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Endocrinology Vol. 139, No. 3 859-866
Copyright © 1998 by The Endocrine Society

ARTICLES

Growth Hormone Stimulates Interferon Regulatory Factor-1 Gene Expression in the Liver1

Catherine Le Stunff2 and Peter Rotwein

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Peter Rotwein, Molecular Medicine Division, Department of Medicine, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, NRC 514, Portland, Oregon 97201-3098. E-mail: rotweinp{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interferon regulatory factor-1 (IRF-1) is a transcription factor first identified as part of the nuclear response to interferons. IRF-1 has been shown to be activated by many cytokines, including PRL, and has been thought to play a role in PRL-regulated gene expression in several experimental systems, including the Nb2 T lymphoma cell line, where it was first characterized as a PRL-responsive gene. We now find that IRF-1 gene expression is rapidly activated in vivo by both PRL and GH treatment. A single ip injection of rat PRL to hypophysectomized female rats caused a transient increase in nascent hepatic nuclear IRF-1 RNA within 15 min of hormone treatment. The rise in IRF-1 transcripts was accompanied by induction of nuclear protein binding to a DNA element from the proximal IRF-1 promoter, as assessed by gel mobility shift assays; this element was shown previously to mediate PRL-activated gene transcription. GH treatment stimulated a greater and more sustained increase in nascent IRF-1 RNA than PRL, leading to accumulation of IRF-1 transcripts for up to 16 h after a single hormone injection. GH also caused a pronounced induction of hepatic nuclear protein binding to the IRF-1 promoter element. Supershift experiments with specific antibodies showed that signal transducer and activator of transcription 1 (STAT1) and to a lesser extent STAT3 were components of the GH-activated protein-DNA complexes. By contrast, these two STATs were not induced in the liver by PRL. Protein binding to the IRF-1 DNA element and IRF-1 gene activation by GH were not blunted by pretreatment with the protein synthesis inhibitor, cycloheximide, indicating that these hormonal effects are primary consequences of GH-activated signal transduction pathways. Our results identify another component of the rapid nuclear response to GH, and support the idea that multiple primary and secondary signaling pathways contribute to the acute actions of GH on gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH and PRL are structurally similar pituitary hormones with diverse biological effects. GH plays a major role in stimulating somatic growth and modulating intermediary metabolism in mammals and other vertebrate species (1, 2), whereas PRL regulates lactation, reproduction, and the immune response (3, 4). Target cells for both hormones are widely distributed (5). GH and PRL receptors also are related to one another (1, 3), and both are members of the cytokine-hematopoietin superfamily, which includes receptors for several hematopoietic growth and differentiation factors and interleukins (1, 6).

Recent studies have demonstrated a remarkable congruence in the earliest actions of GH and PRL. Both hormones induce dimerization of their respective receptors (1, 7), and cause activation of the intracellular tyrosine protein kinase, Janus kinase 2 (JAK2) (8, 9). JAK2 then phosphorylates other intracellular proteins that collectively transduce hormone-activated biological responses. These shared signaling intermediates include adaptor proteins such as insulin receptor substrate-1 (10, 11, 12, 13); enzymes, including phosphatidyl inositol-3 kinase and mitogen-activated protein kinases (11, 12, 13, 14, 15, 16); and transcription factors, principally signal transducers and activators of transcription (STATS) (17, 18, 19, 20, 21, 22, 23, 24, 25, 26), a family of proteins that function as a rapid relay system linking cell-surface receptors to DNA response elements on nuclear target genes (27, 28).

Our laboratory has focused on the early events in GH and PRL action in physiologically relevant settings (19, 20, 29, 30, 31, 32, 33). In previous studies, we found that a single systemic injection of rat GH (rGH) or rPRL to hypophysectomized female rats stimulated rapid changes in nuclear protein phosphorylation and gene expression in the liver (33). Treatment with each hormone acutely enhanced tyrosine phosphorylation of STAT5, induced nuclear protein binding to the GH-responsive DNA element of the serine protease inhibitor (Spi) 2.1 promoter, and activated Spi 2.1 gene transcription (33). Although rGH was substantially more potent than rPRL in stimulating these initial biological effects, and additionally activated STAT1 and STAT3 (33), our results indicated that PRL treatment could rapidly induce a series of biological responses in the liver that were a subset of those stimulated by GH. Because rGH and rPRL bind principally to their cognate receptors in rodent tissues (34), these observations suggested that in the liver the ligand-bound PRL receptor regulated a set of signaling intermediates that were identical to some of those used by the GH receptor.

The transcription factor, interferon regulatory factor-1 (IRF-1), initially identified by its induction after treatment of cells with various interferons (35), also was cloned as a PRL-activated gene from the rat T lymphoma cell line, Nb2 (36). IRF-1 gene transcription is stimulated by PRL in Nb2 cells by mechanisms that involve binding of activated STATS to a DNA element in the proximal IRF-1 promoter (37) that also has been identified as an interferon {gamma} responsive region, or gamma-activated sequence (GAS) (38). In the current studies, we show that IRF-1 gene expression is rapidly induced in the liver after in vivo rPRL treatment, and additionally find that rGH is an even more potent activator of this gene. GH-mediated induction of IRF-1 gene expression is rapid and is sustained for up to 16 h after a single hormone injection. By contrast, PRL-stimulated IRF-1 gene activation is transient. The diminished effects of PRL on IRF-1 gene activation potentially may be attributed to tissue-specific differences in signal transduction between these two related hormone-receptor systems.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Affinity-purified rGH and rPRL were obtained from the National Hormone and Pituitary Program, NIDDK, NIH (rGH, AFP-3699A; rPRL, AFP-3697A). Recombinant human GH (hGH) was obtained from Genentech (South San Francisco, CA). Radionuclides, [{alpha}-32P]deoxycytidine-ATP and [{alpha}-32P]cytidine 5'-triphosphate (CTP), were purchased from Dupont-New England Nuclear (Boston, MA). Monoclonal antibodies to human STAT1 (N-terminus), sheep STAT5 (SH2-SH3 domains), and human STAT6 (N-terminus) were obtained from Transduction Labs. (Lexington, KY). According to the supplier, these antibodies all react with the homologous rat proteins. Polyclonal antihuman C-terminal STAT3 was purchased from Upstate Biotechnology (Lake Placid, NY). This antibody cross-reacts with mouse STAT3. Antirabbit myogenin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cycloheximide (CHX) and antimouse and antirabbit IgG coupled to horseradish peroxidase were purchased from Sigma Chemical Co. (St. Louis, MO). Polyvinylidene fluoride membranes were obtained from Millipore (Bedford, MA). An enhanced chemiluminescence detection system was purchased from Amersham (Arlington Heights, IL). The oligonucleotides listed in Table 1Go were synthesized at the Washington University Protein and Nucleic Acids Chemistry Facility. A recombinant plasmid containing exon 1 of the rat IRF-1 gene and adjacent 5' flanking DNA was obtained from Dr. L. Y. Yu-Lee (Baylor College of Medicine, Houston, TX).


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  		Table 1. Self-annealing oligonucletides used in gel mobility shift experiments

 
Animal studies
Female and male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) were hypophysectomized (hypox) at 10 and 7 weeks of age, respectively, by the supplier. Completeness of anterior pituitary ablation was confirmed by lack of weight gain over the next 2 weeks; the mean weight (±SD) of female rats on arrival was 184.6 ± 1.2 g, and at the time of death was 185.6 ± 1.1 g; the mean weight of male rats was 165 ± 5 g on arrival and 158 ± 6 g at the time of death. Animals were maintained at the Washington University Animal Care Facility on a 12-h light/12-h dark schedule with free access to food and water supplemented with 5% sucrose. Female rats (n = 4/time point) were given a single ip injection of 1.5 µg/g rGH, rPRL, or saline at 15, 30, or 60 min before being killed. One group of male rats (n = 4/time point) received an ip injection of CHX (4 mg/kg) or vehicle, followed 30 min later by one ip injection of recombinant hGH (1.5 µg/g) or vehicle, as previously described (29). Another group of male rats (n = 5/time point) received a single hGH injection at 0.5, 1, 2, 4, 8, or 16 h before being killed (39). Rats were anesthetized by an ip injection of pentobarbital (85 µg/g) before death. Livers were excised, weighed, and placed on ice, and portal veins were flushed with cold PBS containing 1 mM Na3VO4. The livers were dissected free of diaphragmatic remnants and extrahepatic tissues. All animal protocols were approved by the Washington University Animal Welfare Committee.

Nuclear protein extraction
Nuclear isolation and protein extraction were performed as described by us previously (19, 32, 39). Livers were minced and suspended in buffer containing 10 mM HEPES (pH 7.6), 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 mM sucrose, 10% glycerol, 1 mM dithiothreitol (DTT), 20 mM sodium fluoride, 0.4 µM microcystin, 1 µg/ml leupeptin, and 0.5 mM phenylmethylsulfonylfluoride. Nuclei were pelleted by ultracentrifugation at 24,000 rpm in a Beckman (Palo Alto, CA) SW 28 rotor at 4 C for 45 min through a cushion of 2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, and 10 mM Tris-Cl, pH 8.0. The nuclei then were resuspended in buffer containing 100 mM KCl, 10 mM HEPES (pH 7.6), 3 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 10 mM sodium orthovanadate, and 10 mM sodium fluoride, and soluble proteins were extracted by the slow addition of KCl to 400 mM, followed by ultracentrifugation in a Beckman SW 50.1 rotor at 40,000 rpm for 45 min at 4 C. The supernatant was dialyzed for 2 h against two changes of buffer (500 ml each) containing 25 mM HEPES (pH 7.6), 40 mM KCl, 0.1 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride, 10% glycerol, 1 mM DTT, and 1 mM sodium orthovanadate. Protein samples were aliquoted and frozen immediately in liquid nitogen. Protein concentration was quantitated by a Coomassie blue protein assay (Bio-Rad Lab., Richmond, CA).

RNA isolation and analysis
Whole liver and hepatic nuclear RNA were purified by extraction with guanidinium thiocyanate and guanidine hydrochloride as previously described (39). RNA concentrations were determined by spectrophotometry at 260 nm, and the quality was assessed by agarose gel electrophoresis with ethidium bromide staining. Solution-hybridization ribonuclease protection experiments were performed as described by us previously (32, 39), using a [{alpha}-32P]CTP-labeled antisense IRF-1 specific riboprobe derived from exon 1 of the rat IRF-1 gene and its adjacent 5' region. Protected RNA fragments were separated by electrophoresis through 6% polyacrylamide-8.3 M urea gels. Dried gels were exposed to x-ray film at -80 C with intensifying screens, and results were quantitated with a Betascope 603 ß-counter (Betagen, Waltham, MA).

Gel mobility shift assay
Labeled double-stranded probes were prepared by annealing complementary single-stranded oligonucleotides and filling in the overhanging ends using [{alpha}-32P]dATP, unlabeled deoxy-CTP, dGTP, and TTP, and the Klenow fragment of Escherichia coli DNA polymerase I. Unlabeled probes for competition assays were prepared similarly, using unlabeled deoxynucleotide triphosphates. Nuclear protein extracts (5–10 µg) were preincubated for 15 min at 4 C in a 20-µl reaction in binding buffer (50 mM NaCl, 50 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 4 mM spermidine, 17.5% glycerol, 10 mM HEPES, 2% BSA) and 2 µg poly(deoxyinosine-deoxycytosine) phosphates, with or without unlabeled specific or nonspecific competitor DNAs or antibodies. Labeled probe was then added, and the incubation was continued for 30 min at 4 C. All samples were resolved on 5% nondenaturing polyacrylamide gels in 0.5x TBE (45 mM Tris, 44 mM boric acid, 2.5 mM EDTA) after preelectrophoresis for 1 h at 4 C at 10 V/cm. Electrophoresis proceeded under identical conditions for 5 h. Autoradiographs of the dried gels were exposed to x-ray film at -80 C with intensifying screens. In supershift experiments 1 µg antibody was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rapid activation of IRF-1 gene transcription by PRL and GH in vivo
In previously published studies, we showed that GH rapidly activated target gene transcription in the liver of hypox rats after a single hormone injection (39). We also demonstrated that hormonally regulated transcription, as assessed by run-on transcription assays with isolated hepatic nuclei, correlated closely with measurements of nascent nuclear RNA by ribonuclease protection assay (39, 40). Because the latter assays proved to be more sensitive and technically easier, we now used this approach to examine the kinetics of regulation of IRF-1 gene transcription after a single injection of rPRL or rGH, using a riboprobe derived from the 5' end of the rat gene (37). Figure 1Go shows the results of a typical experiment. In hepatic nuclei from hypox female rats, steady-state levels of nascent IRF-1 transcripts are low. Treatment with rPRL caused a transient rise in nuclear IRF-1 RNA, peaking at 15 min after injection at approximately 5-fold above baseline (n = 3 experiments), and returning to initial values by 30 and 60 min. By contrast, rGH stimulated a greater and more sustained increase in nuclear IRF-1 RNA, reaching approximately 30-fold above the starting levels by 30 min and remaining similarly elevated at 60 min (n = 3 experiments). Comparable results were seen with hGH treatment.



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  		Figure 1. GH and PRL activate IRF-1 gene transcription in liver. Top, Autoradiograph shows results of a representative ribonuclease protection experiment with 5 µg nuclear RNA from hypox rats treated with vehicle or with rPRL, rGH, or hGH for indicated times. Protected 181 nt IRF-1 mRNA band is indicated by arrow. Lower mol wt bands seen in lanes 7–9 and 11–12 represent degradation products of protected probe. Gel was exposed to x-ray film with intensifying screens at -80 C for 16 h. Bottom, Map of 5' end of rat IRF-1 gene indicating region used to synthetize antisense RNA probe.

 
Hormonal activation of nuclear protein binding to a DNA site in proximal IRF-1 promoter
In Nb2 T lymphocytes, PRL stimulates IRF-1 gene transcription through mechanisms that involve inducible nuclear protein binding to a GAS-like DNA element in the proximal promoter (26, 37). We therefore looked at the effects of rPRL and rGH on DNA-protein interactions at this site by gel mobility shift assays. As seen in Fig. 2AGo, no binding was detected to a double-stranded oligonucleotide containing the IRF-1 GAS site with nuclear protein extracts from hypox rats. A single DNA-protein complex was induced by rPRL within 15 min of systemic hormone treatment. A stronger gel shift was stimulated by rGH. Two bands were seen, with the upper DNA-protein complex sometimes being resolved into a closely spaced doublet or triplet (see Fig. 2BGo). The broad upper band comigrated with the PRL-regulated gel shifted complex (Fig. 2AGo). The GH- and PRL-activated DNA-protein complexes were induced with similar kinetics, with peak intensity at 30 min and persistence until at least 60 min after hormone injection.



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  		Figure 2. GH and PRL induce nuclear protein binding to a DNA element from proximal IRF-1 promoter. Gel mobility shift assays were performed as described in Materials and Methods using a 32P-labeled double-stranded oligonucleotide derived from proximal IRF-1 promoter (Table 1Go), and hepatic nuclear protein extracts (5 µg/lane) from hypox rats were injected with vehicle or with rGH or rPRL 15, 30, or 60 min before death. A, Time course study. Autoradiograph of a gel shift assay using nuclear extracts from female rats treated with rGH or rPRL for indicated times. Gel-shifted bands are indicated by arrows. Uncomplexed DNA was electrophoresed off gel and is not visible. Dried gel was exposed to x-ray film for 16 h with intensifying screens at -80 C. B, Competition experiments. Each autoradiograph shows results of gel shift assays using nuclear extracts from hypox rats treated with rGH (top) or rPRL (bottom) for 30 min before death. Incubation with indicated unlabeled double-stranded competitor oligonucleotides (see Table 1Go for DNA sequences) at 2- to 10-fold molar excess was performed as described in Materials and Methods. Lane 1 represents free probe. Uncomplexed DNA was electrophoresed off each gel and is not visible. Dried gels were exposed to x-ray film at -80 C for 16 h with intensifying screens. C, Antibody supershift experiments. Autoradiograph shows results of a gel mobility supershift experiment performed after preincubation of hepatic nuclear extracts from rGH treated rats (30 min) with following antibodies: STAT1, lane 3; STAT3, lane 4; STAT5, lane 5; STAT6, lane 6; mouse IgG, lane 7; myogenin, lane 8. Lane 1 represents uncomplexed labeled olinonucleotide probe, and lane 2 shows results with no antibody. Autoradiographic exposure was for 12 h at -80 C with intensifying screens. Thick arrows mark gel-shifted bands, and thin arrows indicate supershifts.

 
A series of competition experiments and antibody supershift assays were performed next to identify the hormonally regulated nuclear proteins interacting with the IRF-1 DNA element. Nuclear protein binding induced by rGH or rPRL was specific, as evidenced by inhibition of complex formation after incubation of hepatic nuclear proteins with an excess of unlabeled IRF-1 oligonucleotide, but not with an unrelated octamer-1 oligomer (Fig. 2BGo). The PRL-inducible complex also was inhibited by incubation with an unlabeled oligonucleotide from the GH-responsive region of the Spi 2.1 gene, which has been shown to bind STAT5 (41), by the high-affinity c-sis-inducible element, which can bind several STATS, particularly 1 and 3 (19), and by the GAS site from the LY6 gene, which also can interact with several different STATS (42). Inhibition of the GH-inducible DNA-nuclear protein complexes presented a more complicated pattern than was observed with the PRL-regulated gel shifted band. Binding was blocked by excess human c-sis-inducible element oligonucleotide, but was not blunted by Spi 2.1 DNA, although the broad upper band was resolved into a minor and major doublet (Fig. 2BGo). Excess LY6 oligonucleotide caused disappearance of the lower gel shifted band, and resolved the upper DNA-nuclear protein complex into a more tightly focused pattern.

The IRF-1 GAS element has been shown previously to be a binding site for several STATS, depending on the cell type and cytokine analyzed (26, 38, 43). We next performed supershift experiments to determine whether similar proteins were components of the hormonally activated DNA-protein complexes (Fig. 2CGo). Antibodies to STAT6, IgG, or myogenin were not effective in altering the gel shifts induced by rGH. An antibody to STAT1 gave rise to a supershift accompanied by a decrease in intensity of the lower band; antiserum to STAT3 also caused a supershift that was accompanied an increase in intensity of the lower band and a decrease in the upper band. Results with an antibody to STAT5 were equivocal. In some experiments, no supershift was detected, as shown in Fig. 2CGo, whereas in other studies, a minor DNA-protein-antibody complex could be seen (data not shown). Similar results were observed with the STAT5 antibody using nuclear proteins from rPRL-treated rats, whereas other antibodies had no effect on the single hormone-regulated DNA-protein complex (data not shown).

Induction of IRF-1 gene expression by GH does not require concurrent protein synthesis
Some acute actions of GH, including activation of insulin-like growth factor I (IGF-I) and Spi 2.1 gene transcription and inhibition of albumin gene expression, have been shown to occur in the absence of ongoing protein synthesis (29, 44), and thus can be considered to be primary hormonal responses. To determine whether new protein synthesis is required for stimulation of IRF-1 transcription by GH, hypox rats were first pretreated with CHX, and then injected with hormone. As reported previously (29), when used at 4 mg/ml, this inhibitor blocked over 98% of incorporation of labeled methionine into hepatic proteins. CHX alone caused an increase in the abundance of nuclear IRF-1 RNA but did not inhibit or potentiate the rise seen with GH (Fig. 3Go). An approximate 30-fold induction of gene expression was observed after hormone treatment in the absence or presence of CHX (n = 3 experiments).



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  		Figure 3. IRF-1 gene activation does not require ongoing protein synthesis. Autoradiograph shows results of a ribonuclease protection experiment with 3 µg nuclear RNA from male hypox rats treated with or without CHX and hGH for indicated times. Protected 181 nt IRF-1 RNA band is indicated by arrow. Gel was exposed to x-ray film with intensifying screens at -80 C for 2 h.

 
CHX also did not alter the pattern of GH-regulated nuclear protein binding to the IRF-1 DNA element (Fig. 4Go). Inducible protein-DNA complexes were observed at 30 and 60 min after pretreatment with drug or vehicle. Therefore, both stimulation of binding of hepatic nuclear proteins to the GAS site in the IRF-1 promoter and IRF-1 gene activation are part of the primary in vivo nuclear response to GH.



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  		Figure 4. GH treatment induces nuclear protein binding to a DNA element from proximal IRF-1 promoter in absence of concurrent protein synthesis. A gel mobility shift assay was performed as described in Materials and Methods using a 32P-labeled double-stranded oligonucleotide derived from proximal IRF-1 promoter, and hepatic nuclear protein extracts (10 µg/lane) from male hypox rats were treated with CHX or vehicle and hGH for indicated times. Autoradiographic exposure was for 16 h at -80 C with intensifying screens. Arrows indicate DNA protein complexes. Free probe was electrophoresed off bottom of gel.

 
GH induces sustained increases in IRF-1 messenger RNA (mRNA)
Some genes such as c-fos (30, 45) are transiently activated by GH, whereas others, including IGF-I or Spi 2.1 (39), show a more persistent pattern of stimulation. To determine whether GH-activated IRF-1 gene expression is transient or sustained, time course studies were performed. Figure 5AGo shows that levels of cytoplasmic IRF-1 mRNA increased markedly in the liver by 2 h after a single systemic GH injection. Fig. 5BGo indicates that IRF-1 gene expression peaked by 4 h after GH and remained above baseline for up to 16 h. This time course is similar to that seen with IGF-I mRNA (39).



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  		Figure 5. GH treatment leads to sustained accumulation of IRF-1 mRNA in liver. A, Autoradiograph shows results of a representative ribonuclease protection experiment with 20 µg total hepatic RNA from hypox and hGH-treated male rats. Gel was exposed with intensifying screens at -80 C for 17 h. Protected 181 nt IRF-1 mRNA band is indicated by arrow. B, Results of four experiments using same groups of rats were quantitated by ß-scannner and are expressed as relative change in IRF-1 gene expression (mean ± SEM) after GH treatment. Results at each time point were significantly different from time 0 (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PRL and GH are related hormones with diverse effects on growth, differentiation, the immune response, and metabolism (1, 2, 3, 4). Our laboratory has used the hypox rat as a model to examine the acute in vivo actions of these hormones. We previously showed that rapid changes in nuclear protein phosphorylation, DNA-protein interactions, and gene expression occurred after a single injection of GH into male or female hypox rats (19, 20, 29, 30, 31, 32, 39), and identified STATS1, -3, and -5, and activating protein-1 as transcription factors responsible for mediating some of the earliest alterations in gene expression that followed hormone treatment (19, 29, 30, 33). More recently, we found that PRL also induced several acute biological responses in the liver, but that these effects were weaker than those observed after a comparable dose of GH (33). We now have examined the in vivo actions of both hormones on the gene encoding the transcription factor, IRF-1, which had been shown to be robustly induced by PRL in the Nb2 rat T cell line (36). We find that both rPRL and rGH are able to stimulate IRF-1 gene expression in the liver within minutes of systemic hormone injection, effects that are temporally correlated with induction of hepatic nuclear protein binding to the GAS-like DNA element in the proximal IRF-1 promoter. Surprisingly, GH is far more potent than PRL in both activating IRF-1 transcription and in potentiating nuclear factor binding to the GAS site. These actions of GH are not blunted by the protein synthesis inhibitor, CHX, indicating that they represent primary hormonal responses.

Yu-Lee and colleagues (36) first showed in the Nb2 cell line that IRF-1 represented the most highly induced gene seen after PRL treatment, and additionally found that transcriptional activation was independent of ongoing protein synthesis. Our results, indicating that PRL also activates IRF-1 gene expression in vivo, thus both confirm and extend their original observations. The relatively small and transient stimulation of IRF-1 gene transcription by PRL that we saw (Fig. 1Go) may be secondary to the low density of long PRL receptors and the predominance in the liver of the short isoform (46), because O’Neal and Yu-Lee (47) found (using a cotransfection assay) that the short receptor did not induce IRF-1 gene activity. Similar results on the transcriptional incompetence of the short PRL receptor have been obtained by other investigators (48). Alternatively, the absence of other pituitary hormones may negatively influence PRL action in vivo, because these factors were not replaced in our experiments.

Additional studies in Nb2 cells identified the GAS site in the proximal IRF-1 promoter as being functionally critical for PRL-stimulated transcription during the G1 phase of the cell cycle, but also showed that sites in the more distal promoter were required for full hormone-regulated gene activation during S phase (37). Even though both STAT1 and STAT5a were found to be components of the protein-DNA complex that assembled on the GAS site after PRL treatment, only STAT1 was able to potentiate IRF-1 promoter function in a reconstituted heterologous cell system (26, 49). In this regard, it is noteworthy that GH, which robustly stimulates IRF-1 gene expression in the liver, also potently activates STAT1 in vivo (19, 20), and induces binding of this protein to the IRF-1 GAS site, effects not seen with PRL treatment (Fig. 2Go).

GH acutely activates several other genes in the liver in addition to IRF-1, including Spi 2.1, IGF-I, and c-fos (30, 39, 50). Spi 2.1 and IGF-I transcription are stimulated by GH even in the presence of CHX (29, 44), indicating that like IRF-1 they represent primary hormone response genes. Both genes also respond to GH treatment with a sustained induction of expression (39, 50), as does IRF-1 (Fig. 5Go). In particular, the kinetics of IGF-I mRNA accumulation after GH closely resemble those of IRF-1, with an increase in transcript abundance being maintained for up to 16 h after a single hormone injection (39). By contrast, c-fos transcription is transiently activated by GH, can be enhanced by CHX alone, and cannot be potentiated further by concurrent GH treatment (29). GH regulates Spi 2.1 gene transcription by activating STAT5 (41), although other as yet uncharacterized hormonally regulated transcription factors also may be involved (51). The mechanisms of stimulation of IGF-I gene expression by GH remain unknown, although in the liver both IGF-I promoters are activated by hormone treatment (32, 52). Thus, even though several genes are rapidly and coordinately induced by GH, the mechanisms of activation are likely to differ greatly.

Multiple classes of transcription factors collaboratively mediate cellular responses to hormones at the level of gene regulation (53). STATS1, -3, and -5 comprise part of the primary response to GH, stimulating target genes after being induced by posttranslational mechanisms that involve changes in tyrosine and possibly serine phosphorylation (54, 55). GH-stimulated activating protein-1 activity represents a secondary hormonal response, because new protein synthesis is required for formation of the active heteromeric c-fos/c-jun complex (29). Because in other contexts IRF-1 functions as an inducible transcription factor that requires ongoing protein synthesis (56), it is likely that it also is part of the secondary response to GH, although in the current study we did not measure changes in IRF-1 protein levels or DNA binding activity.

In summary, we found that GH rapidly induces IRF-1 gene expression in the liver, potentially through a mechanism that is secondary to the acute activation of STAT1 (and other STATS) and binding to a GAS-like element in the proximal IRF-1 promoter. Although PRL treatment also enhances IRF-1 gene expression with initial kinetics of activation that are similar to those seen with GH, and also stimulates nuclear protein binding to the GAS-like element, the weaker response to PRL may be secondary to a more limited number of receptors capable of coupling ligand binding to downstream signal transduction pathways, or to as yet unrecognized differences in cell-surface to nuclear signaling between these two related hormone-receptor systems.


   Acknowledgments
 
We thank the National Hormone and Pituitary Program, NIDDK, NIH for supplying rGH and rPRL, and Dr. Li-Yuan Yu-Lee (Baylor College of Medicine) for providing a plasmid containing the 5' end of the IRF-1 gene.


   Footnotes
 
1 This work was supported by NIH Research Grant 5-RO1-DK-37449 (to P.R.). Oligonucleotide synthesis was supported by NIH Grant DK-20579 (Washington University Diabetes Research and Training Center). Back

2 Recipient of a research fellowship from the European Society for Pediatrics Endocrinology sponsored by Novo-Nordisk A/S. Back

Received September 16, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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