Vitamin D3 cannot revert desensitization of growth hormone (GH)-induced STAT5-signaling in GH-overexpressing mice non-calcemic tissues.

Growth hormone (GH) binding to a membrane receptor dimer triggers multiple intracellular signaling pathways. Signal transducers and activators of transcription are the most relevant of these pathways for GH action. GH also activates several inhibitory mechanisms, particularly suppressors of cytokine signaling (SOCS/CIS) proteins. GH-overexpressing mice exhibit hepatic desensitization of the JAK2/STAT5 GH-signaling pathway, associated with an increased abundance of CIS. Vitamin D3 has been shown to inhibit GH-induced expression of CIS and SOCS-3 and therefore prolong GH signaling in osteoblast-like cells. The purpose of the present study is to determine if vitamin D3 could attenuate CIS expression in GH-overexpressing mice, and consequently allow GH JAK2/STAT5 signaling in GH-responsive tissues in these animals. The abundance of CIS, SOCS-2, SOCS-3, STAT5b and GHR, as well as STAT5b tyrosine phosphorylation after a GH stimulus, were measured in liver and muscle of GHRH-transgenic mice treated with 1alpha,25-dihydroxyvitamin D3 for 7 days. This treatment did not diminish CIS expression in GH-overexpressing mice tissues, nor did the content of SOCS-2 and SOCS-3 significantly vary. GH-induced STAT5b phosphorylation levels were similar to basal values in transgenic mice liver treated with or without vitamin D; the refractoriness to GH was also present in muscle. Therefore, treatment with vitamin D was not sufficient to revert STAT5 GH signaling desensitization in non-calcemic tissues in GH-overexpressing mice.

Differential regulation of membrane associated-growth hormone binding protein (MA-GHBP) and growth hormone receptor (GHR) expression by growth hormone (GH) in mouse liver.

Growth hormone (GH) binding to GH receptor (GHR) is the initial step that leads to the physiological functions of the hormone. Proteolytical cleavage of the GHR in humans and rabbits and alternative processing of the GHR transcript in rodents generates circulating growth hormone binding protein (GHBP). Moreover, other GHR truncated forms that result from alternative processing of the GHR mRNA transcript have been described. These GHR short forms are inserted in the plasma membrane but they are unable to transduce the signal. In rodents, membrane associated-GHBP (MA-GHBP), which accounts for a significant proportion of liver GH binding capacity, represents the main GHR short form found in membranes, and may therefore function as a negative form of the receptor. In the present study, GHR and MA-GHBP content in liver were analyzed using mutant and transgenic mice expressing different concentrations of growth hormone to evaluate the correlation between GH levels, body weight (BW), GHR and MA-GHBP expression. It was found that GH deficiency was associated with diminished BW, GHR and MA-GHBP expression, while increased GH concentration led to increased BW, GHR and MA-GHBP expression, but MA-GHBP upregulation was more pronounced than the observed increase in GHR expression. Since GHR and MA-GHBP both contribute to liver GH binding capacity, GH-induced enrichment of the dominant negative form would represent a compensatory mechanism triggered by high levels of the hormone. This attempt to attenuate the effects of supraphysiological concentrations of GH may be critical to reduce or prevent their plausible damaging effects on the organism.

Increased SH2-Bbeta content and membrane association in transgenic mice overexpressing GH.

Transgenic mice overexpressing GH present a marked GH signaling desensitization, reflected by low basal phosphorylation levels of the tyrosine kinase JAK2, and signal transducer and activator of transcription-5 (STAT5) and a lack of response of these proteins to a high GH dose. To evaluate the mechanisms involved in the regulation of JAK2 activity by high GH levels in vivo, the content and subcellular distribution of SH2-Bbeta were studied in GH-overexpressing transgenic mice. SH2-B is a member of a conserved family of adapter proteins characterized by the presence of a C-terminal SH2 domain, a central pleckstrin homology (PH) domain, and an N-terminal proline rich region. The isoform SH2-Bbeta modulates JAK2 activity by binding to the phosphorylated enzyme, further increasing its activity. However, it may also interact with non-phosphorylated inactive JAK2 via lower affinity binding sites, preventing abnormal activation of the kinase. SH2-Bbeta may also function as an adapter protein, acting as a GH signaling mediator. We now report that, in an animal model of GH excess in which JAK2 is not phosphorylated, although it is increased in the membrane-fraction, both the level of SH2-Bbeta, and especially its association to membranes, are augmented (67% and 13-fold vs normal mice values respectively), suggesting SH2-Bbeta could modulate JAK2 activity in vivo.

Increased sensitivity to GH in liver of Ames dwarf (Prop1df/Prop1df) mice related to diminished CIS abundance.

To investigate the influence of chronic GH deficiency on GH signaling in vivo, we have analyzed Janus kinase (JAK) 2/signal transducers and activators of transcription (STAT) 5 GH signaling pathway, and its regulation by the suppressors of the cytokine signaling SOCS and by the JAK2-interacting protein SH2-Bbeta, in liver of Ames dwarf (Prop1df/Prop1df) mice, which are severely deficient in GH, prolactin and TSH, and of their normal littermates. Prop1df/Prop1df mice displayed unaltered GH receptor, JAK2 and STAT5a/b protein levels. No significant differences in the basal tyrosine-phosphorylation levels of JAK2 and STAT5a/b were found between both groups of animals. After in vivo administration of a high GH dose (5 microg/g body weight (BW)), the tyrosine-phosphorylation levels of JAK2 and STAT5a/b increased significantly, reaching similar values in normal and dwarf mice. However, after stimulation with lower GH doses (50 and 15 ng/g BW) the tyrosine-phosphorylation level of STAT5a/b was higher in dwarf mice. The protein content of CIS, a SOCS protein that inhibits STAT5 signaling, was approximately 80% lower in dwarf mice liver, while SOCS-2 and SOCS-3 levels were unaltered. The content of SH2-Bbeta, a modulator of JAK2 activity, was reduced by approximately 30% in dwarf mice, although this was associated with normal JAK2 response to a high GH dose. In summary, Prop1df/Prop1df mice display increased hepatic sensitivity to GH, an effect that could be related to the lower abundance of CIS in this tissue. Furthermore, the lower CIS content found in this model of GH deficiency suggests that CIS protein levels are regulated by GH in vivo.

Cytokine-inducible SH2 protein up-regulation is associated with desensitization of GH signaling in GHRH-transgenic mice.

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.

Concentration of free growth hormone-binding protein in the serum of mice is not regulated by growth hormone.

Ames dwarf mice that do not express growth hormone (GH) or prolactin (PRL) genes were used to study the effects of GH deficiency on the presence and the characteristics of GH-binding protein (GHBP) in serum. Chromatographic techniques were used to allow characterization of biological rather than immunological activity of GHBP. Two GH-binding fractions were found in dwarf mice serum, one with low affinity and high capacity (GHBPI) and one with high affinity, low capacity and lower molecular mass (GHBPII). Serum concentration of the high-affinity GHBP was 0.73 +/- 0.03 nM with a Kd of 6.3 +/- 1.7 nM. Since Ames dwarf mice have no GH in the circulation, all the GHBP is free. Interestingly, the concentration of GHBP in dwarf mice was similar to the levels of free GHBP measured in normal mice from the same line. Moreover, this value (0.7 nM) closely resembles the concentration of free GHBP in the serum of transgenic mice overexpressing GH, in which peripheral GH levels are grossly elevated. These observations can be interpreted as evidence that the levels of free GHBP in mouse serum are independent of GH concentration, and that GH influences only the levels of bound GHBP in peripheral circulation.

Up-regulation of GH-binding protein by mouse GH in transgenic mice overexpressing GH-releasing hormone.

To study the effects of homologous mouse GH (mGH) on the presence and characteristics of serum GH-binding protein (GHBP) we have used transgenic mice expressing GH-releasing hormone (GHRH) as a model. Chromatographic techniques allowed the characterization of GHBP bioactivity, and immunological techniques were used to determine its concentration and molecular components. Chromatographic separation of labeled human GH or mGH cross-linked to serum GHBPs showed two GH-binding serum fractions in normal as well as in transgenic mice serum. SDS-PAGE of this material revealed a specific band of 66 kDa and another higher molecular weight broad band, which, in the presence of 2-mercapto-ethanol, is converted into the 66 kDa fraction. Since normal mice serum has an mGH concentration of 0. 40+/-0.06 nM and a GHBP concentration of 5.7+/-1.1 nM, while the high-affinity site for mGH has a K(d)

Growth hormone (GH) receptors, binding proteins and IGF-I concentrations in the serum of transgenic mice expressing bovine GH agonist or antagonist.

We have examined the regulation of hepatic growth hormone receptors (GH-R) and serum GH binding proteins (GHBP) in transgenic mice expressing an antagonist of bovine growth hormone (bGH), G119K-bGH, and consequently exhibiting a growth suppressed dwarf phenotype. Specific GHBP could be measured in transgenic dwarf mouse serum only by immunological methods (RIA), because these mice have a very high concentration of mutated bGH in circulation (> 1 microgram/ml) and, therefore, almost all GHBP is bound to G119K-bGH and cannot be quantitated in binding assays. The concentrations of GHBP were 0.6 +/- 0.4 nM and 1.7 +/- 0.4 nM for normal and dwarf mice respectively. The concentrations of free GHBP in normal mice and in transgenic mice expressing wild-type GH can be calculated using chromatographic techniques as the dissociation constant (Kd) and the ratio of bound 125I-GH to free 125I-GH in the serum ([GHBP]free = B/F.Kd). In agreement with the assumption that GHBP reflects GH-R status, liver uptake of injected labeled bGH was greatly reduced in transgenic dwarfs in comparison with normal mice or with transgenic mice expressing wild-type bGH (liver/blood ratio of 0.48 +/- 0.21, 2.7 +/- 0.2, and 1.3 +/- 0.3 respectively) indicating that the high concentration of the mutated bGH (G119K-bGH) prevents labeled bGH uptake, as was expected from the dwarf phenotype. 125I-bGH taken up by the liver of transgenic dwarf mice was found in a smaller molecular species than in normal mice, compatible with the presence of 1:1 [(GH-R):GH] complexes instead of the 2:1 [(GH-R)2:GH] or 2:2 [(GHBP)2:(GH)2] complexes found in normal mice. The concentration of IGF-I, the principal mediator of GH activity, in the G119K-bGH transgenic mice was correlated with the concentration of free GHBP. This allowed us to use free GHBP concentration as a marker of the effects of the active endogenous hormone (mGH) on liver receptors in the presence of different concentrations of the antagonist of GH. The levels of GHBP in serum, as well as the concentration of GH-R in liver microsomes from mice expressing the bGH antagonist, are up-regulated by the high concentration of G119K-bGH (85%), but significantly less so than that which could be expected for the same concentration of native GH (220-275%). This up-regulation suggests that the G119K-bGH antagonist is internalized and induces synthesis of the receptor and of the binding protein.

Growth hormone (GH) and estradiol regulation of membrane-associated GH binding protein and GH receptors in GH releasing hormone transgenic mice.

Growth hormone (GH) releasing hormone (GHRH) transgenic mice were used to examine the influence of GH on GH receptor (GHR) and membrane-associated GH binding protein (MA-GHBP) levels by means of specific radioimmunoassays and Western blot analysis, since MA-GHBP was described as the major constituent of somatogenic binding to liver membranes in mice. In transgenic animals, a 10-fold increment over normal values was found for hepatic somatogenic binding that could be accounted for by a 3--4-fold increase in GHR and a 9-fold augmentation of MA-GHBP levels. The apparent molecular weight of MA-GHBP was smaller than that of serum GHBP, a difference that was partially abolished by endoglycosidase F digestion. In vivo treatment of female mice with 17 beta-estradiol led to an unexpected down-regulation of MA-GHBP and GHR by 60--75% only in transgenic animals. MA-GHBP and GHR levels are strongly up-regulated by GH, although MA-GHBP up-regulation is much more important than that of GHR.

Specific interactions of growth hormone (GH) with GH-receptors and GH-binding proteins in vivo in genetically GH-deficient Ames dwarf mice.

The fate of exogenous radiolabeled growth hormone (125I-hGH) was studied in Ames dwarf mice, which do not express growth hormone (GH) or prolactin (PRL) genes. Labeled GH was injected in low amounts that did not exceed the normal physiological GH concentration in mice. Binding of most of the injected 125I-hGH by the GH-binding proteins (GHBPs) present in plasma represents the first step in the handling of this material in vivo. The decay curve followed a two-compartment model and gave the equation: Conc = 2.807e-0067t + 15301e-0.0647t (coefficient of determination 0.9986+/-0.0019), while in normal mice, GH decay followed a three-compartment model as we have previously reported. The fast compartment with t1/2 of 1-2 min was virtually absent in dwarf mice, and chromatographic studies revealed the disappearance of free GH in these mice. We also present evidence of the labeled GH-forming complexes, presumably with GHBPs under in vivo conditions. The second step of processing labeled GH in vivo is the uptake by the liver, which was slower in dwarf than in normal mice (30-45 vs 15 min). Moreover, a lower GH uptake was found in dwarf than in normal mice (UB ratio of 1.75+/-0.29 [30 min] vs L/B ratio of 3.68+/-0.33 [15 min], respectively) due to lower concentration of free GH in plasma and to the reduced number of GH-receptors (GHRs). The radioactive material present in the liver was compatible with 125I-hGH-GHR complexes with Stokes radius of 59A. In summary, we provide evidence that plasma of dwarf mice contains proteins capable of binding GH in vivo and probably representing GHBPs not complexed with GH. The presence of these proteins modified the pharmacokinetics of 125I-hGH in plasma and its subsequent uptake by the liver. The presence of these binding proteins in the absence of endogenous GH suggests that a fraction of total GHBPs (one class?) is independent of GH concentration.

Identification and initial characterization of serum growth hormone binding protein in the turtle Chrysemys dorbigni.

Proteins that bind growth hormone (GHBP) have been identified in the blood of many mammalian and avian species, but not in reptilian species. We carried out binding studies with the serum of turtles using chromatographic techniques as well as the dextran-charcoal separation method. As in other species, we found at least two different GHBPs: one with high MW and low affinity and the other with lower MW and higher affinity. The high affinity GHBP was partially purified using gel filtration and affinity chromatography, reaching a degree of purification of 11,000 times (0.17 nmol/g of serum protein in the serum vs 1900 nmol/g protein in the purified material). When the high affinity GHBP was characterized, it was found to have a dissociation constant (Kd: 2.6 +/- 0.7 nM) similar to those described for mouse or rat, but lower than those for chicken, rabbit or man. The binding capacity (Bmax) was 120 +/- 43 fmoles/mg of protein, which can be also expressed as 1.08 +/- 0.38 pmol/ml of serum. A preliminary MW estimation of 50-60 kDa was obtained for turtle higher affinity GHBP. The specificity of this high affinity GHBP is somatogenic, since bovine GH competes as well as human GH for 125I-hGH bound to binding protein, while ovine PRL competes only partially and with low affinity. Unrelated hormones, as insulin and glucagon, can not displace the 125I-hGH bound to turtle GHBP. A very important seasonal variation in turtle GHBP activity was observed: maximum binding was found in November (springtime), followed by a continuous decline over March and May.

Prolonged exposure to GH impairs insulin signaling in the heart.

Acromegaly is associated with cardiac hypertrophy, which is believed to be a direct consequence of chronically elevated GH and IGF1. Given that insulin is important for cardiac growth and function, and considering that GH excess induces hyperinsulinemia, insulin resistance, and cardiac alterations, it is of interest to study insulin sensitivity in this tissue under chronic conditions of elevated GH. Transgenic mice overexpressing GH present cardiomegaly and perivascular and interstitial fibrosis in the heart. Mice received an insulin injection, the heart was removed after 2  min, and immunoblotting assays of tissue extracts were performed to evaluate the activation and abundance of insulin-signaling mediators. Insulin-induced tyrosine phosphorylation of the insulin receptor (IR) was conserved in transgenic mice, but the phosphorylation of IR substrate 1 (IRS1), its association with the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), and the phosphorylation of AKT were decreased. In addition, total content of the glucose transporter GLUT4 was reduced in transgenic mice. Insulin failed to induce the phosphorylation of the mammalian target of rapamycin (mTOR). However, transgenic mice displayed increased basal activation of the IR/IRS1/PI3K/AKT/mTOR and p38 signaling pathways along with higher serine phosphorylation of IRS1, which is recognized as an inhibitory modification. We conclude that GH-overexpressing mice exhibit basal activation of insulin signaling but decreased sensitivity to acute insulin stimulation at several signaling steps downstream of the IR in the heart. These alterations may be associated with the cardiac pathology observed in these animals.

Effects of bovine growth hormone (GH) expression in transgenic mice on serum and pituitary immunoreactive mouse GH levels and pituitary GH-releasing factor binding sites.

Pituitary and serum levels of homologous growth hormone (GH) and characteristics of specific GH-releasing factor (GHRF) binding to pituitary homogenates were examined in transgenic mice expressing bovine GH (bGH) gene regulated by different promoters [mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase (PEPCK)] and in their normal littermates. Pituitary GH concentration and GHRF binding were reduced by approximately 50% in transgenic MT-bGH mice in which serum bGH levels were about 20 micrograms/l and by approximately 95% in transgenic PEPCK-bGH mice in which serum bGH levels were tenfold higher. Suppression of plasma immunoreactive mouse GH (mGH) levels was detected in MT-bGH but not in PEPCK-bGH animals, presumably due to cross-reaction of the antiserum employed with bGH. Scatchard plots of GHRF binding to washed homogenates of pituitary glands from normal and young adult MT-bGH transgenic mice were curvilinear, indicating the presence of two types of binding sites, with low and high affinities. Both types of binding sites were reduced in number in MT-bGH transgenic mice without changes in their affinity. In 5-7-month-old MT-bGH transgenic mice there were changes in pituitary GH levels, in GHRF binding levels and in characteristics of GHRF binding that closely resembled the alterations described previously in aging rats. We conclude that over-expression of heterologous GH genes in transgenic mice can lead to partial or virtually complete suppression of somatotroph function, depending on the levels of heterologous GH in the circulation, and that transgenic MT-bGH mice exhibit symptoms of remarkably early onset of neuroendocrine aging.

Growth hormone-binding protein enhances growth hormone activity in vivo.

The decay curve of labeled growth hormone (GH) in the plasma followed a three-compartment model and could be described by the equation: concentration = Ae-alpha t + Be-beta t + Ce-gamma t, where A, B, and C are y-intercepts and alpha, beta, and gamma are compartments. When 125I-labeled ovine prolactin (oPRL) was injected, the decay curve could be described by the equation: concentration = Ae-alpha t + Ce-gamma t. Formation of 125I-labeled bovine-GH-binding protein (GHBP) complexes with somatogenic characteristics was demonstrated in the serum of both normal and GH transgenic mice. In contrast, 125I-oPRL was unable to form complexes of this type in any of the mice studied. Receptor-mediated liver uptake was found to be faster for PRL than for GH (5-6 min vs. 15-20 min). Liver uptake of radioactivity was significantly lower for PRL than for GH [liver to blood ratio (L/B) of 1.7 +/- 0.3 at 6 min vs. L/B of 3.7 +/- 0.6 at 20 min, respectively]. The presence of binding proteins for GH substantially reduces the clearance of this hormone and consequently increases the liver uptake of GH (mediated by GH receptors). This suggests that GHBPs act to increase the biological activity of GH in vivo.

Growth hormone-binding protein in normal mice and in transgenic mice expressing bovine growth hormone gene.

The levels and characteristics of growth hormone (GH)-binding protein (GHBP) and the distribution of GH in peripheral circulation between the free and the bound fractions were studied in three lines of transgenic mice with various degrees of overexpression of bovine (b) GH gene. Two serum fractions bound GH specifically: one with low affinity and high capacity (GHBPI) and one with high affinity and low capacity (GHBPII). The GHBP binding capacity in normal mice (both sexes), transgenic male mice that express the metallothionein-I-hybrid bGH genes, transgenic female mice that express phosphoenolpyruvate carboxykinase (PEPCK)-bGH hybrid genes (PEPCK-bGH-1), and transgenic PEPCK-bGH-5 animals was 1.1 +/- 0.2, 2.0 +/- 0.1, 3.0 +/- 0.1, and 3.9 +/- 0.6 pmol/ml serum, respectively. The amount of GH bound to GHBP in transgenic animals vs. normal siblings was increased 1.8-, 2.5-, and 3.9-fold in these three lines. Consequently, the levels of GH-GHBP complexes in the circulation of PEPCK-bGH-1 transgenic mice were increased approximately 10-fold. Specific GHBP radioimmunoassay confirmed a threefold increase in GHBP in PEPCK-bGH-1 transgenic animals. The levels of GHBP were not significantly correlated to serum GH within or between lines, perhaps due to elevation of serum GH in PEPCK-bGH mice above the level producing maximal response. From these and previous studies, we conclude that life-long exposure to supranormal GH levels leads to major shifts in GH binding in the circulation and in the GH target organs.

Growth hormone (GH) binding and effects of GH analogs in transgenic mice.

Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus, life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb2 cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo.

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