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Endocrinology Vol. 145, No. 1 214-220
Copyright © 2004 by The Endocrine Society

Growth Hormone (GH)-Stimulated Insulin-Like Growth Factor I Gene Expression Is Mediated by a Tyrosine Phosphorylation Pathway Depending on C-Terminal Region of Human GH Receptor in Human GH Receptor-Expressing Ba/F3 Cells

Hideo Yoshizato, Minoru Tanaka, Naoya Nakai, Nobuhiro Nakao and Kunio Nakashima

Department of Materials Science and Engineering (H.Y.), Nagoya Institute of Technology, Nagoya 466-8555; Department of Animal Science (M.T.), Nippon Veterinary and Animal Science University, Tokyo 180-8602; and Department of Biochemistry (N.N., N.N., K.N.) Faculty of Medicine, Mie University, Mie 514-8507, Japan

Address all correspondence and requests for reprints to: Dr. Hideo Yoshizato, Department of Bioscience, Nagoya Institute of Technology, Gokiso-cyo, Showa-ku, Nagaya 466-8555, Japan. E-mail: h-yoshi{at}ks.kyy.nitech.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The signaling pathway of GH-stimulated IGF-I gene expression is still unclear, although it has been reported that the Janus kinase (JAK)-signal transducers and activators of transcription (STAT)5b pathway plays an important role in liver IGF-I expression. In this study, the GH-dependent IGF-I gene expression and its intracellular signaling mechanism have been examined in mouse pro-B, Ba/F3 cells stably expressing human GH receptor (Ba/F3-hGHR). The IGF-I gene expression was stimulated by human GH (0.01-10 nM) in a dose-dependent fashion in Ba/F3-hGHR cells. The specific inhibitors for JAK2 remarkably suppressed the GH-induced IGF-I gene expression, but MAPK or phosphatidylinositol 3 kinase-specific inhibitors failed to block the GH stimulation of the IGF-I gene expression. However, genistein, a nonspecific tyrosine kinase inhibitor that does not inhibit JAK2 and STAT5 phosphorylation, significantly suppressed the GH-induced IGF-I gene expression. Additionally, a Ba/F3-hGHR mutant that contained the truncated C-terminal hGHR up to D351 showed no IGF-I gene expression in response to human GH. The D351 form normally has the GH-induced JAK/STAT5 tyrosine phosphorylation. These results suggest that the JAK-STAT5 pathway and the novel tyrosine phosphorylation pathway, dependent on signaling from the C-terminal region of hGHR, might be involved in the GH-stimulated IGF-I gene expression in Ba/F3 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GROWTH-PROMOTING ACTIVITIES of GH are predominantly exerted by its indirect effect via the production of IGF-I (1). The stimulating action of GH on IGF-I production is known to occur at the level of gene transcription (2, 3).

However, the mechanisms of intracellular signaling leading to the IGF-I gene expression are still unclear, although recently it has been reported that Janus kinase (JAK)/signal transducers and activators of transcription (STAT)5 signaling plays an important role in GH-induced IGF-I gene expression (4).

The effects of GH are manifested through its interaction with a specific cell surface receptor, GH receptor (GHR). GHR belongs to the cytokine receptor superfamily whose intracellular signaling pathway involves tyrosine kinases of the JAK family and the STAT family after the ligand-induced receptor dimerization (5, 6, 7). It has also been reported that the family including GHR may activate signaling pathways involving MAPK, insulin-receptor substrates (IRS-1 and -2), phosphatidylinositol 3 kinase (PI3K) or phospholipase C/protein kinase C (8, 9, 10). To analyze the GH-signaling pathway, GHR has been overexpressed in cells such as COS-7, C6 glioma, and Chinese hamster ovary cells (11, 12, 13, 14). These cells exhibited GH-stimulated proliferation and differentiation after phosphorylation of JAK/STAT and MAPK proteins. However, with these cells, the expression of IGF-I was either not observed or observed only at a very low level (undetectable). Primary cultured hepatic cells responded to GH to induce IGF-I gene expression, but the primary cells gave rise to some difficulties and disadvantages in studying the mechanisms of GH-induced IGF-I gene transcription. For example, it was necessary to isolate some fresh cells from animals under constant physiological conditions, or cells required a high level of glucose, insulin, or other hormones (15, 16, 17, 18). It is, therefore, necessary to establish a cell line that expresses the IGF-I gene in response to GH. Recently we found that IGF-I gene expression is induced by GH in a human GHR (hGHR)-expressing mouse proB cell-derived cell line, Ba/F3-hGHR (19). In this study, we report that the GH-induced IGF-I expression in this cell line is mediated by a novel tyrosine phosphorylation pathway directed from the C-terminal region of hGHR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines
Mouse pro-B, Ba/F3, cells, and Ba/F3 cells stably expressing full-length hGHR (Ba/F3-hGHR) were kindly supplied by Dr. M. Honjo of Mitsui Chemical Inc. (Chiba, Japan). The cDNA encoding truncated hGHR (D351Stop) was synthesized by a PCR technique using an antisense primer (5'-TCAGTCGACGTCACTGCTTAGAAGTCTGTCTGTG-3') to introduce a nonsense mutation into the position 351 aspartate of hGHR. The cDNA was ligated into pCXN2 vector containing chicken ß-actin promoter and neomycin resistant gene. The truncate hGHR-expressing vector was transfected to parental Ba/F3 cells. The cell line stably expressing truncate hGHR was selected by growth in a culture-selection medium [RPMI 1640 medium containing 1 mg/ml G418, 10% fetal bovine serum (FBS), 50 µM 2-mercaptoethanol, 10 nM human GH (hGH), and antibiotics]. The cells were assayed for truncate GH receptor expression by using RT-PCR analysis.

Both hGHR-expressing Ba/F3 cells were grown and maintained in a culture-selection medium until grown to 1 x 106 cell/ml. In the same way, parental Ba/F3 cells were maintained in a culture medium, RPMI 1640, supplemented with 10% FBS, 50 µM 2-mercaptoethanol, 1 ng/ml recombinant mouse IL-3 (PeproTech Ltd., Rocky Hill, NJ), and antibiotics. Before stimulants challenge, these cells (1 x 106 / ml) were incubated in the assay medium (RPMI 1640 medium with 0.5% FBS, 50 µM 2-mercaptoethanol, and antibiotics) for 16–24 h. Then hGH or mouse prolactin (PRL) (final concentration, 10 nM) was added to the plates, the cells were collected at the times needed, washed with ice-cold PBS, and quickly frozen in liquid nitrogen until used. Recombinant hGH and mouse PRL were gifts by Shikibo, Ltd. (Shiga, Japan).

Chemicals
Genistein (Sigma, St. Louis, MO) was used as a nonspecific tyrosine kinase inhibitor, except for inhibition of JAK2 and STAT5 tyrosine phosphorylation. Wortmannin (Sigma) was used as a selective PI3K inhibitor. PD98059 (BIOMOL Inc., Plymouth Meeting, PA) was used a specific inhibitor of MAPK. Genistein and PD98059 were added 1 h before hGH treatment. Wortmannin was repeatedly added every hour from 30 min before hGH treatment until the end of treatment. AG490 (Sigma) was used as a JAK2-specific tyrosine phosphorylation inhibitor and was added 16 h before hGH treatment. All drugs were dissolved in dimethyl sulfoxide (DMSO) and stored at -20 C. Cycloheximide dissolved in water at 10 mg/ml was used as protein synthesis inhibitor and was pretreated for 2 h.

Detection of mouse IGF-I (mIGF-I) mRNA
Total RNA was extracted from 1 x 107 cells by the guanidium isothiocyanate-phenol-chloroform method. mIGF-I or ß-actin mRNA expressions were detected by RT-PCR/Southern blot hybridization. The reverse transcriptase reaction was carried out at 42 C for 1 h in 25 µl of the reaction mixture containing 5 µg total RNA, 50 pmol Oligo-dT primer (Amersham Pharmacia Biotech, Uppsala, Sweden), 75 mM KCl, 50 mM Tris-HCl (pH 7.5), 3 mM MgCl2, 10 mM dithiothreitol, 2 mM deoxynucleoside triphosphates, 10 U of RNase inhibitor (Wako, Osaka, Japan), and 100 U Moloney murine leukemia virus reverse transcriptase (BRL, Bethesda, MD). One microliter of the reaction mixture was amplified by PCR on a thermal cycler (Astec, PC-800, Fukuoka, Japan) in the reaction buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphates, 0.01% gelatin, 50 pM of sense and antisense primer pairs for IGF-I or ß-actin, and 1 U Taq DNA polymerase (Perkin-Elmer, Foster City, CA) in the final volume of 25 µl. The hot-start method was used for this PCR and each cycle consisted of denaturing at 94 C for 45 sec, annealing at 63 C for 1 min, and extension at 72 C for 1 min. The PCR was performed for 25 cycles. The primers used for RT-PCR of IGF-I mRNA were 5'-GAGCTGGTGGATGCTCTTCA-3' (primer 1 for 5' end) and 5'-CCTCCTACATTCTGTAGGTC-3' (primer 2 for 3' end). The predicted length of amplified fragments is 343 bp. For ß-actin mRNA, the primers 5'-TTGTAACCAACTGGGACGATATGG-3' (for 5' end) and 5'-GATCTTGATCTTCATGGTGCTAGG-3' (for 3' end) were used. The predicted length of the amplified fragments is 764 bp. After amplification by PCR, 5 µl of the reaction products were separated by electrophoresis in 1.5% agarose gels, transferred onto nylon membranes (NEN Life Science Products Research Products, Boston, MA) overnight at room temperature in 10x SSC [1.5 M NaCl, 0.15 M sodium citrate (pH 7.4)], and cross-linked to the membranes. The membranes were prehybridized with denatured salmon sperm DNA (0.2 mg/ml) at 37 C for 2 h, and then hybridized with radiolabeled IGF-I or ß-actin cDNA probe in 50% formamide, 6x SSCP [0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA (pH 7.4)] and 0.1% sodium dodecyl sulfate at 42 C for 16 h. After two washings with 2x SSC (1x SSC, 0.15 NaCl, and 15 mM sodium citrate) containing 0.1% sodium dodecyl sulfate at 60 C for 25 min, the membranes were exposed to a x-ray film at room temperature for 16–24 h. The IGF-I probe (a 343-bp mIGF-I cDNA fragment) was labeled with [{alpha}-32P]deoxy ATP using a random primed DNA labeling kit (Amersham Pharmacia Biotech). The primer pairs used for PCR of mIGF-I cDNA detected both mIGF-Ia and Ib fragments with different lengths at the same time. Radioactivities of the Southern blot hybridization signals on the membranes were determined by a bioimage analyzer (BAS2000, Fuji Film, Japan).

The data were analyzed for statistical significance using the Macintosh SuperANOVA program (Apple, Cupertino, CA) and expressed as means ± SD. The significance of differences between the values was determined by Scheffé’s post hoc test, and P < 0.05 was considered significant.

Cell extracts and immunoprecipitation
Cells (1 x 107) were starved for 16 h and then treated with 5 nM hGH for 15 min at 37 C. GH-stimulated Ba/F3-hGHR cells were frozen in liquid nitrogen and lysed in 1 ml of lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM sodium pyrophosphate, 50 mM NaF, 1 mM phenylmethyl sulfonyl fluoride, and 1 mM Na3VO4 at 4 C. Lysates were cleared by centrifugation. Protein concentrations of the lysates were assayed by the modified Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). Cleared lysates (0.5 mg protein) were rotated end over end with Protein A/G PLUS Agarose (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 4 C for 1 h and then centrifuged at 2000 x g. Supernatants were rotated end over end for 1 h with 10 µl of polyclonal rabbit anti-JAK2 or polyclonal rabbit anti-STAT5b (Santa Cruz Biotechnology, Inc.), and then rotated with Protein A/G PLUS Agarose at 4 C for 16 h. The suspension was centrifuged at 2500 x g, and the pellets were washed three times with lysis buffer. The immunoprecipitated complexes were boiled for 5 min in 20 µl of SDS-PAGE sample buffer containing 5% 2-mercaptoethanol. Samples were electrophoresed on SDS-PAGE gels (8% acrylamide) and electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp., Bedford, MA). For direct immunoblotting, twenty micrograms of the cell lysate were mixed 1:1 with 2x SDS-PAGE sample buffer and boiled for 5 min. SDS-PAGE was performed on a 10% acrylamide slab gel and electrotransferred onto polyvinylidene difluoride membranes.

Western blot analysis
The membranes were blocked for 1 h with 5% horse serum in TBS-T [10 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.05% Tween 20] at room temperature. Primary antibodies were as follows: antiphosphotyrosine mouse monoclonal antibody (PY-20, Transduction Laboratories, Lexington, KY), anti-STAT5b rabbit polyclonal antibody, anti-JAK2 rabbit polyclonal antibody, antiphospho-p44/p42 ERK (Thr202/Tyr204) rabbit polyclonal antibody, antiphospho-Akt (Ser473) rabbit polyclonal antibody, anti-Akt rabbit polyclonal antibody (Cell Signaling Technology, Beverly, MA), and anti-p44/p42 ERK rabbit polyclonal antibody (Santa Cruz, Biotechnology, Inc.). The blots were incubated with primary antibodies for 16 h at 4 C in TBS-T with 3% BSA. After incubation with primary antibody, the membranes were washed three times with TBS-T and incubated with antimouse IgG horseradish peroxidase, antirabbit IgG alkaline phosphatase, or antirabbit IgG horseradish peroxidase (Santa Cruz Biotechnology, Inc.) in TBS-T with 5% nonfat dried milk for 1 h at room temperature. Immunoblots were visualized by the enhanced chemiluminescence or the alkaline phosphatase detection system.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH-induced IGF-I gene expression in Ba/F3-hGHR cells
The mIGF-I mRNA expression in the Ba/F3-hGHR cells was detected by RT-PCR/Southern blot hybridization methods. The time course of GH-induced IGF-I mRNA expression in these cells is shown in Fig. 1Go. The IGF-I gene expression in Ba/F3-hGHR cells increased by 2.2-fold after 1 h of hGH treatment and 6.2-fold after 2 h. The maximum IGF-I gene expression was 8.0-fold at 4 h after the treatment (Fig. 1Go). The IGF-I gene expression levels were also dependent on the concentration of hGH (Fig. 2Go). At the lowest dose used (0.01 nM), IGF-I mRNA expression slightly increased; however, this was not statistically significant. With 0.1 nM hGH, the IGF-I mRNA levels were increased by 2.2-fold, and at 10 nM hGH the IGF-I gene expression was increased by more than 10-fold. However, mouse PRL at 1 and 10 nM could not induce the IGF-I gene expression in these Ba/F3-hGHR cells (data not shown). The IGF-I gene expression was not also induced by hGH in the parental Ba/F3 cells that were not transfected with hGHR cDNA (data not shown). Through these experiments, the ß-actin gene expressions as internal control gene were not changed by hGH treatment.



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  		FIG. 1. Time course of hGH-induced increase in mIGF-I gene expression in Ba/F3-hGHR cells. The cells (1 x 106 / ml) were serum-starved for 16 h, then incubated with 1 nM hGH in assay medium for 0, 0.5, 1, 2, 4, 8, and 12 h. Determination of mIGF-I gene expression is described in Materials and Methods. The ethidium bromide staining and an autoradiogram of amplified mIGF-Ia and Ib cDNAs are shown in A and B, respectively. The autoradiogram of mouse ß-actin amplified cDNA is shown in C. The radioactivities of the Southern blot hybridization signals on the membrane in B and C were determined by a BAS2000 bioimage analyzer (Fuji Film) and expressed as percentage of time zero value in D and E. Each value represents the mean ± SD of three separate experiments. *, P < 0.05; **, P < 0.01 vs. the time zero value.

 


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  		FIG. 2. Expression of mIGF-I mRNAs responds to various doses of hGH in Ba/F3-hGHR cells. The starved cells were incubated with a series of concentration (0 or 0.01 to 10 nM) of hGH for 2 h. The ethidium bromide staining and autoradiogram of amplified mIGF-Ia and Ib cDNAs are shown in A and B, respectively. The autoradiogram of mouse ß-actin amplified cDNA is shown in C. The analysis of radioactivities on the membrane was performed as indicated in Fig. 1Go and expressed as percentage of PBS control value in D and E. Each value represents the mean ± SD of three separate experiments. *, P < 0.05; **, P < 0.01 vs. the PBS control.

 
Effects of cycloheximide and inhibitors of protein kinases on GH-induced IGF-I gene expression and JAK2/STAT5b, ERKs, and PI3K phosphorylation in Ba/F3-hGHR cells
Cycloheximide, a protein synthesis inhibitor, did not suppress the GH-induced IGF-I gene expression (Fig. 3Go). The specific inhibitors for MAPK or PI3K (PD98059 and wortmannin, respectively) failed to suppress the GH-induced IGF-I gene expression in Ba/F3-hGHR cells. GH induced the ERK-1/2 (p44/p42) phosphorylation in Ba/F3-hGHR cells, and this reaction was completely suppressed by PD98059 (50 µM) (Fig. 4Go). In addition, Akt was also phosphorylated by GH stimulation in these cells, and wortmannin (100 nM) treatment successfully inhibited GH-induced Akt phosphorylation (Fig. 4Go). On the other hand, the JAK2-specific inhibitor (AG490) and a nonspecific tyrosine kinase inhibitor (genistein) strongly suppressed the GH-induced IGF-I mRNA expression (Fig. 5Go). Genistein could not inhibit the GH-induced JAK2 and STAT5b tyrosine phosphorylation, although AG490 strongly suppressed the GH-induced JAK2 and STAT5b tyrosine phosphorylation (Fig. 6Go). The ß-actin gene expression was not changed by treatments of cycloheximide, each protein kinase inhibitor, or hGH (data not shown).



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  		FIG. 3. Effects of cycloheximide, PD98059, and wortmannin on GH-induced IGF-I gene expression. The inhibitors were added at 30 min to 16 h before the addition of 5 nM hGH, respectively. hGH stimulation was carried out for 2 h. The detail of pretreatment of inhibitors is described in Materials and Methods. Cycloheximide was added at final concentrations of 10 (+) and 100 (++) µg/ml. Wortmannin was added at final concentrations of 0.1 µM (+) and 1.0 µM (++). PD98059 was added at final concentrations of 10 µM (+) and 100 µM (++). The analysis of radioactivities on the membrane was performed as described in Fig. 1Go and expressed as percentage of hGH (-) value. Each value represents the mean ± SD of three separate experiments.

 


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  		FIG. 4. Effects of inhibitor for ERK and Akt phosphorylations in Ba/F3-hGHR cells. Ba/F3-hGHR cells were serum-starved for 16 h, then pretreated with DMSO (0.05%), 50 µM PD98059, or 0.1 µM wortmannin in DMSO for 1 h or 30 min, respectively. After pretreatment, hGH (5 nM) was added and incubated for 15 min before lysis. Twenty micrograms of cell extracts were assayed directly for EKR1/2 and Akt phosphorylation (1:1000), EKR1/2, and Akt (1:5000). Western blot analyses were performed as described in Materials and Methods using antibodies directed against either phospho-ERK1/2 and total ERK1/2, phospho-Akt and total Akt as indicated.

 


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  		FIG. 5. Effects of genistein and AG490 on GH-induced IGF-I gene expression. Two doses of AG490 were added at final concentrations of 50 µM (+) and 100 µM (++), and genistein, 10 µM (+) and 100 µM (++), were pretreated for 16 or 2 h before the addition of 5 nM hGH, respectively. hGH stimulation was carried out for 2 h. The analysis and presentation of radioactivities on the membrane were indicated in Fig. 1Go. Each value represents the mean ± SD of three separate experiments. **, P < 0.01 vs. the GH-treated control.

 


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  		FIG. 6. Effects of inhibitor for JAK2 and STAT5b tyrosine phosphorylations in Ba/F3-hGHR cells. The immunoprecipitations of JAK2 (A) and STAT5b (B) with specific and antiphosphotyrosine antibodies and SDS-PAGE/Western blotting were performed as described in Materials and Methods. AG490 (100 µM) and genistein (100 µM) were incubated for 16 or 2 h before the addition of 5 nM hGH, respectively. hGH was treated for 15 min. The upper panels in A and B each show antiphosphotyrosine antibody (pTy; 1:10,000 dilution), and the blot was stripped and reprobed with specific antibody recognizing JAK2 or STAT5b (1:5,000 dilution) in the lower panels, respectively.

 
GH-induced IGF-I gene expressions and JAK2-STAT5b tyrosine phosphorylation in cells expressing truncated hGHR
The Ba/F3 cells expressing truncated hGHR were constructed as shown in Fig. 7Go. The D351Stop truncated receptor, which includes Box 1, Box 2, and one tyrosine residue Tyr314, lacks 77% of the cytoplasmic domain. Using the RT-PCR method, we confirmed that the truncated GHR molecules are expressed at equal levels compared with the native-form-expressing cell line and are the appropriate size in these cells. D351Stop truncated hGHR-expressing cells did not express the IGF-I gene (Fig. 8AGo); furthermore, the ß-actin gene expression in those cell lines was not changed by hGH (data not shown). However, the truncated D351Stop hGHR expressing cells showed GH-induced JAK2 and STAT5b tyrosine phosphorylation (Fig. 8Go, B and C).



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  		FIG. 7. Schematic presentations of native and truncated hGHR. Numbers indicate the amino and residues in the regions of the extracellular, transmembrane (TM), and cytoplasmic domains. Residue 1 is the first amino acid of the mature protein (20 ). Solid squares indicate box 1 and box 2, respectively. The location of tyrosine residues (Y) in cytoplasmic domain are indicated.

 


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  		FIG. 8. IGF-I gene expressions and JAK2/STAT5b tyrosine phosphorylation in Ba/F3 cells expressing truncated hGHR. A, The total RNA were prepared from each cell line treated with (+) or without (-) 5 nM hGH for 2 h. Detection of IGF-I gene was performed as described in Materials and Methods. The analysis of radioactivities on the membrane was performed as indicated in Fig. 1Go and expressed as a percentage of GH minus control. Each value represents the mean ± SD of three separate experiments. **, P < 0.01; N.S, not significant vs. the GH(-) value. B, JAK2 and C, STAT5b tyrosine phosphorylation in Ba/F3 cells expressing truncated hGHR. Ba/F3-hGHR (full length) and D351Stop cells were treated with (+) or without (-) 5 nM GH for 15 min. The upper panels in B and C each show antiphosphotyrosine antibody immunoblots (1:10,000 dilution), and the blot was stripped and reprobed with specific antibody recognizing JAK2 or STAT5b (1:5,000 dilution) in the lower panels, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGF-I mediates the growth-promoting action of GH and plays a pivotal role in postnatal growth of mammals. Although some hormones such as testosterone, estrogen, glucagon, or other growth factors can induce the IGF-I production (18, 21, 22), GH is the primary regulator of IGF-I production at least in the liver and skeletal muscles (23, 24, 25). However, the signal pathway of the GH-induced IGF-I gene transcription is still unclear. One reason for this has been the lack of appropriate GH-responsive culture cell lines that can produce IGF-I in response to GH. Although IGF-I is predominantly synthesized in the liver, it is also produced in other tissues, including lymphocytes (26, 27, 28). It has been reported that mature B and T cells express endogenous GH receptor and IGF-I (29, 30, 31), but mouse pro-B cells (Ba/F3 cells) could not express IGF-I in response to GH. However, a previous study (32) has shown that the Ba/F3 cells stably expressing human GH receptor (Ba/F3-hGHR) possess a high sensitivity to GH to initiate proliferation. The result prompted us to test this cell line to determine the GH-induced IGF-I gene expression.

In this study, the Ba/F3-hGHR cells showed GH dose- and time-dependent responsiveness on the IGF-I gene expression (Fig. 1Go). These results support our previous report (19). The IGF-I gene expression began to increase within 1 h after administration of hGH (2.2-fold) and significantly increased after 2 h (6.2-fold, Fig. 2Go). Previously, in vivo experiments (32) have shown that the IGF-I gene expression in the liver is elevated within 1 h after administration of GH. We also found that treatment with a protein synthesis inhibitor, cycloheximide, did not inhibit the GH-induced IGF-I gene expression, suggesting that it was not necessary to newly synthesize any proteins in the gene expression. The expression level of IGF-I mRNA in the Ba/F3-hGHR cells was low compared with that in the liver (26), but the GH-induced IGF-I gene expression was clearly demonstrated in these cells (Figs. 1Go and 2Go). The Ba/F3-hGHR cells have exhibited similar responses to GH in the expression of the IGF-I gene to those of the liver in in vivo experiments. Even in hepatic primary culture cells, it took 12–24 h to express IGF-I mRNA after GH stimulation (15, 16, 17, 18). Moreover, the hepatic primary culture cells always require high concentrations of insulin, glucose, and some steroid hormones for their survival and GH responsiveness (15, 16, 17, 18, 33). Ba/F3-hGHR cells showed high responsiveness to GH to induce the IGF-I gene expression in the absence of other hormones at normal glucose level. This may suggest that the Ba/F3-hGHR cells represent a simpler model than that of the cultured liver cells for the GH-induced IGF-I gene expression signaling that works in the absence of other hormones.

The JAK family (JAK1, JAK2, JAK3, and Tyk2) is activated by the ligands binding to cytokine receptor superfamily receptors. JAK2 has been shown to be activated by the GH/GHR system (5, 6), and, subsequently, a variety of signaling pathways including the STAT families, MAPK, PI3K, IRS-1, and IRS-2 are activated (8, 10). Disruption of the STAT5b gene caused a decrease in body weight gain in male mice. STAT5b-/- male mice showed a significant increase in the plasma GH concentration and a 50% decrease in plasma IGF-I level (34, 35). Moreover, hypophysectomized STAT5b-/- male mice did not cause GH-induced IGF-I mRNA expression (4). Additionally, suppressor of cytokine signaling-2 inhibited the GH-stimulated JAK-STAT signaling, and suppressor of cytokine signaling-2-disrupted mice grew significantly larger than their wild-type mice (36). These findings strongly suggest that the JAK2-STAT5b signaling pathway plays an important role in the GH-induced IGF-I gene expression. However, the promotor of the IGF-I gene is unique in that no typical proximal transcriptional element is found. Therefore, it is unclear whether the JAK-STAT pathway is directly involved in the GH-induced IGF-I gene expression.

In this study, we also found that the specific JAK2 tyrosine kinase inhibitor AG490 significantly inhibited the GH-induced IGF-I gene expression and strongly suppressed the GH-induced JAK2 and STAT5b tyrosine phosphorylation. These results led us to suppose that the JAK-STAT5b pathway is important for GH-induced IGF-I gene expression (4). Additionally, other specific inhibitors for protein kinases, PD98059 and wortmannin, could not block GH-induced IGF-I gene expression, although those inhibitors successfully suppressed the GH-induced ERK and Akt phosphorylation, respectively. Our results support recent studies that suggest ERK activity did not contribute to GH-induced IGF-I mRNA expression (37, 38). Moreover, some previous studies also proposed that wortmannin could not contribute to GH-induced IGF-I mRNA expression. However, it is reported that an another PI3K inhibitor, LY294002, clearly suppressed GH-induced IGF-I gene expression, suggesting that the PI3K signaling pathway mediates the LY294002-sensitive pathway (38). Although these inconsistent results cannot be clearly explained, the evidence suggesting that wortmannin and LY294002 inhibit Akt phosphorylation suggests at least that the PI3K/Akt signaling pathways do not contribute to GH-induced IGF-I gene expression. Therefore, our data suggest that the PI3K/Akt and MAPK signaling pathways were not involved in the GH signaling to induce IGF-I gene expression.

Interestingly, the nonspecific tyrosine kinase inhibitor genistein significantly suppressed the GH-induced IGF-I gene expression without inhibition of JAK2 and STAT5b activation, indicating the participation of other type(s) of tyrosine kinase in IGF-I gene expression. Moreover, the cells expressing the D351Stop truncated form of hGHR including the Box 1 and Box 2 region could induce JAK2/STAT5b tyrosine phosphorylation in response to GH, but not IGF-I gene expression. Several studies have suggested that distal GHR tyrosine residues are involved in allowing GH-induced STAT5b activation (39, 40, 41). In contrast to this, some other studies (42, 43) clearly suggested that activation of JAK/STATs by GH does not require a tyrosine to be phosphorylated in the cytoplasmic domain of hGHR. This discrepancy appears to be caused by the cell-specific or possibly species-specific interaction of JAK/STAT with the cytoplasmic domain of the receptor. Moreover, we found that GH induced STAT5b serine phosphorylation in the D351Stop cells (data not shown), leading us to believe that STAT5b might be activated by GH in these cells, and that STAT5b activation is not sufficient for GH-induced IGF-I gene expression. However, in previous studies, it was quite evident that STAT5b activation is necessary for GH-induced IGF-I gene expression to occur; perhaps it possibly plays the initiator of the signaling cascade of IGF-I gene transcription by GH. Furthermore, our results suggest that the novel tyrosine phosphorylation pathway, dependent on the signaling from the C-terminal region of hGHR, might be involved in the GH-stimulated IGF-I gene expression in Ba/F3 cells.

In conclusion, the present data suggest that the GH-stimulated JAK-STAT5b signaling pathway contributes to the expression of the IGF-I gene, but is not a sufficient factor for the transcription. Moreover, our results suggest that a novel or unknown protein-tyrosine kinase (genistein-sensitive pathway) must also associate with a region downstream of D351 in GHR and is also required for the GH-induced IGF-I gene expression.


   Acknowledgments
 
We thank Dr. M. Honjo of Mitsui Chemical Inc. for providing Ba/F3-hGHR cells and Ba/F3 cells.


   Footnotes
 
This work was supported in part by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan.

Abbreviations: DMSO, Dimethyl sulfoxide; FBS, fetal bovine serum; GHR, GH receptor; hGH, human GH; IRS, insulin-receptor substrate; JAK, Janus kinase; mIGF-I, mouse IGF-I; PI3K, phosphatidylinositol 3 kinase; PRL, prolactin; STAT, signal transducers and activators of transcription.

Received June 30, 2003.

Accepted for publication September 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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