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Endocrinology Vol. 142, No. 9 3980-3986
Copyright © 2001 by The Endocrine Society

ARTICLES

LY 294002, an Inhibitor of Phosphatidylinositol 3-Kinase, Inhibits GH-Mediated Expression of the IGF-I Gene in Rat Hepatocytes

Lungile N. N. Shoba, Marsha Newman, Wenli Liu and William L. Lowe, Jr.

Department of Medicine, Veterans Affairs Chicago Healthcare System, Lakeside Division, and Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: William L. Lowe, Jr., M.D., Center for Endocrinology, Metabolism, and Molecular Medicine, Tarry 15-703, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: wlowe{at}northwestern.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The signal transduction pathways that mediate GH-dependent regulation of IGF-I gene expression remain poorly defined. To establish a GH-responsive in vitro model system to study the effect of GH on the expression of the endogenous IGF-I gene, primary hepatocytes from adult male rats were prepared. These cells expressed both the GH receptor and the IGF-I gene, as demonstrated using a ribonuclease protection assay. Western blot analyses using antibodies directed against the phosphorylated forms of the ERKs, signal transducer and activator of transcription-5, and Akt/protein kinase B, a protein kinase that is downstream of PI3K, demonstrated GH-dependent phosphorylation of these signaling molecules. These signaling molecules are components of the major signal transduction pathways that are activated by GH. To determine whether GH-responsive IGF-I gene expression could be demonstrated in these cells, hepatocytes were treated without or with 50 ng/ml GH for 3–48 h. IGF-I mRNA levels increased within 3 h, and a maximal 2.2-fold increase was observed after 24 h of GH treatment. To determine whether ERK activation contributes to GH-induced IGF-I gene expression, hepatocytes were treated for 12 h without or with 50 ng/ml GH and 50 µM PD98059, an inhibitor of MAPK kinase-1 and -2. Treatment with PD98059 did not have a significant effect on GH-induced IGF-I gene expression. Similar studies were performed using 50 µM LY 294002, an inhibitor of PI3K. These studies demonstrated that treatment with LY 294002 completely abrogated GH-induced IGF-I gene expression. In contrast, PI3K-specific doses of another inhibitor of PI3K, wortmannin, failed to inhibit the GH-induced increase in IGF-I mRNA levels. In summary, rat hepatocytes in primary culture provide a good model system to study GH-induced IGF-I gene expression, and the results of these studies suggest that a signaling pathway inhibited by LY 294002, possibly a PI3K-dependent pathway, is important for GH-stimulated IGF-I gene expression in hepatocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I IS A basic 70-amino acid peptide that is expressed in a number of tissues, where it probably acts in an autocrine/paracrine fashion (1, 2, 3). The major site of IGF-I synthesis is the liver, and hepatic IGF-I accounts for the majority of circulating IGF-I, which acts in an endocrine fashion (1, 2, 3). Expression of the IGF-I gene in liver and other tissues is regulated by a number of factors, such as GH, nutrition, and specific hormones (e.g. insulin, estradiol and glucocorticoids), with GH and nutrition being primary regulators (4). GH increases steady state IGF-I mRNA levels in most tissues of adult rats and IGF-I transcription in the liver of adult rats (2, 4, 5, 6). Consistent with GH being a major regulator of IGF-I gene expression, IGF-I mediates many of the growth- promoting effects of GH (1, 2, 3).

Despite an improved understanding of GH receptor signaling, the molecular mechanisms and GH-responsive elements in the IGF-I gene that mediate GH-induced IGF-I gene expression have not been defined. GH binding to its receptor induces tyrosyl phosphorylation and activation of Janus kinase-2 (JAK2), a cytoplasmic kinase that is associated with the GH receptor and mediates the cellular effects of GH (7, 8). Subsequent signaling events include tyrosyl phosphorylation of the GH receptor and phosphorylation and activation of a number of cytoplasmic molecules, including signal transducer and activator of transcription-1 (STAT1), -3, and -5; the MAPKs ERK1 and -2, insulin receptor substrate-1 (IRS1), -2, and -3; and PI3K (7, 8, 9). These molecules are involved in three distinct signal transduction pathways that are activated by GH. The first pathway involves direct tyrosyl phosphorylation and activation of the STATs by JAK2 (7, 8). This leads to STAT dimerization and subsequent translocation into the nucleus, where the STATs activate gene transcription (7, 8, 9, 10). The second major GH-mediated signal transduction pathway involves activation of ERK1 and -2. GH has been shown to induce phosphorylation and nuclear translocation of the ERKs, which results in phosphorylation of Elk-1 and other ternary complex factors and activation of immediate-early gene expression (7, 8, 11, 12, 13). The third pathway involves tyrosyl phosphorylation of IRS1, -2, and -3, with the resulting activation of PI3K (7, 8, 9). To date, the roles of these different pathways in GH-mediated IGF-I gene expression have not been clearly defined.

To address this issue, the present studies were initiated as part of long-term studies to elucidate the molecular mechanisms responsible for the GH-dependent regulation of IGF-I gene expression. The goal of this study was to define the signal transduction pathway(s) that contributes to GH- mediated IGF-I gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of primary hepatocytes
Primary hepatocytes were prepared from 35- to 60-d-old male Sprague Dawley rats (Charles River Laboratories, Inc.) according to the method of Aiken et al. and maintained according to the method of Shih and Towle (14, 15). All studies were approved by the institutional animal care and use committees of Northwestern University and V.A. Chicago Healthcare System. Briefly, the cells were prepared by perfusion and collagenase digestion of the liver and were plated at a density of 8.4 x 106 cells/100-mm dish in dishes coated with type I collagen (Becton Dickinson and Co., New Bedford, MA). The cells were then allowed to attach for 4 h in modified William’s E medium containing 10% FCS and supplemented with 27.5 mM glucose, 23 mM HEPES, 26 mM sodium bicarbonate, 2 mM L-glutamine, 10 nM dexamethasone, 3.84 µg/ml bovine insulin, 50 U/ml penicillin, and 50 U/ml streptomycin. The cell monolayer was washed twice with serum-free modified William’s E medium with 0.25% BSA and cultured for a total of 48 h in serum-free modified William’s E medium supplemented with 0.25% BSA and 500 µg/ml Matrigel (Becton Dickinson and Co.) in the presence or absence of 50 ng/ml human GH for the indicated period of time. For inhibitor studies cells were pretreated with either PD98059 (Calbiochem-Novabiochem Corp., San Diego, CA), LY 294002 (Calbiochem-Novabiochem Corp.), or wortmannin (Calbiochem-Novabiochem Corp.) for 30 min, and then treated without or with GH for the indicated period of time.

Western blot analyses
For Western blot analyses, cells were lysed in 50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0.1% (vol/vol) glycerol, 0.01% (vol/vol) Nonidet P-40, 0.005% (vol/vol) Triton X-100, 0.1 M phenylmethylsulfonylfluoride, 0.4 µg/ml pepstatin A, 0.2 µg/ml leupeptin, 0.2 µg/ml antipain HC, 0.2 µg/ml chymostatin, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, and 25 mM sodium fluoride. Lysates were clarified by centrifuging at 15,000 x g for 5 min at 4 C, and protein concentrations were determined using the Coomassie blue protein assay reagent (Bio-Rad Laboratories, Inc., Richmond, CA). Protein samples were then mixed 1:2 with Laemmli sample buffer with 5% 2-mercaptoethanol and heated at 95 C for 4 min. SDS-PAGE was performed on a 7.5% acrylamide slab gel. Prestained mol wt markers (Bio-Rad Laboratories, Inc.) were used as standards. Electrophoretic transfer of proteins to polyvinylidene difluoride membranes (0.45 µm pore size, Immobilon-P, Millipore Corp., Bedford, MA) was accomplished with a semi dry Trans-Blot transfer system (Bio-Rad Laboratories, Inc.) in a transfer buffer consisting of 25 mM Tris-HCl, 192 mM glycine, and 20% methanol for 1 h at 150 mA. Membranes were blocked for 90 min at room temperature in 20 mM Tris (pH 7.6), 137 mM sodium chloride, and 0.1% Tween-20 (TBST) with 2% nonfat dried milk. Membranes were incubated overnight in TBST at 4 C with primary antibody. Primary antibodies were as follows: antiphospho-ERK, antiphospho-Akt, anti-Akt, and antiphospho-STAT5 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), which were used at a dilution of 1:1,000; anti-ERK2 rabbit polyclonal antibody (New England Biolabs, Inc.), which was used at a dilution of 1:7,500; and anti-STAT5 mouse monoclonal antibody (Transduction Laboratories, Inc., Lexington, KY), which was used at dilution of 1:500. After incubation with primary antibody, blots were incubated with either antimouse or antirabbit (Promega Corp., Madison, WI) IgG horseradish peroxidase-conjugated antibodies at a dilution of 1:7,500 for 90 min at room temperature. After three washes in TBST, immunoreactive bands were detected using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Arlington, IL), according to the manufacturer’s instructions.

RNA extraction
RNA was prepared using the guanidine thiocyanate cesium chloride method as described previously (16, 17). RNA was quantified by measuring absorbance at 260 nm. The accuracy of quantification and the integrity of the RNA were confirmed by size-separating the RNA from different samples by denaturing agarose gel electrophoresis as described previously (16, 17).

Hybridization probes
For quantification of IGF-I mRNA levels by solution hybridization/ribonuclease (RNase) protection assays, a 322-bp rat IGF-I cDNA was subcloned into a pGEM-2 vector (Promega Corp., Madison, WI), and the plasmid DNA was linearized with EcoRI to allow for transcription of antisense IGF-I RNAs (6). This antisense IGF-I RNA distinguishes between IGF-I mRNAs that contain exons 1 and 2 in RNase protection assays. To quantify GH receptor mRNA levels, a 900-bp BglII fragment of the rat GH receptor cDNA was subcloned into the vector pT7T3 (18) (provided by Dr. L. Mathews). For transcription of antisense GH receptor RNAs, the plasmid DNA was linearized with BamHI, which results in transcription of GH receptor antisense RNAs 439 bases in length. This antisense GH receptor mRNA distinguishes between GH receptor mRNAs that encode the GH receptor and the GH-binding protein.

Solution hybridization/RNase protection assay
GH receptor and IGF-I mRNA levels were quantified using a solution hybridization/RNase protection assay as described previously (6). Briefly, 32P-labeled antisense RNAs were transcribed from linearized plasmid DNA and incubated with 20 µg total RNA at 45 C in 75% formamide/0.4 M NaCl. After a 16-h incubation, the samples were digested with RNases A and T1. The protected double stranded hybrids were collected by ethanol precipitation and electrophoresed on an 8% polyacrylamide/8 M urea denaturing gel. All assays were performed in duplicate. Specific mRNA levels were quantified from the gels using either a BAS1000 phosphorimager (Fuji Photo Film Co., Ltd., Stamford, CT) or scanning densitometry.

Statistical analyses
Values are reported as the mean ± SEM. P values were calculated using one-way repeated measures ANOVA with Tukey’s pairwise multiple comparison procedure or Kruskal-Wallis one-way ANOVA on ranks with the Dunnett’s pairwise multiple comparison procedure, as appropriate, using SigmaStat 2.0 software (Jandel Corp., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the IGF-I and GH receptor genes in rat primary hepatocytes
Previous studies have demonstrated evidence of GH-stimulated IGF-I gene expression in hepatocytes (19, 20, 21, 22). To confirm that the IGF-I and GH receptor genes were expressed in rat primary hepatocytes prepared for the current study, an RNase protection assay was performed. IGF-I mRNA was present, and transcripts containing the two different 5'- untranslated regions present in IGF-I were expressed. mRNA encoding the GH receptor was also present, and similar to intact liver, mRNAs encoding both the GH receptor and GH-binding protein were present (data not shown).

GH-dependent phosphorylation of signaling molecules in rat primary hepatocytes
Having established that adult rat hepatocytes in primary culture transcribe both the IGF-I and GH receptor genes, studies were performed to examine GH-mediated signal transduction in the hepatocytes. As described, GH activates a variety of signaling pathways, including ERK1 and -2, STAT5, and PI3K (7, 8). Among the kinases downstream from PI3K is Akt/protein kinase B (PKB) (23). To determine whether GH was able to activate these different signaling pathways in the hepatocytes, cells were treated without or with 50 ng/ml GH for varying periods of time between 5 min and 12 h, and Western blot analyses were performed using antibodies specific for phosphorylated ERK1 and -2, STAT5, and Akt/PKB. GH-dependent tyrosyl phosphorylation of the ERKs occurred within 5 min of GH treatment, but was relatively transient and declined to baseline levels by 30 min (Fig. 1Go, top panel). GH-dependent tyrosyl phosphorylation of STAT5 also occurred within 5 min, and STAT5 levels remained increased after 1 h of GH treatment, but returned to basal levels after 3 h of GH treatment (Fig. 1Go, middle panel). Similar to STAT5, phosphorylation of Akt/PKB was evident within 5 min of GH treatment and began to decline toward basal levels after 1 h of GH treatment (Fig. 1Go, bottom panel).



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  		Figure 1. GH-dependent phosphorylation of signaling molecules in rat primary hepatocytes. Total cellular protein was prepared from rat primary hepatocytes (as described in Materials and Methods) that were treated without or with GH over a period of 12 h. The cellular protein was size-separated using 7.5% SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with antibodies directed against phospho-ERK (top panel), phospho-STAT5 (middle panel), or phospho-Akt (lower panel). Antibody binding was detected using an enhanced chemiluminescence detection system. The blots were stripped and reprobed with antibodies directed against the indicated total protein. These results are representative of the results obtained in three independent experiments, each performed using protein prepared from hepatocytes obtained from a different rat.

 
GH-dependent activation of IGF-I gene expression in rat primary hepatocytes
Having documented that GH activates different signal transduction pathways in hepatocytes, subsequent studies examined whether GH treatment increased IGF-I gene expression in these cells. For these studies the hepatocytes were treated without or with 50 ng/ml GH for 3, 6, 12, 24, and 48 h, and IGF-I mRNA levels were determined. A significant GH-dependent increase in IGF-I mRNA levels was evident within 6 h of treatment with GH, and a maximal 2.2-fold increase in IGF-I mRNA levels was present after 24 h of GH treatment (Fig. 2Go).



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  		Figure 2. Time-course effect of GH treatment on the expression of the IGF-I gene in rat hepatocytes in primary culture. Top panel, Autoradiogram of the results of an RNase protection assay using RNA prepared from hepatocytes treated with 50 ng/ml GH for the indicated periods of time. RNA from hepatocytes treated for varying periods of time with GH was hybridized to a 32P-labeled IGF-I antisense RNA and subjected to solution hybridization/RNase protection analysis. The upper arrow indicates IGF-I mRNAs that contain exon 2 (320 bp), whereas the lower arrow indicates IGF-I mRNAs that contain exon 1 (241 bp). The final lane on the right represents undigested 32P-labeled IGF-I antisense RNA. Bottom panel, Quantification of the time- dependent effect of GH on IGF-I mRNA levels. The values represent the relative level of IGF-I mRNA compared with the level in control cells not treated with GH (0 h) which was defined as 1.0. Each value is the mean ± SEM of duplicate determinations of the IGF-I mRNA level in three different preparations of RNA, each prepared from hepatocytes obtained from a different rat. *, P < 0.05 compared with the level in control cells.

 
Effect of inhibition of GH-dependent ERK and PI 3-kinase activation on IGF-I gene expression
To determine whether the GH-induced increase in IGF-I gene expression was dependent upon activation of the ERKs and/or PI3K, subsequent studies were performed using an inhibitor of these signaling pathways. Initial studies used the inhibitor of MEK1 and -2, PD98059. For these studies, hepatocytes were pretreated for 30 min without or with 50 µM PD98059 and then treated for 12 h without or with 50 ng/ml GH in the absence or presence of 50 µM PD98059. PD98059 caused a small, but insignificant, 24% decrease in GH-stimulated IGF-I gene expression (Fig. 3Go, top and middle panels). To confirm that this concentration of PD98059 was efficacious in inhibiting ERK phosphorylation, cells were pretreated without or with 50 µM PD98059 and then treated for 15 min without or with 50 ng/ml GH (Fig. 3Go, bottom panel). Western blot analyses to determine the level of phospho-ERK were then performed. GH-stimulated ERK phosphorylation was completely abrogated by PD98059. Moreover, in the presence of PD98059, there was no evidence for GH-stimulated ERK phosphorylation for up to 12 h of GH treatment (data not shown).



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  		Figure 3. Effect of PD98905 on GH-induced-induced IGF-I gene expression. Top panel, Autoradiogram of the results of an RNase protection assay using RNA prepared from hepatocytes pretreated for 30 min with 50 µM PD98059 or control medium and then treated without or with 50 ng/ml GH and/or 50 µM PD98059 for 12 h. IGF-I mRNAs that contain exons 1 and 2 are indicated by arrows. The lane on the left represents undigested 32P-labeled IGF-I antisense RNA. Middle panel, Quantification of the effects of GH and PD98059 on IGF-I mRNA levels. The values represent the relative level of IGF-I mRNA compared with the level in control cells treated with neither GH nor PD98059, which was defined as 1.0. Each value is the mean ± SEM of duplicate determinations of the IGF-I mRNA level in three different preparations of RNA, each prepared from hepatocytes obtained from a different rat. *, P < 0.05 compared with the level of IGF-I mRNA in control cells. Bottom panel, Effect of PD98059 on ERK phosphorylation. Total cellular protein was prepared from rat primary hepatocytes that had been pretreated with 50 µM PD98905 or control medium for 30 min and then treated without or with 50 ng/ml GH and/or 50 µM PD98059 for 15 min. Western blot analyses were performed as described in Materials and Methods using an antibody directed against either phospho-ERK or total ERK2 as indicated. These results are representative of the results of three independent experiments, each using protein extracts prepared from hepatocytes obtained from a different rat.

 
The effect of inhibition of PI3K on GH-dependent IGF-I gene expression was examined next. For these studies hepatocytes were pretreated for 30 min without or with 50 µM LY 294002, and then treated without or with 50 ng/ml GH for 12 h in the absence or presence of 50 µM LY 294002 (Fig. 4Go, top and middle panels). In contrast to PD98059, treatment with LY 294002 completely abrogated GH-stimulated IGF-I gene expression and, as expected, treatment with a combination of 50 µM LY 294002 and PD98059 also resulted in complete inhibition of the response to GH treatment. The efficacy of LY 294002 was confirmed using Western blot analysis to examine the effect of 50 µM LY 294002 on GH-stimulated phosphorylation of Akt/PKB (Fig. 4Go, bottom panel). Subsequent studies examined the effect of another PI3K inhibitor, wortmannin, on GH-induced IGF-I gene expression (Fig. 5Go). Like LY 294002, 100 nM wortmannin inhibited GH-stimulated Akt/PKB phosphorylation (Fig. 5Go, bottom panel). In contrast, 100 nM wortmannin had no effect on the stimulation of IGF-I gene expression by GH, whereas 1 µM wortmannin attenuated, but did not abrogate, the GH-induced response.



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  		Figure 4. Effect of LY 294002 on GH-induced IGF-I gene expression. Top panel, Autoradiogram of the results of an RNase protection assay using RNA prepared from hepatocytes pretreated for 30 min with 50 µM LY 294002 or control medium and then treated without or with 50 ng/ml GH and/or 50 µM LY 294002 for 12 h. Also shown are the results using RNA prepared from hepatocytes pretreated for 30 min with 50 µM LY 294002 and 50 µM PD98059 and then treated without or with 50 ng/ml GH in the presence of 50 µM LY 294002 and 50 µM PD98059 for 12 h. IGF-I mRNAs that contain exons 1 and 2 are indicated by arrows. Middle panel, Quantification of the effects of GH and LY 294002 on IGF-I mRNA levels. The values represent the relative level of IGF-I mRNA compared with the level in control cells treated with neither GH, LY 294002, nor PD98059, which was defined as 1.0. Each value is the mean ± SEM of duplicate determinations of the IGF-I mRNA level in three different preparations of RNA, each prepared from hepatocytes obtained from a different rat. *, P < 0.05 compared with the level of IGF-I mRNA in control cells. +, P < 0.05 compared with the level of IGF-I mRNA in cells treated with 50 ng/ml GH. Bottom panel, Effect of LY 294002 on Akt/PKB phosphorylation. Total cellular protein was prepared from rat primary hepatocytes that had been pretreated with 50 µM LY 294002 or control medium for 30 min and then treated without or with 50 ng/ml GH and/or 50 µM LY 294002 for 15 min. Western blot analyses were performed as described in Materials and Methods using an antibody directed against either phospho-Akt/PKB or total Akt/PKB as indicated. These results are representative of the results of three independent experiments, each using protein extracts prepared from hepatocytes obtained from a different rat.

 


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  		Figure 5. Top panel, Quantification of the effects of GH and wortmannin on IGF-I mRNA levels. The values represent the relative level of IGF-I mRNA in cells treated for 6 h as indicated compared with the level in control cells treated with neither GH nor wortmannin (Wort), which was defined as 1.0. Each value is the mean ± SEM of duplicate determinations of the IGF-I mRNA level in three different preparations of RNA, each prepared from hepatocytes obtained from a different rat. *, P < 0.05 compared with the level of IGF-I mRNA in control cells. Bottom panel, Effect of wortmannin on Akt/PKB phosphorylation. Total cellular protein was prepared from rat primary hepatocytes that had been pretreated with 100 nM wortmannin or control medium for 30 min and then treated without or with 50 ng/ml GH and/or 100 nM wortmannin for 15 min. Western blot analyses were performed as described in Materials and Methods using an antibody directed against either phospho-Akt/PKB or total Akt/PKB as indicated. These results are representative of the results of two independent experiments, each using protein extracts prepared from hepatocytes obtained from a different rat.

 
Given the magnitude of the effect of the LY 294002 on GH-stimulated IGF-I gene expression and the differences in the response to LY 294002 and wortmannin, the effect of LY 294002 on GH-stimulated STAT5 phosphorylation was also examined (Fig. 6Go). Treating hepatocytes with LY 294002 had no effect on GH-induced tyrosyl phosphorylation of STAT5, suggesting that inhibition of tyrosyl phosphorylation of STAT5 did not account for the inhibitory effect of LY 294002 on GH-stimulated IGF-I gene expression.



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  		Figure 6. Effect of inhibition of PI3K activity on STAT5 phosphorylation. Total cellular protein was prepared from hepatocytes that had been pretreated with 50 µM LY 294002 or control medium for 30 min and then treated without or with 50 ng/ml GH and/or 50 µM LY 294002 for 15 min. Western blot analyses were performed as described in Materials and Methods using an antibody directed against either phospho-STAT5 or total STAT5 as indicated. These results are representative of the results of two independent experiments, each performed using protein extracts prepared from hepatocytes obtained from a different rat.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The molecular mechanisms responsible for the regulation of IGF-I gene expression remain obscure. Expression of the IGF-I gene is modulated by two promoters, promoters 1 and 2, that regulate the expression of IGF-I mRNAs that contain exons 1 and 2, respectively (4). Previous studies have identified sequences in these promoters responsible for basal promoter activity, the response to cAMP in bone cells, and metabolic regulation of IGF-I gene expression, although sequences that bind proteins in a GH-responsive fashion have not been identified (24, 25, 26, 27, 28, 29, 30, 31, 32). Moreover, despite significant progress in elucidating the signal transduction pathways that are activated by GH, the molecular mechanisms by which GH regulates IGF-I gene expression have not been defined. What is known is that in liver, GH-mediated activation of IGF-I gene expression is rapid and independent of protein synthesis and occurs at the level of gene transcription (5, 33, 34). The goal of the present study was to establish a model system using hepatocytes in primary culture that would allow elucidation of the signaling pathways that contribute to GH-induced IGF-I gene expression. We have demonstrated that adult rat hepatocytes in primary culture express the IGF-I and GH receptor genes and have confirmed the results of previous studies that demonstrated GH-inducible IGF-I gene expression in these cells (19, 20, 21, 22). More importantly, we have now demonstrated that LY 294002, an inhibitor of PI3K, abrogates the GH-induced increase in IGF-I mRNA levels in hepatocytes.

Of interest was the differential effect of LY 294002 and wortmannin on GH-induced IGF-I gene expression. As described, LY 294002 effectively inhibited GH-induced IGF-I gene expression, whereas 100 nM wortmannin, a dose that is relatively specific for the class I PI3K, was able to inhibit GH-induced Akt/PKB phosphorylation, but had no effect on GH-induced IGF-I gene expression. Wortmannin at a dose of 1 µM was able to attenuate the GH-induced increase in IGF-I mRNA levels. There are at least two possible explanations for this observation. One possibility is that the effect of LY 294002 was mediated via inhibition of a GH-induced enzyme distinct from PI 3-kinase that is not inhibited by PI 3-kinase-specific doses of wortmannin. Examples of the differential effects of LY 294002 and wortmannin have been reported previously. In a macrophage cell line, 50–100 µM LY 294002 attenuated phorbol ester-induced ERK activity, whereas 250 nM wortmannin, a dose that fully inhibited PI 3-kinase activity in these cells, had no effect (35). In the present studies GH-induced ERK activity did not contribute significantly to the GH-induced increase in IGF-I mRNA levels. Thus, it is unlikely that a differential effect of LY 294002 on GH-induced ERK activity would explain the differing effects of LY 294002 and wortmannin, but this does not rule out a differential effect of the two inhibitors on another kinase. A more straightforward possibility is differential stabilities of wortmannin and LY 294002. Wortmannin is unstable in aqueous solutions and tends to interact with serum proteins (36); thus, its activity may have been attenuated after a few hours in tissue culture or after interaction with proteins in the Matrigel overlaying the hepatocytes. Although transient activity of wortmannin was evident given its ability to inhibit Akt/PKB phosphorylation, it was ineffective after several hours in culture of inhibiting GH-induced IGF-I gene expression. Interestingly, in other experiments in which 100 nM wortmannin was added every 2 h during 6 h of stimulation with GH, wortmannin attenuated, but did not completely inhibit, the GH-induced increase in IGF-I mRNA levels (data not shown). Future studies will be required to determine the mechanisms for the differences in the ability of LY 294002 and wortmannin to inhibit GH-induced IGF-I gene expression.

The PI3K pathway is one of the three major signaling pathways activated by GH, with the others being the STAT and ERK pathways (7, 8). GH-induced PI3K activation occurs upon its association with an IRS that has been phosphorylated by activated JAK2. IRS1, -2, and -3 are all substrates for JAK2 (7, 8, 9), but in liver the association of PI3K with IRS1 is of primary importance (9). To date, GH activation of PI3K has been shown to mediate several effects in adipocytes, including an antilipolytic effect, activation of cAMP-specific phosphodiesterase-4 via a pathway that requires p70S6kinase, and modulation of GH-induced ERK activity (9, 37, 38, 39). The role of GH-induced activation of PI3K in hepatocytes is less clear. Although PI3K modulates GH-induced ERK activity in adipocytes, the insignificant effect of ERK inhibition on GH-induced IGF-I gene expression suggests that the effect of LY 294002 on GH-induced IGF-I gene expression in hepatocytes occurred primarily via a mechanism distinct from inhibition of ERK activity. Thus, the present studies are the first to report a primary role for GH-induced activation of an LY 294002-inhibitable pathway, presumably a PI3K-dependant pathway, in the regulation of gene expression.

The other major signaling pathway activated by GH is the STATs. GH stimulates phosphorylation and DNA-binding activity of STAT1, -3, and -5 in liver (40, 41, 42). Activation of STAT1 and -3 contributes to GH-induced activation of the c-fos gene via the sis-inducible element present in the c-fos promoter and is important for expression of the gene encoding interferon-regulating factor-1 (43, 44). STAT5 activation is important for GH-mediated expression of genes encoding sexually differentiated hepatic proteins, including several cytochrome P450 genes and the C4-Slp gene as well as the genes encoding the PRL receptor, rat insulin 1, and the serine protease inhibitor, Spi 2.1 (45, 46, 47, 48, 49, 50, 51). In the absence of specific inhibitors of STAT phosphorylation, we were unable to examine directly the role of the STATs in GH-induced IGF-I gene expression, but, interestingly, our finding that LY 294002 abrogated GH-induced IGF-I gene expression without affecting GH-induced STAT5 phosphorylation suggests that STAT5 phosphorylation on tyrosine is not sufficient for GH-induced IGF-I gene expression. These findings do not rule out, however, that STAT5 phosphorylation is necessary for IGF-I gene expression, i.e. that activation of both STAT5 and a PI3K-dependent pathway is required for GH-induced IGF-I gene expression. Future studies will be needed to address that issue.

In summary, we have defined signal transduction pathways that contribute to GH-induced activation of IGF-I gene expression. Specifically, we have demonstrated for the first time that a pathway inhibited by LY 294002, presumably a PI3K-dependent pathway, plays a major role in GH-induced IGF-I gene expression in hepatocytes, although the inability of PI3K-specific doses of wortmannin to reproduce this finding raises the possibility that a pathway distinct from PI3K mediates the effect of GH on IGF-I gene expression. This and many other questions remain about the molecular mechanisms by which GH regulates IGF-I gene expression, but the model system described in these studies should be useful in future studies designed to identify the signal transduction pathways and transcription factors that mediate the effect of GH on IGF-I gene expression.


   Acknowledgments
 
The authors thank Dr. Lawrence Mathews for providing the GH receptor cDNA, Dr. Eun Jig Lee for assistance with liver perfusions, and Dr. Stuart Frank for helpful conversations.


   Footnotes
 
This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.

Abbreviations: IRS, Insulin receptor substrate; JAK, Janus kinase; PKB, protein kinase B; RNase, ribonuclease; STAT, signal transducer and activator of transcription; TBST, 20 mM Tris (pH 7.6), 137 mM sodium chloride, and 0.1% Tween-20.

Received January 30, 2001.

Accepted for publication May 29, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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