Involvement of STAT5 (Signal Transducer and Activator of Transcription 5) and HNF-4 (Hepatocyte Nuclear Factor 4) in the Transcriptional Control of the hnf6 Gene by Growth Hormone

Olivier Lahuna1,2, Mojgan Rastegar2, Dominique Maiter, Jean-Paul Thissen, Frédéric P. Lemaigre and Guy G. Rousseau

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HNF-6 is a tissue-restricted transcription factor that participates in the regulation of several genes in liver. We reported earlier that in adult rats, HNF-6 mRNA concentration in liver drops to almost undetectable levels after hypophysectomy and returns to normal after 1 week of GH treatment. We now show that this results from a rapid effect of GH, and we characterize its molecular mechanism. In hypophysectomized rats, HNF-6 mRNAs increased within 1 h after a single injection of GH. The same GH-dependent induction was reproduced on isolated hepatocytes. To determine whether GH regulates hnf6 expression at the gene level, we studied its promoter. DNA binding experiments showed that 1) the transcription factors STAT5 (signal transducer and activator of transcription 5) and HNF-4 (hepatocyte nuclear factor 4) bind to sites located around -110 and -650, respectively; and 2) STAT5 binding is induced and HNF-4 binding affinity is increased in liver within 1 h after GH injection to hypophysectomized rats. Using transfection experiments and site-directed mutagenesis, we found that STAT5 and HNF-4 stimulated transcription of an hnf6 gene promoter-reporter construct. Furthermore, GH stimulated transcription of this construct in cells that express GH receptors. Consistent with our earlier finding that HNF-6 stimulates the hnf4 and hnf3ß gene promoters, GH treatment of hypophysectomized rats increased the liver concentration of HNF-4 and HNF-3ß mRNAs. Together, these data demonstrate that GH stimulates transcription of the hnf6 gene by a mechanism involving STAT5 and HNF-4. They show that HNF-6 participates not only as an effector, but also as a target, to the regulatory network of liver transcription factors, and that several members of this network are GH regulated.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hepatocyte nuclear factor (HNF)-6 is the prototype of a new class of cut-homeoproteins conserved from Caenorhabditis elegans to humans (1, 2, 3). These proteins are transcription factors that contain a single cut domain and a divergent homeodomain. Two HNF-6 isoforms, {alpha} (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{alpha} 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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH Induces Liver HNF-6 mRNAs by a Direct Effect on Hepatocytes
In rat liver, HNF-6 mRNAs almost disappear after hypophysectomy. When these rats are treated with GH and their liver RNA is analyzed after 1 week of continuous treatment, HNF-6 mRNAs have returned to normal levels (5). We therefore determined the time-course of liver HNF-6 mRNAs induction by GH in vivo. Total RNA was extracted from the liver of hypophysectomized rats before, or at several time intervals after, a single injection of GH. HNF-6 mRNAs were quantified by a ribonuclease (RNase) protection assay using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as a reference. In untreated hypophysectomized animals, HNF-6{alpha} mRNA was barely detectable and HNF-6ß mRNA was below the threshold of sensitivity of the assay (Fig. 1AGo), consistent with the fact that the concentration of HNF-6ß mRNA in the liver of intact rats is lower than that of HNF-6{alpha} mRNA (1). Within 1 h after the injection of GH, the concentrations of HNF-6{alpha} 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. 1Go, A and B).



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Figure 1. Effect of GH on HNF-6 mRNAs Concentration in Rat Liver and in Isolated Hepatocytes

A, RNase protection assays were performed on RNA from the liver of hypophysectomized rats, before (0 h) or after (at the times indicated) a single injection of GH, with the HNF-6 and GAPDH riboprobes and size markers (first three lanes). The other lanes each correspond to a sample from a different rat (four animals per experimental condition). B, Quantitation of relative HNF-6{alpha} mRNA concentration determined in the experiments described in panel A. **, P < 0.01 and ***, P < 0.001 vs. the untreated (0 h) group. C, RNase protection assays were performed (see panel A) on RNA from hepatocytes (four 60-mm plates) incubated with GH (500 ng/ml) for the times indicated. RNA from the liver of an intact male rat was included as a positive control (lane 1). D, Relative concentration of HNF-6{alpha} mRNA determined by RNase protection assay (see panel C) in hepatocytes incubated for 24 h with different concentrations of GH. Data are means ± SEM for three separate experiments.

 
We next investigated whether the GH-dependent regulation of liver HNF-6 mRNAs observed in vivo is a direct effect of the hormone on hepatocytes. To do so, hepatocytes were isolated from intact rats and cultured on a matrix to maintain the expression of liver-specific functions (10). After 48 h, GH (500 ng/ml) was added and the cells were harvested at different time points for quantifying HNF-6 mRNAs as above. As shown in Fig. 1CGo, HNF-6{alpha} 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. 1DGo), 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{alpha} and HNF-6ß mRNA (Fig. 1Go), 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. 1AGo). 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. 2AGo). 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. 2AGo). 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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Figure 2. GH Induction of STAT5 Binding to the hnf6 Gene Promoter

A, DNase I footprinting on the rat hnf6 gene with liver nuclear extracts obtained from hypophysectomized rats at the times indicated after a single GH injection and incubated without or with 50 ng of a STAT-binding oligonucleotide (GRR). B, EMSA with the labeled fragment from -98 to -126 of the hnf6 gene promoter as a probe (H6STAT) without or with 50 ng of the competing cold oligonucleotides or 1.5 µl of the antisera indicated. The nuclear extracts were the same as in panel A.

 
To confirm that the protein involved in the GH-induced footprint was a member of the STAT family, a labeled oligonucleotide corresponding to the footprinted region and containing the hnf6 gene fragment -126 to -98 was used as a probe in electrophoretic mobility shift assay (EMSA) with the nuclear extracts that were used in the footprinting experiments. As shown in Fig. 2BGo, 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{Delta}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. 3AGo, 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 (STAT5{Delta}750Jak2), 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. 3AGo).



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Figure 3. Stimulation of the hnf6 Gene Promoter by STAT5 and by GH

Rat-1 cells (A) or BRL-4 cells (B) were cotransfected transiently with the wild-type or mutated hnf6 promoter-reporter constructs and with vectors for wild-type (pXM-MGF-STAT5), or for constitutively active (STAT5{Delta}750VP16JAK2) or inactive (STAT5{Delta}750JAK2 and pXM-MGF-STAT5{Delta}750) STAT5. In panel B the cells were exposed for 24 h to 100 nM rat GH (solid bars). In the absence of expression vector, the range of firefly and Renilla luciferase activities (see Materials and Methods) was 30- to 100-fold (A) and 100- to 8000-fold (B) the background values. Data are means ± SD for at least three experiments.

 
Since GH induces STAT5 binding to the hnf6 gene promoter and STAT5 stimulates the expression of a reporter gene linked to this promoter, then GH should stimulate this reporter construct in transfection experiments. To verify this prediction, we used BRL-4 hepatoma cells stably transfected with the rat GH receptor (15). When these cells were transiently transfected with the hnf6 promoter-reporter construct, luciferase activity increased 1.8-fold (0.001 < P < 0.01) after exposure to rat GH (Fig. 3BGo). 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. 3BGo).

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. 3BGo, 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. 3BGo). 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. 4AGo). 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. 4BGo) 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. 4AGo, lanes 2 and 3), HNF-4 binding to the hnf-6 promoter did not depend on GH (Fig. 4BGo, 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. 4BGo, lanes 3–7).



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Figure 4. Binding of the HNF-4 Protein to the hnf6 Gene Promoter and Effect of GH

A, DNase I footprinting on the rat hnf6 gene with liver nuclear extracts obtained from hypophysectomized rats before or 3 h after a single GH injection. B, EMSA with the labeled fragment from -633 to -657 of the hnf6 gene promoter as a probe (PH4) without or with 50 ng of the competing cold oligonucleotides or 1 µl of a 1:5 dilution of the antiserum indicated. The nuclear extracts were the same as for the experiments shown in Fig. 2Go. C, Immunoblot, with an anti-HNF-4 antibody, of the extracts used in panel B.

 
In earlier studies we showed that GH treatment of hypophysectomized rats increases the concentration of HNF-6 protein in liver (5) and that HNF-6 binds to, and stimulates the activity of, the hnf4 gene promoter in transiently transfected cells (8). Thus, it was likely that GH increases the expression of the hnf4 gene. To test this, we measured the concentration of HNF-4 mRNA in rat liver after a single injection of GH to hypophysectomized animals. The data in Fig. 5Go, 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. 4CGo, 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. 5BGo) is consistent with the uninduced levels of HNF-4 protein seen by immunoblotting after 12 and 24 h (Fig. 4CGo). 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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Figure 5. Effect of GH on HNF-4 and HNF-3ß mRNA Concentration in Rat Liver

A, Northern blotting of the liver RNA samples obtained as described in Fig. 1AGo. The two lanes per timepoint correspond to samples from different rats. B and C, Relative concentration of HNF-4 mRNA (panel B) and HNF-3ß mRNA (panel C) determined by Northern blotting. Values obtained by densitometry were corrected for variations in RNA loading with reference to the 18 S rRNA values. Data shown in panels B and C are means ± SEM for four rats. *, P < 0.05 and **, P < 0.01 vs. the untreated (0 h) group.

 
We therefore determined by transfection the functionality of this HNF-4 binding site in the context of the intact hnf6 gene promoter. As shown in Fig. 6AGo, 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. 6AGo). These experiments were repeated with BRL-4 cells to evaluate the effect of GH under these conditions (Fig. 6BGo). 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. 3BGo). 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. 6BGo). This GH-independent effect of HNF-4 is consistent with our demonstration by footprinting and by EMSA (Fig. 4Go) 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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Figure 6. Stimulation of the hnf6 Gene Promoter by HNF-4

Rat-1 cells (A) or BRL-4 cells (B) were cotransfected transiently with the wild-type or mutated hnf6 promoter-reporter constructs and with an HNF-4 expression vector, as indicated. See legend of Fig. 3AGo for other details. Data are means ± SD for at least three experiments. In panel B the cells were exposed for 24 h to 100 nM rat GH (solid bars).

 
Implications for the Control of the Hepatic Network of Liver Transcription Factors
Liver transcription factors are organized in a network that involves reciprocal as well as autoregulatory positive and negative feedback loops (9, 17). We had shown earlier that HNF-6 belongs to this network and that it stimulates transcription of the hnf3ß gene in transfected cells (1, 8). The data presented here demonstrate that GH stimulates transcription of the hnf6 gene. In this way, GH could control the expression of the hnf3ß gene in vivo. To investigate this possibility, we measured the concentration of HNF-3ß mRNA in the liver of hypophysectomized rats treated or not with a single injection of GH. As shown in Fig. 5Go, 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. 1BGo).

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{alpha}, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Four-week-old male Wistar rats obtained 7 days after hypophysectomy (IFFA-Credo, Lyon, France) were maintained under standardized conditions of light and temperature with free access to rat chow and to water containing 0.9% (wt/vol) NaCl. Hypophysectomized rats were pretreated during 7 days with a daily subcutaneous injection of L-T4 (Aldrich, Milwaukee, WI) (1 µg/100 g body weight) and cortisol hemisuccinate (Pharmacia & Upjohn, Inc., Kalamazoo, MI) (50 µg/100 g body weight). On day 8, groups of hypophysectomized rats (four per group) were killed before (0 h) or after (at the time indicated) a single subcutaneous injection of purified rat GH (NIDDK rat pituitary hormone distribution program) (100 µg/100 g body weight). These experiments were conducted in accordance with the highest standards of humane animal care.

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{alpha} 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 [{alpha}-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[{alpha}-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{gamma}RI gene promoter) (30); Sp1, 5'-ATTCGATCGGGGCGGGGCGAGC-3' (Promega Corp.). They were labeled with [{gamma}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 STAT5{Delta}750JAK2, STAT5{Delta}750VP16-JAK2, pXM-MGF-STAT5, and pXM-MGF-STAT5{Delta}750 were kindly given by B. Groner. The expression vector HNF-4{alpha}1 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.


    ACKNOWLEDGMENTS
 
We would like to thank P. Lause and M. A. Gueuning for their excellent technical assistance, P. Vandoolaeghe for help with preparation of nuclear extracts, T. Lambert and V. O’ Connor for secretarial work, and all the colleagues mentioned in the text for their generous gifts.


    FOOTNOTES
 
Address requests for reprints to: Guy G. Rousseau, HORM 7529, 75 Avenue Hippocrate, B-1200 Brussels, Belgium.

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. Back

2 Both authors have contributed equally to this work. Back

Received for publication July 21, 1999. Accepted for publication November 16, 1999.


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 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
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