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Endocrinology Vol. 143, No. 2 492-503
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

Regulation of IGF-I mRNA and Signal Transducers and Activators of Transcription-3 and -5 (Stat-3 and -5) by GH in C2C12 Myoblasts

Robert A. Frost, Gerald J. Nystrom and Charles H. Lang

Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: Robert A. Frost, Ph.D., Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey Medical Center, H166, Hershey, Pennsylvania 17033. E-mail: rfrost{at}psu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH and IGF-I are critical hormones for the regulation of longitudinal growth and the maintenance of lean body mass in humans. The regulation of IGF-I expression by GH in hepatocytes is well documented; however less is known about the regulation of IGF-I in peripheral tissues such as muscle. We have examined the regulation of IGF-I mRNA by GH and IGF-I in C2C12 myoblasts. GH stimulated the accumulation of IGF-I mRNA dose- and time-dependently. An elevation of IGF-I mRNA was observed with GH doses as low as 0.75 ng/ml and after exposure to GH for as little as 1 h, and the increase required ongoing transcription and translation. GH applied in a pulsatile fashion for 10 min followed by an 8-h interpulse interval increased IGF-I mRNA to a greater extent than continuous exposure. GH stimulated tyrosine phosphorylation of the GH receptor, signal transducer and activator of transcription-3 (Stat3), and Stat5. Stat5 was resistant to additional phosphorylation if cells were given a GH pulse within 2 h of a previous GH exposure. The refractory period lasted for 4 h, and cells could be maximally stimulated again after 6 h. Stat3 phosphorylation was also enhanced in cells that were allowed to recover from a previous application of GH. The tyrosine kinase inhibitors, genistein, PP1, and AG-490, and the MAPK kinase inhibitor, PD98059, did not block Stat3 or Stat5 phosphorylation. In contrast, WHI-P154, a Janus kinase-3 inhibitor, dose-dependently prevented Stat3, but not Stat5, phosphorylation. GH-inducible nuclear transport of Stat3 was likewise inhibited by WHI-P154. Most importantly, GH-dependent IGF-I mRNA expression was inhibited by WHI-P154. In contrast, IGF-I mRNA expression was inhibited by IGF-I peptide, and the effect of IGF-I was dominant over that of GH. IGF-I mRNA was regulated by both PI3K and MAPK signal transduction pathways, but IGF-I peptide signaled predominantly through a wortmannin-sensitive pathway to down-regulate its own mRNA. Our data suggest that Janus kinases (Jak2 or Jak3) and their downstream targets (Stat3 and Stat5) may play important roles in the expression of IGF-I mRNA and the myoblast response to GH. In addition, C2C12 cells appear to be a good model system to examine GH regulation of Janus kinase/Stat signaling and the regulation of IGF-I mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I IS A 7.5-kDa peptide that mediates many of the actions of GH and has anabolic, mitogenic, and antiapoptotic effects on cells (1). The majority of circulating IGF-I is synthesized by the liver in response to GH, and the two hormones account for nearly all of the postnatal growth in rodents. Mice in which the IGF-I and the GH receptor genes have been deleted by homologous recombination attain a final body weight only 17% of normal (2). In contrast, tissue-specific ablation of the hepatic IGF-I gene reveals the heretofore underappreciated importance of autocrine- and paracrine-derived IGF-I in adult animals. These mice exhibit normal body weight and tissue morphology despite a 75% reduction in circulating IGF-I (3, 4). This phenotype suggests that peripheral tissues, such as skeletal muscle, rely to a large extent on the local synthesis of IGF-I for normal postnatal growth. This conclusion is supported by studies demonstrating that muscle-specific expression of IGF-I is permissive for muscle hypertrophy in both senescent and regenerating skeletal muscle fibers (5).

Many diseases, such as sepsis, thermal injury, diabetes, and chronic alcohol abuse, produce a catabolic condition characterized by a decrease in skeletal muscle mass (6). Sepsis, for example, is characterized by both a decrease in skeletal muscle protein synthesis and an increase in protein degradation (7). The loss of muscle protein is associated with a concomitant decrease in both the plasma concentration of IGF-I and the expression of IGF-I mRNA in skeletal muscle (8, 9). Consequently, there is a deficit of IGF-I peptide in skeletal muscle during catabolic conditions. It is thought that a lack of IGF-I and the overexpression of inhibitory IGF-binding proteins (IGFBPs) contribute to muscle protein catabolism (10). GH has been proposed as a therapeutic adjunct that, because of its anabolic properties, might reverse the muscle wasting that occurs after sepsis and traumatic and thermal injury as well as in acquired immune deficiency syndrome (6, 11).

It is not clear whether GH acts directly on skeletal muscle to stimulate growth, but GH administered in vivo increases the expression of IGF-I mRNA in the musculature (12). Numerous studies have also shown that exogenous IGF-I increases skeletal muscle protein synthesis in mice and humans (13, 14). In vitro experiments with GH have been limited to primary cultures of hepatocytes or cell lines that are manipulated to overexpress the GH receptor (GHR). These systems have been fruitful for delineating GH-responsive signal transduction pathways. For example, GH stimulates the Janus kinase (JAK) and signal transducer and activator of transcription (Stat) pathway (15). Stat5 has been shown to have an essential role in GH signaling. Mice with a mutation in both the Stat5a and -5b genes have a 50% reduction in the plasma concentration of IGF-I, and at 12 wk of age weigh 30–40% less than their wild-type littermates (16). Other members of the Stat family are also activated in response to GH (17). Stat3 is of interest because its overexpression in cardiomyocytes promotes a hypertrophic phenotype (18).

Few skeletal muscle cell lines express endogenous GHR (19), and it has not been determined whether GH necessarily acts similarly on the JAK/Stat signaling pathway in myoblasts as in hepatocytes. In addition, it has been difficult to define conditions under which GH can positively regulate IGF-I mRNA in muscle cells. An understanding of how skeletal muscle responds to GH is important, because GH has been used in clinical trials to reverse muscle wasting. In addition, recent studies suggest that the ability of the liver and peripheral tissues to respond to GH in acquired immune deficiency wasting may vary (20). Moreover, Van den Berghe (21) reported that the inappropriate administration of GH after traumatic injury may result in increased mortality. Studies that increase our knowledge of GH action in peripheral tissues are therefore of both clinical and biochemical importance.

The current experiments were designed to test whether C2C12 cells respond to GH. As such C2C12 cells might be a useful model system for determining the signal transduction pathway(s) by which GH increases the expression of IGF-I in myocytes. In addition, these cells might be useful for determining how cytokines regulate IGF-I mRNA expression. In the present report we show that C2C12 myoblasts respond to GH with an increase in the expression of IGF-I mRNA and Stat3 and Stat5 phosphorylation. C2C12 cells respond best to GH when it is supplied in a pulsatile fashion. The ability of WHI-P154 (a JAK inhibitor) to block GH-induced expression of IGF-I mRNA and Stat3 phosphorylation suggests that JAKs and Stats may be important intermediaries in the GH-induced expression of IGF-I mRNA in C2C12 myoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The C2C12 mouse myoblast cell line was purchased from American Type Culture Collection (Manassas, VA) and used for all studies. Cells were grown in 100-mm petri dishes (Becton Dickinson and Co., Franklin Lakes, NJ) and cultured in MEM containing 5% newborn calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin (25 µg/ml; all from Sigma, St. Louis, MO). Cells were grown to confluence and switched to serum-free MEM for some experiments, in particular those experiments examining the phosphorylation of JAK and Stat proteins. Experiments were performed with recombinant human GH (Serono, Norwell, MA), IGF-I (Genentech, Inc., South San Francisco, CA), PD98059 (20 µM), wortmannin (100 nM), WHI-P154 (1–50 µM; Calbiochem, La Jolla, CA), genistein, AG-490, and an Src family kinase inhibitor developed by Pfizer Pharmaceutical Corp. (PP1; BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA). In some cases inhibitors were added for prolonged periods of time (AG-490, 16 h) or in repetitive doses (wortmannin) to maximize their inhibitory effects. Cell extracts were prepared directly in gel electrophoresis sample buffer unless otherwise noted. Nuclear extracts were prepared by the method of Andrews et al. (22) and were subsequently mixed with sample buffer.

Western blot analysis
Cell extracts were electrophoresed on denaturing polyacrylamide gels and electrophoretically transferred to nitrocellulose with a semidry blotter (Bio-Rad Laboratories, Inc., Melville, NY). The resulting blots were blocked with 5% nonfat dry milk for 1.5 h and incubated with antibodies against either phosphorylated or total ERK, Stat3, Stat5, or phosphotyrosine (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or the GHR (gift from Dr. B. Baumbach, American Cyanamid, Wayne, NJ). Some blots were probed with an antibody against a JAK2 phosphopeptide (Upstate Biotechnology, Inc., Lake Placid, NY). Unbound primary antibody was removed by washing with Tris-buffered saline containing 0.05% Tween 20, and blots were incubated with antirabbit or antimouse Ig conjugated with horseradish peroxidase. Blots were briefly incubated with the components of an enhanced chemiluminescent detection system (Amersham Pharmacia Biotech, Little Chalfont, UK). Dried blots were used to expose x-ray film for 1–3 min.

Immunoprecipitation
Cells were solubilized in HEPES buffer containing 0.1% Triton X-100, 0.1% SDS, and a cocktail of protease and phosphatase inhibitors (Sigma) and were clarified by centrifugation for 15 min at 10,000 x g. The antigens of interest were immunoprecipitated from cell extracts using an excess of precipitating antibody by rotating the sample at 4 C for 12 h. Antibody-antigen complexes were washed and captured with magnetic beads that had been coated with either antimouse or antirabbit Ig (BioMag, Polysciences, Warrington, PA) using a magnetic stand. Protein was displaced from the beads by boiling in electrophoresis sample buffer.

RNA isolation and Northern blotting
Total RNA, DNA, and protein were extracted from C2C12 cells in a mixture of phenol and guanidine thiocyanate (Tri-Reagent, Molecular Research Center, Inc., Cincinnati, OH) using the manufacturer’s protocol. RNA was separated from protein and DNA by the addition of bromocholoropropane and precipitation in isopropanol. Total RNA (25 µg) was electrophoresed under denaturing conditions on a 1.1% agarose gel containing 6% formaldehyde. RNA was transferred to a Nytran Supercharge membrane (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH) using the Turbo Blotting System. After baking, blots were hybridized at 42 C in ULTRAhyb (Ambion, Inc., Austin, TX). The membrane was probed with a 325-bp rat IGF-I cDNA (Peter Rotwein, Portland, OR) that was labeled with [{alpha}-32P]dATP (Amersham Pharmacia Biotech, Arlington Heights, IL) using a random primed DNA labeling kit (Roche, Indianapolis, IN). For normalization of RNA loading, an oligonucleotide directed against rat 18S RNA was radioactively labeled with [{gamma}-32P]ATP (Amersham Pharmacia Biotech) using terminal deoxynucleotidyl transferase. All membranes were washed twice in 2x SSC/0.1% SDS at 42 C for 15 min, followed by an additional wash at 62 C. Membranes were exposed to a PhosphorImager screen (Molecular Dynamics, Inc., Sunnyvale, CA), and the resultant data were quantitated using ImageQuant software.

Statistics
Values are the mean ± SEM. Unless otherwise noted, each experimental condition was tested in sets of six, and each experiment was repeated three times. Data were analyzed by ANOVA, followed by Student-Newman-Keuls test. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH dose- and time-dependently increases IGF-I mRNA expression and Stat activation
GH is a potent stimulus for IGF-I synthesis and secretion in the liver, but less is known about the regulation of IGF-I mRNA in skeletal muscle. We have examined the ability of GH to increase IGF-I mRNA in C2C12 myoblasts. GH increased IGF-I mRNA expression in a time- and dose-dependent manner. GH elevated IGF-I mRNA levels in as little as 1 h, and IGF-I mRNA levels remained elevated for at least 48 h compared with cells grown in the absence of GH (Fig. 1Go, A and B). IGF-I mRNA levels also increased slightly, but significantly, at 24 and 48 h in control cultures compared with the time zero control. Maximal stimulation by GH occurred at a dose of 7.5 ng/ml or more, with an ED50 of 3 ng/ml (Fig. 1CGo). The growth of C2C12 cells to confluence in 5% serum resulted in cultures that contained approximately 98% of the cells as myoblasts. GH treatment did not alter either the number of cells or the number of myotubes in these cultures. It is therefore unlikely that the changes in IGF-I gene expression are secondary to changes in either proliferation or differentiation of the cultures.



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  		Figure 1. Effect of GH on IGF-I mRNA abundance in C2C12 myoblasts. C2C12 myoblasts were grown in 100-mm petri dishes in MEM containing 5% newborn calf serum. After reaching confluence cells were stimulated with 100 ng/ml recombinant human GH or saline for 4, 8, 12, 24, or 48 h. RNA was isolated from triplicate plates and run on a denaturing gel for Northern blotting. The resulting blots were exposed to a PhosphorImager screen and quantified with ImageQuant software. A representative blot is shown in A and quantified in B. Additional cells were treated with increasing concentrations of GH (0.01–100 ng/ml) and isolated after 48 h. IGF-I mRNA was determined as described above. All data are normalized for 18S mRNA as described in Materials and Methods. Values are the mean ± SEM. b, Significantly different from time-matched control value (P < 0.001); c, significantly different than time zero control value (P < 0.05). C, Values with different letters are significantly different from each other.

 
Binding of GH to its cognate receptor triggers receptor dimerization and phosphorylation of Stat5 in hepatocytes, and this represents an early event in the activation of GH-responsive genes (15). In C2C12 myoblasts Stat5 exists in a preformed complex with proteins that were immunoprecipitated with an antiphosphotyrosine antibody (Fig. 2AGo). GH treatment slowed the migration of Stat5 consistent with a change in its phosphorylation status. GH increased the phosphorylation of Stat5 on tyrosine 694, as detected by a phosphospecific antibody. The GHR also became phosphorylated after treatment of C2C12 myoblasts with GH (Fig. 2BGo). Phosphorylation of Stat3 and Stat5 was most efficient in cells that had been withdrawn from serum for at least 2 h, whereas GH was less effective at stimulating Stat phosphorylation in cells that had been exposed to serum for the same amount of time (data not shown). Preincubation of GH with a 5% solution of calf serum for 1 h did not significantly alter the ability of GH to stimulate Stat3 or Stat5 phosphorylation or the total amount of these transcription factors in cell extracts (Fig. 2CGo). Serum by itself also did not alter the basal expression or phosphorylation of Stat3 and Stat5.



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  		Figure 2. Effect of GH on GHR and Stat5 phosphorylation. C2C12 myoblasts were grown as described in Fig. 1Go and subsequently switched to serum-free medium for 2 h. Cells were stimulated with GH (100 ng/ml) for 10 min and isolated either in immunoprecipitation buffer or directly in gel electrophoresis sample buffer. Cell extracts were immunoprecipitated with an antiphosphotyrosine antibody and run on an SDS-PAGE gel for Western blotting for Stat5 (A, top blot). Cell extracts were also probed for pStat5 with a phosphospecific antibody against tyrosine 694 on Stat5 (A, middle blot). An additional blot against total Stat5 in the cell extracts was also performed (A, bottom blot). Phosphotyrosine immunoprecipitates were also probed for GHR, as shown in B. C, Cells were grown in serum-free medium and treated with either GH alone or GH that was preincubated with 5% bovine serum for 1 h. Cell extracts were prepared and probed for pStat5, total Stat5, pStat3, and total Stat3, respectively.

 
C2C12 myoblasts responded to either human or rat GH. Human GH was slightly more potent than rat GH at stimulating the phosphorylation of Stat3 and Stat5 (Fig. 3AGo). The phosphorylation of Stat3, Stat5, and the MAPKs, ERK1 and -2, was transient and exhibited a maximal response approximately 10 min after application of GH to the cells (Fig. 3BGo).



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  		Figure 3. GH stimulates Stat phosphorylation dose- and time-dependently. C2C12 myoblasts were grown as described in Fig. 2Go and exposed to an increasing concentration of either human (H) or rat (R) GH. Cell extracts were isolated after 10 min and run on an SDS-PAGE gel. Western blots were probed with specific antibodies to either pStat5 (Tyr694) or pStat3 (Tyr705) in A, top and bottom blots, respectively. Cells were also isolated at points between 5 and 120 min of hGH treatment (100 ng/ml) and examined for pStat5 (B, top blot), total Stat5 (B, second blot), pStat3 (B, third blot), and pERK (B, fourth blot).

 
C2C12 myoblasts respond optimally to pulsatile GH, and IGF-I mRNA expression requires ongoing transcription and translation
GH is secreted from the pituitary in a pulsatile fashion, and many hepatic genes require a pulsatile exposure to GH for optimal expression (23). We examined the ability of GH applied in a pulsatile or continuous fashion to stimulate Stat3 and Stat5 phosphorylation in C2C12 cells. Cells exposed to GH continuously were unable to respond to a subsequent short exposure to GH (Fig. 4AGo). If the cells were allowed to recover for 2 h in serum-free/GH-free medium, they partially regained their responsiveness to GH. A maximal response to GH was attained in cells that were allowed to recover for 6–8 h. Stat3 exhibited a higher basal level of phosphorylation than Stat5. Yet, both proteins responded to GH stimulation more efficiently in cells that were allowed to recover from a previous GH exposure. The refractory phenomena occurred without an alteration in the concentration of total Stat3 or Stat5 in the cells. Likewise, the concentration of one suppressor of cytokine signaling (SOCS) protein SOCS-3, a known Stat5 inhibitor, was not altered by either continuous or pulsatile exposure to GH (Fig. 4AGo).



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  		Figure 4. Importance of GH pulsatility in stimulating Stat5 phosphorylation and IGF-I mRNA expression. C2C12 myoblasts were grown as described in Fig. 2Go, treated with GH for 10 min, and either isolated immediately (A, GH 10 min, 1 C) or treated continuously (C) for 2, 4, 6, or 8 h and then isolated after an additional 10-min exposure to GH (A). Some cells were treated with GH for 10 min and had their media replaced with fresh media. The cells were subsequently restimulated after 2, 4, 6, or 8 h of recovery (R) with a second pulse of GH. All extracts were run on SDS-PAGE gels and probed for pStat5, total Stat5, pStat3, total Stat3, or SOCS-3 (A, top to bottom blots, respectively). Additional cells were treated either continuously with GH in serum-free media or pulsed with GH for 10 min with an 8-h interpulse interval and a second 10-min GH pulse. RNA from cells receiving two or five such pulses was isolated 8 h after the last GH exposure and run on an agarose gel for Northern blotting as described in Fig. 1Go. A PhosphorImager analysis of the resulting blot was quantified and is plotted in B. All data are normalized for 18S mRNA as described in Materials and Methods. Values are the mean ± SEM. Bars marked with different lowercase letters are significantly different (P < 0.01). An outline of the GH pulse design is shown in C.

 
When cells were grown under serum-free conditions, continuous exposure to GH did not increase IGF-I mRNA expression and thus differed from cells grown in the presence of serum and GH (compare Figs. 1Go and 4Go). Yet, cells that were grown in serum-free medium that were pulsed with GH for 10 min, followed by an 8-h recovery and an additional GH exposure (i.e. two pulses), expressed significantly more IGF-I mRNA than cells continuously exposed to GH (Fig. 4BGo). Likewise, cells receiving five pulses of GH, as outlined in Fig. 4CGo, expressed significantly more IGF-I mRNA than cells receiving only two pulses (Fig. 4BGo).

GH has been shown to increase hepatic expression of IGF-I mRNA via a transcriptional mechanism (24). We examined whether 5,6-dichloro-ß-D-ribofuranosyl-benzimidazole (DRB) or cycloheximide could block the ability of GH to increase IGF-I mRNA abundance. GH treatment for 24 h doubled the expression of IGF-I mRNA, and this response was completely inhibited by DRB (Fig. 5AGo). Cycloheximide also blocked the increase in IGF-I mRNA content in response to GH, suggesting that IGF-I mRNA expression required both ongoing protein synthesis and gene transcription (Fig. 5BGo). Paradoxically, both DRB and cycloheximide increased IGF-I mRNA expression over a 24-h period, suggesting the presence of a labile protein factor that may regulate the stability of IGF-I mRNA.



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  		Figure 5. GH-inducible IGF-I mRNA expression requires ongoing transcription and translation. C2C12 myoblasts were grown as described in Fig. 1Go and treated with either DRB (A) or cycloheximide (B) for 30 min. Cells were subsequently treated with GH for 16 h. RNA was isolated and run on an agarose gel for Northern blotting as described in Fig. 1Go. A PhosphorImager analysis of the resulting blot was quantified and is plotted in A and B. All data are normalized for 18S mRNA as described in Materials and Methods. Values are the mean ± SEM. Bars marked with different lowercase letters are significantly different (P < 0.01).

 
Stats are also phosphorylated on carboxyl-terminal serine residues, and this enhances their ability to activate transcription in some cell types. As GH can activate MAPKs, such as ERK-1 and -2 (25) (see Fig. 3Go), we examined whether ERK activity was necessary for GH-stimulated IGF-I mRNA expression. GH stimulated the phosphorylation of ERK in C2C12 cells, and this response was inhibited by PD98059 (Fig. 6AGo). In contrast, PD98059 did not alter the tyrosine phosphorylation of either Stat3 or Stat5, suggesting that the ERK pathway does not regulate GH signaling at this level. GH stimulated IGF-I mRNA expression 4-fold, and the MAPK kinase (MEK) inhibitor PD98059 did not block this expression (Fig. 6BGo). Indeed, IGF-I mRNA expression was moderately increased by PD98059 alone.



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  		Figure 6. GH-inducible Stat phosphorylation and IGF-I mRNA expression are unaltered by ERK inhibition. Cell extracts were isolated from cells that had been pretreated with PD98059 for 30 min and then stimulated with GH for 10 min. After SDS-PAGE and transfer to nitrocellulose, the resulting blot was probed for phosphorylated ERK (A, top blot), pStat3 (A, second blot), pStat5 (A, third blot), and total Stat5 (A, fourth blot). Cells were also pretreated with PD98059 for 30 min and subsequently treated with GH for 16 h. RNA was isolated and run on an agarose gel for Northern blotting. A PhosphorImager analysis of the resulting blot was quantified, and data are plotted in B. All data are normalized for 18S mRNA as described in Materials and Methods. Values are the mean ± SEM. Bars with different lowercase letters are significantly different (P < 0.01).

 
JAK inhibition blocks GH-induced Stat3 phosphorylation, Stat3 nuclear transport, and IGF-I mRNA expression
Stat3 and Stat5 are both known substrates of JAK2 (26). In turn, JAK2 activation has been shown to rely on Src family kinases that create a docking site for JAK2 on the GHR (27). We have examined the importance of these kinases in the subsequent phosphorylation of Stat3 and Stat5 with specific tyrosine kinase inhibitors. AG-490, a JAK2 inhibitor, had no effect on Stat3, Stat5, or ERK1 and -2 phosphorylation in C2C12 cells at doses up to 100 µM (Fig. 7AGo). Yet, AG-490 also failed to inhibit JAK2 phosphorylation in response to GH, suggesting that this inhibitor is ineffective under the conditions we have examined. Similarly, both genistein and the Src family kinase inhibitor PP1 were without effect on Stat phosphorylation (Fig. 7BGo). Despite the lack of an effect on Stat3 and Stat5, the Src inhibitor did block the ability of GH to stimulate phosphorylation of JAK2. In addition, genistein paradoxically increased phosphorylation of JAK2 under conditions in which neither Stat3 nor Stat5 became phosphorylated. Collectively, the data suggest that tyrosine kinases in addition to (or other than) JAK2 may be involved in the phosphorylation of Stat3 and Stat5 in C2C12 myoblasts. We next tested a potent inhibitor of JAK3 (WHI-P154) to determine whether it could block the ability of GH to stimulate Stat3 and Stat5 phosphorylation. WHI-P154 selectively blocked the ability of GH to stimulate Stat3 phosphorylation, but not Stat5 (Fig. 7CGo). This occurred under conditions where the total amount of Stat3 and Stat5 remained unchanged. WHI-P154 also blocked JAK2 phosphorylation, as detected by a phosphospecific JAK2 antibody. WHI-P154 inhibited Stat3 phosphorylation dose dependently, with an ED50 of 8 µM (Fig. 8Go, A and B). GH stimulated the transport of both Stat3 and Stat5 into a nuclear fraction (Fig. 8CGo). WHI-P154 minimized the GH-inducible nuclear transport of Stat3, but failed to exclude Stat5 from the nucleus. Most importantly, WHI-P154 prevented the GH-induced increase in IGF-I mRNA abundance over a similar concentration range to that observed for the inhibition of Stat3 phosphorylation (Fig. 8DGo).



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  		Figure 7. JAK inhibition blocks GH-inducible Stat3 phosphorylation. C2C12 myoblasts were grown as described in Fig. 2Go and pretreated with 100 µM AG-490 for 15 h (A) or with genistein and PP1 for 30 min (B). Cells were subsequently treated with GH for 10 min and isolated in gel electrophoresis sample buffer. Western blots were probed for pJAK2, pStat5, pStat3, or pERK (A and B, top to bottom blots, respectively). Some cells were also pretreated with WHI-P154 for 30 min and subsequently stimulated with GH for 10 min. Extracts from these cells were probed for pStat3, total Stat3, pStat5, total Stat5, pJak2, total JAK2, pJAK3, and total JAK3, respectively (C).

 


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  		Figure 8. JAK inhibition regulates GH action. C2C12 myoblasts were treated with GH and an increasing concentration of WHI-P154 as described in Fig. 7Go. Cell extracts were subsequently probed for pStat3, total Stat3, and pStat5. Western blots are shown in A and are quantified in B. Some cells were treated as described above and fractionated to obtain nuclear extracts as described in Materials and Methods. Nuclear extracts were probed for total Stat3, pStat3, total Stat5, and pStat5, respectively (C). Additional cells were pretreated with an increasing concentration of JAK3 inhibitor and GH for 16 h. RNA from these cells was isolated and run on an agarose gel as described in Fig. 1Go. The resulting Northern blot was quantified, and the data are presented in D. All data are normalized for 18S mRNA as described in Materials and Methods. Values are the mean ± SEM. Bars with different lowercase letters are significantly different (P < 0.01).

 
IGF-I inhibits expression of its own mRNA
The hepatic expression of IGF-I mRNA in hypophysectomized rats is down-regulated by IGF-I peptide (28). We examined whether IGF-I also modulates the abundance of its own mRNA in C2C12 cells. IGF-I decreased IGF-I mRNA levels dose-dependently, with an ED50 of 5 ng/ml (Fig. 9AGo). Insulin (10 nM) acted similarly to IGF-I in C2C12 cells (data not shown). The effect of IGF-I was dominant over that of GH. In GH-stimulated cells IGF-I decreased IGF-I mRNA to control levels within 4 h, and the continued presence of IGF-I decreased IGF-I mRNA levels below basal (Fig. 9BGo). The ability of IGF-I to down-regulate its own mRNA was blunted by the PI3K inhibitor, wortmannin (Fig. 10AGo). Wortmannin also enhanced the ability of GH to stimulate IGF-I mRNA expression (data not shown). The MEK inhibitor, PD98059, stimulated IGF-I mRNA expression as shown previously (Figs. 6BGo and 10BGo), but failed to block the ability of IGF-I to down-regulate its own mRNA. The effectiveness and specificity of the above inhibitors were tested by probing C2C12 extracts for the phosphorylation of threonine 308 on Akt and threonine 389 on p70S6 kinase after exposure of cells to IGF-I. These two kinases have been shown to lie downstream of PI3K. IGF-I stimulated the phosphorylation of both Akt and p70S6 kinase, and both events were blocked by wortmannin (Fig. 10CGo). PD98059 failed to block the phosphorylation of either kinase, and the inhibitory effect of a combination of the two compounds was not necessarily greater than wortmannin alone over a 1-h time frame. IGF-I also stimulated the phosphorylation of insulin receptor substrate-1 (IRS-1) on tyrosine, and this was not inhibited by either wortmannin or PD98059. This is consistent with IRS-1 phosphorylation preceding the activation of PI3K in IGF-I signal transduction and not being sensitive to wortmannin. A combination of wortmannin and PD98059 stimulated IGF-I mRNA expression, much like PD98059 alone (Figs. 10BGo and 11AGo). The combination of wortmannin and PD98059 was more effective than wortmannin alone in preventing IGF-I mRNA from being down-regulated by IGF-I peptide.



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  		Figure 9. IGF-I inhibition of IGF-I mRNA accumulation. C2C12 myoblasts were grown in the presence of an increasing concentration of IGF-I, and RNA was isolated after 16 h. The resulting Northern blot was quantified, and the data are plotted in A. Alternatively, cells were stimulated with GH for 24 h and treated with IGF-I (75 ng/ml), and RNA was isolated after 2, 4, and 8 h (B). The resulting Northern blots were quantified, and the data are plotted. All data are normalized for 18S mRNA as described in Materials and Methods. Values are the mean ± SEM. Bars with different lowercase letters are significantly different (P < 0.01).

 


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  		Figure 10. Wortmannin restores IGF-I mRNA expression. C2C12 cells were pretreated with wortmannin (WM; 100 nM) or PD98059 (20 µM) for 30 min and subsequently stimulated with IGF-I. RNA was isolated after 16 h. The resulting Northern blots were quantified, and the data are plotted in A and B. All data are normalized for 18S mRNA as described in Materials and Methods. The ability of wortmannin and PD98059 to inhibit IGF-I dependent phosphorylation of threonine 308 on Akt, threonine 389 on p70S6 kinase, and phosphotyrosine on IRS-1 was evaluated by Western blotting (C). Values are the mean ± SEM. Bars with different lowercase letters are significantly different (P < 0.01).

 


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  		Figure 11. Repetitive application of wortmannin, but not rapamycin, restores IGF-I mRNA expression. C2C12 cells were pretreated with a combination of wortmannin and PD98059 for 30 min, followed by IGF-I. RNA was isolated after 16 h. The resulting Northern blot was quantified, and the data are plotted in A. C2C12 cells were also grown in the presence (+) or absence (-) of wortmannin and stimulated with IGF-I 2, 4, 8, or 13 h later. Cells extracts were probed for pAkt (B). Cells were also treated with either a single dose of wortmannin (1x) or three repetitive doses at 3-h intervals (3x). RNA was isolated, the resulting Northern blot was quantified, and the data are plotted in C. Some cells also were pretreated with rapamycin (Rap, 10 ng/ml) alone or in combination with either GH or GH and IGF-I. IGF-I mRNA was analyzed as described above (D).

 
As wortmannin is unstable in aqueous solutions (29), we examined its ability to inhibit Akt phosphorylation as a function of time. Wortmannin blocked the ability of IGF-I to stimulate Akt phosphorylation for up to 4 h after being added to C2C12 cells (Fig. 11BGo). Its effectiveness waned, such that cells treated with wortmannin for 8 or 13 h responded to IGF-I as if they had not been exposed to wortmannin. Based on the above, C2C12 cells were given wortmannin every 3 h to prevent IGF-I from activating PI3K. This protocol completely blocked the ability of IGF-I to down-regulate its own mRNA (Fig. 11CGo).

The mammalian target of rapamycin lies downstream of PI3K, and Stat3 is maximally activated in cells treated with ciliary neurotrophic factor via a rapamycin-sensitive pathway (30). We examined whether rapamycin could block the effect of either GH or IGF-I on IGF-I mRNA expression. We found that rapamycin had no effect on the ability of GH to stimulate IGF-I mRNA expression (Fig. 11DGo). Rapamycin also did not block the ability of IGF-I to down-regulate its own mRNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that GH stimulates the expression of IGF-I mRNA in C2C12 myoblasts. Although GH has been shown to increase IGF-I mRNA in primary cultures of hepatocytes and in rat skeletal muscle in vivo, this is one of the first studies demonstrating that GH can also increase IGF-I mRNA in the C2C12 cell line (31). C2C12 myoblasts express endogenous GHR, JAK2, JAK3, Stat3, and Stat5, and most of these proteins become phosphorylated in response to GH. WHI-P154, a JAK inhibitor, blocks the ability of GH to stimulate both Stat3 phosphorylation and IGF-I mRNA expression over the same concentration range. In addition, WHI-P154 minimizes the nuclear accumulation of Stat3 in response to GH. Collectively, these data suggest that Stat3 may play an important role in the expression of IGF-I mRNA in myoblasts.

Stat3 and Stat5 become phosphorylated on tyrosine residues within minutes of exposing C2C12 cells to GH, and both proteins are rapidly dephosphorylated thereafter. Stat5 exists in a preformed complex with other proteins that are phosphorylated on tyrosine, and upon GH exposure Stat5 is phosphorylated on tyrosine 694. Continuous exposure to GH makes C2C12 cells resistant to subsequent stimulation of Stat5 phosphorylation. If the cells are allowed to recover under serum-free/GH-free conditions for 4–8 h, they regain their ability to be stimulated by GH. The refractory period in C2C12 cells is therefore similar to that which has been observed in the hepatic CWSV-1 cell line (32). One possible explanation for the phenomena would be an increased expression of a SOCS protein such as SOCS3. SOCS3 mRNA and protein are rapidly expressed in response to GH. In addition, SOCS3 is a potent intracellular inhibitor of GH action in hepatocytes (33). In contrast to hepatocytes, GH had no effect on the expression of SOCS3 protein in C2C12 cells. This suggests that another mechanism must explain the GH refractoriness of C2C12 myoblasts. Such mechanisms may include GHR down-regulation, inhibition by another suppressor of cytokine signaling such as the cytokine-inducible SH2 protein (CIS) (34), or the induction of a tyrosine phosphatase that inactivates the JAK/Stat pathway. The application of GH in 10-min pulses with an 8-h interpulse interval allowed for progressively more IGF-I mRNA to be expressed with each subsequent pulse. Pulsatile exposure to GH in this system may more accurately mimic in vivo conditions (35).

The manner in which C2C12 cells are exposed to GH seems to be critical for observing a maximal response to the hormone. Cells grown in the absence of serum respond to GH with a more robust stimulation of Stat3 and Stat5 phosphorylation. GH-binding proteins in serum do not alter this effect, as preincubation of GH with 5% bovine calf serum did not inhibit Stat phosphorylation. Paradoxically, cells grown in the presence of serum respond better than cells grown in serum-free medium to a single application of GH when measuring the expression of IGF-I mRNA. This suggests that accessory factors in serum may enhance the ability of GH to stimulate IGF-I mRNA expression. Rycyzyn et al. (36, 37) have made similar observations in GH- and PRL-dependent cells lines where cyclophilin B enhances proliferation in response to both hormones. As C2C12 cells respond to pulsatile GH exposure in serum-free medium, this mode of delivery may negate the need for accessory serum factors. IGF-I mRNA expression or GH responsiveness may also vary at different stages of the cell cycle. Continuous exposure to GH may also increase IGF-I peptide to a level that becomes inhibitory for subsequent IGF-I mRNA expression. IGFBPs in serum would be expected to blunt this effect, whereas serum-free medium would not. Additional experiments are necessary to delineate components of serum that both positively and negatively regulate IGF-I mRNA expression in C2C12 cells.

The ability of GH to stimulate IGF-I mRNA expression was abrogated in the presence of either the RNA polymerase II inhibitor DRB or the protein synthesis inhibitor cycloheximide. This suggests that GH-inducible IGF-I mRNA expression requires both ongoing protein synthesis and transcription. Thus, Stat activation on its own may not be sufficient to activate IGF-I gene transcription, but may require the synthesis of an intermediary transcription factor. Indeed, the CCAAT/enhancer binding protein-{delta} (C/EBP{delta}) has been shown to be required for transcriptional activation of the IGF-I gene in osteoblasts in response to PGE2 (38).

WHI-P154 blocked nuclear accumulation of Stat3, but not Stat5, in response to GH. This suggests that the binding of Stat3 to either the IGF-I promoter or the promoter of an intermediary transcription factor may be important for the subsequent induction of IGF-I mRNA. More importantly, the JAK3 inhibitor also blocked IGF-I mRNA expression in C2C12 cells. The mouse IGF-I gene has been reported to have an interferon-{gamma} regulatory element, but the importance of this element has not been determined (39). It is also possible that a GH-inducible Stat transcription factor binds to and activates the promoter of a secondary transcription factor such as C/EBP{delta}, which, after its transcription and translation, can activate IGF-I transcription. Hutt et al. (40) have recently shown that Stat3 activates C/EBP{delta} gene transcription in mouse mammary epithelial cells, and C/EBP{delta} is a critical regulator of IGF-I gene transcription in osteoblasts (41). Although C2C12 myoblasts share many transcription factors with osteoblasts and hepatocytes, further experiments are necessary to test their GH responsiveness at the transcriptional level. In particular, experiments in which an IGF-I reporter gene construct is used to test whether the IGF-I gene is directly stimulated by either Stats or C/EBPs in C2C12 cells would be informative.

Paradoxically, both cycloheximide and DRB stimulated the accumulation of IGF-I mRNA on their own. Cycloheximide has previously been shown to increase IGF-I mRNA abundance in osteoblasts (42), C6 glioma cells (43), and U937 cells ( 44). Cycloheximide treatment may regulate a labile protein factor that normally functions to degrade IGF-I mRNA. Other messages, such as IGFBP-1 mRNA, can also be stabilized by cycloheximide treatment, a phenomenon that can be attributed to multiple AUUUA motifs found in the 3'-untranslated region of IGFBP-1 mRNA (45). Alternatively, cycloheximide has been shown to prolong the phosphorylation of JAK-1, -2, and -3 and Stat5, and this response may explain the ability of cycloheximide to enhance IGF-I mRNA expression (32, 46).

The ability of C2C12 cells to respond to GH appears to differ from that of other cell types. A tyrphostin-based JAK2 inhibitor was ineffective at inhibiting GH-stimulated JAK2, Stat3, and Stat5 phosphorylation. This occurred despite attempts to maximize the effectiveness of AG-490 by working with the compound at high doses (up to 200 µM) for a variety of preincubation times (30 min to 16 h) and handling this light-sensitive compound in the dark. To further define what kinases might regulate Stat3 and Stat5 phosphorylation, we tested whether PP1 (a Src family kinase inhibitor) and genistein (a general tyrosine kinase inhibitor) were able to block GH-induced phosphorylation of Stat3 and Stat5. Neither inhibitor blocked GH-induced changes in Stat phosphorylation. Paradoxically, genistein increased the phosphorylation of JAK2 without altering Stat3 or Stat5 phosphorylation. PP1 also blocked JAK2 phosphorylation without altering GH-induced Stat3 and -5 phosphorylation. Both observations suggest that the phosphorylation state of JAK2 as a putative upstream kinase does not necessarily correlate with the phosphorylation of Stat3 and Stat5 as two potential substrates.

In a test of other JAKs we found that a JAK3 inhibitor (WHI-P154) blocked the ability of GH to stimulate Stat3 phosphorylation. This effect was specific, as Stat5 phosphorylation was unaffected. WHI-P154 also blocked JAK2 phosphorylation, as detected by an antibody raised against the JAK2 autophosphorylation site. From our experiments, it is not clear whether WHI-P154 inhibits JAK2 autophosphorylation or the ability of JAK3 or another kinase to transphosphorylate JAK2. A limitation of these studies is that the mouse JAK2 and JAK3 phosphorylation sites share some homology and may not be differentiated by this antibody. For example, we can detect both JAK2 and JAK3 in immunoprecipitates made with the pJAK2 antibody. In addition, although the class of inhibitors to which WHI-P154 belongs shows a greater ability to inhibit JAK3 than JAK2 in vitro, it is still possible that WHI-P154 may not be completely specific and thereby inhibit JAK2 in C2C12 cells. Lastly, autophosphorylation of JAK3 is a complex event in which tyrosine 980 is critical for phosphorylation of its neighboring tyrosine (Y981) (47). As Y981 acts as a negative regulatory site, alterations in the total phosphotyrosine content of JAK3 are a poor surrogate marker for the activity of the enzyme. We observed that C2C12 myoblasts express JAK3, but that the total phosphotyrosine content of JAK3 does not change in cells treated with either GH or WHI-P154. Experiments that specifically eliminate JAK2 or JAK3 by either an antisense RNA approach or expression of a dominant negative kinase are necessary to delineate whether either kinase is necessary for Stat3 phosphorylation and IGF-I mRNA expression.

While this paper was under review, Sadowski et al. (31) also reported that GH stimulates Stat5a and Stat5b phosphorylation and IGF-I mRNA expression in C2C12 cells. These researchers likewise were unable to block IGF-I mRNA expression with either wortmannin or PD98059. They also demonstrated that GH could activate a Stat5 reporter construct in C2C12 cells, but it is not clear whether Stat5 alone is sufficient to activate the IGF-I promoter in myoblasts.

We have found that IGF-I peptide negatively regulates IGF-I mRNA expression in C2C12 cells as it does in liver. The effect of IGF-I on IGF-I mRNA expression is dominant over that of GH. This may be a circumstance where overexpression of IGF-I can be prevented via a negative feedback loop. The ability of IGF-I to down-regulate its own mRNA is PI3K dependent. A single dose of wortmannin blunted the ability of IGF-I to down-regulate its own mRNA by 30%, whereas PD98059 was ineffective. A combination of wortmannin and PD98059 was more effective than either inhibitor alone, but PD98059 had a significant stimulatory effect on IGF-I mRNA by itself. In short-term experiments wortmannin blocked the phosphorylation of Akt and p70S6 kinase, two kinases that lie downstream of PI3K. Yet, the inhibitory effect of wortmannin waned with time. When cells were given multiple doses of wortmannin at 3-h intervals, wortmannin completely blocked the ability of IGF-I to down-regulate its own mRNA.

Although GH stimulates both IRS-1 phosphorylation and PI3K activity (48), wortmannin did not block the ability of GH to stimulate IGF-I expression. This is consistent with the inability of wortmannin to inhibit IGF-I expression in primary cultures of rat hepatocytes, but differs from the effect of a second PI3K inhibitor, LY294001, on IGF-I mRNA expression (49). Rapamycin, which inhibits the mammalian target of rapamycin, also failed to antagonize the abilities of GH and IGF-I peptide to alter IGF-I mRNA expression. It is not clear how IGF-I down-regulates IGF-I mRNA, but our preliminary data suggest that IGF-I pretreatment blunts the ability of GH to stimulate Stat3, but not Stat5, phosphorylation. IGF-I may decrease JAK3 activity or increase the activity of a phosphatase that can dephosphorylate Stat3. Additional studies will be necessary to ascertain whether IGF-I changes the half-life of IGF-I mRNA or directly affects IGF-I gene expression.

In summary, the results of the present study indicate that C2C12 myoblasts are a good model for examining the effects of GH and IGF-I on IGF-I mRNA expression in a physiologically relevant cell type. These cells contain endogenous GH receptors and JAK-Stat proteins and therefore facilitate studies without the need for exogenous expression of the pathway components. Although both Stat3 and Stat5 become phosphorylated in response to GH, they appear to be regulated by different kinases. Phosphorylation of Stat3 is inhibited by WHI-P154, whereas Stat5 phosphorylation is not. As WHI-P154 also blocks the ability of GH to stimulate IGF-I mRNA expression, it is likely that Stat3 plays a critical role in the regulation of either the IGF-I gene or the expression of an intermediary transcription factor that regulates IGF-I transcription. IGF-I has a dominant effect over GH and negatively regulates the expression of its own mRNA. The JAK/Stat pathway and IGF-I mRNA expression in C2C12 cells therefore share some common signaling intermediates with hepatocytes, but also exhibit regulatory features that may be unique to myoblasts.


   Acknowledgments
 
We thank the National Pituitary Program and the NIDDK for providing rat GH, and Genentech, Inc. for providing recombinant human IGF-I.


   Footnotes
 
This work was supported in part by NIH Grants GM-38032 and AA-11290.

Abbreviations: C/EBP, CCAAT/enhancer binding protein; DRB, 5,6-dichloro-ß-D-ribofuranosyl-benzimidazole; GHR, GH receptor; IGFBP, IGF-binding protein; IRS-1, insulin receptor substrate-1; JAK, Janus kinase; MEK, MAPK kinase; SOCS, suppressor of cytokine signaling; Stat, signal transducer and activator of transcription.

Received June 4, 2001.

Accepted for publication October 17, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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