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Endocrinology Vol. 142, No. 9 3890-3900
Copyright © 2001 by The Endocrine Society

ARTICLES

GH Regulation of IGF-I and Suppressor of Cytokine Signaling Gene Expression in C2C12 Skeletal Muscle Cells

Cynthia L. Sadowski, Thomas T. Wheeler, Lu-Hai Wang and Henry B. Sadowski

Departments of Microbiology (C.L.S., L.-H.W.) and Biochemistry and Molecular Biology (H.B.S.), Mount Sinai School of Medicine, New York, New York 10029; and Dairy Science Group (T.T.W.), AgResearch, Hamilton, Private Bag 3123 New Zealand

Address all correspondence and requests for reprints to: Henry B. Sadowski, Ph.D., Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029. E-mail: henry.sadowski{at}mssm.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH is required for normal postnatal growth and metabolism. GH stimulates postnatal growth through induction of IGF-I gene expression. Although the liver is the major site of GH-regulated IGF-I, recent evidence indicates that GH-regulated IGF-I expression in nonhepatic tissues is sufficient for normal postnatal growth. One potentially important nonhepatic site of GH-stimulated IGF-I expression is skeletal muscle, as injection of GH into animals leads to increased IGF-I mRNA in this tissue. Nevertheless, direct effects of GH in skeletal muscle cells in culture have not been reported. We therefore tested the C2C12 myogenic cell line for its response to GH and demonstrate that C2C12 skeletal muscle cells rapidly respond to physiological levels of GH with increased tyrosine phosphorylation of the GH receptor, Janus kinase 2, signal transducer and activator of transcription-5a and -5b, insulin receptor substrate-1, and activation of MAPKs/ERKs and protein kinase B/Akt. In these cells, GH stimulates the expression of IGF-I and two members of the suppressors of cytokine signaling family, cytokine-inducible SH2-containing protein and suppressor of cytokine signaling-2. Treatment of C2C12 myoblasts with either the MAPK kinase inhibitor PD98059 or the PI3K inhibitor wortmannin results in higher levels of GH-induced IGF-I and suppressor of cytokine signaling-2 mRNA expression, suggesting that activation of MAPK and PI3K pathways has an inhibitory role in IGF-I and suppressor of cytokine signaling-2 gene regulation. Therefore, C2C12 cells provide the first in vitro model system to study various aspects of GH action in skeletal muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH REGULATES postnatal growth and diverse metabolic pathways (1). GH elicits its pleiotropic effects by binding to the GH receptor (GHR). As a member of the cytokine receptor superfamily, the GHR lacks intrinsic tyrosine kinase activity. Instead, Janus kinase-2 (JAK2), a cytoplasmic tyrosine kinase, is found associated with the GHR (2). Upon GH binding to the GHR, JAK2 is activated and phosphorylates tyrosine residues on multiple targets, including itself, the GHR, and several members of the signal transducer and activator of transcription (Stat) family of transcription factors, including Stat1, -3, -5A, and -5B (3, 4, 5, 6). Phosphorylation of the activating tyrosine residue in the Stat proteins results in the nuclear translocation of Stat dimers and confers the ability of the Stat dimers to specifically bind to promoter sequences of target genes and to trans-activate those genes (7).

Other substrates for GH-activated JAK2 have been proposed. Observations that GH induces acute insulin-like effects, such as glucose uptake, amino acid transport, glycogenesis, and lipogenesis (8), have suggested that GH mediates these effects through the same molecules and pathways employed by the insulin receptor. Consistent with these observations, GH has been shown to induce tyrosine phosphorylation of insulin receptor substrate-1 and -2 (IRS-1 and IRS-2) as well as Shc in several animal tissues (9, 10) and cultured cell lines such as 3T3-F442A cells (11, 12, 13). Like insulin, GH has been shown to activate both the PI3K (9, 14) and MAPK/ERK (3, 4, 5) signaling cascades.

Although significant progress has been made in identifying the signaling components and pathways that are activated in response to GH, the exact mechanisms of GH regulation of postnatal body growth remain an active area of research. Several mechanisms of GH action on postnatal growth have been proposed. For over 2 decades, it was postulated that GH exerts its action by stimulating the liver to synthesize and secrete IGF-I and that hepatically derived IGF-I mediates the growth-promoting effects of GH in true endocrine fashion (the somatomedin hypothesis) (15). Consistent with this hypothesis, the GHR is expressed at high levels in the liver, and in vivo experiments have demonstrated that GH rapidly stimulates transcription of the IGF-I gene in this tissue (16). Data from targeted disruption of the genes for IGF-I (17, 18, 19) and the GHR (20) in mice further supported this model. Nevertheless, the GHR is also expressed in several nonhepatic tissues, including kidney, heart, intestine, lung, bone, pancreas, fat, and skeletal muscle (21), and several reports have suggested that GH can regulate IGF-I expression in these peripheral tissues that could act in an autocrine/paracrine fashion (22, 23).

The relative contribution of liver-derived IGF-I vs. locally produced IGF-I to postnatal growth has therefore been a matter of debate. To address this question, two groups have generated liver-specific targeted disruption of the IGF-I gene (24, 25). Surprisingly, the body growth rates of the liver IGF-I null mice are normal despite circulating IGF-I levels that are reduced by more than 75%. Taken together, these results indicate that liver-derived IGF-I is not essential for postnatal body growth and support a model of GH action in which GH promotes postnatal body growth predominantly through the local production of IGF-I in nonhepatic tissues [the modified somatomedin hypothesis (24)].

Consistent with this model, injection of GH into animals leads to increased IGF-I mRNA in skeletal muscle (26, 27). Furthermore, increases in the tyrosine phosphorylation of JAK2, Stat5, IRS-1, IRS-2, and SHC have been observed in skeletal muscle within 5 min of GH injection, suggesting direct effects of GH in this tissue (10, 28). Nevertheless, evidence for direct effects of GH in skeletal muscle cells in culture have not been reported. We therefore characterized the responses to GH in myogenic C2C12 cells. Here, we provide evidence that the responses observed in skeletal muscle in vivo represent direct effects of GH. We demonstrate that C2C12 skeletal muscle cells respond rapidly to physiological levels of GH with increased tyrosine phosphorylation of the GHR, JAK2, Stat5a, Stat5b, and IRS-1; transient activation of ERKs and protein kinase B (PKB); and increases in IGF-I mRNA expression. Analysis of GH regulation of the IGF-I promoter has been difficult due to both the complexity of the IGF-I gene and the lack of appropriate cell model systems that express IGF-I in a GH-regulated manner (29). We provide the first in vitro model system for the study of IGF-I regulation in skeletal muscle. We also demonstrate that treatment of C2C12 cells with GH leads to increases in the expression of two members of the suppressors of cytokine signaling family, cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signaling-2 (SOCS-2) (30). The induction of SOCS-2 mRNA expression by GH in C2C12 cells is particularly interesting, as SOCS-2 interacts with the IGF-I receptor (IGF-IR) in a yeast two-hybrid system and in mammalian cells (31). Therefore, in myogenic C2C12 cells, GH induces the expression of IGF-I and a protein, SOCS-2, that may modulate IGF-IR function. Indeed, SOCS-2 knockout mice become significantly larger than their wild-type littermates during the postnatal growth phase, demonstrating an important role for SOCS-2 as a negative regulator of the GH/IGF-I axis (32).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies
Recombinant human GH was purchased from Genentech, Inc. (San Francisco, CA). Insulin was a gift from Eli Lily & Co. (Indianapolis, IN). 1,4-Diazabicyclo(2.2.2)octane (DABCO), 4,6-diamino-2-phenylindole (DAPI), and BSA were obtained from Sigma (St. Louis, MO). PD98059 and wortmannin were obtained from Calbiochem(San Diego, CA). Matrigel was purchased from Collaborative Biomedical Products (Bedford, MA). Chick embryo extract, HEPES, and TRIzol were obtained from Life Technologies, Inc. (Grand Island, NY). The Luciferase Assay Reagent Kit was obtained from Promega Corp. (Madison, WI). The lactogenic hormone-responsive reporter (LHRR) construct was a gift from Fabrice Gouilleux. The constructs containing the cDNAs for the SOCS/CIS genes were gifts from Douglas Hilton. The polyclonal anti-GHR antibody (anti-GHRcyt-AL47) was a gift from Stuart Frank. The antiphosphotyrosine monoclonal antibody 4G10, anti-JAK2, and anti-IRS-1 polyclonal antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The polyclonal antibodies specifically recognizing mouse Stat5a or mouse Stat5b used for immunoprecipitations and supershift analysis were purchased from Zymed Laboratories, Inc. (San Francisco, CA). For immunoblot analysis, the monoclonal antibody recognizing both Stat5a and Stat5b was obtained from Transduction Laboratories, Inc. (Belmont, CA). Biotinylated IgG and streptavidin- conjugated Cy3 were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Caltag Laboratories, Inc. (Burlingame, CA). Monoclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody, polyclonal antibody recognizing phospho-Akt (Ser473), and polyclonal anti-Akt antibody were obtained from New England Biolabs, Inc. (Beverly, MA). Polyclonal antibody recognizing p44/p42 MAPK was obtained from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA).

Generation and characterization of phospho-Stat5 monoclonal antibodies
The generation of the phospho-Stat5 monoclonal antibody used in these studies (18E5, IgG2b), has been described previously (33). This monoclonal antibody is highly specific for the tyrosine-phosphorylated forms of Stat5a and Stat5b in both Western blotting and immunoprecipitation assays and displays no reactivity against tyrosine-phosphorylated GHR, Stat1, Stat3, or JAK family members.

Cell culture and transfection
Mouse skeletal muscle C2C12 myoblasts were cultured in a 37 C, 6% CO2 incubator in growth medium (DMEM containing 15% heat-inactivated FBS, 0.5% chick embryo extract, 25 mM HEPES, and 0.2% gentamicin). For myoblast cultures, C2C12 cells were grown to 80% confluence on tissue culture dishes coated with Matrigel, washed with PBS, and placed in placed in serum starvation medium (SSM; DMEM containing 0.2% BSA and 25 mM HEPES) for 16 h before treatment with GH or diluent (PBS). For the generation of myotubes, C2C12 cells were grown to confluence on tissue culture dishes coated with Matrigel, washed with PBS, and placed in differentiation medium (DMEM containing 2% heat-inactivated horse serum and 25 mM HEPES) for 3 d. Well differentiated cultures (>60% of the plate multinucleated myotubes) were then washed with PBS and placed in SSM for 16 h before treatment with GH or diluent (PBS). For transfections, C2C12 cells were plated at 1.5 x 105 cells/35-mm tissue culture dish (Matrigel-coated). After 16 h, the cells were washed and transfected with 0.1 µg LHRR luciferase reporter plasmid, 0.25 µg cytomegalovirus-ß-galactosidase, and 0.75 µg pUC119 with 6 µl Fugene (Roche, Indianapolis, IN) according to the manufacturer’s directions. For myoblasts, 24 h after transfection the cells were placed in SSM for 16 h before treatment with GH or diluent (PBS). For myotubes, 24 h after transfection, the cells were washed with PBS and placed in DM. After 3 d, the cells were washed and placed in SSM for 16 h. After GH stimulation, cells were harvested in 1 x Reporter Lysis Buffer (Promega Corp., Madison, WI). After normalization for ß-galactosidase activity, lysates were assayed for luciferase activity using the Luciferase Assay Kit (Promega Corp.).

Immunoprecipitation and immunoblotting
After treatment, cell cultures were placed on ice and washed twice with ice-cold PBS/0.5 mM NaVO3 before lysis in the appropriate buffer. For immunoprecipitations of the GHR or JAK2, the cells were lysed in JAK lysis buffer [200 mM NaCl, 50 mM NaF, 50 mM Tris (pH 7.5), 1% Triton-X-100, 1 mM EDTA, 1 mM NaVO3, 0.5 mM phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM benzamidine, and 5 µg/ml pepstatin A]. For immunoprecipitations of Stat5a and Stat5b and for direct analysis of proteins, the cells were lysed in RIPA buffer [200 mM NaCl, 50 mM NaF, 50 mM Tris (pH 7.5), 1% Triton-X-100, 1% sodium deoxycholate, 1 mM EDTA, 1 mM NaVO3, 0.5 mM phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM benzamidine, and 5 µg/ml pepstatin A] for 20 min at 4 C. Lysates were cleared of insoluble material by centrifugation. Protein concentration of the lysates was determined by the modified Bradford assay (Bio-Rad Laboratories, Inc., Richmond, CA) using bovine {gamma}-globulin to generate a standard curve. Cleared lysates (protein amounts indicated in the figure legends) were immunoprecipitated overnight at 4 C with the respective antibodies (volumes of antibodies indicated in the figure legends), incubated with protein A-Sepharose, washed extensively, boiled in SDS-PAGE sample buffer containing 100 mM dithiothreitol, and subjected to SDS-PAGE (7.5% acrylamide). Alternatively, for direct immunoblotting of cell lysates, cleared RIPA or JAK lysis buffer lysates containing equal protein were boiled in SDS-PAGE sample buffer and subjected to SDS-PAGE (7.5% acrylamide). After electrophoretic transfer of proteins to nitrocellulose, the membranes were blocked overnight at 4 C in either BSA blocking buffer (3% BSA in PBS/0.1% Tween 20) for antiphosphotyrosine Western blots or milk blocking buffer (4% nonfat dried milk in PBS/0.1% Tween 20) for all other Western blots. Blocked membranes were incubated with primary antibodies in the appropriate blocking buffer for 2 h at 25 C. The membranes were washed extensively, incubated with antimouse IgG2b-horseradish peroxidase or antirabbit IgG-horseradish peroxidase in milk blocking buffer for 1 h at 25 C, washed extensively, developed with Supersignal ECL reagent from Pierce Chemical Co. (Rockford, IL), and exposed to film (Kodak XAR, Eastman Kodak Co., Rochester, NY). Blots were stripped by three 10-min washes with stripping buffer [62.5 mM Tris (pH 6.8), 2% SDS, and 143 mM 2-mercaptoethanol] at 62 C and then washed extensively to remove the stripping buffer.

EMSA
For preparation of whole cell extracts, cells were collected in ice-cold PBS containing 0.5 mM NaVO3, pelleted briefly, and resuspended in 3x packed cell volumes of high salt buffer [20 mM HEPES (pH 7.9), 400 mM NaCl, 20% glycerol, 20 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM Na4P2O7, 1 mM NaVO3, 0.4 µM microcystin, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM benzamidine, and 5 µg/ml pepstatin A] containing 0.1% Triton-X-100. After rocking for 30 min at 4 C, the lysates were clarified by centrifugation and assayed for protein concentration. Nuclear extracts were prepared as described previously (34). Briefly, cells were collected as described above and gently lysed by resuspension in 5x packed cell volumes of hypotonic buffer [20 mM HEPES (pH 7.9), 20 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM NaP2O7, 1 mM NaVO3, 0.4 µM microcystin, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM benzamidine, and 5 µg/ml pepstatin A] containing 0.1% Triton X-100. Crude nuclei were collected by low speed centrifugation (3000 x g) and resuspended in 2.5x packed cell volumes of high salt buffer. Nuclear proteins were extracted by rocking for 30 min at 4 C, and the extracts were clarified by centrifugation and assayed for protein concentration. For EMSA, two complementary, single stranded oligonucleotides comprising either a high affinity sis-inducible element (SIE) derived from the c-fos promoter (35) (m67SIE: upper, 5'-gtcgaCATTTCCCGTAAATC-3'; lower, 5'-tcgacGATTTACGGGAAATG-3') or a high affinity PRL-inducible element (PIE) derived from the rat ß-casein promoter (36) (upper, 5'-gtcgaGATTTCTAGGAATTT-3'; lower, 5'-tcgacAAATTCCTAGAAATC-3') were annealed, radiolabeled using [32P]deoxy-CTP and Klenow fragment of DNA polymerase I, and purified by nondenaturing PAGE. The m67 SIE represents a high affinity binding site for homo- and heterodimers of Stat1 and Stat3 (37), and the PIE represents a high affinity binding site for homo- and heterodimers of Stat5a and Stat5b (36). EMSA was performed as previously described (34). For supershift analysis, 3 µg anti-Stat5a or anti-Stat5b antibodies (Zymed Laboratories, Inc.) were incubated with extracts for 30 min on ice before incubation with probe.

Immunofluorescence
C2C12 myoblasts or myotubes were serum-starved for 16 h. After treatment with GH or diluent, the cells were fixed and permeabilized in 95% ethanol/5% acetic acid at -20 C for 10 min. The fixed cells were blocked in PBS/5% goat serum/0.5 mM sodium vanadate for 60 min at 25 C and incubated with antiphosphoStat5 hybridoma culture supernatant for 60 min. After washing with PBS, the cells were incubated with biotinylated antimouse IgG diluted 1:800 in blocking solution for 1 h, washed with PBS, and then incubated with streptavidin-conjugated Cy3 diluted 1:200 in PBS/1.5% BSA. The cells were counterstained with DAPI for 5 min, washed in PBS, and mounted with DABCO. Stained cells were viewed with a Zeiss Axiophot fluorescent microscope (Carl Zeiss, New York, NY), and pictures were taken at 400x magnification.

RNA extraction and Northern analysis
After treatment with GH, C2C12 cells were rinsed twice with ice-cold PBS, and total RNA was extracted using TRIzol (Life Technologies, Inc.). Total RNA was fractionated by electrophoresis on agarose-formaldehyde gel, transferred to Hybond XL membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) using the Northern Max kit (Ambion, Inc., Austin, TX), and fixed by UV cross-linking. Membranes were hybridized at 68 C overnight (except for SOCS-1 at 75 C) with 32P-radiolabeled antisense riboprobe derived from full-length cDNA inserts encoding CIS, SOCS-1, SOCS-2, or SOCS-3 (30) prepared with the Strip-EZ RNA kit (Ambion, Inc.). The IGF-I riboprobe was derived from a 1.0-kb genomic fragment containing exon 3 (17). Membranes were washed at high stringency (0.1% SDS in 0.1 x SSC; 1 x SSC = 150 mM NaCl and 15 mM sodium citrate) at 68 C and exposed to Kodak XAR-5 film with an intensifying screen at -70 C. For reprobing, membranes were stripped according to the manufacturer’s directions for the Strip-EZ RNA kit (Ambion, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH stimulates GHR, JAK2, and Stat5 tyrosine phosphorylation in C2C12 myoblasts and myotubes
GH has been shown to activate JAK2 and Stat5 in skeletal muscle in vivo (10, 28). To determine whether GH activates the JAK/Stat pathway in skeletal muscle cells in vitro, we performed experiments with the myogenic C2C12 cell line. These cells are maintained in culture as myoblasts/satellite cells in the presence of high concentrations of serum. When C2C12 cells are grown to confluence and serum-containing medium removed, the myoblasts withdraw from the cell cycle and begin a myogenic program of differentiation. The myoblasts fuse to form multinucleated myotubes that express muscle-specific genes at high levels. We analyzed both myoblasts and myotubes. First, we tested for GH activation of the GHR. As shown in Fig. 1AGo, GH induces rapid tyrosine phosphorylation of its receptor to nearly equivalent levels in both myoblasts and myotubes. GH also induces rapid and robust tyrosine phosphorylation of JAK2 in the myoblasts (Fig. 1BGo). Surprisingly, the GH-stimulated tyrosine phosphorylation of JAK2 in myotubes is significantly lower. Although the slightly lower level of GHR in myotubes may contribute to this effect, the reduced tyrosine phosphorylation of JAK2 in myotubes also correlates to the reduced expression of JAK2 protein in myotubes. Interestingly, JAK2 expression in skeletal muscle of rodents progressively declines after birth (38). This may reflect the progressive decline in the number of myoblasts within the muscle compartment after birth, where committed myoblasts continue to become myotubes and join myofibers (39). Despite the reduction in both protein expression and tyrosine phosphorylation of JAK2 in differentiated cells, saturating levels of GH stimulate strong tyrosine phosphorylation of Stat5a and Stat5b in both myoblasts and myotubes (Fig. 1CGo). It is possible that the reduced JAK2 expression in myotubes is compensated by increases in the expression of both Stat5 proteins in myotubes.



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  		Figure 1. GHR, JAK2, Stat5a, and Stat5b are tyrosine phosphorylated in response to GH in C2C12 myoblasts and myotubes. A, Proliferating (myoblasts) or differentiating (myotubes) cultures of C2C12 cells were serum-starved for 16 h, then incubated in the absence or presence of 500 ng/ml GH for the indicated time points before lysis. Extracts (5 mg protein) were immunoprecipitated with 2 µl anti-GHR polyclonal antiserum, and three quarters of the immunoprecipitation was assayed for tyrosine phosphorylation by immunoblotting with an antiphosphotyrosine antibody (4G10; 1 µg/ml). The remaining one quarter of the immunoprecipitation was assayed for GHR by immunoblotting with the anti-GHR polyclonal antiserum (1:2500). B, C2C12 cells were serum-starved for 16 h, then incubated in the absence or presence of 500 ng/ml GH for 5 min before lysis. Extracts (3 mg protein) were immunoprecipitated with 2 µl anti-JAK2 polyclonal antiserum and assayed for tyrosine phosphorylation by immunoblotting with an antiphosphotyrosine antibody (4G10; 1 µg/ml). The blots were stripped and reprobed with anti-JAK2 polyclonal antiserum (1:6000). C, C2C12 cells were serum-starved for 16 h, then incubated in the absence (-) or presence (+) of 500 ng/ml GH for 10 min before lysis. Extracts (0.8 mg protein) were immunoprecipitated with 3 µg Stat5a-specific or 3 µg Stat5b-specific antibodies and assayed for Stat5 tyrosine phosphorylation by immunoblotting with an antiphospho-Stat5 antibody (18E5; 1:20) that recognizes tyrosine-phosphorylated Stat5a and Stat5b. The blot was stripped and reprobed with antiserum recognizing both Stat5a and Stat5b (1 µg/ml). *, Coimmunoprecipitated pp125 band that is likely to be JAK2. Data are the results of two or three independent experiments.

 
Rapid activation of Stat5 occurs at physiological doses of GH in C2C12 myoblasts and myotubes
In vivo, GH release from the pituitary is pulsatile and blood GH concentrations range from 2–100 ng/ml in mice (40). To determine whether the activation of Stat5 in skeletal muscle occurs at physiological levels of GH, we treated C2C12 myoblasts or myotubes with varying concentrations of GH for 15 min. We analyzed cell lysates for Stat5 tyrosine phosphorylation by direct immunoblot analysis with a phosphorylation state-specific Stat5 monoclonal antibody. As shown in Fig. 2AGo, the myoblasts were extremely sensitive to low levels of GH, as Stat5 tyrosine phosphorylation was observed at 0.8 ng/ml GH, the lowest dose tested, and peaked at 20 ng/ml. The myotubes were less sensitive to GH, as Stat5 activation was detectable at 4 ng/ml GH, but still peaked by 20 ng/ml. These results indicate that both myoblasts and myotubes respond to physiological levels of GH with Stat5 activation and that the myoblasts are particularly sensitive to GH. The reduced sensitivity of the myotubes to subsaturating doses of GH is likely to be the result of lower JAK2 expression (Fig. 1BGo) as well as slightly lower levels of GHR expression (Fig. 1AGo).



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  		Figure 2. Stimulation of Stat5 tyrosine phosphorylation by physiological doses of GH is rapid and sustained. A, C2C12 myoblasts or myotubes were serum-starved for 16 h, then incubated with the indicated concentrations of GH for 15 min before lysis. Extracts (25 µg protein) were assayed directly for Stat5 tyrosine phosphorylation by immunoblotting with antiphospho-Stat5 antibody (18E5; 1:20) or assayed directly for Stat5 content by immunoblotting with antibodies that recognize both Stat5a and Stat5b (1 µg/ml). B, C2C12 myoblasts or myotubes were serum-starved for 16 h, then incubated in the presence of 500 ng/ml GH for the indicated times before lysis. Extracts (25 µg protein) were assayed directly for Stat5 tyrosine phosphorylation by immunoblotting with antiphospho-Stat5 antibody (18E5; 1:20) or assayed directly for Stat5 content by immunoblotting with antibodies that recognize both Stat5a and Stat5b (1 µg/ml). Data are the results of two independent experiments.

 
To determine the kinetics of Stat5 activation in response to GH, we treated C2C12 myoblasts and myotubes with GH for various periods and assayed cell lysates for Stat5 tyrosine phosphorylation. As shown in Fig. 2BGo, we detected Stat5 activation in both myoblasts and myotubes at the earliest time point tested (5 min). This response peaked at 10 min and started to decline by 20 min. Interestingly, the decline in Stat5 tyrosine phosphorylation in the continuous presence of GH is consistently more rapid in myotubes than in the myoblasts and may reflect proteolysis of activated Stat5 proteins in the differentiated cells. Nevertheless, tyrosine-phosphorylated Stat5 proteins are still detectable in both myotubes and myoblasts 6 h after the addition of GH (data not shown).

GH induces the nuclear accumulation of DNA-binding competent Stat5
To directly demonstrate that GH stimulates Stat5 activation in both myoblasts and myotubes we performed immunofluorescence with the antiphospho-Stat5 antibody on C2C12 myoblasts or myotubes treated with GH for 10 min before fixation. The cells were counterstained with DAPI to identify nuclei. The results shown in Fig. 3Go demonstrate that in the majority of cells, GH stimulates the appearance of tyrosine-phosphorylated Stat5 in the nuclei of both myoblasts and myotubes.



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  		Figure 3. GH induces nuclear translocation of phosphoStat5. C2C12 myoblasts (left panels) or myotubes (right panels) were serum-starved for 16 h, then incubated in the absence (-) or presence (+) of 500 ng/ml GH for 10 min. After fixation, cells were immunostained with an antiphosphoStat5 antibody (upper panels) and counterstained with DAPI (lower panels). Data are the results of three independent experiments.

 
The activation of JAK2 kinase and subsequent stimulation of Stat5 tyrosine phosphorylation and DNA binding by GH has been consistently observed in several cell types (5, 6). As shown in Fig. 4AGo, we observed a dramatic induction of Stat5 DNA-binding activity in C212 myoblasts and myotubes as determined by EMSA with a high affinity Stat5-binding site from the rat ß-casein gene promoter (36). Incubation of extracts with either Stat5a- or Stat5b-specific antibodies before the addition of probe leads to nearly complete loss of this GH-inducible DNA-binding complex and the appearance of supershifted complexes. These results suggest that this GH-inducible DNA-binding activity is composed predominately of Stat5a/Stat5b heterodimers. Consistent with this interpretation, low levels of Stat5b homodimers can be observed in the extracts treated with anti-Stat5a antibody (5b/5b migrates faster than 5a/5b dimer) and Stat5a homodimers in the extracts treated with the Stat5b antibody (5a/5a migrates slower than the 5a/5b dimer). In contrast to Stat5, the activation of Stat1 and Stat3 in response to GH appears to be more variable and, in some cases, cell type specific (5). We therefore tested whether GH activated Stat1 and Stat3 DNA binding in both myoblasts and myotubes, and the results are shown in Fig. 4BGo. We consistently found higher basal Stat3 DNA-binding activity in myotubes compared with myoblasts. The basal Stat3 DNA-binding activity in both myoblasts and myotubes, however, was also dependent on the serum starvation conditions (data not shown). Nevertheless, GH treatment results in a moderate induction of Stat3 DNA-binding activity in both myoblasts and myotubes. In contrast, we detected Stat1 DNA-binding activity in response to GH in myoblasts, but not in myotubes. Consistent with the pattern of Stat1 and Stat3 homodimer formation, Stat1:Stat3 heterodimer formation in response to GH was significant in myoblasts, but essentially undetectable in myotubes. Therefore, the ability of GH to stimulate Stat1 and Stat3 DNA-binding activities changes during differentiation, whereas the ability of GH to stimulate Stat5 DNA-binding activity is largely insensitive to the differentiation state. The myoblast-restricted activation of Stat3 and Stat1 by GH may be related to the very strong activation of JAK2 in myoblasts compared with myotubes. Unlike Stat5, which needs to be recruited to tyrosine phosphorylation sites in the GHR for activation by GH (5, 6), activation of Stat3 and Stat1 by GH does not require tyrosine phosphorylation sites in the GHR, as these Stats appear to be recruited to tyrosine phosphorylation sites on JAK2 (5, 6).



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  		Figure 4. Differential induction of Stat1, Stat3, and Stat5 DNA-binding activity in response to GH in C2C12 myoblasts and myotubes. C2C12 myoblasts or myotubes were serum-starved for 16 h, then incubated in the absence (-) or presence (+) of 500 ng/ml GH for 10 min before native whole cell lysates or nuclear extracts were prepared. Fifteen micrograms of native whole cell extracts (myoblasts) or 9 µg nuclear extract (myotubes) were assayed for either Stat5 (ß-casein PIE; A) or Stat1 and Stat3 (m67SIE; B)-binding activity by EMSA as described in Materials and Methods. Three micrograms of anti-Stat5a or anti-Stat5b antibody were used for the supershift (SS) analysis in A. Stat3:3, Homodimers of Stat3; Stat1:3, heterodimers of Stat1 and Stat3; Stat1:1, homodimers of Stat1 in B. Data are the results of three independent experiments.

 
Activation of a Stat5 reporter gene by GH in myoblasts and myotubes
To date, we have shown that in myoblasts and myotubes treated with GH, Stat5 proteins become phosphorylated on the activating tyrosine residues, migrate into the nucleus, and are DNA binding competent. To determine whether these activated Stat5 proteins are capable of trans-activation, we tested for the ability of GH to activate a Stat5 luciferase reporter construct in both myoblasts and myotubes. C2C12 cells were transiently transfected with LHRR, a construct containing a luciferase gene driven by six copies of the Stat5-binding site from the rat ß-casein promoter upstream of a minimal thymidine kinase promoter. After stimulation of the transfected myoblasts or myotubes with GH, cell lysates were assayed for luciferase activity, and the results are shown in Fig. 5Go. In response to GH, we observed a 7.3-fold activation of the Stat5 reporter in myoblasts and a 2.2-fold activation in the myotubes. The lower level of activation in the myotubes is due to a combination of reduced activated expression of the reporter in the GH-treated myotubes and a higher basal expression of the reporter in untreated myotubes compared with the myoblasts. The lower activated expression of the reporter in GH-treated myotubes may reflect the more rapid decline in activated Stat5 proteins in the myotubes compared with the myoblasts (Fig. 2BGo). An alternative possibility is that activated nuclear localized Stat5 proteins in myotubes are less able to trans-activate the synthetic promoter. The higher basal expression of the reporter in GH-treated myotubes may reflect some of the reporter activity that is derived from integrated plasmid, as these cells were first transfected as myoblasts and then differentiated for 4 d before treatment with GH. Nevertheless, it is clear that GH stimulates trans-activation of a Stat5 reporter gene in both C2C12 myoblasts and myotubes.



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  		Figure 5. GH induction of a Stat5 reporter construct with endogenous Stat5 and GHR. C2C12 myoblasts were transiently cotransfected with 0.2 µg LHRR, a Stat5 luciferase reporter construct, and 0.25 µg cytomegalovirus-ß-galactosidase. Twenty-four hours after transfection, the myoblasts were serum-starved for 16 h, then treated with or without 500 ng/ml GH for 24 h. Cell lysates were prepared and then assayed for ß-galactosidase and luciferase activities. For the myotube transfections, the transfected myoblasts were placed in differentiation medium 24 h after transfection. After 3 d in differentiation medium, myotubes were serum-starved for 16 h, then treated with or without 500 ng/ml GH for 24 h. Cell lysates were prepared and then assayed for ß-galactosidase and luciferase activities. Relative light units (lysis buffer blank subtracted) represent the mean ± SEM of triplicate transfections for each condition. Fold activation (x) is given as the ratio of induced/uninduced. The results are representative of four independent transfection experiments.

 
GH increases CIS and SOCS-2 mRNA expression in myoblasts and myotubes
Several GH-inducible genes have been identified, and many of these are likely to represent Stat target genes. Of particular interest is a novel family of cytokine-inducible genes, the CIS/SOCS genes, which were initially isolated as suppressors of cytokine signaling (30). GH has been reported to induce the expression of one or more of the SOCS family members in liver and mammary gland in vivo (41, 42, 43) and at least two different cell lines in vitro (41, 43). In addition, the expression of SOCS-2 and CIS mRNAs are reduced significantly in the skeletal muscle of hypophysectomized mice, whereas SOCS-3 mRNA expression is unaffected by this treatment (44). This result suggests that SOCS-2 and CIS are probably GH-regulated in skeletal muscle. To determine whether GH induces the expression of one or more members of the SOCS family in myoblasts and myotubes, we performed Northern analysis with antisense riboprobes for CIS, SOCS-1, SOCS-2, and SOCS-3. In myoblasts, we observed dramatic induction of CIS and SOCS-2 mRNA expression in response to GH, with less prominent increases in SOCS-3 mRNA levels (Fig. 6Go). SOCS-1 mRNA levels showed little or no increase in response to GH. Although the response of these genes to GH in the myotubes is weaker, it matches the overall pattern of GH induction observed with the myoblasts.



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  		Figure 6. GH induction of SOCS/CIS family members in C2C12 myoblasts and myotubes. C2C12 myoblasts or myotubes were serum-starved for 16 h, then incubated in the presence of 500 ng/ml GH for the indicated times before harvesting for RNA. Fifteen micrograms of total RNA from each time point were fractionated on a 1% denaturing formaldehyde agarose gel and transferred to a nylon membrane. The membrane was hybridized first with a 32P-labeled antisense riboprobe transcribed from the full-length cDNA for SOCS-2. After exposure to film, the membrane was stripped and sequentially hybridized and stripped in this order: SOCS-1, SOCS-3, then CIS. The exposure times were 3 h (SOCS-2), 48 h (SOCS-1), 3 h (SOCS-3), and 48 h (CIS). Data are the results of two independent experiments.

 
GH induces IGF-I mRNA expression in C2C12 myoblasts and myotubes
In vivo studies have suggested that GH stimulates IGF-I mRNA expression in skeletal muscle (16, 26, 27). To determine whether this is a direct effect of GH, we examined the ability of GH to regulate IGF-I gene expression in C2C12 cells. C2C12 myoblasts and myotubes were serum-starved and then stimulated with GH for varying lengths of time before harvest. RNA was prepared and analyzed by Northern analysis with an antisense riboprobe corresponding to exon 3 of the IGF-I gene (17). GH rapidly stimulated increases in the levels of the 7.4-kb IGF-I transcripts in both myoblasts and myotubes (Fig. 7Go). The GH-stimulated increases in IGF-I mRNA transcripts were detectable within 30 min and peaked between 1–2 h. This is the first report of GH-activated IGF-I mRNA expression in a skeletal muscle model in vitro.



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  		Figure 7. GH induction of IGF-I mRNA expression in C2C12 myoblasts and myotubes. C2C12 myoblasts (subconfluent) or myotubes were serum-starved for 16 h, then incubated in the presence of 500 ng/ml GH for the indicated times before harvesting for RNA. Fifteen micrograms of total RNA from each time point were fractionated on a 1% denaturing formaldehyde agarose gel and transferred to a nylon membrane. The nylon membrane was probed with a 32P-labeled antisense riboprobe transcribed from a 1.0-kb fragment containing exon 3. After washing, the membrane was exposed to film for 3 h. Data are the results of three independent experiments.

 
Stat-independent GH signaling pathways in C2C12 cells
In addition to the JAK/Stat pathway, GH activates a variety of signaling pathways, including the Ras/Raf/MAPK kinase (MEK)/MAPK and the PI3 kinase/PKB/PDK1/pp70s6k pathways (3, 4, 5). The ability of GH to activate these pathways requires JAK2 (4, 5) and is likely to involve tyrosine phosphorylation of IRS molecules and Shc (9, 10, 11, 12). To determine whether GH activates these pathways in C2C12 cells, we treated serum-starved myoblasts and myotubes with GH for various periods and prepared cell lysates. We assayed these lysates for tyrosine phosphorylation or activation of several known GH signaling proteins, including IRS-1, ERK-1, ERK-2, and PKB. First, we measured ERK activation by immunoblotting with a phosphorylation-state specific antibody that specifically recognizes the activated forms of ERK-1 and ERK-2. As shown in Fig. 8Go, GH induced transient activation of ERK-1 and ERK-2 in myoblasts. For comparison, treatment of myoblasts with insulin for 5 min resulted in a greater activation of both ERK-1 and ERK-2. In contrast, in the myotubes the activation of ERK-1 was undetectable even for the insulin control. In addition, the activation of ERK-2 by either GH or insulin was considerably reduced in myotubes. Immunoblotting with a phosphorylation state-specific antibody that specifically recognizes activated PKB revealed that the activation of PKB by GH in myoblasts was transient and modest compared with the level observed in response to insulin. Unlike the myoblasts, however, activation of PKB in myotubes was refractile to stimulation by GH, but not by insulin. Immunoprecipitation of lysates with antiserum recognizing IRS-1 followed by immunoblotting with antiphosphotyrosine antibody shows that increases in the tyrosine phosphorylation of IRS-1 in response to GH were transient and modest compared with the insulin control value in both myoblasts and myotubes. From these results we conclude that many of the signaling pathways that are activated by GH in skeletal muscle in vivo (10) are also activated in C2C12 myoblasts and myotubes. Although the significance remains to be determined, the reduced activation of ERKs and PKB in response to GH in myotubes compared with myoblasts is probably related to the lower levels of GH-activated JAK2 in the myotubes compared with the myoblasts.



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  		Figure 8. GH signaling via the MAP kinase, PI3K, and IRS-1 pathways in C2C12 myoblasts and myotubes. C2C12 myoblasts or myotubes were serum-starved for 3 h, then incubated in the absence or presence of either 500 ng/ml GH or 1 µg/ml insulin for the indicated times before lysis. Top four panels, Fifty micrograms of extracts were assayed directly for ERK1 and ERK2 phosphorylation (1 µg/ml), ERK1 and ERK2 (1:3000), PKB phosphorylation (1:1500), or PKB (1:1000) as indicated. Bottom two panels, Five hundred micrograms of each extract were immunoprecipitated with 2.5 µg antiserum recognizing IRS-1 and immunoblotted with antiphosphotyrosine antibody (4G10; 1 µg/ml). The blot was stripped and reprobed with antiserum recognizing IRS-1 (0.25 µg/ml; bottom panel). Data are the results of three independent experiments.

 
Stimulation of IGF-I and SOCS-2 mRNA expression by GH in myoblasts does not require the MEK/MAPK or PI3K pathways
As GH causes transient activation of PKB and ERKs in C2C12 cells, we investigated whether these pathways are required for GH stimulation of IGF and SOCS-2 mRNA expression in C2C12 myoblasts. Therefore, we pretreated myoblasts with the PI3 kinase inhibitor wortmannin, the MEK inhibitor PD98059, or vehicle [dimethylsulfoxide (DMSO)] before stimulation with GH for 2 h. RNA was harvested and analyzed by Northern analysis for IGF-I and SOCS-2 expression. As shown in Fig. 9AGo, neither wortmannin nor PD98059 inhibited the GH-stimulated increase in IGF-I or SOCS-2 mRNA expression. Surprisingly, inhibition of PI3 kinase with wortmannin or inhibition of MEK with PD98059 led to higher levels of GH-stimulated IGF-I and SOCS-2 mRNA expression than vehicle alone. To demonstrate the efficacy and specificity of the inhibitors, parallel cultures were pretreated identically, and cell lysates were prepared after 10-min stimulation with GH. Immunoblotting with the antiphospho-ERK (Fig. 9BGo) indicates that pretreatment with PD98059 reduces the basal levels of activated ERK1 and ERK2 and abolishes nearly all of the GH stimulation. At the concentration of wortmannin used (50 nM), however, some reduction in both basal and GH-stimulated levels of activated ERKs was observed. Immunoblotting with antiphospho-PKB (Fig. 9BGo) demonstrates that the pretreatment with wortmannin very effectively reduced the amount of activated PKB to undetectable levels, whereas PD98059 had no effect on the basal or GH-stimulated level of activated PKB. Overall, these results indicate that the MAPK and PI3K pathways are not required for GH stimulation of IGF-I and SOCS-2 mRNA expression in C2C12 myoblasts. Moreover, these pathways appear to exert an inhibitory influence on GH stimulation of IGF-I and SOCS-2 mRNA expression in these cells.



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  		Figure 9. GH induction of IGF-I and SOCS-2 mRNA expression does not require PI3K or MEK kinase activity. C2C12 myoblasts were serum-starved for 16 h, then pretreated with DMSO (0.05%), 50 nM wortmannin in DMSO, or 10 µM PD98059 in DMSO for 1 h (A and B). A, After pretreatment, the cells were incubated in the absence or presence of 500 ng/ml GH for 2 h before lysis. Fifteen micrograms of total RNA from each sample were assayed by Northern analysis, and the membrane was hybridized with a 32P-labeled antisense riboprobe transcribed from a 1.0-kb genomic fragment containing exon 3 of IGF-I. The membrane was exposed to film for 3 h after washing. After exposure to film, the membrane was stripped and probed with a 32P-labeled antisense riboprobe transcribed from the full-length cDNA for SOCS-2. The membrane was exposed to film for 3 h after washing. B, Parallel cultures pretreated as above were incubated in the absence or presence of 500 ng/ml GH for 10 min before lysis. Extracts (75 µg) were assayed directly for ERK1 and ERK2 phosphorylation or PKB phosphorylation as indicated. Data are the results of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The somatomedin hypothesis proposed that IGF-I is a hepatically derived circulating mediator of GH and a crucial factor for postnatal growth. Nevertheless, injection of GH into animals leads to increases in IGF-I mRNA expression in several nonhepatic tissues, including skeletal muscle. Increases in tyrosine phosphorylation of JAK2, Stat5, IRS-1, IRS-2, and Shc have been detected in skeletal muscle within 5 min of injection of GH into animals, suggesting direct effects of GH in this tissue (10, 28). Therefore, we examined the response to GH in the myogenic C2C12 cell line. We demonstrate that both the myoblasts and myotubes respond rapidly to physiological levels of GH with increased tyrosine phosphorylation of the GHR, JAK2, Stat5a, Stat5b, and IRS-1, whereas activation of ERKs and PKB is largely restricted to myoblasts. IGF-I mRNA expression is also induced in response to GH in these cells by a mechanism that does not require MEK or PI3K activity. These data suggest that GH is capable of directly stimulating the expression of IGF-I in skeletal muscle in vivo that could potentially act in an autocrine/paracrine mode to stimulate postnatal growth.

Targeted disruption of three genes in mice has clearly demonstrated important roles for IGF-I, GHR, and Stat5a/b in postnatal growth (17, 18, 19, 20, 45). Taken together, these studies indicated that GH-stimulated expression of IGF-I is required for normal postnatal growth and that Stat5 is likely to be involved in the GH regulation of IGF-I expression. At issue is the site of IGF-I expression in response to GH that is required for postnatal growth. In the liver-specific IGF-I knockout mice, body growth rates are normal despite circulating IGF-I levels that are 25% of the levels in wild-type mice (24, 25). In addition, the nonhepatic production of IGF-I in these mice is not increased significantly as a compensatory mechanism for loss of liver IGF-I, suggesting that the autocrine/paracrine production of IGF-I is responsible for postnatal growth. Thus, a model for postnatal growth emerges where GH stimulates the autocrine/paracrine production of IGF-I in nonhepatic tissues such as skeletal muscle that is mediated in part by the JAK2/Stat5 pathway.

Although it encodes a small peptide, the IGF-I gene spans over 70 kb and contains two promoters that direct transcription from multiple start sites and six exons that are differentially spliced, leading to different forms of IGF-I mRNAs (46, 47). Consistent with the complexity of the IGF-I gene, its transcription appears to be regulated by multiple transcription factors in a complex manner (46, 47). Although GH injection has been shown to induce transcription of the IGF-I gene in several tissues in vivo, the mechanism by which GH stimulates IGF-I transcription is still unresolved. This is due in part to the fact that GH-responsive cell lines that transcribe the IGF-I gene in a GH-regulated fashion have been difficult to find. We have now identified a physiologically relevant model system to dissect the mechanism by which GH stimulates IGF-I transcription in skeletal muscle cells. We have shown that the JAK2/Stat5 pathway is activated in response to GH in C2C12 skeletal muscle cells and that expression of IGF-I mRNA is induced. Although further studies are necessary to determine whether Stat5 is involved in the GH regulation of IGF-I in these cells, it is clear that MAPK and PI3K pathways are not required.

It is not clear why it has been difficult to observe GH effects in myogenic cell lines (48). We performed our studies with C2C12 cells cultured in the presence of laminin-rich Matrigel. Interestingly, we observed at least a 2-fold reduction in the ability of GH to activate Stat5 in cells cultured in the absence of Matrigel (Sadowski, C. L., unpublished observations). A positive role for laminin-rich extracellular matrix in GH signaling in primary hepatocytes (49, 50) and PRL signaling in primary mouse mammary epithelial cells has been well established (51, 52). This effect is not observed with collagen-rich extracellular matrix. It is conceivable that in the absence of laminin-rich extracellular matrix, GH signaling in most myogenic cell lines may be inefficient.

There are several target genes for GH other than IGF-I (6), including members of the SOCS/CIS family of proteins (30). This family of proteins (CIS and SOCS1–7) has been defined by the presence of a variable N-terminal region, a centrally positioned SH2 domain, and a conserved C-terminal domain of unknown function called the SOCS box (53). The expression of CIS and SOCS-1, -2, and -3 is induced by various cytokines through the JAK/Stat pathway leading to SOCS protein-mediated inhibition of cytokine signaling. SOCS-1 and SOCS-3 have been consistently shown to act as potent inhibitors of GH signaling in a variety of cell systems through a combination of their SH-2 and N-terminal domains (6). On the other hand, SOCS-2 and CIS weakly inhibit, exert no effect, or augment GH signaling, depending on their expression levels and the cell system (6). We examined the GH regulation of four members of the SOCS/CIS family in C2C12 cells, CIS, and SOCS-1, -2, and -3. Two of these genes, CIS and SOCS-2, have been suggested to be GH regulated in skeletal muscle (44). Although GH strongly induces SOCS-3 and, to a lesser extent, SOCS-1 expression in 3T3-F442A preadipocytes (41), in GH-treated C2C12 myoblasts, we detect only weak and transient increases in SOCS-1 and SOCS-3 expression, with strong and prolonged increases in CIS and SOCS-2 expression. This pattern of SOCS/CIS gene expression may be responsible for the prolonged kinetics of Stat5 activation we observed in GH-stimulated myoblasts. GH also stimulates SOCS-2 and CIS mRNA expression in the myotubes, but to a lesser extent than in the myoblasts over the time period examined (4 h). Like IGF-I, inhibitors of MAPK and PI3K pathways augment the GH-stimulated expression of SOCS-2. Unlike C2C12 cells, GH treatment of 3T3-F442A preadipocytes leads to robust activation of ERKs and PI3K (3, 4, 14) and may explain why GH does not strongly induce SOCS-2 in the 3T3-F442A preadipocytes (41).

The induction of SOCS-2 by GH in myoblasts is of particular interest for several reasons. First, SOCS-2 is expressed in skeletal muscle in vivo (31). Second, the SOCS-2 protein has been reported to interact with the autophosphorylated IGF-I receptor (IGF-IR) in a yeast two-hybrid screen as well as by coimmunoprecipitation in mammalian cells (31). This interaction suggests the possibility that GH-induced SOCS-2 may also modulate IGF-IR signaling. Targeted disruption of the SOCS-2 gene in mice has recently been reported (32). The SOCS-2 null mice are gigantic and display a phenotype that is consistent with the hypothesis that SOCS-2 functions to negatively regulate GH or IGF-I signaling in an organ- specific context. Postnatal growth of skeletal muscle is characterized by increases in both the length and girth of myofibers (39). This involves 1) expansion of myoblast and satellite cell populations, 2) differentiation of committed myoblasts into myotubes that either form new myofibers or join existing myofibers, and 3) increases in the cytoplasmic content of existing fibers (39). As GH (indirectly) and IGF-I (directly) are known to stimulate all of these processes (48, 54), negative regulation of GH or IGF-I signaling by SOCS-2 might be essential for normal postnatal growth of skeletal muscle by preventing an inappropriate response to locally produced IGF-I.

We can envision a model of postnatal growth of skeletal muscle where GH-induced IGF-I acts locally in an autocrine/paracrine manner. In this model, GH-activated Stat5 could play an important role in mediating the increased expression of IGF-I as well as potentially modulating GHR and IGF-IR function through the induction of SOCS-2 protein. Future studies will be directed toward examining the validity of this model.


   Acknowledgments
 
We thank T. S. Choi for her expert technical assistance, I. Wolf for synthesis and purification of the phospho- and apo-Stat5 peptides, H. Park and T Moran at the MSSM hybridoma facility for the generation and initial screening of the antiphopho-Stat5-producing hybridomas, and A. Bergemann for the use of his fluorescence microscope. We thank S. Frank for the generous gift of GHR antibody. We also thank D. Hilton and F. Gouilleux for their generous gifts of plasmids.


   Footnotes
 
This work was supported in part by research awards from the American Diabetes Association and NIH (DK-53000; to H.B.S.) and a National Research Scientist Award from the NIH (NS-10499; to C.L.S.).

Abbreviations: CIS, Cytokine-inducible SH2-containing protein; DABCO, 1,4-diazabicyclo(2.2.2)octane; DAPI, 4,6-diamino-2-phenylindole; DMSO, dimethylsulfoxide; GHR, GH receptor; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; JAK, Janus kinase; LHRR, lactogenic hormone- responsive reporter; MEK, MAPK kinase; PIE, PRL-inducible element; PKB, protein kinase B; SIE, sis-inducible element; SOCS-2, suppressor of cytokine signaling-2; SSM, serum starvation medium; Stat, signal transducer and activator of transcription.

Received February 8, 2001.

Accepted for publication May 8, 2001.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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