1 Institut für Krebsforschung, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria
2 Research Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria
3 To whom correspondence should be addressed Email: bettina.grasl-kraupp{at}univie.ac.at
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Abstract |
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Abbreviations: HGF, hepatocyte growth factor; LI, labelling index; PB, phenobarbital; pro-TGF+, positive for pro-TGF
; pro-TGF
-, negative for pro-TGF
; TGF
, transforming growth factor
; TGF-ß1, transforming growth factor ß1
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Introduction |
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We have shown recently that hepatocytes in the intact liver and in primary culture synthesize pro-forms of TGF, that translocate to the nucleus in G1 where they appear to serve in a novel function for mitogenic signalling (4). Nuclear location of pro-TGF
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 TGF
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 TGF
, EGF, amphiregulin, ß-cellulin and others (18,19). Like pro-TGF
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 ß1 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 TGF
and/or erbb-receptors mayat least under certain conditionsbypass cytoplasmic phosphorylation cascades, widely thought to be essential in transducing mitogenic stimuli. Because TGF
is up-regulated in different types of human malignancies, such as hepatocellular carcinoma, novel therapeutic approaches concentrate on the possible benefit of blocking TGF
-evoked signal transduction, e.g. by blockade of the receptor or ligand receptor interactions on the cell surface (2428). However, human hepatocellular adenoma and carcinoma often express pro-TGF
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 TGF
gene products.
In the present study we investigated whether the high association between nuclear pro-TGF and DNA replication in hepatocytes depends on an active c-jun. For this purpose, the inducible cre-loxP recombination system was used to delete c-jun in mouse hepatocytes during postnatal life; the growth potential of these hepatocytes was tested in an ex vivo system. We found that c-jun
liver mouse hepatocytes show a lower basal rate of DNA replication in primary culture but are stimulated to DNA synthesis by HGF and mature TGF
to a much higher extent than control hepatocytes. DNA synthesis was detected almost exclusively in mouse hepatocyte nuclei expressing nuclear pro-TGF
and erbb-1. Thus, the nuclear pro-TGF
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.
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Materials and methods |
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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.
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 2x SDS buffer and were applied to one-dimensional SDSpolyacrylamide 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 TGF (Oncogene Science, Uniondale, NY; Ab-1, clone 134A-2B3; 1:300), against the C-terminal amino acids 144160 of pro-form of wild-type TGF
(InnoGenex, San Ramon, CA; 1:300), against the extracellular domain of erbb-1 (1:500, Genosys Biotechnologies, Pampisford, UK), or against residues 985996 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/µl, was applied at a final concentration of 10 ng/ml medium. Recombinant human mature TGF (UBI, Lake Placid, NY) was prepared as a stock of 10 µg/10 µl in 10 mM acetic acid and added as final concentrations of 20 and 50 ng/µl.
Histology
Liver tissue of untreated C57/B6 mice, fixed in Carnoy's solution, was processed as described (36,37); three serial sections, 2 µm thick, were cut; one of the sections was stained with haematoxylin and eosin, the second one for pro-TGF and the third one for erbb-1 (4).
Immunostaining for pro-TGF 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 TGF (originally clone 213-4.4, Oncogene Science) or the C-terminus of human pro-TGF
(Innogenex); rabbit polyclonal IgG against mature TGF
(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 TGF and erbb-1; confocal laser scanning microscopy
The procedure followed published descriptions with the following modifications (4,35): primary antisera (anti-TGF 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 [3H]thymidine (sp. act. 6080 Ci/mmol; added at 0.5 µCi/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 µg/ml Hoechst 33258 (Riedel de Haen, Seelze, FRG) and mounted. To determine DNA synthesis a total of 1000 TGF- and 300400 pro-TGF
+ hepatocyte nuclei were evaluated per plate. For the evaluation of 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.
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Results |
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Basal and induced DNA replication in cultured mouse hepatocytes is highly associated with nuclear pro-TGF
In untreated primary cultures the incidence of pro-TGF+ nuclei was
3-fold higher in controls (23.6 ± 10.7%) than in c-jun
liver hepatocytes (7.9 ± 4.8%); 83.5 ± 9.8% of the mouse hepatocytes with pro-TGF
+ nuclei and only 4.1 ± 3.1% with negative nuclei replicated DNA, which was observed in both control and c-jun
liver hepatocytes (Figures 2B and C and 4E). As 84.2% of the pro-TGF
+ nuclei also expressed erbb-1, at least 67.7% of the pro-TGF
+ nuclei in DNA synthesis should be positive for erbb-1. In conclusion, DNA replication in pro-TGF
- nuclei was a rare event and nuclear pro-TGF
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-TGF is involved in the growth response of control and c-jun
liver hepatocytes in primary culture. Forty-eight hours after addition of HGF the percentage of cultured hepatocytes with pro-TGF
+ 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-TGF
+ within the same nuclei, correlated highly (Pearson's test: r2 = 0.7127; significant at P < 0.0001). Treatment of cultures with mature TGF
dose-dependently induced a similar effect. Thus, the induction of DNA synthesis by HGF and TGF
was highly associated with de novo occurrence of nuclear pro-TGF
; this mechanism was active in both control and c-jun
liver hepatocytes.
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Discussion |
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Suppressed basal DNA replication after inactivation of c-jun
C-junliver 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-jun
liver hepatocytes might be due to inefficient activation of cyclin-dependent kinases, which are essential for a proliferative response.
Mitogen-induced DNA synthesis occurs in the absence of c-jun
The effects of PB or TGF 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 TGF
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 (57,9). 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-junliver hepatocytes, stimulation was not compromised at all in the present study. In contrast, the relative induction of DNA replication was more pronounced in c-jun
liver 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-TGF 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 TGF are present in the nuclei of both, control and c-jun
liver mice. We have shown recently that a subfraction of the isolated primary hepatocytes synthesize pro-TGF
. Immediately after seeding of the cells pro-TGF
is distributed evenly throughout the cytoplasm and soon thereafter starts to translocate to the nucleus; 16 h later hepatocytes show intranuclear accumulation of pro-TGF
without cytoplasmic residues and after a further lag phase of
22 h almost all of the positive nuclei replicate DNA (4). Addition of mature TGF
to the medium increased DNA synthesis exclusively in pro-TGF
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-TGF
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-TGF in hepatocytes scattered throughout the liver lobule. This again is followed by DNA replication in the fraction of pro-TGF
positive nuclei. In the resting liver, however, the constitutive occurrence of nuclear pro-TGF
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-TGF
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 TGF
to erbb-receptors at the cell membrane (4).
One advantage of a direct action of pro-TGF 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 TGF
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 TGF or HGF were associated with induction of nuclear pro-TGF
in cultured mouse hepatocytes of both controls and and c-jun
liver mice. In vivo various hepatomitogenic signals led to pronounced induction of nuclear occurrence of pro-TGF
. In vitro the number of pro-TGF
+ hepatocyte nuclei was elevated by treatment with hepatomitogenic steroid hormones or prostaglandins (4). A further interesting finding is, that treatment with mature TGF
induces the nuclear pro-TGF
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 TGF
and the pathway involving nuclear pro-TGF
are interdependent.
Possible interaction of pro-TGF and erbb-1
How pro-TGF 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-TGF
+ hepatocyte nuclei also were positive for erbb-1. It is currently under investigation whether the large TGF
precursor may attach within the binding site of any of the erbb-receptors suited for the small molecule of mature TGF
and whether pro-TGF
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 125I-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 (4447). 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 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-TGF 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-TGF
and that link nuclear pro-TGF
to DNA replication.
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Acknowledgments |
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References |
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