Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.     

Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway

Nitric oxide (NO) has been invoked as an important pathogenic factor in a wide range of immunologically mediated diseases. The present study demonstrates that macrophage-derived NO may conversely function to fine tune T cell-mediated inflammation via reversible dephosphorylation of intracellular signaling molecules, which are involved in the control of T cell proliferation. Thus, T cells activated in the presence of alveolar macrophages are unable to proliferate despite expression of IL-2R and secretion of IL-2. This process is reproduced by the NO generator S-nitroso-N-acetylpenicillamine and is inhibitable by the NO synthase inhibitor N(G)-methyl-L-arginine. Analysis of T cell lysates by immunoprecipitation with specific Abs and subsequent immunoblotting indicated marked reduction of tyrosine phosphorylation of Jak3 and STAT5 mediated by NO. Further studies indicated that NO-mediated T cell suppression was reversible by the guanylate cyclase inhibitors methylene blue and LY-83583 and was reproduced by a cell-permeable analogue of cyclic GMP, implicating guanylate cyclase activation as a key step in the inhibition of T cell activation by NO.     

Normal growth hormone secretion in growth hormone insufficient children retested after completion of linear growth

Tuberculosis of the cervical spine is rare, comprising 3-5% of cases of tuberculosis of the spine. Eight patients with tuberculosis of the cervical spine seen during 1989-1992 were reviewed. They all presented with neck pain. The 4 children presented with a kyphotic deformity. In all the children the disease was extensive, with a large prevertebral abscess formation, while in the adults it was localised to one or two motion segments. Cord compression was present in 4 of the 8 patients. All the patients were treated with antituberculosis drugs and 6 underwent surgery. There was full neurological recovery in all patients. The kyphosis was improved though not fully corrected. There was a problem in stabilisation of severe involvement of the body and dens of C2. Surgery seems to play a major role in the treatment of tuberculosis of the cervical spine.     

Effect of growth hormone therapy on bone metabolism of growth hormone deficient children

The effects of human growth hormone (hGH) therapy on biochemical markers of bone metabolism were studied in 17 children (10 boys and 7 girls, aged 3.7-13.1 years old) with idiopathic GH deficiency, before and 1 and 6 months after GH therapy (0.5 0.7 IU/kg weekly SC). Serum levels of calcium, phosphate, alkaline phosphatase osteocalcin, parathyroid hormone, 1,25 dihydroxyvitamin D, insulin-like growth factor I (IGF-I) and renal phosphate per 100 ml glomerular filtrate (TPO4/GFR) were assessed. During therapy with hGH a significant decrease of serum calcium levels and increases of phosphate, osteocalcin, parathyroid hormone 1,25 dihydroxyvitamin D and IGF-I were observed. TPO4/GFR was also significantly increased. Growth response (increment in HV) was positively related with changes in alkaline phosphatase and IGF-I levels after 6 months of hGH therapy. There was also a significant positive correlation between increment in HV and increment in TPO4/GFR after 1 month of GH therapy, whereas no correlation between HV and changes in osteocalcin levels was found. CONCLUSION: GH treatment significantly influences mineral metabolism and the measurement of TPO4/ GFR after 1 month of GH therapy may serve as a useful predictor of growth response to hGH therapy in GH-deficient children.     

Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.     

Oral administration of arginine enhances the growth hormone response to growth hormone releasing hormone in short children

We have evaluated the effect of oral administration of arginine chlorhydrate on the growth hormone response to growth hormone releasing hormone in a group of nine short prepubertal children (six boys and four girls). Arginine chlorhydrate 10 g, administered orally 60 min before an i.v. bolus injection of growth hormone releasing hormone 1-29, 1 microgram/kg, significantly enhanced the growth hormone response to the neuropeptide, confirming the results of previous studies which used the i.v. route. Furthermore, our data strengthen the view that the effects of arginine chlorhydrate on growth hormone secretion are mediated by inhibition of endogenous somatostatin release.     

Effects of sumatriptan on growth hormone releasing hormone-stimulated growth hormone secretion in acromegaly

Specific inhibition of mammalian genes is possible through the use of antisense oligonucleotides (AS ODNs) or ribozymes. These strategies have led to a better understanding of several cellular and molecular mechanisms, among which cancer development. Recently, these strategies have been applied also for therapeutical purposes in diseases such as AIDS and cancer. In some of these therapeutical trials the antisense strategy is combined with gene transfer technology: the AS ODN or the ribozyme are expressed within the cell by the use of adenoviral or retroviral vectors. However, many difficulties have still to be overcome before ODNs and ribozymes can be used routinely in the clinic.     

Urinary growth hormone following exercise to assess growth hormone production in adults

OBJECTIVE: The insulin stress test (IST) is the most commonly used test to assess the GH reserve in children and adults. It is a time-consuming, expensive and potentially dangerous test. We investigated whether measurement of urinary growth hormone excretion following exercise would prove on reliable method to diagnose adult GH deficiency. DESIGN: Healthy volunteers underwent a standard IST to confirm GH secretion. Using a standardized exercise protocol on a treadmill, the urinary excretion of GH was measured. Three patients confirmed as GH deficient by an IST were exercised during the same exercise protocol and their urinary excretion of GH was measured. PATIENTS: Ten healthy volunteers and three patients with hypopituitarism were evaluated. MEASUREMENTS: A standard IST was performed on both healthy volunteers and patients, with measurements of plasma GH and plasma cortisol. Urinary growth hormone and urinary GH/creatinine (GH/CR) ratios were measured before and after IST. On a separate visit, healthy volunteers and patients were exercised on the treadmill with measurements of plasma GH and cortisol. Urinary GH and GH/CR ratios were measured before and after exercise. RESULTS: There was at least a two-fold increase in urinary GH and GH/CR ratios following exercise in all healthy adults. By contrast, patients with GH deficiency showed no rise in urinary GH or urinary GH/CR ratios following exercise. CONCLUSIONS: Measurements of urinary GH following exercise can distinguish between GH-deficient adults and healthy volunteers. Urinary GH excretion can be measured over a timed interval following exercise or can be expressed as the GH/CR ratio. This can be measured on a single sample following exercise and can be used to diagnose GH deficiency. The exercise test employed for this study is arduous. We are therefore performing further studies with a less strenuous exercise protocol with a view to designing a 'patient-friendly' exercise test for GH deficiency in adults.     

Tripeptide growth hormone secretagogues

The purpose of the present study was to compare in untreated patients suffering from moderate to severe periodontitis the efficacy of dental floss (DF) and interdental brushes (IDB) in the reduction of plaque, gingival inflammation, and probing depth in a 6-week period prior to subgingival debridement. Twenty-six patients (12 female, 14 male; mean age 37.4 years; range 27 to 72 years) were instructed to use DF for one side of the dentition and IDB for the other side as an adjunct to the daily toothbrushing for 6 weeks. Oral hygiene instructions for toothbrushing and the use of the two devices were given at baseline and at week 3. Measurements were carried out at baseline and at 6 weeks including plaque scores, probing depth, and 2 bleeding scores (periodontal pocket bleeding index and angulated bleeding index). With the IDB, the approximal plaque score at baseline of 3.09 reduced to 2.15 at 6 weeks and with DF from 3.10 to 2.47, respectively. IDB proved to remove significantly more plaque than DF. Baseline probing depth of 5.84 mm for IDB sites and 5.59 mm for DF sites was reduced to 5.01 mm at 6 weeks for both regimens. Analysis showed that the use of IDB resulted in a greater pocket reduction. Both bleeding indices were slightly reduced with IDB and DF, but no differences between devices were found. In relation to patient acceptance, more problems were observed with DF, and IDB were felt to be more efficacious. In conclusion, the results of the present study indicate that in combination with a manual toothbrush, the use of interdental brushes is more effective in removal of plaque and results in a larger reduction of probing depth than the use of dental floss. Although the differences were small, they indicate, in combination with patient preferences, that interdental brushes are to be considered preferable to floss for interdental plaque removal in patients suffering from moderate to severe periodontitis.     

Interaction between estradiol-17b and growth hormone in control of food intake in weanling rats.

Discusses previous studies indicating that estradiol reduces the food intake of adult female rats, while prepubertal females are refractory to its appetite-depressing action unless they have been hypophysectomized. In an experiment with 30 ovariectomized and hypophysectomized Sprague-Dawley female rats, treatment of weanlings with pituitary growth hormone restored the refractoriness of the neural feeding system to estradiol. It is suggested that growth hormone masks ventromedial hypothalamic restraint of food intake. Because estradiol affects eating by acting on the ventromedial hypothalamus, it is ineffective in young animals, which are highly responsive to growth hormone. (17 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A history of growth hormone

After the debacle of Brown-Séquard testicular extracts, hormone replacement therapy proper began in 1891 with the use of sheep thyroid extract to treat myxoedema. The second success in the field was "bovine' insulin to treat human diabetes in 1992. In contrast, the first successful use of growth hormone in a human pituitary dwarf did not come until 1958. Growth hormone preparations of reasonable purity had been made in the 1920s and were shown to be effective in rats and dogs, but the need for primate growth hormone in primates was not recognised until the late 1940s.