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[CANCER RESEARCH54, 6065-6068, December 1, 19941
Advances in Brief
Expression of Helix-Loop-Helix
Factor Id-i Is Dependent on the Hepatocyte
Proliferation and Differentiation Status in Rat Liver and
in Primary Culture1
Catherine
Le Jossic,
Gennady
P. Ilyin,2 Pascal
Loyer,
Denise
Glaise,
Sandrine
Cariou,
and
Christiane Guguen-Guillouzo
INSERM U49. Unite de RecherchesHepatologiques.HôpitalPontchaillou, 35033 Rennes,France
Abstract
Id proteins are known as negative regulators of differentiation in
various cell types. In this report, we show that the Id-i gene was down
regulated during the development of rat liver. No Id-i transcripts were
detected in terminal differentiated hepatocytes. We have studied Id-i
expression
in proliferating
hepatocytes
using an in vivo model of liver
regeneration after partial hepatectomy and an in vitro growth factor
fetal life; and (c) maturation of neonatal liver and acquisition of a
stimulated hepatocyte culture system. Strong activation of Id-i was oh
served in mid-late G1 of the hepatocyte cell cycle at a time corresponding
to a mitogen restriction point. These observations suggest that Id-i is
involved in the control of proliferation and differentiation in liver cells.
Introduction
vivo with E2A proteins (5, 6). Generally, high levels of Id mRNA in
proliferative and undifferentiated cells decrease as they are induced to
differentiate (3, 4). On the other hand, the expression of Id genes is
rapidly induced in a diverse range of cell lines activated by growth
factors or phorbol 12-myristate 13-acetate. Finally, overexpression of
human Id-related gene HLH 1r21 produced a morphologically trans
formed phenotype in NIH 3T3 cells (7). These observations suggest
that Id proteins can act as negative regulators of a differentiation
program in various cell types and are involved in the control of cell
proliferation and possibly of transformation.
The regulation of Id gene expression has been mainly studied in
Received 8/18/94; accepted 10!17f94.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
research
was
supported
by lnstitut
National
de la Santa
et de Ia Recherche
Médicale,
EEC (BIOT-CT9O—0l89)and the Association pour Ia Recherche contre Ic
Cancer.
2 To
3 The
whom
requests
abbreviations
terminally differentiated phenotype. Quiescent hepatocytes constitute
one of the few terminally differentiated cell types in the adult body
which retain their proliferating capacity. Both in viva and in vitro
models of proliferating hepatocytes are available. In the present re
port, we show the sequence of Id-i expression over the course of liver
The HLH3 proteins are known to play an important role in the
regulation of differentiation in various cell-specific lineages as well as
in cell proliferation and transformation. Members of this regulatory
gene family share two functional motifs: the region of basic amino
acids involved in DNA binding and the HLH domain which is
essential for dimerization (1). The HLH proteins recognize the nude
otide consensus sequence CANNTG, known as the E-box. It is sug
gested that HLH proteins can be divided into different classes. Class
A contains the ubiquitously expressed proteins Ei2 and E47. Class B
comprises the tissue-specific factors such as the MyoD family, which
can form heterodimers with class A molecules (2).
A distinct class of HLH transcriptional regulators includes Id pro
teins, which lack the basic DNA-binding region, and are able to
inhibit the binding of several HLH proteins to DNA by forming
biologically inactive hetero-oligomers (3, 4). In particular, Id-i can
inhibit differentiation of muscle and myeloid cells by associating in
I This
tissues and cells of mesodermal origin. To gain insight into the
potential role of Id proteins in proliferation and differentiation of
endoderm-derived tissues, we determined the expression of the Id-i
gene in rat liver in relation to hepatocyte proliferation and differen
tiation status. Liver development in mammals involves at least three
major steps: (a) commitment of embryonic cells to become hepato
cytes; (b) modulation of liver-specific gene expression during late
for
used
reprints
are:
should
HLH,
be
addressed.
helix-loop-helix;
P1-fr,
partial
hepatectomy;
EGF,
epidermal growth factor; RLEC, rat liver epithelial cells.
development and in proliferating adult hepatocytes.
Materials and Methods
Animals. Pregnantand normalfemale Sprague-Dawleyrats (180—200
g)
were obtained from Charles River Laboratories (Cléon,France). Breeding was
done by placing female rats with males of the same strain overnight; noon of
the next day was considered as 0.5 days postcoitum. On the appropriate days
of gestation, rats were anesthetized, embryos were removed, and their livers
were minced and washed with PBS to reduce the number of hemopoietic cells.
A partial (two-thirds) hepatectomy was performed as described by Higgins and
Anderson (8). Control animals underwent a sham operation comprising lapa
rotomy and liver manipulation without tissue removal. At different times after
PHT, animals were sacrificed, and their livers were washed with PBS by
perfusion
through
the portal
vein to eliminate
blood
cells.
Livers
were
then
minced, frozen in liquid nitrogen, and kept at —80°C
until further processing.
CeHIsolationandCulture. Hepatocytes
fromadultmaleSprague-Dawley
rats were isolated by the classical two-step collagenase perfusion procedure.
Hepatocytes were seeded at 7.5 X 10―
cells/cm2on a 75-cm2flask in a mixture
of 75% MEM and 25% medium 199, supplemented with 10% FCS and, per ml,
100 units penicillin, 100 mg streptomycin sulfate, 1 mg bovine serum albumin,
and 5 mg bovine insulin. After cell attachment (4 h later), the medium was
renewed with the same medium deprived of FCS and supplemented with EGF
(50 ngfml) and pyruvate (20 mM) or with 1.4 X i0_6 M hydrocortisone
hemisuccinate. It was changed every day thereafter. Faza 967 and FAO,
well-differentiated rat hepatoma cells derived from H4IIEC3, were maintained
in a mixture ofSO% Ham's F-12 and 50% NCI'C 135 with 10% FCS. NIH 3T3
embryonic fibroblasts were grown in MEM supplemented with 10% FCS.
RLEC,SDVI, were obtainedas describedpreviously(9), and BRL 3A cells
were grown in Williams' E medium supplemented with 10% FCS.
RNA Isolation and Northern Blot Analysis. Total RNA was extracted by
the thiocyanate guanidium procedure. Twenty ,.@gof RNA were separated by
electrophoresis
and transferred onto a nylon membrane (Hybond N + ; Amer
sham). Hybridization was performed with [ca-32P]dC'I'P-labeledprobe for 16 h
at 65°C.A 910-base pair cDNA fragment of clone pRID9.1, spanning the
full-length rat Id-i mRNA coding sequence, was used as a probe. This plasmid
was kindly provided by Jeremy P. Springhorn (Brigham and Women's Hos
pital, Boston, MA) (10). In some experiments, we used a 5'-truncated 550-base
pair fragment
of rat Id-I cDNA
lacking
sequences
encoding
the NH2-terminal
6065
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ID-i EXPRESSION
A
region and HLH domain. The blots were rehybridized with rat glyceraldehyde
3-phosphate dehydrogenase
IN LIVER
RLEC
Livar4 6' 6 8 10 12 12 18 24' 24 hours
cDNA as a control for gel loading and transfer.
.-
Gel Mobifity Shift Assay. Preparation of nuclear extracts and gel mobility
assay were performed as described by Cereghini et a!. (11), except that the
nuclear extracts were not dialyzed. Binding reactions were carried out in a
15-pi volume
containing
1 mM sodium
phosphate
(pH 7.5), 0.1 mM EDTA,
.
0.5
mM EGTA, 0.5 mM DTT, 10% (v/v) glycerol, 0.5 ,.@gpolydeoxyinosinic
deoxycytidylic acid, 1 mt@iMgC12, 10 m@ spermidine, and 0.1—0.2 ng of
5 ‘-end32P-labeled double-stranded oligonucleotide. The oligonucleotide con
taming the E-box was derived from position —618to —598of the 5'-flanking
B
RLECF.I.H.4
7 17 24 31 48 65 72 80 96 102115l20hours
,
region ofthe rat a-fetoprotein gene (12) and had the sequence of 5'-ACCCAT
GCATCTGTGACATACAT-3'.The oligonucleotide probe, containing the
UAE site of the adenovirus major late promoter, had the sequence of 5'CGGTAGGCCACGTGACCGGOT-3'. Five @gof nuclear extracts were
added to the reaction mixture and incubated for 10 mm on ice. The DNA
C
F.I.H.4 18 24 2730
z
42 4$ 54 60 66 l2hours
protein complexes were separated by 6% acrylamide gel in 0.5 X ThE (45 mr@i
Tris-borate/l.25 mMEDTA). The gel was then fixed, dried, and subjected to
autoradiography.
N.,,,,
Results
We have examined the Id-i expression by Northern blot analysis
with full-length rat Id-i cDNA probe in several liver-derived cell lines
in comparison with proliferating cultures of NIH 3T3 cells. Id-i
D
36 48 00 66 l2hosws
transcripts were observed in the differentiated rat hepatoma FAO and
Faza 967 cells in moderate levels, similar to those found in NIH 3T3
cells, whereas Id-i messengers were abundant in RLEC (Fig. iA).
Therefore, RLEC were used as a reference in Northern blot analysis.
Fig. 2. Expression of Id-I in proliferating rat hepatocytes. Total RNA was extracted
from liver biopsies at different times after PHT (A) and from cultured hepatocytes
stimulated with EGF (B and C) or maintained without growth factor (D). Twenty @g
of
Analysis of Id-i expression over the course of rat liver development
revealed the presence of high amounts of Id transcripts in fetal and
RNAs were analyzed
neonatal rat livers. From 2 weeks after birth, the levels of Id-i RNA
by Northern
blot hybridization
with full-length
rat Id-i cDNA
probe
(A andB) or with probespecificfor Id-I, whichlackednucleotides
encodingNH2terminal region and HLH domain (C and D). °,sham-operated animals; FIH. freshly
isolated hepatocytes.
decreased and were either very low or undetectable after day 28 (Fig.
1B). In the adult rat liver, hepatocytesare blockedin a quiescentstate
but can be easily stimulated to proliferate in response to loss of tissue
mass. We found a rapid induction of Id-i from 6 h after PHT.
mRNA remained through the cell cycle, with a gradual decrease after
80 h (Fig. 28). To define more precisely the appearance ofld-i during
the G@phase and to confirm the specificity of hybridization, we
Thereafter, the levels of Id-i mRNA increased, reaching a maximum
at 18 h. No significant induction of Id-i was observed in sham
operated animals (Fig. 14). It was previously shown that EGF/pyru
performed a detailed analysis of id-I expression in rat hepatocytes
maintained in culture, with and without EGF, using a rat Id-i probe
vate-stimulated rat hepatocytes in primary culture represent a suitable
model to study mechanisms of cell cycle progression (i3). Only a very
lacking nucleotides encoding the NH2-terminal region and conserva
tive HLH domain. The strong activation of Id-I occurred at 42 h of
culture; it reached a maximum starting at 54 h (Fig. 2C). In contrast,
low level of [3Hjthymidine incorporation could be observed in rat
hepatocytes, maintained in the basal medium without growth factors.
In contrast, DNA synthesis occurred in most of the rat hepatocytes
stimulated with EGF/pyruvate, at the third day of culture. No Id-i
transcripts were detected in freshly isolated rat hepatocytes and during
the first hours in culture. A drastic increase of Id-I transcripts was
observed between 31 and 48 h of culture. Then, high amounts of Id-i
only low levels of Id-i transcripts were detected in nonstimulated
hepatocytes (Fig. 2D).
The Id-i protein can selectively inhibit the binding to the E-box
oligonucleotide probes of one set of the HLH proteins, such as E12,
E47, and MyoD, but not of the other set, including USF and AP-4 (3,
A
RLEC313 FAZAFAOBRL
Fig. 1. Northern blot analysis of Id-i expression
@
in various liver-derived cell lines (A) and during
development of rat liver (B). Total RNA was cx
tractedfromcellsincultureorlivertissue,and20
,.i.gof RNA were applied to gels. Northem blots
were sequentially hybridized with rat Id-i and
glyceraldehyde 3-phosphate dehydrogenase cDNA
B
probes.
Idi
@EC14.5 16.5 18.5 0.Sd 3d
d.p.c. d.p.c. d.p.c.
GAPDH
•
0
0
0
0
s
3d
7d
Sd
lid
13d
iSd
1$d
21d 24d
@d
0
•s•.•.O.O.Oos•ô
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ID-I EXPRESSION IN LIVER
Fig. 3. E-box-binding
activities in proliferating
hepatocytes.Nuclearextractswerepreparedfrom
liver biopsies or from cultured rat hepatocytes stim
A
I
2
3
comp.-
-
+ +
4
B
R.L
ulated with EGF. Gel mobilityshift assay was
,._.
performed with 32P-labeled E-box probe as de
scribed in “Materials
and Methods.―
A, competition
experiments were carried out by addition of 100fold excess of unlabeled E-box probe (Lane 3) or
•@s
4
14
24
• %
• S
• %
48
72
96
@• •@%
• .
120 hours
•
1$
an unrelated probe containing a UAE site (Lane 4).
Lane 1, labeled probe only; Lane 2, labeled probe
with rat liver nuclear extracts. B, labeled E-box
probe was incubated with nuclear extracts from
EGF-stimulated
F.I.H.
rat hepatocytes at different times
in culture. RI.. rat liver; FIll, freshly isolated hepa
tocytes.
4). Although it is not known what kind of HLH proteins are present
in the hepatocytes, we analyzed E-box binding activity of nuclear
extracts prepared from adult rat liver and from cultured hepatocytes
stimulated with EGF by gel mobility shift assay. Several E-box-bound
complexes were observed in adult liver nuclear extracts. All bindings
were efficiently suppressed by addition of 100-fold molar excess of
the unlabeled probe. Competition performed with another E-box
probe containing the UAE site of adenovirus major late promoter,
known to bind the ubiquitous cellular transcriptional factor USF (14),
resulted in partial blocking of binding activities of fast-migrating
In this laboratory, it has been recently shown that, under the
complexes (Fig. 3A). The slow-migrating complexes were drastically
reduced in freshly isolated hepatocytes, partly reappeared in hepato
cytes from 4—24h of culture, and were again suppressed later through
the cell cycle (Fig. 3B). By contrast, fast-migrating complexes were
constantly present in proliferating hepatocytes with variations in
intensity.
conditions used here, an increase of DNA synthesis took place at
around 54—60 h and peaked at 72—78h of culture. Furthermore, a
restriction point has been defined at 42—45h as a step in G1 beyond
which the cells cannot progress without EGF stimulation.4 Activation
of Id-i precisely started at 42 h, with the highest levels from 54 h of
culture. The fact that the occurrence of Id-i was strictly correlated
with the EGF restriction point strongly argues for a particular role of
Id-iinlate
G1.
By gel mobility shift assay, we demonstrated that adult rat liver
Discussion
Cell proliferation and differentiation are the essential but alternative
events of living organisms. For instance, in muscle cells, the differ
entiation program is closely coupled to the cell cycle. Identification of
the myogenic HLH transcriptional factors has provided insight into
the mechanisms of cross-talk between the regulatory pathways that
control myoblast proliferation and differentiation (15). Members of
the MyoD family of transcriptional regulators can activate the muscle
differentiation program as well as induce growth arrest (16, 17).
Conversely, Id proteins inhibit myogenesis and, at the same time, act
as the essential regulators of cell cycle progression (5, 18). In the liver,
cessation of DNA synthesis in postnatal hepatocytes correlates with
hepatocyte
replicate initially, whereas nonparenchymal cells divide after a lag of
2—3days; and (c) a long-lasting G1 phase characterizes the hepatocyte
cell cycle. DNA synthesis begins at 19—20h after PHT and peaks at
22—24h (21, 22). We have observed that ld-i was activated from 6 h
after PHT and increased between 12 and 18 h, which corresponded to
the mid-late G1 phase. Moreover, analysis of Id-i expression in
growth factor-stimulated rat hepatocytes confirmed the beginning of
Id-i expression at the mid-late G1 phase of the hepatocyte cell cycle.
maturation
and
acquisition
of the adult
phenotype.
In
contrast, stimulation of hepatocyte proliferation by PHT leads to
partial retrodifferentiation, which is characterized by reexpression of
fetal liver markers, including plasma proteins like a-fetoprotein and
intracellular isoenzymes (19, 20). These observations suggest that the
molecular mechanisms which link these two biological phenomena
might be common in different cell types.
Our data demonstrate the presence of Id-i transcripts in fetal and
neonatal rat liver. From 2 weeks after birth, the levels of Id-i mRNA
decreased, reflecting the gradual maturation of the liver. Terminally
differentiated adult rat liver contained very low levels, if any, of Id-i
transcripts. To establish the potential role of Id-i in the hepatocyte cell
cycle progression, we analyzed the levels of Id-i mRNA in prolifer
ating hepatocytes using both an in vivo model of hepatocyte prolifer
ation triggered by resection of part of the organ and an in vitro
EGF/pyruvate-stimulated rat hepatocyte culture system. Liver regen
eration after PHT provides a unique system to study the proliferating
response of nontumoral, highly differentiated cells in a solid tissue
proteins to the E-box, the activation of Id-i in proliferating hepato
cytes would be reflected in the suppression of E-box-binding activi
ties. Indeed, slow-migrating complexes were reduced in freshly iso
lated hepatocytes and in EGF/pyruvate-stimulated
hepatocytes from
48 h in culture, which is correlated with activation of Id-i. However,
no Id-i transcripts were detected in freshly isolated hepatocytes,
suggesting participation of other proteins in restricting E-box-binding
activities. Isolation of hepatocytes was accompanied by a rapid and
transient activation of several immediate-early genes, like c-los and
c-jun. Expression of these genes was shown to begin during the
dissociation of liver cells, to peak in freshly isolated hepatocytes and
to decrease thereafter (23). This raises the possibility that c-jun may
interact with liver HLH proteins as reported with MyoD (24). Alter
natively, other members of the Id family could be involved in regu
lation of E-box-binding activities. In line with this possibility, we
have found that Id-2 was activated in freshly isolated hepatocytes,
decreased thereafter, and also drastically increased in parallel with
Id-i in mid-late G@of the hepatocyte cell cycle.5
Taken together, our data suggest a potential role of Id-i in differ
entiation and proliferation of hepatic cells. Identification of Id-binding
proteins may clarify the molecular mechanisms which couple these
mutually exclusive biological phenomena. In addition, since Id-i
4 P. Loyer,
and is characterized by several properties: (a) the cell growth response
is a perfectly regulated synchronous
nuclear extracts contained several proteins that can bind the E-box. At
least some of the E-box-bound species were distinct from the ubiq
uitous nuclear factor USF. Indeed, an oligonucleotide, known to bind
USF preferentially, only partly suppressed fast-migrating complexes.
Since the Id-i protein acts as an inhibitor of the binding of HLH
process; (b) only hepatocytes
S. Cariou,
D.
Glaise,
M.
Biodec,
G.
Baffet,
and
C.
Guguen-Guillouzo.
Growth factor dependence of entry and progression through Gi and S phases of adult rat
hepatocytes in vitro, submitted for publication.
5 G. P. Ilyin,
C. Le Jossic,
and C. Guguen-Guillouzo,
manuscript
in preparation.
6067
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ID-l EXPRESSION IN LIVER
11. Cereghini, S., Blumenfeld, M., and Yaniv, M. A liver-specific factor essential for
appeared to be expressed in various hepatic cell lines with the highest
albumin transcription differs between differentiated and dedifferentiated rat hepatoma
levels observed in RLEC, which have been reported as a “facultative
cells. Genes Dcv., 2: 957—974,1988.
hepatocyte stem cell―population (25), the involvement of Id-i protein
12. Nahon, J-L., Danan, J-L., Poiret, M., Tratner, I., Jose-Estanyol, M., and Sala-Trepat,
J-M.The rat a-fetoproteinand albumingenes.Transcriptional
controlandcompar
in liver carcinogenesis might be suggested.
ison of the sequence and promoter region. J. Biol. Chem., 262: 12479—12487,
1987.
13. McGowan, J. A. Hepatocyte proliferation in culture. In: A. Guillouzo and C. Guguen
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Guillouzo (eds.), Isolated and Cultured Hepatocytes, pp. 13—38.INSERM, Paris and
rat Id-i.
John Libbey Eumtext, London, 1986.
14. Gregor, P. D., Sawadogo, M., and Roeder, R. G. The adenovirus major late tran
scription factor USF is a member of the helix-loop-helix group of regulatory proteins
and bind to DNA as a dimer. Genes Dcv., 4: 1730—1740,1990.
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6068
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research.
Expression of Helix-Loop-Helix Factor Id-1 Is Dependent on the
Hepatocyte Proliferation and Differentiation Status in Rat Liver
and in Primary Culture
Catherine Le Jossic, Gennady P. Ilyin, Pascal Loyer, et al.
Cancer Res 1994;54:6065-6068.
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