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Hormone and Metabolic Research Unit (O.L., M.R., F.P.L., G.G.R.) Christian de Duve Institute of Cellular Pathology Unité de Diabétologie (D.M., J.-P.T.) Université catholique de Louvain B-1200 Brussels, Belgium
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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(465 residues) and ß (491 residues), have been cloned in the rat.
They differ only by the length (27 or 53 amino acids) of the linker
between the cut domain and the homeodomain (2). These two isoforms
originate from the same gene by differential splicing (4). They differ
in affinity for DNA target sequences, but both behave as
transcriptional activators in transient transfection assays (2). The
hnf6 gene is strongly expressed in the liver (1).
Transfection experiments performed with HNF-6 showed that it
stimulates the transcription of liver-expressed genes that code for
proteins such as 6-phosphofructo-2-kinase, an enzyme involved in
glucose metabolism (1), CYP2C12, an enzyme of steroid metabolism (5),
transthyretin, a plasma transport protein (2), protein C, which
controls coagulation (6), and the transcription factors HNF-4 and
HNF-3ß (7, 8). These two transcription factors are involved in the
differentiation of hepatocytes and the maintenance of liver-specific
functions (9). Thus, the HNF-6 family participates to the regulatory
network of factors that controls liver development and
differentiation. How the hnf6 gene is regulated is therefore an important issue. One candidate is GH. Indeed, we found in adult rats that the liver concentration of HNF-6 mRNAs drops dramatically after hypophysectomy and returns to normal after administration of GH (5). The aim of the present work was to determine whether this effect results from a direct action of GH on the hepatocyte and on the hnf6 gene. We show here that this is the case. We also identify transcription factors that mediate the effect of GH on the hnf6 gene promoter. Our data show that GH rapidly induces the binding of signal transducer and activator of transcription (STAT)5 and increases the binding of HNF-4 to the hnf6 gene promoter. This results in a stimulation of the promoter. Finally, we provide evidence that GH controls the network of liver-enriched transcription factors and that HNF-6 participates not only as an effector, but also as a target, to this regulatory network.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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mRNA was barely
detectable and HNF-6ß mRNA was below the threshold of sensitivity of
the assay (Fig. 1A
), consistent with the
fact that the concentration of HNF-6ß mRNA in the liver of intact
rats is lower than that of HNF-6 mRNA (1). Within 1 h after the
injection of GH, the concentrations of HNF-6 and -ß mRNAs
increased 6-fold, to reach 50-fold after 3 h and return to basal
levels by 9 h after the injection (Fig. 1, A and B).
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, HNF-6 and ß mRNAs concentration
increased between 30 min and 1 h after addition of GH, to reach a
maximum after 2 h. The experiment was repeated with different
concentrations of GH. This yielded a typical dose-response curve (Fig. 1D), with a near-maximal response at a concentration of 50 ng/ml (2
nM) GH, which is physiological. We concluded that
the hnf6 gene is expressed at a low level in the absence of
GH and that GH increases the concentration of liver HNF-6 mRNAs by a
direct effect on the hepatocyte.
Involvement of STAT5 in the Transcriptional Stimulation of the
hnf6 Gene by GH
The experiments reported above suggested that GH directly
stimulates the transcription of the hnf6 gene in liver. This
was consistent with the observed coordinate effect of GH on HNF-6
and HNF-6ß mRNA (Fig. 1), which both originate from the same gene
(4). Upon binding to its receptor at the surface of the hepatocyte, GH
induces receptor dimerization and association with the tyrosine kinase
Jak-2. The latter phosphorylates the receptor, which then serves as a
docking site for STAT factors. These become phosphorylated, dimerize,
and bind to regulatory regions of GH-responsive genes (11). We
therefore searched for such regions in the hnf6 gene
promoter. Nuclear extracts were prepared from the rat livers that were
used to demonstrate an effect of GH on HNF-6 mRNAs (see Fig. 1A). These
extracts were incubated with labeled fragments of the hnf6
gene promoter to conduct deoxyribonuclease I (DNase I) footprinting
assays. These experiments showed GH-dependent protein binding to the
region from -105 to -124 of the promoter (Fig. 2A). This footprint was not seen with
liver extracts from hypophysectomized rats. The footprint appeared
within 1 h of GH treatment and had disappeared after 6 h
(Fig. 2A). The underlying sequence, TTCTAAGAA (from -116 to -108), is
compatible with the binding consensus for STAT factors (12), among
which STAT1, STAT3, and STAT5 are known to mediate several actions of
GH in the liver (13).
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, the protein-DNA complex
was seen exclusively with the liver extracts obtained from rats that
had been killed 1 or 3 h after GH injection (lanes 3 and 4),
consistent with the footprinting data. This complex disappeared with
excess of cold probe (lane 8) or cold STAT-binding oligonucleotide
(lane 9), but not with cold Sp1-binding oligonucleotide (lane 10).
Moreover, the migration of the complex was retarded by an anti-STAT5,
but not by an anti-STAT3, antibody (lanes 11 and 12). We concluded from
these experiments that GH induces the binding of STAT5 to the
hnf6 gene promoter within 1 h.
To test the role of STAT5 in the control of the hnf6 gene
promoter, we overexpressed a constitutively active form of STAT5
(STAT5
750VP16Jak2) in transfected cells. This chimeric protein
includes the receptor-binding (SH2) and DNA-binding domains of STAT5
and the transactivation domain of VP16 fused to the kinase domain of
Jak2, which ensures the phosphorylation-dependent dimerization and
nuclear translocation required for gene targeting of the chimera (14).
The cells were cotransfected with a luciferase reporter gene linked to
0.75 kb of the hnf6 promoter. As shown in Fig. 3A, constitutively active STAT5
stimulated transcription from the hnf6 gene promoter. This
did not occur when the cells were transfected with a transcriptionally
inactive form of STAT5 (STAT5750Jak2), which lacks the
transactivation domain of VP16, or with a reporter construct in which
the STAT5-binding site in the hnf6 promoter had been
destroyed by mutation (Fig. 3A).
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). The amplitude of this GH effect was comparable to that
reported in the same cells for another GH-inducible gene (15). Our
experiments with the hnf-6 promoter-reporter construct also
showed that the STAT-binding site in this promoter is absolutely
required for the stimulation by GH, as GH had no effect when this
binding site had been destroyed (Fig. 3B).
The demonstration that GH can transactivate the hnf6 gene
promoter and that this depends on the integrity of the STAT-binding
site allowed us to provide evidence for the role of STAT5 in the GH
effect. BRL-4 cells were now transiently cotransfected with the
hnf6 reporter construct and an expression vector for
wild-type STAT5. As shown in Fig. 3B, overexpression of STAT5 clearly
amplified the stimulation of hnf6 promoter activity by GH.
Overexpressed STAT5 had little effect in the absence of GH, which is
consistent with the notion that the transcriptional action of STAT
factors requires their ligand (i.e. GH)-dependent
phosphorylation by Jak proteins. The amplification of the GH effect by
exogenous STAT5 did not occur with an expression vector lacking the
transactivation domain of STAT5 (Fig. 3B). Taken together, our data
demonstrate that GH can stimulate transcription of the hnf6
gene through an induction of STAT5 binding to its cognate site in the
hnf6 promoter.
Involvement of HNF-4 in the Transcriptional Stimulation of the
hnf6 Gene by GH
Another DNase I footprint, from -633 to -670, was detected in
the hnf6 gene promoter with liver nuclear extracts (Fig. 4A). This region encompasses a sequence,
CGGGCAAAGGCCA (-652 to -640), compatible with the binding consensus
for HNF-4 (4, 16). To identify the protein involved in this footprint,
EMSA were performed with the corresponding oligonucleotide probe and
with the liver extracts used to demonstrate GH-dependent STAT5 binding
to the hnf6 promoter. The data (Fig. 4B) indeed showed
specific binding of HNF-4, as demonstrated by competition with the cold
probe (lane 8) and with an HNF-4-binding oligonucleotide (lane 9), but
not with an Sp1-binding oligonucleotide (lane 10). A supershift was
observed with an anti-HNF-4 antibody (lane 11). A complex exhibiting
the same properties was seen in EMSA with this hnf6 promoter
probe when using, instead of liver extracts, extracts from Cos-7 cells
that had been transfected with an HNF-4 expression vector (data not
shown). Consistent with the footprinting data (Fig. 4A, lanes 2 and 3),
HNF-4 binding to the hnf-6 promoter did not depend on GH
(Fig. 4B, lane 2). However, HNF-4 binding increased strongly within the
hour after the injection of GH and returned to uninduced levels between
6 and 12 h (Fig. 4B, lanes 3–7).
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, A and B, show that liver HNF-4 mRNA
increased only slightly. This took place 6 h after the GH
injection and returned to uninduced levels by 9 h. Obviously, this
late and minor change in HNF-4 mRNA concentration could not account for
the rapid and intense GH-induced increase in HNF-4 binding. Therefore,
the signaling cascade triggered by GH in rat liver most probably
targets the HNF-4 protein itself, thereby increasing its stability or
its affinity for the hnf6 gene promoter. To investigate
these possibilities, we determined by immunoblotting the concentration
of HNF-4 in the nuclear extracts used for testing HNF-4 binding after
GH treatment. As shown in Fig. 4C, the concentration of HNF-4 did not
change over the 6 h that followed the GH injection, a period
during which HNF-4 binding increased dramatically. Thus, GH treatment
confers to HNF-4 a higher affinity for its DNA target in the
hnf-6 promoter. The fact that HNF-4 mRNA had returned to
normal levels between 6 and 9 h after the injection of GH (Fig. 5B) is consistent with the uninduced levels of HNF-4 protein seen by
immunoblotting after 12 and 24 h (Fig. 4C). We concluded from
these experiments that HNF-4 binds to the hnf6 promoter and
that this interaction is increased by GH treatment.
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, the
activity of the hnf6 promoter-luciferase reporter construct
was stimulated by the cotransfection of an HNF-4 expression vector.
This effect was abolished when the HNF-4-binding site in the
hnf6 promoter was destroyed by mutation (Fig. 6A). These
experiments were repeated with BRL-4 cells to evaluate the effect of GH
under these conditions (Fig. 6B). The 1.8-fold increase in wild-type
promoter activity induced by GH was reduced to 1.3-fold (0.001<
P< 0.01) after destruction of the HNF-4 binding site. The
residual activity was probably due to the activation by GH of
endogenous STAT5. We could not test this with a promoter in which both
the STAT5 and the HNF-4 sites had been destroyed, as destruction of the
STAT5 site alone abolishes the response to GH (see Fig. 3B).
Overexpression of HNF-4 in the BRL-4 cells amplified 2.4-fold (0.001<
P <0.01) promoter activity in the absence of GH, and this
effect was lost after destruction of the HNF-4 binding site (Fig. 6B).
This GH-independent effect of HNF-4 is consistent with our
demonstration by footprinting and by EMSA (Fig. 4) that, unlike for
STAT5, the binding of HNF-4 to the hnf6 gene promoter does
not depend on GH. In keeping with this interpretation, GH did not
significantly stimulate the promoter after transfection of HNF-4,
presumably because overexpressed HNF-4 saturated its binding site on
the promoter, without need for the GH-induced increase in binding
affinity that takes place with endogenous HNF-4. These results,
together with the binding data, show that HNF-4 participates in, but is
not essential to, the stimulatory action of GH on the
transcription of the hnf6 gene.
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, A and
C, the concentration of HNF-3ß mRNA was increased 3 h after the
GH injection, and it decreased later to values below those in untreated
rats. The time course of induction of HNF-3ß mRNA is compatible with
an HNF-6-mediated effect of GH, since hnf6 gene expression
increases 6-fold within the hour after the injection of GH (see Fig. 1B). In conclusion, our data demonstrate that GH signaling stimulates the expression of the hnf6 gene in liver by a direct action on the hepatocyte. They provide evidence that the effect of GH described here involves 1) GH-dependent binding of STAT5 to the promoter and a GH-induced increase in the affinity of HNF-4 for the promoter, and 2) increased transcription from the hnf6 gene promoter. The mechanism by which GH triggers STAT5 binding to target genes is well documented. In contrast, a GH-dependent increase in the affinity of HNF-4 for a DNA target sequence has not, to our knowledge, been reported before. Consistent with its rapid kinetics, this effect could result from GH-induced tyrosine phosphorylation, e.g. by Jak2, of HNF-4. Indeed, HNF-4 is a tyrosine phosphoprotein, and dephosphorylation of its tyrosine residues in vivo or in vitro decreases its affinity for DNA (18). We cannot exclude the possibility that regions of the hnf6 gene other than the one studied in this paper are involved in the stimulatory effect of GH on hnf6 gene expression seen in vivo. Still, the STAT binding site identified here was absolutely necessary for the transcriptional stimulation of the hnf6 gene promoter by GH. An identical phenomenon has been described for the ß-casein gene, whose transcriptional activation by GH depends on STAT5 binding (19).
The experiments described in this paper show that GH controls the network of hepatocyte transcription factors in three ways. First, GH increases the transcription of the hnf6 gene by the mechanisms discussed above. Second, GH increases the affinity of HNF-4 for DNA. Whether this holds true for HNF-4 target sequences other than the one studied here remains to be established. Third, GH increases the amount of HNF-3ß mRNA. Since HNF-6, HNF-4, and HNF-3ß in turn control the transcription of a number of genes, the latter might in this way be indirectly regulated by GH. Insofar as these three transcription factors are tissue-restricted, they should have a key role in the tissue specificity of the action of GH on gene expression.
Finally, the demonstration that HNF-4 controls the transcription of the
hnf6 gene adds a new loop to the regulatory network of liver
transcription factors. This, together with the fact that HNF-6
stimulates the activity of the hnf4 gene promoter in
transfected cells (8), would predict the existence of a positive
feedback mechanism involving HNF-4 and HNF-6. However, such an
autoregulatory loop must be kept in check in vivo by
negative control mechanisms. These might explain why, in the animal, GH
treatment leads to a spectacular increase in hnf6 gene
expression, but only to a modest increase in hnf4 gene
expression. The sharp drop in liver HNF-6 mRNA concentration after
3 h was not surprising in view of the existence of mechanisms that
rapidly terminate GH-induced STAT5 signaling (20, 21). Experiments on
transfected cells and on embryoid bodies have shown that HNF-3ß can
induce HNF-3, which inhibits the HNF-4 gene both directly, and
indirectly via inhibition of the expression of HNF-1, which is an
inducer of the hnf4 gene (17, 22). In these ways, induction
of HNF-3ß by HNF-6 could eventually turn off the hnf4
gene. The kinetics and cell type specificity of such positive and
negative regulatory loops should be taken into account to fully
understand how GH controls gene expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Cultures and Transfections
For hepatocyte cultures, matrigel was prepared (23) from
Engelbreth-Holm-Swarm sarcoma cells propagated in C57BL/6 female mice.
Hepatocytes were obtained by nonrecirculating collagenase perfusion
through the portal vein of normal male rats anesthetized with
pentobarbital (6 mg/100 g body weight), as described (24). Cells were
seeded at a density of 1.8 x 106 per 60-mm
plates and incubated for 48 h at 37 C in 5%
CO2 in serum-free DMEM/Ham’s F-12 medium
supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml),
hydrocortisone (50 nM), insulin (175 nM),
L-ornithine (0.4 mM), L-lactic acid
(17.7 µM), selenium (25 nM), and ethanolamine
(1 µM). After 48 h of culture, cells were incubated
with rat GH as indicated. Cos-7 cells were cultured and transfected as
described (3). Rat-1 cells, kindly provided by J. Wyke, were grown in
DMEM with 10% FCS. For transient transfection, 3 x
105 cells were plated on 60-mm dishes and
incubated with N-[1-(2,
3-dioleoyloxy)propyl]-N,N,N-triethyl-ammonium methylsulfate
(DOTAP, Roche Molecular Biochemicals, Indianapolis,
IN) and 7 µg of reporter construct, 50 ng to 2 µg of
expression vector, and 1 µg of pRL138 as internal control. After
16 h, the cells were washed with PBS and further incubated for
24 h before luciferase activities were measured with the
Dual-Luciferase kit (Promega Corp., Madison, WI) and a
TD20/20 Luminometer (Promega Corp.). The data were
expressed as the ratio of firefly luciferase (reporter activity) to
Renilla luciferase (internal control).
BRL-4-GHR1-638 cells (15), from a rat hepatoma
cell line stably transfected with the rat GH receptor cDNA and kindly
provided by G. Norstedt, were grown in DMEM with 10% FCS and cultured
to confluence in six-well plates. Transient transfection was performed
in serum-free DMEM with DOTAP according to the manufacturer’s
instructions. One microgram of reporter plasmid and 1 µg of pRL138 as
internal control were transfected per well. After 8 h the medium
was changed to serum-free DMEM with or without 100
nM rat GH, and the cells were further incubated
for 24 h.
Detection of mRNA
RNase protection assays were performed as described (1) with 20
µg of total RNA isolated from individual livers or from cultured
hepatocytes by the guanidine thiocyanate/cesium chloride method (25)
and with an HNF-6 probe (290 b) that allows detection of two
specifically protected fragments (254 and 215 b) originating from
HNF-6 and HNF-6ß mRNA, respectively. A rat GAPDH antisense RNA
probe (Ambion, Inc., Beverly, MA) was cohybridized (4
x 104 cpm) with the HNF-6 probe (300 x
104 cpm) as an internal reference to correct for
variations in RNA concentration. After digestion with RNase and
separation of the protected fragments on a 6% polyacrylamide
denaturing gel, the GAPDH and HNF-6 mRNAs were quantified with a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale,
CA). For Northern blot analysis, total RNA (20 µg) was size
fractionated on a denaturing 1% agarose gel and transferred to nylon
membranes (Hybond-N, Amersham Pharmacia Biotech,
Buckinghamshire, UK) by overnight vacuum blotting (VacuGeneXL blotting
system, Pharmacia LKB, Uppsala, Sweden). After UV
cross-linking (Stratalinker, Stratagene, La Jolla, CA),
the membranes were hybridized to 32P-labeled
probes specific for rat HNF-3ß or HNF-4. HNF-3ß and HNF-4 mRNAs
were detected with random-primed rat cDNA fragments containing
nucleotides 947-2218 (26) and 614-1424 (27), respectively. Each
Northern blot was rehybridized with a 32P-labeled
oligonucleotide specific for 18 S rRNA to correct for variations in RNA
concentration. The relative concentration of mRNA in each lane was
quantified by scanning autoradiograms (8-day exposure at -80 C with
two intensifying screens) with a LKB Ultroscan XL
laser densitometer (Pharmacia Biotech, Uppsala, Sweden).
Results were expressed by assigning a value of 1 arbitrary
densitometric unit to liver mRNA from hypophysectomized rats killed at
time 0. Statistical analysis was performed with an ANOVA test.
DNase I Footprinting and EMSAs
For the DNase I footprint of STAT5, a
BamHI-ClaI fragment of the rat hnf6
gene promoter was labeled on one strand at the BamHI site
(-44) with 32P [-dGTP] and cleaved at the
SacI site (-323). For the DNase I footprint of HNF-4, a
XhoI-StuI fragment of the rat hnf6
gene promoter was labeled on one strand at the XhoI site
(-196) with 32P[-dGTP] and cleaved at the
StuI site (-756). The incubations, which contained 15 µg
of rat liver nuclear protein and the probe (100–150 counts per
second), were carried out as described previously (28) and were
followed by analysis on 6% polyacrylamide-8 M
urea sequencing gels. For EMSA, nuclear extracts were prepared from rat
livers as described (29). The following double-stranded
oligonucleotides were used as probes: PH4,
5'-GCGAACGGGCAAAGGCCATGGCATA-3' (from -657 to -633 of the rat
hnf6 gene promoter); HNF-4,
5'-AAGGCTGAAGTCCAAAGTTCAGTCCCTTC-3' (HNF-4-binding oligonucleotide,
-71 to -43 of the rat hnf1 gene promoter); H6STAT,
5'-GGCAGCAGGATTCTAAGAAAGAGAGGGGC-3' (-126 to -98 of the rat
hnf6 gene promoter); GRR, 5'-ATGTATTTCCCAGAAA-3'
(STAT-binding oligonucleotide from the Fc
RI gene
promoter) (30); Sp1, 5'-ATTCGATCGGGGCGGGGCGAGC-3' (Promega Corp.). They were labeled with
[32P]-ATP (Amersham Pharmacia Biotech) by T4 polynucleotide kinase (United States Biochemical Corp., Cleveland , OH) and EMSAs were performed as
described (5). Antibodies were added to the liver nuclear extracts on
ice 45 min before addition of the labeled probe. Incubation with the
labeled probe was then allowed to proceed for 45 min on ice before
electrophoresis. The anti-STAT3 and anti-STAT5 antibodies were from
Santa Cruz Biotechnology, Inc., (Santa Cruz, CA), and the
anti-HNF-4 antibody was kindly provided by M. Pontoglio.
Immunoblotting
Liver nuclear extracts (20 µg of protein) from
hypophysectomized male rats that had received a single injection of GH
as indicated were loaded on an 8% acrylamide gel. After SDS-PAGE the
proteins were transferred to a polyvinylidene fluoride membrane
(Amersham Pharmacia Biotech) that was incubated overnight
with the anti-HNF-4 antiserum (1:15,000) used for EMSA.
Protein-antibody complexes were visualized using the Enhanced
Chemiluminescence Detection System of Roche Molecular Chemicals
(Indianapolis, IN).
Expression Vectors and Reporter Constructs
The expression vectors STAT5750JAK2,
STAT5750VP16-JAK2, pXM-MGF-STAT5, and
pXM-MGF-STAT5750 were kindly given by B. Groner. The expression
vector HNF-41 was kindly given by B. Laine. The reporter construct
pNF/0.75 luc has been described previously (4). pRL138, used as an
internal control, contains the pfk2 gene promoter (-138 to +86) cloned
in pRLnull (Promega Corp.). The pNF/0.75 (HNF-4 mut)luc
was made by PCR amplification with two sets of primers (the
mutated oligonucleotides are underlined): PH6IIIS,
5'-TTGTGAGGGTCATGGATACCAGTTCTA-3' (-803 to -777 of the rat
hnf6 gene promoter, sense strand) and GAH4NAS,
5'-AAAAGTACTCCGCCATTGGGCTTTATTCCC-TGG-3' (the 3'-end
corresponds to -678 of the rat hnfh6 gene promoter,
antisense strand), GAH4S,
5'-AAAAGTACTGTCCTCCGATGGCATAGTCTCCAGCTCC-3' (the 3'-end
corresponds to -621 of the rat hnf6 gene promoter, sense
strand) and SacAS, 5'-CCGCTGCCCACCCTCACGCCC-3' (-273 to - 253
of the rat hnf6 gene promoter, antisense strand). The first
fragment was digested with ScaI and
StuI, while the second fragment was digested with
SacI and ScaI. The digested fragments were gel
purified and ligated with pNF/0.75 luc opened at the StuI
and SacI sites. In this way the HNF-4 site was replaced with
a GAL-4 binding site. To prepare the pNF/0.75 (STATmut)luc construct,
two PCR reactions were carried out. The first PCR was done with the
primers H6STATM,
5'-CTCGCCCCTCTCTTGAATTCAATCCTGCTGCCCCC-3'
(-129 to -95 of the rat hnf6 gene promoter, antisense
strand) and HNF-6–3'Sac, 5'-CTACCGAATCTCAGCCACAG-3' (-240 to -221 of
the rat hnf6 gene promoter, sense strand). In the second PCR
the amplified fragment from the first PCR was used as a primer with GL
Primer 2, 5'-CTTTATGTTTTTGGCGTCTTCC-3' (standard primer of pGL3 basic
vector of Promega Corp.). The product of this PCR was
digested with XhoI and cloned in the XhoI site of
pNF/0.75 luc.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants from the Belgian State Program on Interuniversity Poles of Attraction, Prime Minister’s Office, Federal Office for Scientific, Technical and Cultural Affairs; from the Délégation Générale Higher Education and Scientific Medical Research, French Community of Belgium; from the Fund for Scientific Medical Research (Belgium); from the National Fund for Scientific Research (Belgium); from the Fonds de Développement Scientifique (Louvain University); and from the Danone Institute (Belgium). J.-P.T is Research Associate and F.P.L. is Senior Research Associate of the National Fund for Scientific Research (Belgium).
1 Present address: Unité INSERM 135, Hôpital
de Bicêtre, 78 Rue du Général Leclerc, 94275 Le
Kremlin Bicêtre, France. 
2 Both authors have contributed equally to this work.
Received for publication July 21, 1999. Accepted for publication November 16, 1999.
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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M. G. Holloway, G. D. Miles, A. A. Dombkowski, and D. J. Waxman Liver-Specific Hepatocyte Nuclear Factor-4{alpha} Deficiency: Greater Impact on Gene Expression in Male than in Female Mouse Liver Mol. Endocrinol., May 1, 2008; 22(5): 1274 - 1286. [Abstract] [Full Text] [PDF]
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D. J. DiGirolamo, A. Mukherjee, K. Fulzele, Y. Gan, X. Cao, S. J. Frank, and T. L. Clemens Mode of Growth Hormone Action in Osteoblasts J. Biol. Chem., October 26, 2007; 282(43): 31666 - 31674. [Abstract] [Full Text] [PDF]
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E. V. Laz, M. G. Holloway, C.-S. Chen, and D. J. Waxman Characterization of Three Growth Hormone-Responsive Transcription Factors Preferentially Expressed in Adult Female Liver Endocrinology, July 1, 2007; 148(7): 3327 - 3337. [Abstract] [Full Text] [PDF]
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H. Jiang, Y. Wang, M. Wu, Z. Gu, S. J. Frank, and R. Torres-Diaz Growth Hormone Stimulates Hepatic Expression of Bovine Growth Hormone Receptor Messenger Ribonucleic Acid through Signal Transducer and Activator of Transcription 5 Activation of a Major Growth Hormone Receptor Gene Promoter Endocrinology, July 1, 2007; 148(7): 3307 - 3315. [Abstract] [Full Text] [PDF]
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O. M. Vidal, R. Merino, E. Rico-Bautista, L. Fernandez-Perez, D. J. Chia, J. Woelfle, M. Ono, B. Lenhard, G. Norstedt, P. Rotwein, et al. In Vivo Transcript Profiling and Phylogenetic Analysis Identifies Suppressor of Cytokine Signaling 2 as a Direct Signal Transducer and Activator of Transcription 5b Target in Liver Mol. Endocrinol., January 1, 2007; 21(1): 293 - 311. [Abstract] [Full Text] [PDF]
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D. J. Waxman and C. O'Connor Growth Hormone Regulation of Sex-Dependent Liver Gene Expression Mol. Endocrinol., November 1, 2006; 20(11): 2613 - 2629. [Abstract] [Full Text] [PDF]
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M. G. Holloway, E. V. Laz, and D. J. Waxman Codependence of Growth Hormone-Responsive, Sexually Dimorphic Hepatic Gene Expression on Signal Transducer and Activator of Transcription 5b and Hepatic Nuclear Factor 4{alpha} Mol. Endocrinol., March 1, 2006; 20(3): 647 - 660. [Abstract] [Full Text] [PDF]
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D. J. Chia, M. Ono, J. Woelfle, M. Schlesinger-Massart, H. Jiang, and P. Rotwein Characterization of Distinct Stat5b Binding Sites That Mediate Growth Hormone-stimulated IGF-I Gene Transcription J. Biol. Chem., February 10, 2006; 281(6): 3190 - 3197. [Abstract] [Full Text] [PDF]
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 A. V. Poll, C. E. Pierreux, L. Lokmane, C. Haumaitre, Y. Achouri, P. Jacquemin, G. G. Rousseau, S. Cereghini, and F. P. Lemaigre A vHNF1/TCF2-HNF6 Cascade Regulates the Transcription Factor Network That Controls Generation of Pancreatic Precursor Cells Diabetes, January 1, 2006; 55(1): 61 - 69. [Abstract] [Full Text] [PDF]
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 J. Xu, S. Ji, D. Y Venable, J. L Franklin, and J. L Messina Prolonged insulin treatment inhibits GH signaling via STAT3 and STAT1 J. Endocrinol., March 1, 2005; 184(3): 481 - 492. [Abstract] [Full Text] [PDF]
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C. A. Wiwi and D. J. Waxman Role of Hepatocyte Nuclear Factors in Transcriptional Regulation of Male-specific CYP2A2 J. Biol. Chem., February 4, 2005; 280(5): 3259 - 3268. [Abstract] [Full Text] [PDF]
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 J. E. Rowland, A. M. Lichanska, L. M. Kerr, M. White, E. M. d'Aniello, S. L. Maher, R. Brown, R. D. Teasdale, P. G. Noakes, and M. J. Waters In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts Mol. Cell. Biol., January 1, 2005; 25(1): 66 - 77. [Abstract] [Full Text] [PDF]
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S Eleswarapu and H Jiang Growth hormone regulates the expression of hepatocyte nuclear factor-3 gamma and other liver-enriched transcription factors in the bovine liver J. Endocrinol., January 1, 2005; 184(1): 95 - 105. [Abstract] [Full Text] [PDF]
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 K.-C. Leung, G. Johannsson, G. M. Leong, and K. K. Y. Ho Estrogen Regulation of Growth Hormone Action Endocr. Rev., October 1, 2004; 25(5): 693 - 721. [Abstract] [Full Text] [PDF]
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C. A. Wiwi, M. Gupte, and D. J. Waxman Sexually Dimorphic P450 Gene Expression in Liver-Specific Hepatocyte Nuclear Factor 4{alpha}-Deficient Mice Mol. Endocrinol., August 1, 2004; 18(8): 1975 - 1987. [Abstract] [Full Text] [PDF]
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 D. J. Waxman and J. L. Celenza Sexual dimorphism of hepatic gene expression: novel biological role of KRAB zinc finger repressors revealed Genes & Dev., November 1, 2003; 17(21): 2607 - 2613. [Full Text] [PDF]
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 F. Clotman, V. J. Lannoy, M. Reber, S. Cereghini, D. Cassiman, P. Jacquemin, T. Roskams, G. G. Rousseau, and F. P. Lemaigre The onecut transcription factor HNF6 is required for normal development of the biliary tract Development, March 6, 2003; 129(8): 1819 - 1828. [Abstract] [Full Text] [PDF]
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Y. E. Timsit and D. S. Riddick Stimulation of Hepatic Signal Transducer and Activator of Transcription 5b by GH Is Not Altered by 3-Methylcholanthrene Endocrinology, September 1, 2002; 143(9): 3284 - 3294. [Abstract] [Full Text] [PDF]
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H. Helander, J.-A. Gustafsson, and A. Mode Possible Involvement of Truncated Signal Transducer and Activator of Transcription-5 in the GH Pattern-Dependent Regulation of CYP2C12 Gene Expression in Rat Liver Mol. Endocrinol., July 1, 2002; 16(7): 1598 - 1611. [Abstract] [Full Text] [PDF]
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 J. E. Dumont, S. Dremier, I. Pirson, and C. Maenhaut Cross signaling, cell specificity, and physiology Am J Physiol Cell Physiol, July 1, 2002; 283(1): C2 - C28. [Abstract] [Full Text] [PDF]
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 H. Schrem, J. Klempnauer, and J. Borlak Liver-Enriched Transcription Factors in Liver Function and Development. Part I: The Hepatocyte Nuclear Factor Network and Liver-Specific Gene Expression Pharmacol. Rev., March 1, 2002; 54(1): 129 - 158. [Abstract] [Full Text] [PDF]
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S.-H. Park and D. J. Waxman Inhibitory Cross-talk between STAT5b and Liver Nuclear Factor HNF3beta . IMPACT ON THE REGULATION OF GROWTH HORMONE PULSE-STIMULATED, MALE-SPECIFIC LIVER CYTOCHROME P-450 GENE EXPRESSION J. Biol. Chem., November 9, 2001; 276(46): 43031 - 43039. [Abstract] [Full Text] [PDF]
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 A. Bailly, M. E. Torres-Padilla, A. P. Tinel, and M. C. Weiss An enhancer element 6 kb upstream of the mouse HNF4{alpha}1 promoter is activated by glucocorticoids and liver-enriched transcription factors Nucleic Acids Res., September 1, 2001; 29(17): 3495 - 3505. [Abstract] [Full Text] [PDF]
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J. Eeckhoute, P. Formstecher, and B. Laine Maturity-Onset Diabetes of the Young Type 1 (MODY1)-Associated Mutations R154X and E276Q in Hepatocyte Nuclear Factor 4{{alpha}} (HNF4{{alpha}}) Gene Impair Recruitment of p300, a Key Transcriptional Coactivator Mol. Endocrinol., July 1, 2001; 15(7): 1200 - 1210. [Abstract] [Full Text] [PDF]
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H. K. Choi and D. J. Waxman Plasma Growth Hormone Pulse Activation of Hepatic JAK-STAT5 Signaling: Developmental Regulation and Role in Male-Specific Liver Gene Expression Endocrinology, September 1, 2000; 141(9): 3245 - 3255. [Abstract] [Full Text] [PDF]
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M. Rastegar, G. G. Rousseau, and F. P. Lemaigre CCAAT/Enhancer-Binding Protein-{alpha} Is a Component of the Growth Hormone-Regulated Network of Liver Transcription Factors Endocrinology, May 1, 2000; 141(5): 1686 - 1692. [Abstract] [Full Text] [PDF]
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V. J. Lannoy, A. Rodolosse, C. E. Pierreux, G. G. Rousseau, and F. P. Lemaigre Transcriptional Stimulation by Hepatocyte Nuclear Factor-6. TARGET-SPECIFIC RECRUITMENT OF EITHER CREB-BINDING PROTEIN (CBP) or p300/CBP-ASSOCIATED FACTOR (p/CAF) J. Biol. Chem., July 14, 2000; 275(29): 22098 - 22103. [Abstract] [Full Text] [PDF]
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G. B. Ehret, P. Reichenbach, U. Schindler, C. M. Horvath, S. Fritz, M. Nabholz, and P. Bucher DNA Binding Specificity of Different STAT Proteins. COMPARISON OF IN VITRO SPECIFICITY WITH NATURAL TARGET SITES J. Biol. Chem., February 23, 2001; 276(9): 6675 - 6688. [Abstract] [Full Text] [PDF]
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N. Delesque-Touchard, S.-H. Park, and D. J. Waxman Synergistic Action of Hepatocyte Nuclear Factors 3 and 6 on CYP2C12 Gene Expression and Suppression by Growth Hormone-activated STAT5b. PROPOSED MODEL FOR FEMALE-SPECIFIC EXPRESSION OF CYP2C12 IN ADULT RAT LIVER J. Biol. Chem., October 27, 2000; 275(44): 34173 - 34182. [Abstract] [Full Text] [PDF]
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R. S. Streeper, L. A. Hornbuckle, C. A. Svitek, J. K. Goldman, J. K. Oeser, and R. M. O'Brien Protein Kinase A Phosphorylates Hepatocyte Nuclear Factor-6 and Stimulates Glucose-6-phosphatase Catalytic Subunit Gene Transcription J. Biol. Chem., May 25, 2001; 276(22): 19111 - 19118. [Abstract] [Full Text] [PDF]
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