Toxicol., 12, 79±82.
14. Hilberg,F., Aguzzi,A., Howells,N. and Wagner,E.F. (1993) c-jun is
essential for normal mouse development and hepatogenesis. Nature,
365, 179±181.
15. K
uhn,R., Schwenk,F., Aguet,M. and Rajekski,K. (1995) Inducible gene
targeting in mice. Science, 269, 1427±1429.
16. Eferl,R., Sibilia,M., Hilberg,F., Fuchsbichler,A., Kufferath,I., Guertl,B.,
Zenez,R., Wagner,E.F. and Zatloukal,K.J. (1999) Functions of c-Jun in
liver and heart development. J. Cell Biol., 145, 1049±1061.
Induction of DNA synthesis in primary mouse hepatocytes
17. Behrens,A., Sibilia,M., David,J.-P., M
ohle-Steinlein,U., Tronche,F.,
Schutz,G. and Wagner,E.F. (2002). Impaired postnatal hepatocyte
proliferation and liver regeneration in mice lacking c-jun in the liver.
EMBO J., 21, 1782±1790.
18. Olayioye,M.A., Neve,R.M., Lane,H.A. and Hynes,N.E. (2000) The ErbB
signaling network: receptor heterodimerization in development and
cancer. EMBO J., 19, 3159±3167.
19. Yarden,Y. and Sliwkowski,M.X. (2001) Untangling the ErbB signalling
network. Nature Rev. Mol. Cell Biol., 2, 127±137.
20. Lin,S.Y., Makino,K., Xia,W., Matin,A., Wen,Y., Kwong,K.Y.,
Bourguignon,L. and Hung,M.C. (2001) Nuclear localization of EGF
receptor and its potential new role as a transcription factor. Nature Cell
Biol., 3, 802±808.
21. Offterdinger,M., Sch
ofer,C., Weipoltshammer,K. and Grunt,T.W. (2002)
C-erbB-3: a nuclear protein in mammary epithelial cells. J. Cell Biol., 157,
929±939.
22. Ni,C.Y., Murphy,M.P., Golde,T.E. and Carpenter,G. (2001) Gammasecretase cleavage and nuclear localization of ErbB-4 receptor tyrosine
kinase. Science, 294, 2179±2181.
23. Wells,A. and Marti,U. (2002) Signalling shortcuts: cell-surface receptors
in the nucleus? Nature Rev. Mol. Cell Biol., 3, 1±6.
24. Yamaguchi,K., Carr,B.I. and Nalesnik,M. A. (1995). Concomitant and
isolated expression of TGFa and EGF-R in human hepatoma cells
supports the hypothesis of autocrine, paracrine and endocrine growth of
human hepatoma. J. Surg. Oncol., 58, 240±245.
25. Pitot,H.C., Dragan,Y.P., Teeguarden,J., Hsia,S. and Campbell,H. (1996)
Quantitation of multistage carcinogenesis in rat liver. Toxicol. Pathol., 24,
119±128.
26. Kiss,A., Wang,N.J., Xie,J.P. and Thorgeirsson,S.S. (1997) Analysis of
transforming growth factor (TGF)-alpha/epidermal growth factor receptor,
hepatocyte growth factor/c-met, TGF-b receptor type II and p53
expression in human hepatocellular carcinomas. Clin. Cancer Res., 3,
1059±1066.
27. Levitzki,A. and Gazit,A. (1995) Tyrosine kinase inhibition: an approach to
drug development. Science, 267, 1782±1788.
28. Mendelsohn,J. (1997) Epidermal growth factor receptor inhibition by
a monoclonal antibody as anticancer therapy. Clin. Cancer Res., 276,
60±66.
29. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
30. Tata,J.R. (1974) Isolation of nuclei from rat liver and other tissues.
Methods Enzymol., 31, 253±262.
31. Gerner,C., Holzmann,K., Grimm,R. and Sauermann,G. (1998) Similarity
between nuclear matrix proteins of various cells revealed by an improved
isolation method. J. Cell Biochem., 71, 363±374.
32. Gerner,C., Frohwein,U., Gotzmann,J., Bayer,E., Gelbmann,D.,
Bursch,W. and Schulte-Hermann,R. (2000) The fas-induced apoptosis
analyzed by high throughput proteome analysis. J. Biol. Chem., 275,
39018±39026.
33. Seglen,P.O. (1976) Preparation of isolated rat liver cells. Methods Cell
Biol., 13, 29±83.
34. Parzefall,W., Kainzbauer,E., Qin, H.M., Chabicovsky, M. and
Schulte-Hermann, R. (2002) Response of isolated hepatocytes from
carcinogen sensitive (C3H) and insensitive (C57BL) mice to signals
inducing replication or apoptosis. Arch. Toxicol., 76, 699±706.
35. L
ow-Baselli,A., Hufnagl,K., Parzefall,W., Schulte-Hermann,R. and GraslKraupp,B. (2000) Initiated rat hepatocytes in primary culture: a novel tool
to study alterations in growth control during the first stage of
carcinogenesis. Carcinogenesis, 21, 79±86.
36. Grasl-Kraupp,B., Bursch,W., Ruttkay-Nedecky,B., Wagner,A., Lauer,B.
and Schulte-Hermann,R. (1994) Food restriction eliminates preneoplastic
cells through apoptosis and antagonizes carcinogenesis in rat liver. Proc.
Natl Acad. Sci. USA, 91, 9995±9999.
37. Grasl-Kraupp,B., Rossmanith,W., Ruttkay-Nedecky,B., M
ullauer,L.,
Kammerer,B., Bursch,W. and Schulte-Hermann,R. (1998) Levels of
transforming growth factor b and transforming growth factor b receptors
in rat liver during growth, regression by apoptosis and neoplasia.
Hepatology, 28, 717±726.
38. Oberhammer,F.A., Pavelka,M., Sharma,S., Tiefenbacher,R., Purchio,A.F.,
Bursch,W. and Schulte-Hermann,R. (1992) Induction of apoptosis in
cultured hepatocytes and in regressing liver by transforming growth factor
b1. Proc. Natl Acad. Sci. USA, 89, 5408±5412.
39. Schulte-Hermann,R., Bursch,W. and Grasl-Kraupp,B. (1995) Active cell
death (apoptosis) in liver biology and disease. In Boyer,J.L. and
Ockner,R.K. (eds) Progress in Liver Diseases. Saunders, Philadelphia,
pp. 1±35.
40. Wu,H., Wade,M., Krall,L., Grisham,J., Xiong,Y. and Van Dyke,T.
(1996) Targeted in vivo expression of the cyclin-dependent kinase
inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver
development and regeneration. Genes Dev., 10, 245±260.
41. Jochum,W., Passegue,E. and Wagner,E.F. (2001) AP-1 in mouse
development and tumorigenesis. Oncogene, 20, 2401±2412.
42. Passegue,E., Jochum,W., Behrens,A., Ricci,R. and Wagner,E.F. (2002).
JunB can substitute for jun in mouse development and cell proliferation.
Nature Genet., 30, 158±166.
43. Raper,S.E., Burwen,J.S., Barker,M.E. and Jones,A.L. (1987) Translocation
of epidermal growth factor to the hepatocyte nucleus during rat liver
regeneration. Gastroenterology, 9, 1243±1250.
44. Ichii,S., Yoshida,A. and Hoshikawa,T. (1988) Binding sites of epidermal
growth factor in nuclear fraction from rat liver. Endocrinologica Japonica,
35, 567±575.
45. Marti,U., Burwen,J.S., Wells,A., Barker,M.E., Huling,S., Feren,A.M. and
Jones,A.L. (1991) Localization of epidermal growth factor receptor in
hepatocyte nuclei. Hepatology, 13, 15±20.
46. Holt,S.J., Alexander,P., Inman,C.B. and Davies,D.E. (1994) Epidermal
growth factor induced tyrosine phosphorylation of nuclear proteins with
translocation of epidermal growth factor receptor into the nucleus.
Biochem. Pharm., 47, 117±126.
47. Payrastre,B., Nievers,M., Boonstra,J., Breton,M., Verkleij,A.J. and Van
Bergen en Henegouwen,P.M.P. (1992) A different localization of
phosphoinositide kinases, diacylglycerol kinase and phospholipase C in
the nuclear matrix. J. Biol. Chem., 267, 5078±5084.
48. David,M., Wong,L., Flavell,R., Thompson,S.A., Wells,A., Larner,A.C.
and Johnson,G.R. (1996) STAT activation by epidermal growth factor
(EGF) and amphiregulin. Requirement for the EGF receptor kinase but
not for tyrosine phosphorylation sites or JAK1. J. Biol. Chem., 271,
9185±9188.
Received October 17, 2002; revised February 10, 2003;
accepted February 11, 2003
841