Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

Growth hormone secretagogues: characterization, efficacy, and minimal bioactive conformation.

Another class of growth hormone (GH) secretagogues has been discovered by altering the backbone structure of a flexible linear GH-releasing peptide (GHRP). In vitro and in vivo characterization confirms these GH secretagogues as the most potent and smallest (M(r) < 500) reported. Anabolic efficacy is demonstrated in rodents with intermittent delivery. A convergent model of the bioactive conformation of GHRPs is developed and is supported by the NMR structure of a highly potent cyclic analog of GHRP-2. The model and functional data provide a logical framework for the further design of low-molecular weight secretagogues and illustrate the utility of an interdisciplinary approach to elucidating potential bound-state conformations of flexible peptide ligands.

Rat growth hormone gene introns stimulate nucleosome alignment in vitro and in transgenic mice.

Average hepatic expression (mRNA per cell per gene) of a metallothionein-rat growth hormone (rGH) gene with its natural introns was about 15-fold higher than an intronless version when tested in transgenic mice. We examined the idea that intron removal leads to an alteration in chromatin structure that might be responsible for this effect. Using an in vitro chromatin assembly system, we observed that nucleosomes were aligned in a characteristic ordered array over the gene and promoter when all introns were present. Linker histones were necessary for this alignment to occur. In contrast, nucleosome alignment was perturbed in constructs lacking some or all of the introns. A similar disruption of nucleosome alignment was observed when comparing chromatin from livers of transgenic mice carrying rGH transgenes with or without introns. In vitro, sequences at the 3' end of the rGH gene position nucleosomes and facilitate nucleosome alignment upstream; however, nucleosome alignment does not occur on the approximately 3 kb of downstream flanking rat sequence. These observations suggest that signals present in genomic rGH DNA may serve to establish appropriate nucleosome alignment during development and, possibly, to restore nucleosome alignment to the transcribed region after disruption incurred by the passage of an RNA polymerase molecule, thereby facilitating subsequent rounds of transcription.

Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone.

Augmentation of vertebrate growth by growth hormone (GH) is primarily due to its regulation of insulin-like growth factor I (IGF I) and IGF II levels. To characterize the effect of GH on the levels of IGF I and IGF II mRNA in a teleost, 10 micrograms of bovine GH (bGH) per g of body weight was administered to juvenile rainbow trout (Oncorhynchus mykiss) through i.p. injection. The levels of IGF I and IGF II mRNA were determined simultaneously, by using RNase protection assays, in the liver, pyloric ceca, kidney, and gill at 0, 1, 3, 6, 12, 24, 48, and 72 hr after injection. In the liver, IGF I mRNA levels were significantly elevated at 6 and 12 hr (approximately 2- to 3-fold, P < or = 0.01), while IGF II mRNA levels were significantly elevated at 3 and 6 hr (approximately 3-fold, P < or = 0.01). In the pyloric ceca, IGF II mRNA levels were significantly elevated at 12, 24, and 48 hr (approximately 3-fold, P < or = 0.01), while IGF I mRNA was below the limits of assay accuracy. GH-dependent IGF mRNA appearance was not detected in the gill and kidney. Serum bGH levels, determined by using a radioimmunoassay, were significantly elevated at 3 and 6 hr (P < 0.005). In primary hepatocyte culture, IGF I and IGF II mRNA levels increased in a bGH dose-dependent fashion, with ED50 values of approximately 45 and approximately 6 ng of bGH per ml, respectively. The GH-dependent appearance of IGF II mRNA in the liver and pyloric ceca suggests important roles for this peptide hormone exclusive of IGF I.

Design and biological activities of L-163,191 (MK-0677): a potent, orally active growth hormone secretagogue.

A potent, orally active growth hormone (GH) secretagogue L-163,191 belonging to a recently synthesized structural class has been characterized. L-163,191 releases GH from rat pituitary cells in culture with EC50 = 1.3 +/- 0.09 nM and is mechanistically indistinguishable from the GH-releasing peptide GHRP-6 and the prototypical nonpeptide GH secretagogue L-692,429 but clearly distinguishable from the natural GH secretagogue, GH-releasing hormone. L-163,191 elevates GH in dogs after oral doses as low as 0.125 mg/kg and was shown to be specific in its release of GH without significant effect on plasma levels of aldosterone, luteinizing hormone, thyroxine, and prolactin after oral administration of 1 mg/kg. Only modest increases in cortisol were observed. Based on these properties, L-163,191 has been selected for clinical studies.

Growth hormone, insulin-like growth factor I and cell proliferation in the mouse blastocyst

The role of growth hormone (GH) in embryonic growth is controversial, yet preimplantation embryos express GH, insulin-like growth factor I (IGF-I) and their receptors. In this study, addition of bovine GH doubled the proportion of two-cell embryos forming blastocysts and increased by about 25% the number of cells in those blastocysts with a concentration-response curve showing maximal activity at 1 pg bovine GH ml(-1), with decreasing activity at higher and lower concentrations. GH increased the number of cells in the trophectoderm by 25%, but did not affect the inner cell mass of blastocysts. Inhibition of cell proliferation by anti-GH antiserum indicated that GH is a potent autocrine or paracrine regulator of the number of trophectoderm cells in vivo. Type 1 IGF receptors (IGF1R) were localized to cytoplasmic vesicles and plasma membrane in the apical domains of uncompacted and compacted eight-cell embryos, but were predominantly apparent in cytoplasmic vesicles of the trophectoderm cells of the blastocyst, similar to GH receptors. Studies using alphaIR3 antiserum which blocks ligand activation of IGF1R, showed that IGF1R participate in the autocrine or paracrine regulation of the number of cells in the inner cell mass by an endogenous IGF-I-IGF1R pathway. However, alphaIR3 did not affect GH stimulation of the number of trophectoderm cells. Therefore, CH does not use secondary actions via embryonic IGF-I to modify the number of blastocyst cells. This result indicates that GH and IGF-I act independently. GH may selectively regulate the number of trophectoderm cells and thus implantation and placental growth. Embryonic GH may act in concert with IGF-I, which stimulates proliferation in the inner cell mass, to optimize blastocyst development.

Growth hormone and epidermal growth factor in salivary glands of giant and dwarf transgenic mice

Epidermal growth factor (EGF) in rat salivary glands is regulated by testosterone, thyroxin, and growth hormone (GH). Salivary glands of 45-day-old giant and dwarf male and female transgenic mice were examined histologically and by immunohistochemistry (IHC) for EGF. Male giants showed no significant differences from wild-type (WT) parotid and submandibular glands. However, their sublingual glands expressed EGF diffusely and strongly in granular cells within the striated ducts, where they were not found in WT mice. Submandibular gland ducts of female WT were different, having individual granular cells strongly positive for EGF and distributed sporadically along the striated duct walls. Neither female GH-antagonist dwarf mice nor GH-receptor knockout mice had any granular cells expressing EGF in any gland. Obvious presence of granular duct cells in the sublingual glands of giant male mice suggests GH-upregulated granular cell EGF expression. Furthermore, absence of granular duct cells from all glands in female GH-antagonist and GH-receptor knockout transgenic mice suggests that GH is necessary for the differentiation of the granular cell phenotype in female salivary glands.

Influence of growth hormone on the mandibular condylar cartilage of rats

Growth hormone (GH) stimulates mandibular growth but its effect on the mandibular condylar cartilage is not well. understood. Objective: This study was designed to understand the influence of GH on mitotic activity and on chondrocytes maturation. The effect of GH on cartilage thickness was also determined. Design: An animal model witt differences in GH status was determined by comparing mutant Lewis dwarf rats with reduced pituitary GH synthesis (dwarf), with normal rats and dwarf animals treated with GH. Six dwarf rats were injected with GH for 6 days, while other six normal rats and six dwarf rats composed other two groups. Mandibular condylar tissues were processed and stained for Herovici's stain and immunohistochemistry, for proliferating cell nuclear antigen (PCNA) and alkaline phosphatase (ALP). Measurements of cartilage thickness as well as the numbers of immunopositive cells for each antibody were analysed by one-way analysis of variance. Results: Cartilage thickness was significantly reduced in the dwarf animals treated with GH. PCNA expression was significant lower in the dwarf rats, but significantly increased when these animals were treated with GH. ALP expression was significant higher in the dwarf animals, while it was significantly reduced in the dwarf animals treated with GH. Conclusions: The results from this study showed that GH stimulates mitotic activity and delays cartilage cells maturation in the mandibular condyte. This effect at the cellular Level may produce changes in the cartilage thickness. (C) 2004 Elsevier Ltd. All rights reserved.

Mouse cellular cementum is highly dependent on growth hormone status

Cementum is known to be growth-hormone (GH)-responsive, but to what extent is unclear. This study examines the effects of extremes of GH status on cementogenesis in three lines of genetically modified mice; GH excess (giant), GH antagonist excess (dwarf), and GH receptor-deleted (GHR-KO) (dwarf). Age-matched mandibular molar tissues were processed for light microscope histology. Digital images of sections of first molar teeth were captured for morphometric analysis of lingual root cementum. Cross-sectional area of the cellular cementum was a sensitive guide to GH status, being reduced nearly 10-fold in GHR-KO mice, three-fold in GH antagonist mice, and increased almost two-fold in giant mice (p

Blunted growth hormone response to clonidine in post-traumatic stress disorder

Hyperactivity of the sympathetic and noradrenergic systems is thought to be a feature of post-traumatic stress disorder (PTSD). Assessment of noradrenergic receptor function can be undertaken by measuring the growth hormone (GH) response to the alpha(2)-agonist clonidine. The aim of this study was to examine whether subjects with combat-related PTSD (with or without co-morbid depression) have a blunted growth hormone response to clonidine, compared to a combat-exposed control group. Twenty-three Vietnam veterans suffering from PTSD alone, 27 suffering from PTSD and co-morbid depression, and 32 veteran controls with no psychiatric illness were administered 1.5 mug/kg clonidine i.v. Plasma growth hormone was measured every 20 min for 120 min. The growth hormone response to clonidine was significantly blunted in the non-depressed PTSD group compared to both the depressed PTSD group and the control group as measured by peak growth hormone, delta growth hormone and AUC growth hormone. Subjects with PTSD and no co-morbid depressive illness show a blunted growth hormone response to clonidine. This suggests that post-synaptic alpha(2)-receptors are subsensitive. This finding is consistent with other studies showing increased noradrenergic activity in PTSD. (C) 2003 Elsevier Ltd. All rights reserved.

Comparative analysis of CNS populations in knockout mice with altered growth hormone responsiveness

Recently we have shown that growth hormone (GH) inhibits neuronal differentiation and that this process is blocked by suppressor of cytokine signalling-2 (SOCS2). Here we examine several cortical and subcortical neuronal populations in GH hyper-responsive SOCS2 null (-/-) mice and GH non-responsive GH receptor null (GHR-/-) mice. While SOCS2-/- mice showed a 30% decrease in density of NeuN positive neurons in cortex compared to wildtype, GHR-/- mice showed a 25% increase even though brain size was decreased. Interneuron sub-populations were variably affected, with a slight decrease in cortical parvalbumin expressing interneurons in SOCS2-/- mice and an increase in cortical calbindin and calretinin and striatal cholinergic neuron density in GHR-/- mice. Analysis of glial cell numbers in cresyl violet or glial fibrillary acidic protein (GFAP) stained sections of cortex showed that the neuron: glia ratio was increased in GHR-/- mice and decreased in SOCS2-/- mice. The astrocytes in GHR-/- mice appeared smaller, while they were larger in SOCS2-/- mice. Neuronal soma size also varied in the different genotypes, with smaller striatal cholinergic neurons in GHR-/- mice. While the size of layer 5 pyramidal neurons was not significantly different from wildtype, SOCS2-/- neurons were larger than GHR-/- neurons. In addition, primary dendritic length was similar in all genotypes but dendritic branching of pyramidal neurons in the cortex appeared sparser in GHR-/- and SOCS2-/- mice. These results suggest that GH, possibly regulated by SOCS2, has multiple effects on central nervous system (CNS) development and maturation, regulating the number and size of multiple neuronal and glial cell types.

The role of growth hormone replacement in a growth hormone deficient patient with underlying cardiomyopathy and severe congestive heart failure

It has been reported that-growth hormone (GH) deficiency induced cardiomyopathy responds to growth hormone replacement therapy. We describe the case of a middle-aged male with cardiomyopathic heart failure and growth hormone deficiency of the adult secondary to surgical panhypopituitarism. We demonstrate clinical and hemodynamic improvement of cardiac function with growth hormone replacement therapy despite underlying structural heart disease. Copyright (C) 2005 by the International Society for Heart and Lung Transplantation.

Influence of growth hormone on the craniofacial complex of transgenic mice

Growth hormone (GH) secretion affects bone and cartilage physiology. This study investigated the effect of GH on the size of the craniofacial structures and their angular relationship. Three different models of mice with a genetically altered GH axis were used: GH excess (giant), dwarf GH antagonist (dwarf-Ant), and dwarf GH receptor knockout (dwarf-KO) mice. Each model was compared with the corresponding wild type (Wt). Five craniofacial distances were analysed: craniofacial length, upper face height, mandibular anterior height, mandibular ramus length, and mandibular corpus length. In addition, upper and lower incisor lengths and four angular relationships, nasal bone with cranial base, maxillary plane with cranial base, mandibular plane with cranial base, and the angle of the mandible, were determined. Data were analysed by one-way ANOVA. Craniofacial length, upper face height and mandibular corpus length were significantly increased in the giant mice and significantly reduced in the dwarf mice. Mandibular anterior height and mandibular ramus length were significantly affected in the dwarf-KO mice but not in the giant mice. The length of both the upper and lower incisors was significantly increased and reduced in the giant and dwarf-KO mice, respectively. In addition, the angle of the mandible was significantly increased in the giant mice and significantly reduced in the dwarf mice. It is concluded that GH plays a major role in the growth and development of the craniofacial complex by directly and indirectly modulating the size and the angular relationships of the craniofacial structures, including the incisor teeth.