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Endocrinology, doi:10.1210/en.2003-1498
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Endocrinology Vol. 145, No. 6 2824-2832
Copyright © 2004 by The Endocrine Society

Suppression of Growth Hormone (GH) Janus Tyrosine Kinase 2/Signal Transducer and Activator of Transcription 5 Signaling Pathway in Transgenic Mice Overexpressing Bovine GH

Johanna G. Miquet, Ana I. Sotelo, Andrzej Bartke and Daniel Turyn

Instituto de Química y Fisicoquímica Biológicas (University of Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas) (J.G.M., A.I.S., D.T.), Facultad de Farmacia y Bioquímica, Junín 956 (1113) Buenos Aires, Argentina; and Department of Geriatrics (A.B.), School of Medicine, Southern Illinois University, Springfield, Illinois

Address all correspondence and requests for reprints to: Dr. Daniel Turyn, Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 (1113) Buenos Aires, Argentina. E-mail: dturyn{at}qb.ffyb.uba.ar.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High continuous GH levels in vivo produce desensitization of the Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway of GH signaling in the liver. To evaluate the mechanisms involved in this desensitization, transgenic mice overexpressing bovine GH were used. In these animals, GH receptor and membrane-associated JAK2 kinase are increased 4.5- and 6-fold, respectively. However, JAK2. STAT5a and –5b do not become tyrosine phosphorylated in response to GH stimulus, nor are these STAT proteins recruited to membranes, suggesting that they cannot bind to the receptor. The content of the suppressor cytokine-inducible src homology 2 (SH2)-containing protein (CIS), both total and membrane-associated, is markedly increased in the liver of GH transgenic mice. This could account for the inhibition of STAT5 activation, because CIS competes with STAT5 for GH receptor docking sites. Existence of an alternative mechanism of negative regulation of this signaling pathway by chronically elevated GH levels is suggested by the low level of JAK2 phosphorylation that transgenic mice exhibit. Whereas total SH2-containing phosphatase 2 (SHP-2) content is the same in both kinds of mice, membrane-associated SHP-2 protein levels increase 4.5-fold in GH transgenic animals. This could explain the dramatic inhibition of JAK2 phosphotyrosine level, thus contributing to the suppression of GH signaling observed in these transgenic mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS PRODUCED AND SECRETED by cells of the anterior pituitary gland; it exerts growth-promoting and metabolic effects regulating important physiological processes in liver and other tissues. GH signaling is initiated by hormone binding to membrane GH receptor (GHR), inducing its dimerization (1). GHR lacks intrinsic tyrosine kinase activity but is associated with the Janus tyrosine kinase 2 (JAK2) (2). Receptor dimerization leads to autophosphorylation and activation of the associated kinase, which in turn phosphorylates GHR on multiple intracellular tyrosine residues (2, 3, 4). These phosphorylated residues on GHR and JAK2 form docking sites for different intracellular transduction mediators, including signal transducers and activators of transcription (STATs) and those leading to the activation of MAPK and phosphatidylinositol 3'-kinase among others (2, 4, 5). In liver, the transcriptional activators STAT5a and –5b are the most prominent mediators activated by GH. Upon GH stimulus, both STAT5a and –5b bind to the phosphorylated GHR and undergo rapid tyrosine phosphorylation, dimerization, and nuclear translocation, where they promote transcription of different genes (2, 4).

The activation of GH signal transduction also initiates negative modulation and termination pathways, among which phosphotyrosine phosphatases and suppressors of cytokine signaling/cytokine-inducible src homology 2 (SH2)-containing protein (SOCS/CIS) can be included (4, 5, 6). GH signaling may also be terminated by internalization/degradation of receptor complexes (7). The inhibition of GH signaling by SOCS/CIS was described in different cell types and tissues (8, 9, 10, 11, 12, 13). GH induces transcriptional activation of SOCS-1, –2, and –3 as well as CIS, this induction being dependent on activation of STAT proteins (10, 14). SOCS/CIS proteins bind to GHR or JAK2 via their phosphotyrosine binding domain SH2 (src homology 2). SOCS-1 and SOCS-3 inhibit JAK2 kinase activity, whereas CIS competes with STAT5 for receptor docking sites (15). They also have a SOCS box motif that mediates targeting of associated proteins to proteasome degradation (15).

As GH signaling is mediated by JAK2-catalyzed protein tyrosine phosphorylation, another cascade of attenuation/termination comprises different phosphatases, as SH2containing phosphatases 1 and 2 (SHP-1 and SHP-2). Whereas SHP-2 is ubiquitously expressed, SHP-1 is mainly restricted to hematopoietic cells (16). SHP-2 can bind to phosphorylated GHR-JAK2 complexes through its SH2 domain, and it can be phosphorylated by JAK2 as well (17, 18, 19). Although SHP-2 can play a positive role and act as an adaptor protein for the MAPK pathway (17, 18, 20), it also negatively regulates GHR/JAK2/STAT5 signaling (19). SHP-2 is a cytoplasmic enzyme, but its membrane localization is important for its activity (21). Signal-regulatory protein {alpha}1 (SIRP{alpha}1), a receptor-like transmembrane protein constitutively associated with JAK2 (22), may act as a scaffold for SHP-2. In response to GH, JAK2 phosphorylates SIRP{alpha}1 (17). SHP-2 binding to the phosphorylated residues of SIRP{alpha}1 results in its recruitment to the vicinity of the cellular membrane and in the activation of the phosphatase. By suppression of JAK2 activity, the SIRP{alpha}1/SHP-2 complex would negatively regulate GHR/JAK2 signaling (22).

The temporal pattern of plasma GH secretion governs many of the physiological responses to GH (4, 23). The JAK2/STAT5 pathway is down-regulated in hepatocytes continuously exposed to GH (24, 25) as well as in female rats (26, 27), whose GH secretion pattern is frequent and irregular leading to persistent GH levels. Recently, we reported that the JAK2/STAT5 signaling pathway is desensitized in transgenic mice overexpressing GHRH. This lack of response to GH was associated with an 18-fold increase of CIS. This suppressor would compete with STAT5 for the same docking sites on GHR or target the GHR/JAK2 complex to degradation by the proteasome (28). To confirm and extend the knowledge on the effects of high and continuous levels of GH on hepatic GH signaling in vivo, transgenic mice overexpressing bovine GH (bGH) were used. With this model we intend to confirm the relationship between CIS and GH desensitization and to explore the influence of another regulatory protein, the phosphatase SHP-2, in the desensitization of the signal caused by high GH secretion in vivo.

In the current report, the important rise in hepatic CIS levels induced by continuous high GH levels in vivo is confirmed, and a higher binding of CIS to membranes is described. This may allow the formation of GHR-JAK2-CIS complexes instead of GHR-JAK2-STAT5 complexes, which could account for the inhibition of STAT5 binding to GHR and its activation in transgenic animals. Increased SHP-2 binding to microsomes in transgenic mouse liver is also found; this could explain the dramatic inhibition of JAK2 phosphotyrosine level and may contribute to the extinguishing of the GH signal.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
PEPCK-bGH mice containing the bGH gene fused to control sequences of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene were derived from animals kindly provided by Dr. Thomas E. Wagner and Jeung S. Yun (Ohio University, Athens, OH). The hemizygous transgenic mice were derived from a founder male and were produced by mating transgenic males with normal C57BL/6 x C3H F1 hybrid females purchased from the Jackson Laboratory (Bar Harbor, ME). Matings produced an approximately equal proportion of transgenic and normal progeny. Normal siblings of transgenic mice were used as controls. Transgenic animals had markedly accelerated postweaning growth, leading to a significant increase in body weight. Female adult animals (3–6 months old) were used. The mice were housed three to five per cage in a room with controlled light (12 h light per day) and temperature (22 ± 2 C). The animals had free access to food (Lab Diet Formula 5008, containing a minimum of 23% protein and 6.5% fat and a maximum of 4% fiber; Purina Mills Inc., St. Louis, MO) and tap water. The appropriateness of the experimental procedure, the required number of animals used, and the method of acquisition were in compliance with federal and local laws and with institutional regulations.

bGH determination
bGH was determined by RIA as described in Sotelo et al. (29) using a RIA kit obtained through the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD. Ovine GH (oGH) was labeled using limiting amounts of chloramine T; specific activity ranged from 70–120 µCi/µg.

Preparation of liver extracts
The mice were fasted overnight, and then 5 mg of oGH per kg of body weight in 0.2 ml of 0.9% NaCl were injected ip. Normal and transgenic mice were injected with saline to evaluate basal conditions. Mice were killed 7.5 min after GH injection. Livers were then removed and homogenized in 10 vol of solubilization buffer [1% Triton, 100 mM HEPES, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.035 TIU/ml aprotinin (pH 7.4)] at 4 C. Liver homogenates were centrifuged at 100,000 x g for 50 min at 4 C to remove insoluble material. The protein concentration of supernatants was determined by the method of Bradford (30). An aliquot of solubilized liver, diluted in Laemmli buffer, boiled for 5 min, and stored at –20 C until electrophoresis, was used to determine SOCS-1, SOCS-2, and SOCS-3 total content.

Immunoprecipitation
Ten milligrams of solubilized total liver protein were incubated at 4 C overnight with 10 µl of anti-GHR antiserum ({alpha}GHR, 1/100), 5 µl of anti-JAK2 antiserum ({alpha}JAK2, 1/200), or 4–5 µg of anti-STAT5a antibody ({alpha}STAT5a, 1/50), anti-STAT5b antibody ({alpha}STAT5b, 1/50), anti-CIS antibody ({alpha}CIS, 1/50), anti-SHP-2 antibody ({alpha}SHP-2, 1/100), or anti-SIRP{alpha}1 antibody ({alpha}SIRP{alpha}1, 1/100) in a final volume of 1 ml. After incubation, 25 µl of protein A-Sepharose or protein G-Sepharose (50%, vol/vol) was added to the mixture. The preparation was further incubated with constant rocking for 2 h at 4 C and then centrifuged at 3000 x g for 1 min at 4 C. The supernatant was discarded and the precipitate was washed three times with washing buffer (50 mM Tris, 10 mM vanadate, 1% Triton X-100, pH 7.4). The final pellet was resuspended in 35 µl of Laemmli buffer, boiled for 5 min, and stored at –20 C until electrophoresis.

Preparation of liver microsomes
Livers were homogenized in buffer containing protease and phosphatase inhibitors (0.25 M sucrose, 50 mM Tris/HCl, 1 mM PMSF, 10 mM EDTA, 1 mg/ml bacitracin, 10 mM sodium orthovanadate, 100 mM sodium fluoride, 0.032 TIU/ml aprotinin, pH 7.4). The homogenates were centrifuged at 12,000 x g for 30 min, and the resulting supernatants were centrifuged at 100,000 x g for 1 h at 4 C. Pellets containing the membrane fraction were resuspended in 50 mM Tris/HCl, 2 mM PMSF, 10 mM sodium vanadate, pH 7.4 buffer; protein concentration was determined by the method of Lowry (31). The samples were boiled in Laemmli buffer for 10 min and stored at –20 C until electrophoresis.

Immunoblotting
Samples were subjected to electrophoresis on 7.5 or 10% SDS-polyacrylamide gels using a Bio-Rad Mini Protean apparatus (Bio-Rad Laboratories, Hercules, CA). Electrotransference of proteins from gel to nitrocellulose membranes was performed for 1 h at 100 V (constant) using the Bio-Rad miniature transfer apparatus in 25 mM Tris, 192 mM glycine, 20% (vol/vol) methanol, pH 8.3. To reduce nonspecific antibody binding, membranes were incubated 2 h at room temperature in T-TBS blocking buffer (10 mM Tris/HCl, 150 mM NaCl, and 0.02% Tween 20, pH 7.6) containing either 3% BSA for phosphotyrosine detection or 5% nonfat powdered milk for protein detection. The membranes were then incubated overnight at 4 C with antiphosphotyrosine ({alpha}PY) (1:500), {alpha}GHR (1:500), {alpha}JAK2 (1:1000), {alpha}STAT5a (1:400), {alpha}STAT5b (1:400), {alpha}CIS (1:200), {alpha}SHP-2 (1:2500), {alpha}SIRP{alpha}1 (1:250), {alpha}SOCS-1 (1:200), {alpha}SOCS-2 (1:200), or {alpha}SOCS-3 (1:200). Bound antibodies were detected by enhanced chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ) using preflashed Kodak XAR film (Eastman Kodak, Rochester, NY). Band intensities were quantified by optical densitometry (densitometer model CS-930, Shimadzu, Japan) of the developed autoradiographs. To reprobe with other antibodies, the membranes were incubated in stripping buffer (2% SDS, 100 mM 2-mercaptoethanol, 62.5 mM Tris/HCl, pH 6.7) for 40 min at 50 C while shaking.

Reagents
oGH was obtained through the National Hormone and Pituitary Program, NIDDK, NIH.

BSA fraction V, Kodak X-OMAT XAR 5 films, protein G-Sepharose, protein A-Sepharose, and nitrocellulose membranes were obtained from Sigma Chemical Co. (St. Louis, MO). Antibodies {alpha}STAT5a (L-20, catalog no. sc-1081), {alpha}STAT5b (C-17, no. sc-835), {alpha}CIS (N-19, no. sc-1529), {alpha}PY (PY-99, no. sc-7020), {alpha}SOCS-1 (H-93, no. sc-9021), {alpha}SOCS-2 (H-74, no. sc-9022), and {alpha}SOCS-3 (H-103, no. sc-9023) were purchased from Santa Cruz Biotechnology Laboratories (Santa Cruz, CA); {alpha}JAK2 (no. 06–255) and {alpha}SIRP{alpha}1 (no. 06–729) antibodies were from Upstate Laboratories (Lake Placid, NY); {alpha}SHP-2 (no. 610621) was from Transduction Laboratories (Lexington, KY). {alpha}GHR antiserum was obtained in our laboratory, according to Camarillo et al. (32). All other chemicals were of reagent grade.

Statistical analysis
Experiments were performed analyzing all groups of animals in parallel, with n representing the number of different individuals used in each group. Results are presented as mean ± SEM of the number of samples indicated in Table 1Go and mean ± SD in the figures. Statistical analyses were performed by ANOVA followed by the Tukey-Kramer test using the InStat statistical program by GraphPad Software, Inc. (San Diego, CA). Student’s t test was used when the values of two groups were analyzed. Data were considered significantly different when P < 0.05.


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  		TABLE 1. Body weight and serum GH concentration of normal and transgenic mice

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PEPCK-bGH transgenic mice were used as a model to study GH signaling desensitization by high continuous GH levels. These animals exhibit chronically elevated bGH, which is approximately 70-fold higher than normal mouse GH values [20 ng/ml was considered as a mean value for mouse GH (mGH) levels in normal mice, according to our previous findings (33, 34)], resulting in a 70% body weight increase when compared with normal littermates (Table 1Go). To evaluate GH signaling, normal and transgenic mice received a massive dose of oGH (5 mg/kg) or saline ip 7.5 min before harvesting tissues for analysis. The JAK2/STAT5 pathway was studied in liver because this organ is an important target for GH, and the JAK2/STAT5 signaling pathway was chosen as it is the most prominent for this hormone.

In solubilized livers of normal and transgenic mice, GHR, JAK2, STAT5a, and STAT5b content was analyzed by immunoprecipitation and Western blotting with specific antibodies. Densitometric analysis of autoradiographs showed that GHR levels were 2.5-fold higher in transgenic mice over normal animals, whereas neither JAK2, STAT5a, nor STAT5b protein content varied in these animals (Fig. 1Go). Because the GH-stimulating dose was given 7.5 min before liver extraction, this treatment did not affect the content of the studied proteins, as could have been expected. Activation of the JAK2/STAT5 pathway was estimated by tyrosine phosphorylation of these signaling proteins. Whereas basal phosphorylation was barely detectable in normal mice, exogenous GH administration produced a dramatic increase of JAK2, STAT5a, and STAT5b phosphorylation levels. Transgenic mice not only did not respond to the GH stimulus, but basal phosphorylation also was comparable to that of normal animals, despite the high circulating bGH concentration (Fig. 2Go).



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  		FIG. 1. Hepatic GHR, JAK2, STAT5a, and STAT5b abundance in liver from normal (N) and PEPCK-bGH transgenic (T) mice. Animals were injected ip with normal saline [nonstimulated (–)] or oGH (5 mg/kg) [GH-stimulated (+)], killed after 7.5 min, and livers were removed. Extracts were prepared, and equal amounts of solubilized liver protein were immunoprecipitated (IP) with {alpha}GHR, {alpha}JAK2, {alpha}STAT5a, or {alpha}STAT5b antibodies, separated by SDS-PAGE, and subjected to immunoblot analysis (WB) using the same IP antibodies. Quantification of protein abundance in liver was performed by scanning densitometry and expressed as a percentage of values measured in nonstimulated normal mice. Data are the mean ± SD of the indicated number of subsets (n) of different individuals, run in separate experiments: GHR and JAK2, n = 4; STAT5a, n = 7; STAT5b, n = 6. *, P < 0.05 vs. normal control mice.

 


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  		FIG. 2. Hepatic JAK2, STAT5a, and STAT5b tyrosine phosphorylation in normal mice (N) and PEPCK-bGH transgenic (T) mice. Animals were injected ip with normal saline [nonstimulated (–)] or oGH (5 mg/kg) [GH-stimulated (+)], and after 7.5 min, livers were excised. Extracts were prepared, and equal amounts of solubilized liver protein were immunoprecipitated (IP) with {alpha}JAK2, {alpha}STAT5a, or {alpha}STAT5b antibodies, separated by SDS-PAGE, and subjected to immunoblot analysis (WB) using {alpha}PY. Protein phosphorylation was quantified by scanning densitometry and expressed as a percentage of the corresponding values in GH-stimulated normal mice. Data are the mean ± SD of four subsets of different individuals run in separate experiments (n = 4). *, P < 0.001 vs. saline-treated normal mice.

 
Because JAK2/STAT5 signaling is desensitized in these transgenic animals, the negative regulators of this pathway, CIS, SOCS-1, SOCS-2, SOCS-3, and SHP-2, were evaluated by immunoprecipitation and Western blotting of whole-liver solubilizates. CIS protein levels were 6.7-fold elevated in transgenic mice, whereas no changes were seen in SHP-2 content when compared with nontransgenic littermates (Fig. 3AGo). SOCS-2 protein levels did not significantly vary, whereas transgenic animals exhibited a 40% decrease of SOCS-3 abundance (Fig. 3BGo). SOCS-1 abundance could not be detected either in normal or in transgenic mice liver (data not shown). GH treatment did not alter the content of these proteins in normal or in transgenic animals.



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  		FIG. 3. A, Hepatic CIS and SHP-2 abundance in liver from normal (N) and PEPCK-bGH transgenic (T) mice. Animals were injected ip with normal saline [nonstimulated (–)] or oGH (5 mg/kg) [GH-stimulated (+)]. After 7.5 min, livers were excised and extracts were prepared. Equal amounts of solubilized liver protein were immunoprecipitated (IP) with {alpha}CIS or {alpha}SHP-2 antibodies, separated by SDS-PAGE, and subjected to immunoblot analysis (WB) using the same antibodies. Quantification of CIS and SHP-2 abundance in liver was performed by scanning densitometry and expressed as a percentage of the corresponding values in nonstimulated normal mice. Data are the mean ± SD of the indicated number of subsets of different individuals (n) run in separate experiments: SHP-2, n = 4; CIS n = 5. *, P < 0.001 vs. normal control mice. B, Hepatic SOCS-2 and SOCS-3 abundance in liver from normal (N) and PEPCK-bGH transgenic (T) mice. Equal amounts of solubilized liver protein were separated by SDS-PAGE and subjected to immunoblot analysis (WB) using {alpha}SOCS-2 or {alpha}SOCS-3. Quantification was performed by scanning densitometry and expressed as a percentage of the mean value measured in normal mice. Data are the mean ± SD of eight subsets of different individuals run in two separate experiments (n = 8). *, P < 0.01 vs. normal mice.

 
Subcellular localization of signaling proteins is another approach to evaluate the functional state of signaling pathways. Even when total protein content may not vary, protein association with membranes can change upon hormone stimulation due to recruitment of signaling mediators to receptor complexes or to other related proteins at the plasma membrane. To study the membrane-associated JAK2/STAT5 signaling proteins as well as the negative regulators SOCS/CIS and SHP-2, liver microsomes of transgenic mice and their control littermates were analyzed by Western blotting. GHR content displayed a 350% increase in transgenic mice compared with normal animals (Fig. 4Go), in accordance with results obtained for solubilized whole-tissue values. Membrane-associated JAK2 levels were increased 6-fold in transgenic mice (Fig. 4Go), whereas total protein content did not vary in these animals (Fig. 1Go). In normal mice, GH stimulation induced an approximately 2-fold increase in membraneassociated STAT5a and –5b levels (Fig. 4Go), probably reflecting recruitment of STAT5 molecules from the cytoplasmic pool because total protein content did not vary in solubilized liver (Fig. 1Go). In contrast, STAT5a and –5b levels in transgenic mice were similar to those of normal nonstimulated mice, either under basal or GH-stimulated conditions (Fig. 4Go).



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  		FIG. 4. Membrane-associated GHR, JAK2, STAT5a, and STAT5b abundance in liver microsomes from normal (N) and PEPCK-bGH transgenic (T) mice. Animals were injected ip with normal saline [nonstimulated (–)] or oGH (5 mg/kg) [GH-stimulated (+)]. Mice were killed after 7.5 min, and livers were removed. Microsomes were prepared, separated by SDS-PAGE, and subjected to immunoblot analysis (WB) using {alpha}GHR, {alpha}JAK2, {alpha}STAT5a, or {alpha}STAT5b antibodies. Quantification of protein abundance in liver microsomes was performed by scanning densitometry and expressed as a percentage of the mean value measured in nonstimulated normal mice. Data are the mean ± SD of four subsets of different individuals run in two separate experiments (n = 4). {dagger}, P < 0.05 vs. normal control mice; #, P < 0.01 vs. nonstimulated normal mice; *, P < 0.001 vs. normal control mice.

 
The regulatory proteins CIS/SOCS and SHP-2 in liver microsomes of transgenic mice and in their control littermates were determined. Membrane-associated CIS exhibited a 5-fold increase in transgenic mice when compared with normal animals (Fig. 5AGo), whereas no significant differences were found for SOCS-2 and SOCS-3 proteins (Fig. 5BGo). A 4.5-fold increase in membrane-associated SHP-2 levels was also detected in transgenic in comparison with normal mice (Fig. 5AGo), even when total SHP-2 content in solubilized livers did not vary (Fig. 3AGo). SIRP{alpha}1, a transmembrane protein that may act as scaffolding for SHP-2, was evaluated in total solubilized liver and in the microsomal fraction. No differences were found between normal and transgenic mice (data not shown). Under the experimental conditions used, no tyrosine phosphorylation of either SHP-2 or SIRP{alpha}1 could be detected (data not shown). GH treatment did not alter the association of the analyzed regulatory proteins to membranes.



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  		FIG. 5. A, Membrane-associated CIS and SHP-2 abundance in liver microsomes from normal (N) and PEPCK-bGH transgenic (T) mice. Animals were injected ip with normal saline [nonstimulated (–)] or oGH (5 mg/kg) [GH-stimulated (+)], and after 7.5 min, livers were excised. Microsomes were prepared, separated by SDS-PAGE, and subjected to immunoblot analysis (WB) using {alpha}CIS or {alpha}SHP-2 antibodies. Quantification of protein abundance in liver was performed by scanning densitometry and expressed as a percentage of the mean value determined for nonstimulated normal mice. Data are the mean ± SD of five subsets of different individuals run in three separate experiments (n = 5) for SHP-2 and four subsets of different individuals run in two separate experiments (n = 4) for CIS. *, P < 0.05; {dagger}, P < 0.001 vs. normal control mice. B, Membrane-associated SOCS-2 and SOCS-3 abundance in liver microsomes from normal (N) and PEPCK-bGH transgenic (T) mice. Samples were obtained and processed as described in A. Immunoblot analysis (WB) was carried out using {alpha}SOCS-2 or {alpha}SOCS-3 antibodies. Quantification of protein abundance in liver was performed by scanning densitometry and expressed as a percentage of the mean value determined for nonstimulated normal mice. Data are the mean ± SD of four subsets of different individuals run in two separate experiments (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transgenic mice overexpressing bGH from the line used in the present study reach adult body weights approximately 70% greater than those of their normal nontransgenic littermates. However, this size increment is not comparable to the extremely high GH levels in these animals, approximately 70-fold higher than in normal mice, a difference further amplified by the higher affinity of bGH for mouse GHR. This would imply that there must be attenuating mechanisms regulating GH action in these mice. Circulating GH binding protein (GHBP), which structurally corresponds to the GHR extracellular domain, can prolong GH half-life, but it can also serve as a negative regulator of GH action as it competes with GHR for GH (35). When GHBP is associated with membranes (MA-GHBP), its inhibitory role can be potentiated, because short receptor forms may inhibit receptor activation (36). GH induces up-regulation of the related proteins GHBP and GHR. Pregnant mice exhibit a gradual increase in GH levels, accompanied by an increment of GHBP and hepatic MA-GHBP levels and a modest increase in liver GHR levels (32, 37). GHRH transgenic mice overexpressing mGH display a similar pattern (38, 39). Transgenic mice overexpressing bGH (PEPCK-bGH) used in this work also exhibit high circulating GHBP levels (29) as well as high liver MA-GHBP levels (40). However, up-regulation of binding proteins does not seem sufficient to counteract the high GH levels that transgenic mice overexpress. Therefore, other inhibitory mechanisms must be involved, probably within the cell, to attenuate this GH excess.

GH induces GHR dimerization, which leads to the activation by autophosphorylation of the associated tyrosine kinase JAK2, the crucial step for GH signaling. JAK2, in turn, phosphorylates GHR and other signaling molecules, among which STATs are the most relevant. In turn, GH-induced signaling is terminated by different mechanisms, such as activation of phosphatases (SHP-1 and SHP-2), induction of suppressors of cytokine signaling (SOCS/CIS), and internalization/degradation of the GH-GHR complex (4, 5).

GH stimulates the expression of several SOCS/CIS proteins. SOCS-1 and SOCS-3 are rapidly and transiently induced; SOCS-2 exhibits a slower kinetics, presenting a moderate and prolonged rise; CIS, in turn, displays dual kinetics, with a transient rise followed by a more persistent one (8, 9, 41). CIS and SOCS-2 have a predominant expression in liver; SOCS-3 is expressed to a lesser extent in this tissue, whereas SOCS-1 seems to be restricted to the thymus and spleen (9, 42, 43). Recently we found that CIS content is increased 18-fold in the liver of GHRH transgenic mice overexpressing mGH, without accompanying changes in SOCS-2 and a slight decrease in SOCS-3 abundance (28). The prominent induction of CIS suggested that this protein could be implicated in the desensitization of GHR/JAK2/STAT5 signaling by continuous high levels of GH observed in these mice. This inhibition could be explained in terms of the competition between CIS and STAT5 for the docking site on GHR, because CIS and STAT5b bind to the same phosphotyrosine residues on GHR via their SH2 domain (26, 44), or by CIS targeting the GHR/JAK2 complex to proteasome degradation (15, 13).

To further explore the mechanisms of attenuation of GH signaling by continuous high concentration of GH in vivo, we studied basal and GH-induced activation of JAK2, STAT5a, and STAT5b in another transgenic mouse line, the PEPCK-bGH. In these transgenic animals, basal phosphorylation of these proteins was not significantly different from values measured in normal mice, despite the very high GH concentration in their circulation. After GH administration, normal mice exhibited significantly increased tyrosine phosphorylation of these proteins, but transgenic mice did not respond to the massive GH stimulus (Fig. 2Go). GH receptor content was elevated in transgenic mouse liver, both in whole-tissue solubilizate and in the microsomal fraction. This result was expected, because GHR is an integral membrane protein, and GH induces its synthesis. Total liver concentration of JAK2, STAT5a, and STAT5b proteins was similar in normal and transgenic mice, but different results were found when their membrane association was determined in the liver microsomal fraction. Membrane-associated JAK2 content was 6-fold higher in transgenic than in normal mice liver (Fig. 4Go), probably due to recruitment of JAK2 molecules from other cellular pools, because JAK2 total abundance in solubilized liver did not change (Fig. 1Go). This increment can be related to the higher GHR levels observed in these mice, suggesting higher GHR/JAK2 complex formation. Membrane-associated STAT5a and –5b content increased 2-fold in normal mouse liver after GH administration (Fig. 4Go). This increment reflects recruitment of cytoplasmic STAT5 to a receptor-activated complex in plasma membrane, because there were no changes in total cellular content of these proteins (Fig. 1Go). Transgenic mice, either with or without GH stimulus, had membrane-associated STAT5 levels similar to those of nonstimulated normal mice (Fig. 4Go), despite the higher GHR levels in GH transgenic animals. Although the time interval of 7.5 min after GH administration is not sufficient to expect de novo protein synthesis (9, 4, 28), it is adequate for GH-dependent membrane recruitment of signaling proteins, as can be observed in normal mice.

In PEPCK-bGH transgenic mice, the JAK2/STAT5 pathway is desensitized, because there is no response to a massive stimulus with GH and no increase in the basal phosphorylation of the implicated proteins can be detected, despite the elevated GH levels. CIS is proposed to be a major factor responsible for the down-regulation of STAT5b signaling in the liver (26). The constitutive induction of total cellular CIS (Fig. 3AGo) suggests that this could account for the desensitization of JAK2/STAT5 signaling by GH in vivo in PEPCK-bGH transgenic mice. The important increase of total CIS levels observed in these transgenic mice as well as in GHRH transgenic mice, and the current finding that the association of this protein with membranes is higher (Fig. 5AGo), probably forming GHR-JAK2-CIS complexes, supports the idea that CIS is crucial for the desensitization of the GH signaling pathway in mice with chronically elevated GH levels. The mechanism by which CIS renders the GHR/JAK2 complex inactive would imply competition with STAT5 for the phosphorylated residues on the GHR, because membrane-associated CIS is increased. Targeting to proteasome degradation of protein complexes cannot be excluded, but increased membrane-associated GHR/JAK2 levels in transgenic mice livers would argue against the prevalence of this inhibitory mechanism.

In whole solubilized liver, SOCS-2 content did not significantly vary between normal and transgenic mice, whereas SOCS-3 was 40% less abundant in transgenic animals, in agreement with our previous findings (28). Moreover, there were no differences in the association of these SOCS proteins with liver membranes between normal and transgenic animals. These results suggest that SOCS-2 is not directly involved in GH desensitization under conditions of chronic exposure in vivo. The decrease in total SOCS-3 levels seen in transgenic mice agrees with the rapid induction kinetics exhibited by this protein. Normal mice, subjected to a pulsatile GH release pattern, may present higher SOCS-3 levels induced by GH peaks. In contrast, SOCS-3 expression may be blunted in transgenic mice with a continuous GH release pattern. Although we consider this kinetic hypothesis more likely, we cannot exclude SOCS-3 degradation by targeting signaling proteins to proteasome. The protein doublet observed for SOCS-3 in Fig. 3BGo may be due to posttranslational modifications. SOCS proteins can be ubiquitinated or tyrosine phosphorylated (13, 15, 45, 46); however, the small molecular weight difference between the two bands in the doublet does not seem compatible with monoubiquitination. SOCS-1 could not be detected either in normal or in transgenic mouse liver, in accordance with reported data indicating this protein is not expressed in this organ (42).

The competition between CIS and STAT5 for the docking sites on GHR could account for the lack of STAT5 phosphorylation but not for the low JAK2 phosphotyrosine level, even when CIS overexpression has been related to a partial inhibition of JAK2 phosphorylation (44). Because activation of most of the GH signaling components is mediated by phosphorylation of tyrosine residues by JAK2, the phosphatase-mediated attenuation/termination of GH signal transduction was addressed. SHP-2 interacts with GHR by binding to a carboxyl-terminal specific phosphotyrosine on GHR (19). GHR-bound SHP-2 negatively regulates GHR/JAK2/STAT5 signaling by removing the phosphate of tyrosine residues from these signaling molecules (19, 47). In the current work, a significant increase of SHP-2 binding to liver membranes in transgenic mice overexpressing bGH is described (Fig. 5AGo). This increase of SHP-2 binding to microsomal membranes occurs without a significant change in total SHP-2 content (Fig. 3AGo), indicating contribution from the cytoplasmic pool of this phosphatase. The recruitment of SHP-2 to membranes could be due to either phosphatase binding to GHR or JAK2, or it could be mediated by the signal-regulatory protein SIRP{alpha}1. However, the levels of SIRP{alpha}1 did not vary between normal and transgenic mice (data not shown). Because SHP-2 binds to phosphotyrosine residues of SIRP{alpha}1, phosphorylation of SIRP{alpha}1 could reflect its scaffold activity. Nevertheless, its phosphorylation could not be detected. These results suggest that SIRP{alpha}1 would not play an important role in this model of GH overexpression. The high levels of GHR and membrane-associated JAK2 observed in these animals would be sufficient for the observed phosphatase recruitment. The ability of SHP-2 to dephosphorylate multiple signaling molecules, such as GHR, JAK2, or STAT5, may provide another control point for GH signaling desensitization. SHP-2 binding to microsomal membranes could account for the low tyrosine phosphorylation level detected in JAK2, STAT5a, and STAT5b.

The present work was carried out in liver because this organ is an important target of GH and it is the main tissue expressing GHR. However, it would be very interesting to determine whether these phenomena occur in other GH target tissues, to achieve a broader understanding of the impact of GH on the organism as a whole. In summary, the continuous high levels of GH in vivo produce desensitization of GH signaling of the JAK2/STAT5 pathway in the liver of bGH transgenic mice. Insensitivity to an exogenous GH stimulus is reflected by lack of JAK2 and STAT5 phosphorylation and inhibition of STAT5 recruitment to membrane-associated GHR/JAK2 complex. This inhibition would depend on constitutively high CIS protein levels and its recruitment to GHR, inhibiting STAT5 binding to its docking sites. A second mechanism of negative regulation would involve tyrosine phosphatase SHP-2, which dephosphorylates JAK2 and STAT5 to shut off GH signal, because this enzyme is increased in the hepatic membrane fraction of GH-overexpressing mice.


   Acknowledgments
 
Transgenic and normal mice used in this work were derived from animals kindly provided by Drs. T. E. Wagner and J. S. Yun. We thank Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, and NIDDK for oGH and reagents for bGH RIA.


   Footnotes
 
This work was supported by University of Buenos Aires (UBA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Antorchas, and Agencia Nacional de Promoción Científica y Tecnológica Convocatoria (Argentina) to D.T. and by the National Institutes of Health via Grants AG19899 and AG16622 to A.B. D.T. and A.I.S. are Career Investigators of CONICET, and J.G.M. is supported by a Fellowship from UBA.

Abbreviations: bGH, Bovine GH; CIS, cytokine-inducible SH2containing protein; GHBP, GH binding protein; GHR, GH receptor; {alpha}GHR, anti-GHR antiserum; JAK2, Janus kinase 2; MA-GHBP, membrane-associated GHBP; mGH, mouse GH; oGH, ovine GH; PEPCK, phosphoenolpyruvate carboxykinase; PMSF, phenylmethylsulfonyl fluoride; PY, phosphotyrosine; SH2, src homology 2; SHP, SH2-containing phosphatase; SIRP, signal-regulatory protein; STAT, signal transducer and activator of transcription.

Received November 5, 2003.

Accepted for publication March 3, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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