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The Cooperative Research Centre for Cellular Growth Factors and the Walter and Eliza Hall Institute of Medical Research (C.J.G., D.M., J.E.C., N.A.N., D.J.H., W.S.A.) Post Office, Royal Melbourne Hospital, Victoria, Australia; Centenary Institute of Cancer Medicine and Cell Biology (P.B.), Sydney 2042, Australia; Departments of Pathology, University Health Network and Department of Laboratory Medicine and Pathobiology (S.L.A.), University of Toronto, Ontario M5G 2M9, Canada; Commonwealth Scientific and Industrial Research Organization Health Sciences and Nutrition (T.E.A.), Parkville Laboratory, Parkville, Victoria 3052, Australia; AgResearch (H.W.D), Ruakura Research Centre, Hamilton, New Zealand
Address all correspondence and requests for reprints to: Dr. Christopher Greenhalgh, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia. E-mail: greenhalgh{at}wehi.edu.au.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A growing body of evidence suggests that SOCS proteins act within the GH and IGF-I signaling pathways. SOCS-1, SOCS-2, and SOCS-3 have been shown to bind to the IGF-I receptor in vitro (8, 9, 10), although it is unknown whether IGF-I induces SOCS expression, and it is unclear whether these molecules actually inhibit IGF-I receptor signaling. GH induces the expression of CIS, SOCS-1, -2, and -3, each of which has been shown to inhibit GH receptor signaling to varying degrees in STAT5 reporter and gel shift assays (11, 12, 13, 14, 15, 16). It was anticipated that gene deletion studies would better define the physiological roles of these proteins and what function, if any, they may play in regulating growth. SOCS-1-/- mice suffer from deregulated interferon-
signaling (17, 18) but do not appear to have any significant growth abnormality, whereas mice lacking SOCS-3 die in utero from placental insufficiency (19), impeding investigation of a potential role for SOCS-3 in regulating GH signaling. CIS transgenic mice suffer from growth retardation and lactational defects that are comparable to those observed in STAT5a- and STAT5b-deficient animals and are thought to be caused by the suppression of STAT5 phosphorylation (20, 21, 22, 23); however, CIS-/- mice are reported to have no abnormal growth phenotype (24). Interestingly, SOCS-2-/- mice grow significantly larger than normal mice, reaching 1.3 to 1.5 times the weight of wild-type mice. SOCS-2-deficient mice exhibit increased bone growth, enlargement of most organs, collagen deposition in the skin and some ducts and vessels, lower major urinary protein levels, and elevated IGF-I mRNA levels in some tissues but no significant change in serum IGF-I protein levels (25). This phenotype shares some features of the phenotypes described for GH and IGF-I transgenic animals (26, 27) and suggests that SOCS-2 acts to regulate the somatotrophic axis.
GH is released from the pituitary into the circulation where it has direct, as well as indirect, effects on tissue growth (reviewed in Refs. 28 and 29). GH is capable of inducing IGF-I mRNA in a number of postnatal tissues (30, 31) but, as recently shown by Lupu et al. (32), does not control all peripheral (i.e. other than the liver) IGF-I mRNA production. Consequently, it has been difficult to quantitatively determine what contribution and by what mechanism each of these mitogenic factors makes to organ growth, and exactly how these growth-promoting stimuli are attenuated in individual tissues. It is of interest to determine whether SOCS-2 plays a negative regulatory role in this complex signal cascade, as suggested by in vitro and in vivo studies.
In this paper we investigate the role of SOCS-2 in growth control using biochemical, genetic, and histological techniques. We show that SOCS-2 is not required for normal pituitary gland structure or GH production, is not induced by IGF-I, and appears not to control IGF-I signaling in primary embryonic fibroblasts. Studies of GH responses in cells lacking SOCS-2 and in mice lacking both SOCS-2 and STAT5b suggests a role for SOCS-2 in regulating the GH signaling pathway.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). To characterize the GH secretory system further, we examined the pituitary glands of embryonic d 17 (E17) and 8-wk-old male and female wild-type and SOCS-2-/- mice. The pituitaries at E17 showed normal development for stage of gestation and had normal GH immunoreactivity (data not shown). The pituitaries of 8-wk-old female SOCS-2-/- mice had normal architecture with intact anterior lobes and intermediate lobes (Fig. 1B). The adenohypophyses were composed of a normal number of acidophils, basophils, and chromophobes that formed acini. The features were not different from those of age-matched female controls (Fig. 1C). Immunolocalization of GH in the SOCS-2-/- mice (Fig. 1D) revealed numerous somatotrophs with diffuse cytoplasmic GH immunoreactivity; the GH positivity and somatotroph morphology were not different from that of age-matched control mice (Fig. 1E). No differences were noted in immunostaining for PRL, ß-TSH, ß-FSH, ß-LH, and ACTH (data not shown).
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). Examination of total STAT5 phosphorylation over a longer period after GH stimulation also confirmed this difference (Fig. 2B). The observations of prolonged STAT5a and STAT5b phosphorylation were confirmed in four independent experiments.
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, A and B). The responsiveness of the cells to IGF-I and GH was confirmed by blotting for early growth response factor-I (egr-1) that is induced by both of these growth factors (33, 34).
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Given that IGF-I is a key determinant in embryonic growth (35, 36) we also examined whether SOCS-2 was expressed in the fetal mice. Northern blotting of total RNA from whole embryos indicated that SOCS-2 was highly expressed in all stages examined (Fig. 3D). This is intriguing as no obvious phenotype is observed in SOCS-2-deficient animals until postnatal growth begins, and raises the possibility that SOCS-2 may have a role in the regulation of cytokine and growth factor receptor signaling pathways before GH-driven postnatal growth begins at 2.5–3 wk of age. Deletion of SOCS-2 caused significant elevation of heart IGF-I mRNA levels in 6-wk-old mice (25), and it was of interest to determine whether this alteration in IGF-I mRNA production was present in prenatal mice. To investigate this we performed ribonuclease (RNAse) protection assays to quantitate IGF-I mRNA levels in the hearts of 11-d-old male wild-type and SOCS-2-/- mice (Fig. 3E). Phosphoimaging quantitation revealed that SOCS-2-/- heart IGF-I mRNA levels were 210% that of wild-type mice, an increase that is comparable to the cardiac IGF-I mRNA levels observed in 6-wk-old SOCS-2-/- mice (25).
Normal IGF-I Signaling in Primary Embryonic Fibroblasts (PEFs) from SOCS-2-/- Mice
The high level of expression of SOCS-2 mRNA in embryos, evidence for higher IGF-I mRNA levels in fetal SOCS-2-/- hearts, and the fact that IGF-I signaling is known to be important for embryonic growth and embryonic fibroblast proliferation (35, 36, 37) led us to analyze a number of IGF-I signaling parameters in PEFs to determine whether IGF-I signaling was perturbed in SOCS-2-/- PEFs. PEFs were stimulated with IGF-I for various times and analyzed for phosphorylation of IGF-I signaling components. The IGF-I receptor was rapidly phosphorylated upon addition of IGF-I, with phosphorylation reaching a peak after 2 h and declining to near basal levels by 24 h. There were no significant differences in the kinetics and magnitude of IGF-I receptor phosphorylation in wild-type and SOCS-2-/- PEFs (Fig. 4A). Similarly, there were no obvious differences in the phosphorylation status of insulin receptor substrate-1 (IRS-1) or MAPK in response to IGF-I in similar experiments (Fig. 4B and data not shown). To gain a more complete understanding of IGF-I signaling, we used the fact that protons are exported from the cellular cytoplasm into the medium in response to growth factor signaling. A Cytosensor was employed to measure proton efflux (extracellular pH) but failed to detect significant differences between wild-type and SOCS-2-/- PEFs when pulsed with IGF-I for 6 min, pulsed again 5 h later, or continuously exposed to IGF-I (data not shown). Together, these data suggest that IGF-I does not induce SOCS-2 expression in the cell lines and tissue examined, and at least some facets of IGF-I signaling are not perturbed in SOCS-2-deficient PEFs.
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) (21, 22, 25). SOCS-2-/- STAT5b-/- male mice were larger than STAT5b-/- mice, as were the carcasses of male and female double knockout mice compared with those of STAT5b-/- mice, but the magnitude of these increases was not as profound as those observed when comparing SOCS-2-/- and wild-type mice. However, with the exception of body length, which was only marginally greater in SOCS-2-/- STAT5b-/- mice compared with STAT5b-/- controls, no other significant differences were observed in bone lengths or organ weights in mice of these genotypes. This was in contrast to SOCS-2-/- mice, which had significantly larger liver, heart, lung, salivary gland, and carcass weights, and longer body and bone lengths when compared with wild-type mice, as observed previously (25) (Table 1 and Fig. 6 and data not shown).
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). STAT5b-/- mice showed no organ pathology except for a prominent layer of vacuolation (possibly in adipocytes) occupying either the whole dermal layer or the inner region of the dermis, although skin appeared thinner than that of wild-type mice. In SOCS-2-/- STAT5b-/- mice there was a reduction in the magnitude of collagen deposition compared with SOCS-2-/- mice and amelioration of the dermal abnormality seen in STAT5b-/- mice. Collectively, these data show that the effects of SOCS-2 deletion on growth in mice are very largely dependent on STAT5b, suggesting a key role for SOCS-2 in the negative regulation of the GH/STAT5b pathway.
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DISCUSSION |
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Analysis of GH serum levels indicated that the excessive growth of SOCS-2-/- mice is not due to a consistent elevation of circulating GH levels. These results were reinforced by histological examination of pituitary glands that found no significant alterations in structure or immunostaining profiles. It has been reported that the increased postnatal growth in hg mice may be accompanied by lower levels of circulating GH (39). Although our own data suggest that the removal of SOCS-2 does not grossly elevate serum or pituitary GH levels in mice, it should be noted that single point measurements of GH levels alone are not a conclusive demonstration of GH levels and do not indicate whether the pattern of GH secretion may be altered.
Recent studies showed that SOCS-2 can interact with the IGF-I receptor both in yeast two-hybrid assays and after cotransfection of 293 human embryonic kidney cells (8, 9). The interaction was hormone dependent and required a functional receptor kinase domain, implying a possible role for SOCS-2 in control of IGF-I signal transduction. Our studies in a range of cell lines and primary tissues yielded no evidence for SOCS-2 mRNA induction by IGF-I but confirmed previous observations of GH-stimulated SOCS-2 mRNA production (11, 14). It should be noted that in the present study, human GH (hGH) was used both in vivo and in vitro as a somatotropic hormone. Although it is widely appreciated that hGH can bind to both somatogenic and lactogenic (PRL) receptors (40), its ability to activate components of the Jak-Stat signaling pathway in rodent liver is mediated principally via the GH receptor (41). In the other target tissues analyzed after hGH treatment of mice (lung, heart, skeletal muscle), expression of PRL receptor mRNA is very low or absent (42). We are therefore confident that the results obtained using hGH reflect activation of the somatotrophic axis.
The finding of no significant SOCS-2 induction by IGF-I does not preclude the possibility that SOCS-2 is induced by other cytokines/growth factors, such as GH, with subsequent blockade of IGF-I signaling. However, we observed no significant differences in IGF-I signaling in wild-type cells and those lacking SOCS-2, in assays including time courses of activation of IGF-I receptor as well as downstream signaling components. Because these studies were performed in PEFs, it is difficult to formally exclude a role for SOCS-2 in IGF-I signaling in all tissues, particularly as definitive analysis of IGF-I signaling in other cell types and organs is complicated by difficulties in deriving and culturing primary cells of interest, and the low level of IGF-I receptor expression in many cell types. However, in all analyses performed, there was no evidence for significant actions of SOCS-2 in the regulation of IGF-I signal transduction.
In contrast, modest but reproducible differences in GH receptor signaling emerged from analyses of STAT5 phosphorylation in primary hepatocytes from SOCS-2-/- mice. Prolonged STAT5a and STAT5b, and total STAT5, phosphorylation was observed in GH-stimulated SOCS-2-/- cells. Although the levels of STAT5 phosphorylation steadily declined in wild-type cells after the addition of GH, STAT5 phosphorylation appeared to be sustained in SOCS-2-deficient hepatocytes at later time points (6–12 h). These kinetics correlate well with the pattern of SOCS-2 induction by GH in the liver and isolated hepatocytes, where SOCS-2 mRNA transcripts accumulate slowly over several hours (11, 14). Consequently, SOCS-2 may not play a significant role in controlling GH signaling until a number of hours after initial GH exposure when SOCS-2 mRNA and protein concentrations are elevated.
Although the differences in STAT5 activation appear modest, the accumulated effects of small changes in GH receptor/STAT5 activity throughout the postnatal growth phase may be sufficient to establish the SOCS-2-/- phenotype. We have attempted to examine STAT5 phosphorylation in tissues freshly removed from SOCS-2-/- mice; whereas there appeared to be more phosphorylated STAT5 in the muscles of male SOCS-2-/- mice relative to wild type (data not shown), interpretation was complicated by the large variation in STAT5 phosphorylation observed among animals, presumably caused by the pulsatile release of GH from the pituitary gland. To circumvent this problem we are currently generating SOCS-2-/- mice that also carry the little mutation in the GHRH receptor (43) that results in minimal circulating GH. This should allow definitive analysis of exogenous GH-induced STAT5 phosphorylation in a number of primary tissues in the presence and absence of SOCS-2.
The precise mechanism by which SOCS2 might act to regulate STAT5 phosphorylation remains to be determined. SOCS-2 has been shown to bind in a tyrosyl phosphorylation-dependent manner to a region of a glutathione-S-transferase-GH receptor fusion protein that contains three STAT5 binding sites (12, 16, 44) and can inhibit up to 50% of STAT5 activity in cellular overexpression studies (12, 13). It is possible that SOCS-2 may bind to one or more of these sites and repress STAT5 signaling from the GH receptor by competing for STAT5 binding. A similar mechanism has been proposed for the regulation of erythropoietin signaling by CIS, which binds to one of two STAT5 binding sites of the erythropoietin receptor and inhibits some, but not all, signaling pathways (45, 46).
Our biochemical analyses in SOCS-2-deficient cells imply that prolonged activation of STAT5 contributes to the phenotype in SOCS-2-/- mice. To examine directly the role of STAT5b in the SOCS-2-/- phenotype, we generated SOCS-2-/- STAT5b-/- double knockout mice. If the model that SOCS-2 controls growth by the regulation of STAT5b activation is correct, then the absence of SOCS-2 on a STAT5b-null background should have little or no effect on growth. Consistent with this model, SOCS-2-/- STAT5b-/- mice were not significantly different from STAT5b-/- mice in the majority of parameters measured, including organ weights and the length of most bones, whereas the increases observed in carcass and body weights were minimal compared with the magnitude of increases observed in wild-type and SOCS-2-/- mice. This implies that STAT5b mediates a large proportion of the excess growth in SOCS-2-deficient mice of both sexes. This is intriguing as STAT5b is not thought to be an important determinant of the growth in female mice as only male STAT5b-deficient mice exhibit growth retardation (21, 22). Female rodent GH secretion differs from that of males and leads to desensitization and cessation of most STAT5b phosphorylation in the female liver (47, 48). It is possible then, that SOCS-2 has distinct roles in growth control in male and female mice. Although in male mice SOCS-2 may regulate growth by simply controlling STAT5b activity, in the female it may also contribute to the desensitization process. In this way, in the absence of SOCS-2, STAT5b would contribute to excess growth in mice of both sexes. This interpretation is supported by the observation that the levels of SOCS-2 mRNA expression in the liver, muscle, and fat of normal male and female mice are the same (if not greater in females) (11). This latter observation establishes that although STAT5b appears to mediate SOCS-2 expression to some degree (49, 50), SOCS-2 is still available to act in female mice in which STAT5b activity is thought to be minimal.
Although many growth parameters in SOCS-2-/- STAT5b-/- mice were not significantly altered compared with STAT5b-/- mice, some differences were observed. The double knockout mice exhibited longer bodies and evidence of increased collagen deposition compared with the STAT5b-null mice, and some increases in body and carcass weights were evident. If these phenotypes reflect the increased activity of GH signaling, these data suggest that SOCS-2 might also regulate some STAT5b-independent aspects of GH signaling. This may involve STAT5a, given the observations of prolonged STAT5a phosphorylation observed in SOCS-2-/- hepatocytes upon GH stimulation. Thus, although the precise role of SOCS-2 in growth control appears complex, we believe that the present studies establish that SOCS-2 does not act to control pituitary GH function or GH serum levels, but acts within the GH signaling cascade. Our data implicate STAT5b, a key mediator of GH responses, as a central player in the gigantism resulting from SOCS-2 deficiency and suggests that an important role of SOCS2 in growth control is the regulation of STAT5b activity.
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MATERIALS AND METHODS |
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All animal experimentation described in this paper was conducted in accord with accepted standards of care, as outlined in the National Health and Medical Research Council Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Reagents and Antibodies
hGH (Genotropin) was obtained from Amersham Pharmacia Biotech, Melbourne, Australia), whereas rat IGF-I was purchased from GroPep Pty. Ltd. (Adelaide, Australia). Rabbit and mouse anti-STAT5a and STAT5b antibodies were obtained from Zymed Laboratories, Inc. (South San Francisco, CA), mouse monoclonal antibodies against phosphotyrosine (4G10) and phosphorylated STAT5 were from Upstate Biotechnology, Inc. (Lake Placid, NY), whereas agarose-conjugated monoclonal antibodies against phosphorylated tyrosine (agarose-PY99, sc-7020 AC) and rabbit polyclonal anti-IGF-IRß (sc-713) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies against the Ig heavy and light chains of rabbit and mouse were obtained from Australian Medical Research and Development Corp. (Melbourne, Australia) and Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), respectively, whereas protein A- and protein G-Sepharose were obtained from Amersham Pharmacia Biotech.
Embryonic Fibroblast Derivation and Culture
PEFs were produced as described (51). Briefly, embryos at 14 d of gestation were dissected to remove the head and internal organs before they were treated with collagenase to disrupt cell adhesion. First- and second-passage cells were plated at a density of 4 x 105 cells/ml in DMEM containing 10% FCS, grown to 80% confluency, washed with PBS, and placed in DMEM for at least 8 h at 37 C before addition of IGF-I.
Serum GH Measurement
Serum concentrations of GH were determined using a GH RIA kit according to the instructions of the manufacturer (rat GH assay kit, RPA551, Amersham Pharmacia Biotech). Mouse serum samples were taken from 25 wild-type and 25 SOCS-2-/- 6-wk-old male and female mice and assayed in duplicate.
Hepatocyte Derivation and Culture
Primary hepatocytes were derived from 7- to 8-wk-old male mice using a collagenase perfusion method as described (52). Isolated hepatocytes were plated in RPMI 1640 medium, 10% FCS at 5 x 106 cells per 175-cm2 flask, and allowed to attach to the flask for 2 h. Cells were then washed once with PBS to remove FCS and dead cells before RPMI 1640 medium containing 0.5% BSA and 50 µM ß-mercaptoethanol was added to the flasks. Cells were allowed to stand for 4–12 h at 37 C before GH stimulation.
Immunoprecipitation and Western Blotting
Hepatocytes and fibroblasts were lysed in KALB buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.5; 1% Triton X-100; 1 mM EDTA; 1 mM sodium orthovanadate; 0.5 mM phenylmethylsulfonyl fluoride; 1 mM sodium fluoride; and protease inhibitors (Roche Molecular Chemicals, Mannheim, Germany) for 30 min at 4 C. Lysates were cleared of insoluble material by centrifugation at 10,000 x g for 10 min and then precleared with protein A-Sepharose for 30 min at 4 C. Lysates were removed and incubated for 3 h with 4 µg of either rabbit anti-STAT5a, rabbit anti-STAT5b, rabbit anti-IRS-1, or rabbit anti-IGF-I receptor antibodies with protein A- or G-Sepharose. Sepharose was washed three times with KALB buffer before being boiled in 30 µl 2x SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8; 20% glycerol; 4% SDS) containing 5% ß-mercaptoethanol and subjected to SDS-PAGE (7.5% or 10% acrylamide).
After electrophoresis, proteins were transferred to nitrocellulose and then blocked with either BSA blocking buffer [5% (wt/vol) BSA in PBS/0.1% (vol/vol) Tween-20] for antiphosphotyrosine probing or Blotto [5% (wt/vol) nonfat dried skim milk powder] for all other Western blots for at least 2 h at room temperature. Primary antibodies were diluted in the appropriate blocking buffer and incubated with the membrane for 1 h before being extensively washed and then incubated with antimouse IgG or antirabbit IgG in PBS/0.1% Tween-20 for 1 h at room temperature. Membranes were thoroughly washed and then developed with SuperSignal Lumino/Enhancer reagents (Pierce Chemical Co., Rockford, IL) and exposed to Hyperfilm MP (Amersham Pharmacia Biotech). Blots were stripped by incubating them with 200 mM Tris-HCl (pH 8.8) 1.5 M glycine with 200 mM ß-mercaptoethanol for 15 min at room temperature before extensive washing and reblocking overnight.
RNA Extraction and Northern Blot Analysis
Fibroblast cell lines were maintained as described in Ref. 14 . 3T3-F442A (53) and p6 cells (54) were stimulated for various times with either rat IGF-I (150 ng/ml), hGH (500 ng/ml), or murine LIF (104 U/ml) and then washed with PBS and dissolved with TRIzol (Life Technologies, Inc., Gaithersburg, MD). Mice were injected iv with either 50 µg hGH or 15 µg rat IGF-I and organs were collected at various time points after animals were killed. Organs were frozen in liquid nitrogen before total RNA was extracted using TRIzol. Northern blotting was performed as described in Ref. 55 , except 10 µg of total RNA were loaded per lane, and radiolabeled DNA probes were separated from unincorporated nucleotides using MicroSpin S-300 HR columns (Amersham Pharmacia Biotech). Probes were derived by excising the coding regions from plasmids containing SOCS-2, SOCS-3, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and egr-1.
RNAse Protection Assays
RNAse protection assays (RPA III) were performed according to the manufacturer’s instructions (Ambion, Inc., Austin, TX) using IGF-I probes described previously (25, 56).
Histological Examination
Sections were prepared from the tissues of 12-wk-old mice that had been fixed in 10% saline-buffered formalin by standard techniques, stained with hematoxylin and eosin or Masson Trichome, and examined by light microscopy. For examination of pituitary morphology, 8-wk-old male/female mice of each group and equal numbers of age-matched littermate controls were killed by decapitation. At autopsy, the pituitaries were removed and weighed; the other organs were carefully inspected, weighed, and measured. Male and female embryos at d 17 gestation were dissected from the uterus. The heads were cut off center longitudinally to allow penetration of fixative. For light microscopy, tissues were fixed in buffered formalin and embedded in paraffin; sections 4- to 5-mm thick were stained with hematoxylin and eosin and with the Gordon-Sweet silver method to demonstrate the reticulin fiber network. Immunocytochemical stains to localize adenohypophysial hormones were performed using the streptavidin-biotin-peroxidase complex technique. Primary polyclonal antisera directed against rat pituitary hormones were used as described previously (57) to localize GH, PRL, ß-TSH, ß-FSH, ß-LH, and ACTH.
Statistical and Densitometry Analysis
Statistical analysis was performed using the t test, and significant differences were determined to be present when P < 0.05. Western blots of STAT5a, STAT5b, and phosphorylated STAT5 were scanned by a densitometer before phosphorylated STAT5 levels were normalized to the corresponding STAT5 loading and the time zero value.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CIS, Cytokine-induced SH2-containing protein; egr-1, early growth response factor 1; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; hGH, human GH; IRS-1, insulin receptor substrate 1; JAK, Janus family of tyrosine kinases; LIF, leukemia-inhibitory factor; PEF, primary embryonic fibroblast; RNase, ribonuclease; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.
Received for publication October 29, 2001. Accepted for publication January 31, 2002.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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F. Gu, N. Dube, J. W. Kim, A. Cheng, M. d. J. Ibarra-Sanchez, M. L. Tremblay, and Y. R. Boisclair Protein Tyrosine Phosphatase 1B Attenuates Growth Hormone-Mediated JAK2-STAT Signaling Mol. Cell. Biol., June 1, 2003; 23(11): 3753 - 3762. [Abstract] [Full Text] [PDF]
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K. C. Leung, N. Doyle, M. Ballesteros, K. Sjogren, C. K. W. Watts, T. H. Low, G. M. Leong, R. J. M. Ross, and K. K. Y. Ho Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2 PNAS, February 4, 2003; 100(3): 1016 - 1021. [Abstract] [Full Text] [PDF]
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C. J. Greenhalgh, D. Metcalf, A. L. Thaus, J. E. Corbin, R. Uren, P. O. Morgan, L. J. Fabri, J.-G. Zhang, H. M. Martin, T. A. Willson, et al. Biological Evidence That SOCS-2 Can Act Either as an Enhancer or Suppressor of Growth Hormone Signaling J. Biol. Chem., October 18, 2002; 277(43): 40181 - 40184. [Abstract] [Full Text] [PDF]
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