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Endocrinology Vol. 143, No. 2 386-394
Copyright © 2002 by The Endocrine Society

GRH-SOMATOSTATIN-GH

Cytokine-Inducible SH2 Protein Up-Regulation Is Associated with Desensitization of GH Signaling in GHRH-Transgenic Mice

L. González1, J. G. Miquet2, A. I. Sotelo, A. Bartke and D. Turyn3

Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica (L.G., J.G.M., A.I.S., D.T.), Junín 956, 1113 Buenos Aires, Argentina; and Department of Physiology, Southern Illinois University School of Medicine (A.B.), Carbondale, Illinois 62901-6512

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
 
The effects of continuous high GH levels on GH signal transduction through the GH receptor (GHR)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway as well as the desensitization of this pathway by suppressors of cytokine signaling (SOCS) were studied in transgenic mice overexpressing GHRH.

In transgenic mice, hepatic GHR levels were 4.5-fold higher than in normal animals, whereas the protein contents of JAK2, STAT5a, and STAT5b did not vary. This same pattern was found for basal tyrosine phosphorylation (PY-): PY-GHR was 4.5-fold increased in transgenic mice, whereas there were no differences in PY-JAK2 and PY-STATs between normal and transgenic animals. After GH administration, tyrosine phosphorylation of GHR, JAK2, and STAT5s increased 3- to 7-fold in normal mice, but no significant changes were found in transgenic mice, indicating a decreased GH sensitivity in these animals.

The content of cytokine-inducible SH2 protein, a member of the SOCS family, was 18-fold higher in GHRH-transgenic than in normal mice. Conversely, SOCS-3, present in normal mice, was hardly seen in transgenic animals, whereas SOCS-2 levels did not vary. These findings suggest that cytokine-inducible SH2 protein, significantly induced by continuously elevated GH levels, may be the SOCS protein responsible for the GH signaling desensitization in transgenic animals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH REGULATES IMPORTANT physiological processes in the liver. Its actions include direct mitogenic effects, insulin-like and insulin-antagonizing metabolic effects, as well as gene regulatory actions (1). GH signaling is initiated by hormone-induced GH receptor (GHR) dimerization (2), leading to tyrosine phosphorylation (PY-) and activation of the associated Janus tyrosine kinase (JAK2), which, in turn, phosphorylates GHR on multiple intracellular tyrosine residues (3). These phosphorylated tyrosines residues on GHR and JAK2 form docking sites for the transcriptional activators signal transducer and activator of transcription 5a (STAT5a) and STAT5b as well as for other intracellular signaling molecules (4, 5, 6). Several studies showed that STATs are key intracellular mediators of the effects of plasma GH on several liver-expressed genes (6, 7, 8). It has also been demonstrated that in rodents, STAT5, mainly hepatic STAT5b, is a key mediator of the differential effects of the sex dimorphic patterns of GH secretion, which lead to differences in GH-regulated liver gene expression between males and females (7, 9, 10).

The activation of GH signal transduction initiates other pathways for its termination (6, 11, 12, 13). One negative regulatory pathway is proposed to involve tyrosine phosphatases, such as SH2 domain containing protein-tyrosine phosphatase-1 (6, 14, 15). Another negative regulatory pathway of GH signaling, which involves new protein synthesis, includes cytokine receptor-JAK2 kinase signal inhibitory molecules, the SOCS/cytokine-inducible SH2 protein (CIS) proteins (6, 11, 12, 13). The inhibition of GH signaling by SOCS/CIS was described in different cell types and tissues (12, 16). Transcriptional activation of these proteins is induced by GH (11, 12, 13, 17, 18), an action dependent upon activation of STAT proteins (18, 19). The mechanism of action of each SOCS protein is different. While SOCS-1 and SOCS-3 interact with phosphorylated JAK2 inhibiting its activity, CIS and SOCS-2 interact directly with the phosphorylated GHR (12). Moreover, the kinetics of induction of these proteins by GH is also different. In rat liver (11), SOCS-3 is rapidly and transiently induced, but it is nearly undetectable at longer intervals after GH exposure. In these animals, CIS shows an early pattern of induction similar to SOCS-3, but it is subsequently induced in a slower, although more persistent, mode, resulting in a prolonged CIS elevation. SOCS-2 exhibits only the latter pattern, i.e. a slow but persistent, induction (11). In mouse liver, GH induces a strong and transient expression of SOCS-3, whereas the elevation of mRNA levels for CIS, SOCS-1, and SOCS-2 is less pronounced (17). Although GH-activated STAT5s can activate the expression of SOCS/CIS genes, the specific roles of these proteins in terminating GH signaling are only partially understood, especially in vivo.

In the present study transgenic mice expressing human GHRH (hGHRH) were used as a model to investigate the effects of high and continuous levels of homologous (mouse) GH on GH signaling in vivo as well as the involvement of certain SOCS proteins in the desensitization of GH signaling caused by such pattern of GH secretion. The results suggest that CIS is associated with the inhibition of GH-stimulated tyrosine phosphorylation of GHR, JAK2, STAT5a, and STAT5b, leading to a desensitization of the GH signaling pathway, whereas SOCS-2 and SOCS-3 are not involved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Transgenic Mt-hGHRH animals were produced in our colony, which was derived from animals originally produced by Dr. K. Mayo (20) and provided by Dr. J. Hyde. Adult female transgenic mice and their normal female siblings were produced by mating hemizygous male carriers of the Mt-hGHRH gene with normal C57BH6JxC3H/J F1 females. The mice were housed three to five per cage in a room with controlled light (12 h light/d) and temperature (22 ± 2 C). The animals had free access to food (Lab Diet Formula 5008, containing a minimum of 23% protein, 6.5% fat, and a maximum of 4% fiber; PMI, Inc., St. Louis, MO) and tap water. The protocol of these studies complied with applicable laws and regulations and was approved by institutional committee.

Hormones
Ovine GH (oGH) was obtained through the National Hormone and Pituitary Program, NIDDK, NIH.

Radioiodination of hormones and proteins
Mouse GH (mGH) was labeled using limiting amounts of chloramine T, as previously described (21); specific activity ranged from 70 to 120 mCi/mg. Protein A and protein G were radiolabeled according to the same procedure (30 mCi/mg).

mGH determination
mGH was determined by RIA as previously described (22). The mGH RIA kit was obtained through the National Hormone and Pituitary Program, NIDDK, NIH.

Preparation of liver extracts
Female mice (5 ± 1-month-old) were starved overnight and injected ip with 5 mg oGH/kg BW in normal saline (0.9% NaCl) in a final volume of 0.2 ml. Animals injected with saline were used to evaluate basal conditions. The animals were killed 7.5 min after injection, the livers were removed and homogenized in 10 vol solubilization buffer [1% Triton X-100, 100 mM HEPES (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulfonylfluoride, and 0.1 mg/ml aprotinin] at 4 C. Liver homogenates were centrifuged at 100,000 x g at 4 C in a 90 Ti rotor (Beckman Coulter, Inc., Palo Alto, CA) for 40 min to remove insoluble material. The protein concentration of the supernatants was determined by the method of Bradford (23).

Immunoprecipitation
Ten to 12 mg solubilized liver total protein were incubated at 4 C overnight with anti-PY antibody ({alpha}PY), anti-JAK2 antibody ({alpha}JAK2), anti-STAT5a antibody ({alpha}STAT5a), anti-STAT5b antibody ({alpha}STAT5b), anti-CIS antibody ({alpha}CIS), anti-SOCS-2 antibody ({alpha}SOCS-2), or anti-SOCS-3 antibody ({alpha}SOCS-3). After incubation, 100 µl protein A-Sepharose (50%, vol/vol) or 80 µl protein G-Sepharose (50%, vol/vol) were added to the mixture. The preparation was further incubated with constant rocking for 2 h and then centrifuged at 3,000 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, and 1% Triton X-100, pH 7.4). The final pellet was resuspended in 35 µl Laemmli buffer, boiled for 5 min, and stored at -80 C until electrophoresis.

Immunoblotting
Samples were subjected to electrophoresis on 7.5% or 10% SDS-polyacrylamide gels using a Mini Protean apparatus (Bio-Rad Laboratories, Inc., Richmond, CA). Electrotransference of proteins from gel to nitrocellulose membranes was performed for 1.5 h at 100 V (constant) using the Bio-Rad Laboratories, Inc., miniature transfer apparatus in 25 mM Tris, 192 mM glycine, 20% (vol/vol) methanol, and 0.02% SDS, pH 8.3. To reduce nonspecific antibody binding, membranes were incubated for 2 h at room temperature in blocking buffer [10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.02% Tween 20, 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 {alpha}PY, {alpha}STAT5a, {alpha}STAT5b, {alpha}CIS, {alpha}SOCS-2 or {alpha}SOCS-3, 1 µg/ml in each case, or with {alpha}GHR or {alpha}JAK2 antisera (1:500 diluted in the corresponding blocking buffer), after which they were subjected to four 5-min washes in blocking buffer [10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.02% Tween 20], incubated with [125I]protein A or [125I]protein G (0.125 µCi/ml) in blocking buffer for 1 h at room temperature, and then washed again for 60 min. [125I]Protein A or [125I]protein G bound to antibodies was detected by autoradiography of membranes using preflashed Kodak XAR film (Eastman Kodak Co., Rochester, NY) at -70 C for 6–72 h. Band intensities were quantitated by optical densitometry (densitometer model CS-930, Shimadzu, Japan) of the developed autoradiographs.

Tyrosine phosphorylation of proteins was determined by immunoprecipitation of specific proteins, followed by Western blotting with antiphosphotyrosine antibodies, or by immunoprecipitation with anti-PY antibody, followed by Western blotting with the protein in question, achieving the same results with either order. The results shown refer to the strategy cited.

Chemicals
BSA (fraction V), Kodak X-OMAT XAR 5 films, protein G, and protein A were obtained from Sigma. Nitrocellulose membranes were purchased from Schleicher \|[amp ]\| Schuell, Inc. (Keene, NH), and Na125I was purchased from DuPont (Boston, MA). Antibodies anti-STAT 5a, anti-STAT5b, anti-CIS, anti-SOCS-2, and anti-SOCS-3 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-JAK2 antiserum was obtained from Upstate Laboratories (Lake Placid, NY). Anti-GHR antiserum was provided by Drs I. Camarillo and F. Talamantes (24). All other chemicals were of reagent grade.

Statistical analysis
Results are presented as the mean ± SEM. Experiments were performed by analyzing all groups of animals in parallel. Statistical analyses were performed by ANOVA, followed by the Tukey-Kramer test using the InStat statistical program from GraphPad Software, Inc. (San Diego, CA). A t test was used when the values of two groups were analyzed. The level of significance used was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The characteristics of female Mt-hGHRH transgenic and their normal females siblings used in the present study are shown in Table 1Go. GHR immunoreactivity in solubilized livers was analyzed by Western blotting with {alpha}GHR. One main broad band was detected in solubilized samples from both normal and transgenic mice with an apparent Mr of 113 kDa (Fig. 1BGo) in agreement with our previous report (25). By densitometric analysis of autoradiographs, the GHR concentration was estimated to be 4.5-fold increased in transgenic compared with normal animals (Fig. 1BGo; 434 ± 80% and 497 ± 77% of nonstimulated normal values for nonstimulated and stimulated transgenic mice, respectively; P < 0.001; n = 8/group).


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  		Table 1. Body weight and serum mGH concentration in normal and Mt-hGHRH-transgenic mice

 


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  		Figure 1. Hepatic GHR phosphorylation and abundance in liver from normal control and GHRH-transgenic mice. Animals were injected ip with normal saline (-) or oGH (5 mg/kg; +). After 7.5 min, livers were excised, and extracts were prepared as described in Materials and Methods. A, Equal amounts of solubilized liver protein (10–12 mg) were immunoprecipitated (IP) with {alpha}PY, separated by SDS-PAGE, and subjected to immunoblot analysis (blot) with {alpha}GHR. GHR phosphorylation was quantified by scanning densitometry and expressed as a percentage of the GH-stimulated control mean. Data are the mean ± SEM of seven different experiments (n = 7). **, P < 0.001 vs. saline-treated control mice. B, One hundred micrograms of the same samples were electrophoresed and immunoblotted with the {alpha}GHR antibody as described in Materials and Methods. Quantitation of GHR abundance in liver was performed by scanning densitometry. Data are the mean ± SEM of eight separate experiments (n = 8). **, P < 0.001 vs. control mice.

 
Solubilized livers were also incubated with antiphosphorylated tyrosine ({alpha}PY), and the resulting immunoprecipitates were subjected to immunoblotting with {alpha}GHR. Receptor phosphorylation was quantified, and data were expressed as a percentage of the mean value measured in GH-stimulated normal mice. Basal GHR phosphorylation was increased by 350% in transgenic mice livers compared with nonstimulated normal subjects (130 ± 23% vs. 29 ± 9%; P < 0.001; n = 7/group; Fig. 1AGo). Exogenous administration of GH produced a 242% increase in receptor phosphorylation levels in normal mice (P < 0.001). Although an 18% increase in GHR tyrosine phosphorylation was observed in transgenic mice after GH administration, this change did not reach statistical significance (Fig. 1AGo), indicating a decreased sensitivity of the GHR to GH in these animals.

Basal PY of JAK2 was not significantly different in normal and transgenic mice (21 ± 4% and 28 ± 5% of stimulated control, respectively; Fig. 2AGo). GH administration caused a 375% increase in JAK2 phosphorylation in normal mice (P < 0.001; n = 9), whereas no significant changes in JAK2 tyrosine phosphorylation were detected after GH treatment in livers from transgenic animals (Fig. 2AGo). When JAK2 immunoreactivity in solubilized livers from normal and transgenic mice was analyzed by Western blotting with {alpha}JAK2, one main broad band was observed in both hepatic extracts with apparent Mr of 130 kDa (Fig. 2BGo). GH treatment did not significantly change JAK2 protein content in either group (Fig. 2BGo).



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  		Figure 2. Hepatic JAK2 phosphorylation and abundance in normal control and GHRH-transgenic mice. Animals were injected ip with normal saline (-) or 5 mg/kg oGH (+). After 7.5 min, the livers were excised, and extracts were prepared as described in Materials and Methods. A, Equal amounts of solubilized liver protein were immunoprecipitated (IP) with {alpha}PY, separated by SDS-PAGE, and subjected to immunoblot analysis using {alpha}JAK2. JAK2 phosphorylation was quantified by scanning densitometry and expressed as a percentage of the GH-stimulated control mean. Data are the mean ± SEM of nine different experiments (n = 9). **, P < 0.001 vs. saline-treated control mice. B, Liver extracts were immunoprecipitated with {alpha}JAK2 and immunoblotted with the same antibody. Quantitation of JAK2 abundance was performed by scanning densitometry. Data are the mean ± SEM of four separate experiments (n = 4).

 
STAT5a was immunoprecipitated from liver homogenates of control and transgenic mice with a specific {alpha}STAT5a antibody. Quantitative analysis of the specific band showed that the STAT5a protein concentration did not significantly change in transgenic and normal mice (Fig. 3BGo). After GH administration, tyrosine phosphorylation of STAT5a increased by approximately 488% in normal mice (17 ± 6% vs. 100% of stimulated control; P < 0.001; n = 6/group; Fig. 3AGo), whereas no significant changes were detected in transgenic mice livers after the same treatment (Fig. 3AGo). In these transgenic animals, basal tyrosine phosphorylation of STAT5a was slightly, but not significantly, higher than the values measured in normal mice (Fig. 3AGo).



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  		Figure 3. Hepatic STAT5 phosphorylation and abundance in normal control and GHRH-transgenic mice. Animals were injected ip with normal saline (-) or 5 mg/kg oGH (+). After 7.5 min, livers were excised, and extracts were prepared as described in Materials and Methods. A, Equal amounts of solubilized liver protein were immunoprecipitated (IP) with {alpha}STAT5a, separated by SDS-PAGE, and subjected to immunoblot analysis using {alpha}PY. STAT5a phosphorylation was quantified by scanning densitometry and expressed as a percentage of the GH-stimulated control mean. Data are the mean ± SEM of six different experiments (n = 6). **, P < 0.001 vs. saline-treated control mice. B, Liver extracts were immunoprecipitated with {alpha}STAT5a and immunoblotted with the same antibody. Data are the mean ± SEM of six (n = 6) separate experiments. C, Equal amounts of solubilized liver protein were immunoprecipitated (IP) with {alpha}STAT5b, separated by SDS-PAGE, and subjected to immunoblot analysis using {alpha}PY. STAT5b phosphorylation was quantified by scanning densitometry and expressed as a percentage of the GH-stimulated control mean. Data are the mean ± SEM of six different experiments (n = 6). **, P < 0.001 vs. saline-treated control mice. D, Liver extracts were immunoprecipitated with {alpha}STAT5b and immunoblotted with the same antibody. STAT5b abundance was estimated by scanning densitometry. Data are the mean ± SEM of six separate experiments (n = 6).

 
Tyrosine phosphorylation of STAT5b increased by 566% in normal mice after GH administration (15 ± 2% vs. 100% of stimulated control; P < 0.001; n = 7/group; Fig. 3CGo), whereas in transgenic mice GH injection did not cause a significant increase in STAT5b phosphorylation. Basal tyrosine phosphorylation in transgenic animals was numerically higher than that in normal mice, but this apparent difference was not statistically significant. There were no important changes in STAT5b protein content between normal and transgenic mice (Fig. 3DGo).

CIS immunoreactivity in solubilized livers from normal and transgenic mice was analyzed by Western blotting with {alpha}CIS. One main band was observed in autoradiographs (Fig. 4Go), which allowed the detection of an 18-fold increase in CIS concentration in transgenic mice over normal animals. In contrast, SOCS-3 was present in normal mice, but it was difficult to detect in transgenic animals (Fig. 5Go). SOCS-2 abundance did not change between normal and GHRH-transgenic animals (Fig. 6Go). As could have been expected (6, 11), the interval of 7.5 min after GH administration was not sufficient to produce detectable changes in SOCS protein levels in any of the mice (Figs. 4–6GoGoGo).



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  		Figure 4. Hepatic CIS abundance in normal control and GHRH-transgenic mice. Animals were injected ip with normal saline (-) or 5 mg/kg of oGH (+). After 7.5 min, livers were excised, and extracts were prepared as described in Materials and Methods. Liver extracts were immunoprecipitated with {alpha}CIS and immunoblotted with the same antibody. The abundance of CIS in livers from control and transgenic mice was obtained by scanning densitometry quantitation. Data are the mean ± SEM of six separate experiments (n = 6). **, P < 0.001 vs. normal control mice.

 


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  		Figure 5. Hepatic SOCS-3 abundance in normal control and GHRH-transgenic mice. Animals were injected ip with normal saline (-) or 5 mg/kg of oGH (+). After 7.5 min, livers were excised, and extracts were prepared as described in Materials and Methods. Liver extracts were immunoprecipitated with {alpha}SOCS-3 and immunoblotted with the same antibody. The abundance of SOCS-3 in liver from control and transgenic mice was obtained by scanning densitometry quantitation. Data are the mean ± SEM of three separate experiments (n = 3). **, P < 0.001 vs. normal control mice.

 


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  		Figure 6. Hepatic SOCS-2 abundance in normal control and GHRH-transgenic mice. Animals were injected ip with normal saline (-) or 5 mg/kg oGH (+). After 7.5 min, livers were excised, and extracts were prepared as described in Materials and Methods. Liver extracts were immunoprecipitated with {alpha}SOCS-2 and immunoblotted with the same antibody. The abundance of SOCS-2 in liver from control and transgenic mice was obtained by scanning densitometry quantitation. Data are the mean ± SEM of three separate experiments (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In rodents, GH is secreted in an intermittent manner, with an important sex difference in the pulsating pattern (9, 14, 26). In female rats the secretion is frequent, resulting in a continuous presence of GH in circulation even between pulses, which is different from the intermittent presence of GH seen in male rats (6, 14, 26). A relationship between the almost continuous GH levels, such as those observed in female rats, and down-regulation of the GHR/JAK2-dependent signaling to STAT5b was proposed (6, 15, 26). This down-regulation of the signaling from GHR/JAK2 to STAT5b has also been demonstrated in hepatocytes continuously exposed to GH (15, 27).

To analyze the effects of high and continuous levels of GH on GH signaling in vivo, both basal and GH-induced activation at the GHR, JAK2, and STAT5 levels were studied in GHRH-transgenic mice, which express high levels of endogenous GH. The levels of STAT5a, STA5b, and JAK2 proteins were similar in normal and transgenic mice, whereas GHR protein levels were 4.5-fold higher in transgenic than in normal animals. In these transgenic mice, there was an important increase in basal GHR phosphorylation, whereas the level of phosphorylation of GHR after GH treatment was barely altered in transgenic animals, but was significantly increased in normal mice. The increase in GHR protein content could explain the high basal phosphorylation of this protein in transgenic mice. In fact, when hepatic GHR phosphorylation was related to GHR concentration (phosphotyrosine/protein), as shown in Fig. 7Go, the values obtained for normal mice and transgenic mice in the basal condition and for transgenic mice under stimulation were similar.



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  		Figure 7. PY-GHR/GHR was determined as the ratio between tyrosine phosphorylated GHR and GHR protein content. Data are the mean ± SEM of the results shown in Fig. 1Go. **, P < 0.001 vs. normal control mice.

 
Basal JAK2 phosphorylation appeared to be slightly higher in transgenic than in normal mice, but this apparent difference was not statistically significant. After GH administration, tyrosine phosphorylation of JAK2 increased significantly in normal, but not in transgenic, animals. Basal phosphorylation levels of STAT5a and STAT5b as well as their activation after GH injection were also examined, and the results were similar to those obtained for JAK2. This lack of activation by GH could have been predicted considering the reported STAT5b desensitization in female rats. However, an increase in basal phosphorylation of the proteins involved in this pathway was expected in transgenic mice because of the very high levels of GH. Not only GHR, but also serum GH-binding protein and membrane-associated GH-binding protein that would be involved in the attenuation of GH action (28), are increased in these transgenic animals (22, 25). However, as the increase in the levels of these binding proteins is smaller than the increase in the levels of GH (22, 25), higher basal phosphorylation levels than the measured values were expected. Taken together, these results indicate that the components of the hepatic GH signaling pathway here described show decreased sensitivity to GH in GHRH-transgenic mice.

A recently described negative regulatory pathway of GH signaling includes the SOCS/CIS proteins (11, 12, 13, 16, 17, 19, 29). Among the proteins that belong to this family, it was of particular interest to study CIS and SOCS-3. CIS has been implicated in the desensitization of the signal from GHR/JAK2 to STAT5b by continuous levels of GH (6, 13, 30). SOCS-3 is the SOCS protein preferably induced by GH in mouse liver (17). Moreover, CIS and SOCS-3 interact with different phosphorylated signaling molecules, such as GHR and JAK2, respectively, suggesting that their physiological function is likely to be different (12). In addition, the kinetics of GH induction of these proteins is also different: SOCS-3 is rapidly and transiently induced, whereas CIS is initially induced in the same fashion but afterward shows a second rise that is slower and more persistent (11, 17). The rapid and transient induction of SOCS-3 would suggest involvement of this protein in the desensitization process between GH pulses, whereas the longer-lasting induction of CIS would relate it to the desensitization caused by chronic stimulation with GH. Both of these proteins were measured in solubilized liver from normal and GHRH-transgenic mice. CIS was mainly observed in transgenic mice. In contrast, SOCS-3 immunoreactivity prevailed in normal mouse liver extracts, whereas it was very weak in transgenic animals. These results suggest that SOCS-3 protein is not directly related to the desensitization observed in the liver of transgenic mice, an animal in which pulses of GH would be attenuated to render the continuous pattern of GH secretion. However, elevation of CIS in GHRH-transgenic mice could be linked with the inhibition of GH-stimulated tyrosine phosphorylation of GHR, JAK2, STAT5a, and STAT5b, resulting in a desensitization of the GH signaling pathway. This inhibitory mechanism would imply competition between the high levels of CIS and STAT5 for the phosphorylated residues on the GHR (6, 12). This could account for the inhibition of the phosphorylation of STAT5, but not for that of JAK2. Partial inhibition of JAK2 phosphorylation due to CIS overexpression has been demonstrated in transfected cells (12), where it was proposed that CIS binds to the GHR/JAK2 complex in a manner that would interfere with the receptor dimerization step or with any other event required for JAK2 activation.

CIS expression is induced by activation of the JAK2/STAT5 pathway (19). However, there is CIS expression in STAT5b knockout mice (18), suggesting that CIS is not exclusively regulated by STAT5. As STAT5 is desensitized in the GHRH-transgenic mouse model described here, it cannot be ruled out that the overexpression of CIS found could be attributed to other transcription factors, such as STAT1 or STAT3, acting on CIS induction.

Ram and Waxman (30) related CIS to desensitization of the GH signal transduction caused by continuous stimulation with GH in cells. The proposed mechanism by which CIS produced such desensitization depended on the duration of exposure to continuous GH levels. Short periods of exposure led to increased levels of CIS, which could compete with STAT5b for PY-GHR sites, therefore not allowing its activation. Longer exposure periods led to decreased levels of CIS, presumably due to degradation of the GHR/JAK2 complex targeted by CIS to the proteosome. Karlsson et al. (13) measured CIS mRNA levels after GH stimulation for longer periods, describing the second CIS induction. The present results agree with increased and sustained levels of CIS after prolonged exposure to continuous levels of GH. CIS would therefore be acting by a dual mechanism; due to its high levels it should be competing with STAT5 for binding to GHR, whereas it could also be targeting the GHR-JAK2 complex to degradation by the proteosome. In fact, preliminary results reveal an ubiquitinated protein coimmunoprecipitating with CIS in transgenic mouse liver extracts, but not in normal ones, although the identity of such a protein remains to be elucidated.

Other members of the SOCS family, namely SOCS-1 and SOCS-2, are also induced by GH in mouse liver, but the degree of induction of these proteins is not as important as that of CIS and SOCS-3 (17). SOCS-1 has been shown to be maximally inhibitory at 15–60 min after GH treatment (30). Therefore, as SOCS-1 is poorly and transiently induced by GH in mouse liver, it was not considered to be involved in desensitization of the signal by continuous and high GH levels. On the other hand, SOCS-2 is also poorly induced in mouse liver, but its mRNA pattern of induction is persistent. A recent study set aside the role of SOCS-2 in the desensitization of GH signaling in the rat (13). To further prove that this protein was not involved in the desensitization of GH caused by continuous and high levels of GH, the level of expression of this protein was determined in normal and GHRH-transgenic mice. The results suggest that SOCS-2 is not related to the GH desensitization mechanism found in GHRH-transgenic mice.

In summary, it is concluded that continuously elevated levels of GH produce desensitization of GH signaling at every level of the GHR/JAK2/STAT5 pathway in vivo. Insensitivity to GH administration is related to CIS up-regulation and SOCS-3 down-regulation, with no change in SOCS-2 levels. Therefore, GH desensitization would depend on CIS protein synthesis, in agreement with previous reports (6, 13, 30), although other known mechanisms of GH signaling termination or attenuation, such as phosphatases, other members of the SOCS family, or internalization and degradation of the GHR, cannot be excluded. The mechanism by which CIS causes such desensitization of GH signaling in vivo could involve competition between overexpressed CIS and STAT5a and -5b for phosphorylated docking sites in GHR and targeting of GHR/JAK2 complex to degradation by the proteosome.


   Acknowledgments
 
Transgenic and normal mice used in this work were derived from animals developed by Drs. K. Mayo and J. Hyde. We thank Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, and the NIDDK for oGH, mGH, and reagents for mGH RIA. We thank Drs. F. Talamantes and I. G. Camarillo for providing the GHR antiserum.


   Footnotes
 
This work was supported by University of Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, and Ministerio de Salud de la República Argentina (Carrillo-Oñativia; to D.T.) and by Illinois Council for Food and Agricultural Research and NIH Grant HD-20001 (to A.B.).

1 Supported by a fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas. Back

2 Fellow from University of Buenos Aires. Back

3 Career investigator. Back

Abbreviations: CIS, cytokine-inducible SH2 proteins; GHR, GH receptor; hGHRH, human GHRH; JAK, Janus kinase; mGH, mouse GH; oGH, ovine GH; PY, tyrosine-phosphorylated; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription.

Received July 23, 2001.

Accepted for publication October 16, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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