Functional characterization of growth hormone releasing hormone and its receptor in amphioxus with implication for origin of hypothalamic-pituitary axis.

Growth hormone-releasing hormone (GHRH) has been widely shown to stimulate growth hormone (GH) production via binding to GHRH receptor GHRHR in various species of vertebrates, but information regarding the functional roles of GHRH and GHRHR in the protochordate amphioxus remains rather scarce. We showed here that two mature peptides, BjGHRH-1 and BjGHRH-2, encoded by BjGHRH precursor, and a single BjGHRHR protein were identified in the amphioxus Branchiostoma. japonicum. Like the distribution profiles of vertebrate GHRHs and GHRHRs, both the genes Bjghrh and Bjghrhr were widely expressed in the different tissues of amphioxus, including in the cerebral vesicle, Hatschek's pit, neural tube, gill, hepatic caecum, notochord, testis and ovary. Moreover, both BjGHRH-1 and BjGHRH-2 interacted with BjGHRHR, and triggered the cAMP/PKA signal pathway in a dose-dependent manner. Importantly, BjGHRH-1 and BjGHRH-2 were both able to activate the expression of GH-like gene in the cells of Hatschek's pit. These indicate that a functional vertebrate-like GHRH-GHRHR axis had already emerged in amphioxus, which is a seminal innovation making physiological divergence including reproduction, growth, metabolism, stress and osmoregulation possible during the early evolution of vertebrates.

Constitutive signal bias mediated by the human GHRHR splice variant 1.

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone-releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to ß-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus ß-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward ß-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in a Cohort of Hungarian Pediatric Patients with Hematological and Oncological Disorders: A Pilot Study.

Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.

Experimental Autoimmune Encephalomyelitis Influences GH-Axis in Female Rats.

Inflammation, demyelination, and axonal damage to the central nervous system (CNS) are the hallmarks of multiple sclerosis (MS) and its representative animal model, experimental autoimmune encephalomyelitis (EAE). There is scientific evidence for the involvement of growth hormone (GH) in autoimmune regulation. Previous data on the relationship between the GH/insulin like growth factor-1 (IGF-1) axis and MS/EAE are inconclusive; therefore, the aim of our study was to investigate the changes in the GH axis during acute monophasic EAE. The results show that the gene expression of Ghrh and Sst in the hypothalamus does not change, except for Npy and Agrp, while at the pituitary level the Gh, Ghrhr and Ghr genes are upregulated. Interestingly, the cell volume of somatotropic cells in the pituitary gland remains unchanged at the peak of the disease. We found elevated serum GH levels in association with low IGF-1 concentration and downregulated Ghr and Igf1r expression in the liver, indicating a condition resembling GH resistance. This is likely due to inadequate nutrient intake at the peak of the disease when inflammation in the CNS is greatest. Considering that GH secretion is finely regulated by numerous central and peripheral signals, the involvement of the GH/IGF-1 axis in MS/EAE should be thoroughly investigated for possible future therapeutic strategies, especially with a view to improving EAE disease.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of human malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with exposure to asbestos, with poor prognosis and no effective therapies. The strong inhibitory activities of growth hormone-releasing hormone (GHRH) antagonists have been demonstrated in different experimental human cancers, including lung cancer; however, their role in MPM remains unknown. We assessed the effects of the GHRH antagonists MIA-602 and MIA-690 in vitro in MPM cell lines and in primary MPM cells, and in vivo in MPM xenografts. GHRH, GHRH receptor, and its main splice variant SV1 were found in all the MPM cell types examined. In vitro, MIA-602 and MIA-690 reduced survival and proliferation in both MPM cell lines and primary cells and showed synergistic inhibitory activity with the chemotherapy drug pemetrexed. In MPM cells, GHRH antagonists also regulated activity and expression of apoptotic molecules, inhibited cell migration, and reduced the expression of matrix metalloproteinases. These effects were accompanied by impairment of mitochondrial activity and increased production of reactive oxygen species. In vivo, s.c. administration of MIA-602 and MIA-690 at the dose of 5 µg/d for 4 wk strongly inhibited the growth of MPM xenografts in mice, along with reduction of tumor insulin-like growth factor-I and vascular endothelial growth factor. Overall, these results suggest that treatment with GHRH antagonists, alone or in association with chemotherapy, may offer an approach for the treatment of MPM.

Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in Primary Human Endometrial Carcinomas: Novel Therapeutic Approaches.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various tumors, including endometrial carcinomas (EC). However, tumoral receptors that mediate the antiproliferative effects of GHRH antagonists in human ECs have not been fully characterized. In this study, we investigated the expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors (GHRH-R) in 39 human ECs and in 7 normal endometrial tissue samples using RT-PCR. Primers designed for the PCR amplification of mRNA for the full length GHRH-R and SVs were utilized. The PCR products were sequenced, and their specificity was confirmed. Nine ECs cancers (23%) expressed mRNA for SV1, three (7.7%) showed SV2 and eight (20.5%) revealed mRNA for SV4. The presence of SVs for GHRH-Rs could not be detected in any of the normal endometrial tissue specimens. The presence of specific, high affinity GHRH-Rs was also demonstrated in EC specimens using radioligand binding studies. Twenty-four of the investigated thirty-nine tumor samples (61.5%) and three of the seven corresponding normal endometrial tissues (42.9%) expressed mRNA for GHRH ligand. Our findings suggest the possible existence of an autocrine loop in EC based on GHRH and its tumoral SV receptors. The antiproliferative effects of GHRH antagonists on EC are likely to be exerted in part by the local SVs and GHRH system.

Disruption of the GHRH receptor and its impact on children and adults: The Itabaianinha syndrome.

Since 1994, we have been studying an extended kindred with 105 subjects (over 8 generations) residing in Itabaianinha County, in the Brazilian state of Sergipe, who have severe isolated GH deficiency (IGHD) due to a homozygous inactivating mutation (c.57 + 1G > A) in the GH releasing hormone (GHRH) receptor (GHRHR) gene. Most of these individuals have never received GH replacement therapy. They have low GH, and very low and often undetectable levels of serum IGF-I. Their principal physical findings are proportionate short stature, doll facies, high-pitched-voice, central obesity, wrinkled skin, and youthful hair with delayed pigmentation, and virtual absence of graying. The newborns from this cohort are of normal size, indicating that GH is not needed for intra-uterine growth. However, these IGHD individuals exhibit a myriad of phenotypic changes throughout the body, with a greater number of beneficial than harmful consequences. This GHRH signal disruption syndrome has been a valuable model to study the GH roles in body size and function. This reviews summarized the findings we have reported on this cohort.

The effect of miR-93 on GH secretion in pituitary cells of Yanbian yellow cattle.

Yanbian yellow cattle breeding is limited by slow growth. We previously found that the miRNA miR-93 was differentially expressed between the blood exosomes of Yanbian yellow cattle and Han Yan cattle, which differ in growth characteristics. In this experiment, we evaluated the effects of miR-93 on growth hormone (GH) secretion by pituitary cells of Yanbian yellow cattle using qPCR, Western blot, Targetscan and RNA hybrid analysis software and Dual-Luciferase reporter gene system. The results showed that miR-93 targeted 3' UTR of GHRHR(growth hormone releasing hormone receptor); GH mRNA and protein levels in pituitary cells of Yanbian yellow cattle were significantly lower in the miR-93-mi group than in the NC control group (p < 0.01), while GH mRNA and protein levels were higher in the miR-93-in group than in the iNC control group, but the difference was not significant (p > 0.05); GHRHR mRNA and protein levels were significantly lower in the miR-93-mi group than in the NC control group (p < 0.01), while GHRHR protein levels were significantly higher in the miR-93-in group than in the iNC control group (p < 0.05), but there was no significant difference about GHRHR mRNA level between two groups (p > 0.05). These results prove that miR-93 regulates GH secretion in pituitary cells via GHRHR.

Regulation of Vascular Calcification by Growth Hormone-Releasing Hormone and Its Agonists.

RATIONALE: Vascular calcification (VC) is a marker of the severity of atherosclerotic disease. Hormones play important roles in regulating calcification; estrogen and parathyroid hormones exert opposing effects, the former alleviating VC and the latter exacerbating it. To date no treatment strategies have been developed to regulate clinical VC. OBJECTIVE: The objective of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) and its agonist (GHRH-A) on the blocking of VC in a mouse model. METHODS AND RESULTS: Young adult osteoprotegerin-deficient mice were given daily subcutaneous injections of GHRH-A (MR409) for 4 weeks. Significant reductions in calcification of the aortas of MR409-treated mice were paralleled by markedly lower alkaline phosphatase activity and a dramatic reduction in the expression of transcription factors, including the osteogenic marker gene Runx2 and its downstream factors, osteonectin and osteocalcin. The mechanism of action of GHRH-A was dissected in smooth muscle cells isolated from human and mouse aortas. Calcification of smooth muscle cells induced by osteogenic medium was inhibited in the presence of GHRH or MR409, as evidenced by reduced alkaline phosphatase activity and Runx2 expression. Inhibition of calcification by MR409 was partially reversed by MIA602, a GHRH antagonist, or a GHRH receptor-selective small interfering RNA. Treatment with MR409 induced elevated cytosolic cAMP and its target, protein kinase A which in turn blocked nicotinamide adenine dinucleotide phosphate oxidase activity and reduced production of reactive oxygen species, thus blocking the phosphorylation of nuclear factor κB (p65), a key intermediate in the ligand of receptor activator for nuclear factor-κ B-Runx2/alkaline phosphatase osteogenesis program. A protein kinase A-selective small interfering RNA or the chemical inhibitor H89 abolished these beneficial effects of MR409. CONCLUSIONS: GHRH-A controls osteogenesis in smooth muscle cells by targeting cross talk between protein kinase A and nuclear factor κB (p65) and through the suppression of reactive oxygen species production that induces the Runx2 gene and alkaline phosphatase. Inflammation-mediated osteogenesis is thereby blocked. GHRH-A may represent a new pharmacological strategy to regulate VC.

Pituitary miRNAs target GHRHR splice variants to regulate GH synthesis by mediating different intracellular signalling pathways.

Growth hormone (GH), whose synthesis and release are mainly regulated by intracellular signals mediated by growth hormone-releasing hormone receptor (GHRHR), is one of the major pituitary hormones and critical regulators of organism growth, metabolism, and immunoregulation. Pig GHRHR splice variants (SVs) may activate different signalling pathways via the variable C-terminal by alternative splicing, and SVs have the potential to change microRNA (miRNA) binding sites. In this study, we first confirmed the existence of pig GHRHR SVs (i.e., GHRHR, GHRHR SV1 and SV2) and demonstrated the inhibitory effects of critical pituitary miRNAs (i.e., let-7e and miR-328-5p) on GH synthesis and cell proliferation of primary pituitary cells. The SVs of GHRHR targeted by let-7e and miR-328-5p were predicted via bioinformatics analysis and verified by performing dual-luciferase reporter assays and detecting the expression of target transcripts. The differential responses of let-7e, and miR-328-5p to GH-releasing hormone and the changes in signalling pathways mediated by GHRHR suggested that let-7e and miR-328-5p were involved in GH synthesis mediated by GHRHR SVs, indicating that the two miRNAs played different roles by different ways. Finally, results showed that the protein coded by the GHRHR transcript regulated GH through the NO/NOS signalling pathway, whereas that coded by SV1 and SV2 regulated GH through the PKA/CREB signalling pathway, which was confirmed by the changes in signalling pathways after transfecting the expression vectors of GHRHR SVs to GH3 cells. To the best of our knowledge, this paper is the first to report pituitary miRNAs regulate GH synthesis by targeting the different SVs of GHRHR.

Contribution of functionally assessed GHRHR mutations to idiopathic isolated growth hormone deficiency in patients without GH1 mutations.

Isolated growth hormone deficiency (IGHD) is a rare condition mainly caused by mutations in GH1. The aim of this study was to assess the contribution of GHRHR mutations to IGHD in an unusually large group of patients. All GHRHR coding exons and flanking intronic regions were sequenced in 312 unrelated patients with nonsyndromic IGHD. Functional consequences of all newly identified missense variants were assessed in vitro (i.e., study of the expression of recombinant GHRHRs and their ability to activate the cyclic adenosine monophosphate (cAMP) signaling pathway). Genotype-phenotype correlation analyses were performed according to the nature of the identified mutation. We identified 20 different disease-causing GHRHR mutations (truncating and missense loss-of-function mutations), among which 15 are novel, in 24 unrelated patients. Of note, about half (13/24) of those patients represent sporadic cases. The clinical phenotype of patients with at least one missense GHRHR mutation was found to be indistinguishable from that of patients with bi-allelic truncating mutations. This study, which unveils disease-causing GHRHR mutations in 8% (24/312) of IGHD cases, identifies GHRHR as the second IGHD gene most frequently involved after GH1. The finding that 8% of IGHD cases without GH1 mutations are explained by GHRHR molecular defects (including missense mutations), together with the high proportion of sporadic cases among those patients, has important implications for genetic counseling.

Growth hormone-releasing hormone receptor mediates cytokine production in ciliary and iris epithelial cells during LPS-induced ocular inflammation.

The receptor for growth hormone-releasing hormone (GHRH-R) has been shown to upregulate specifically in the ciliary and iris epithelial cells and infiltrating cells in the aqueous humor in a rat model of acute anterior uveitis. Treatment with GHRHR-R antagonist alleviates significantly these inflammatory responses. Herein we investigated whether the ciliary and iris epithelial cells can respond directly to lipopolysaccharide (LPS) without the influences of circulating leukocytes to produce inflammatory mediators through a GHRH-R mediated mechanism. In explant cultures of rat ciliary body and iris, LPS caused a substantial increase of GHRH-R in 24 h. Immunohistochemistry showed a localization of TLR4, the receptor for LPS, and an elevated expression of IL-6 and IL-1ß in ciliary and iris epithelial cells after LPS treatment. LPS also elevated the level of IL-1ß, IL-6, and iNOS and increased secretion of IL-1ß and IL-6 from the explants. The GHRH-R antagonist, MIA-602, suppressed the elevated expression of IL-1ß and IL-6, and reduced the release of IL-6. Such effects were not seen for the GHRHR agonist, MR-409. When co-cultured with leukocytes, expression of GHRH-R in the ocular explants was further enhanced during LPS treatment. Our results demonstrate a direct action of LPS on ciliary and iris epithelial cells to produce pro-inflammatory factors through a GHRH-R mediated mechanism, and suggest a role of these epithelial cells, in addition to the resident antigen presenting cells, in immune surveillance of the eye. Infiltrating leukocytes may enhance these inflammatory responses by regulating GHRH-R in ciliary and iris epithelial cells, in addition to their functions of synthesizing proinflammatory cytokines.

Cell-Autonomous and Non-Cell-Autonomous Regulation of a Feeding State-Dependent Chemoreceptor Gene via MEF-2 and bHLH Transcription Factors.

Food and feeding-state dependent changes in chemoreceptor gene expression may allow Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state.

Association between the GHR, GHRHR and IGF1 gene polymorphisms and milk coagulation properties in Sarda sheep.

We investigated whether variation of the sheep Growth Hormone Receptor (GHR), Growth Hormone Releasing Hormone Receptor (GHRHR) and Insulin-Like Growth Factor 1 (IGF1) genes were associated with milk coagulation properties (MCP) in sheep. The GHR, GHRHR and IGF1 genes are part of the GH system, which is known to modulate metabolism, growth and reproduction as well as mammogenesis and galactopoiesis in dairy species. A total of 380 dairy Sarda sheep were genotyped for 36 SNPs mapping to these three genes. Traditional MCP were measured as rennet coagulation time (RCT), curd-firming time (k20) and curd firmness at 30 m (a30). Modeling of curd firming over time (CFt) was based on a 60 m lactodynamographic test, generating a total of 240 records of curd firmness (mm) for each milk sample. The model parameters obtained included: the rennet coagulation time as a result of modeling all data available (RCTeq, min); the asymptotic potential value of curd firmness (CFP, mm) at an infinite time; the CF instant rate constant (kCF, %/min); the syneresis instant rate constant (kSR, %/min); the maximum value of CF (CFmax, mm) and the time at achievement of CFmax (tmax, min). Statistical analysis revealed that variation of the GHR gene was significantly associated with RCT, kSR and CFP (P < 0.05). No other significant associations were detected. These findings may be useful for the dairy industry, as well as for selection programs.

Association between the GHR, GHRHR, and IGF1 gene polymorphisms and milk yield and quality traits in Sarda sheep.

The growth hormone receptor (GHR), the growth hormone releasing hormone receptor (GHRHR), and the insulin-like growth factor 1 (IGF1) genes are known to modulate growth, reproduction, and lactation traits in livestock. The aim of the current work was to investigate if the variation of the sheep GHR, GHRHR, and IGF1 genes is associated with milk yield and quality traits. Three hundred eighty dairy Sarda sheep were genotyped for 36 single nucleotide polymorphisms (SNP) mapping to these 3 loci, and records for milk yield and daily fat and protein yield, as well as for fat, protein, casein, lactose, and milk urea contents, pH, somatic cell score, logarithmic bacterial count, and milk energy were obtained. The linkage disequilibrium analysis was performed only for GHR, as both GHRHR and IGF1 had only 1 polymorphic SNP. Haplotype analysis revealed the existence of 7 haplotype blocks in GHR. Two haplotype blocks, including part of the intron 1 and the upstream region, were clearly separated from the remaining 5 blocks by SNP rs412986330, which may be a recombination hotspot. The latter 5 blocks were contiguous, spanning from intron 2 to exon 10. Statistical analysis revealed that the GHR polymorphism is significantly associated with milk traits for daily fat and protein yield and fat, milk urea, and lactose content. Moreover, variation in IGF1 was associated with milk protein and casein content. Data generated in this research provide new insights into the allelic effects of the ovine GHRHR, GHR, and IGF1 genes on milk production and quality traits, information that may be useful in gene-assisted selection programs.

Next generation sequencing-based mutation screening of 86 patients with idiopathic short stature.

Although mutations in ACAN, FGFR3, NPR2, and SHOX typically lead to skeletal dysplasia, and mutations in GHRHR, GH1, GHR, STAT5B, IGF1, IGFALS, and IGF1R usually underlie hormonal defects of the growth hormone (GH)-insulin-like growth factor 1 (IGF1) axis, such mutations have also been identified in patients with idiopathic short stature (ISS). Of these, SHOX abnormalities are known to account for a certain percentage of ISS cases, whereas the frequency of mutations in the other 10 genes in ISS cohorts remains unknown. Here, we performed next-generation sequencing-based mutation screening of the 10 genes in 86 unrelated Japanese ISS patients without SHOX abnormalities. We searched for rare protein-altering variants. The functional significance of the identified variants was assessed by in silico analyses. Consequently, we identified 18 heterozygous rare variants in 19 patients, including four probable damaging variants in ACAN, six pathogenicity-unknown variants in FGFR3, GHRHR, GHR, and IGFALS, and eight possible benign variants. Pathogenic variants in NPR2, GH1, and IGF1 were absent from our cohort. Unlike previously reported patients with ACAN mutations, our four patients with ACAN variants manifested non-specific short stature with age-appropriate or mildly delayed bone ages, and had parents of normal stature. These results indicate that ACAN mutations can underlie ISS without characteristic skeletal features, and that such mutations are possibly associated with de novo occurrence or low penetrance. In addition, our data imply that mutations in FGFR3, NPR2, and GH-IGF1 axis genes play only limited roles in the etiology of ISS.

Agonists of growth hormone-releasing hormone stimulate self-renewal of cardiac stem cells and promote their survival.

The beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R) in heart failure models are associated with an increase in the number of ckit(+) cardiac stem cells (CSCs). The goal of the present study was to determine the presence of GHRH-R in CSCs, the effect of GHRH-R agonists on their proliferation and survival, and the mechanisms involved. We investigated the expression of GHRH-R in CSCs of different species and the effect of GHRH-R agonists on their cell proliferation and survival. GHRH-R is expressed in ckit(+) CSCs isolated from mouse, rat, and pig. Treatment of porcine CSCs with the GHRH-R agonist JI-38 significantly increased the rate of cell division. Similar results were observed with other GHRH-R agonists, MR-356 and MR-409. JI-38 exerted a protective effect on survival of porcine CSCs under conditions of oxidative stress induced by exposure to hydrogen peroxide. Treatment with JI-38 before exposure to peroxide significantly reduced cell death. A similar effect was observed with MR-356. Addition of GHRH-R agonists to porcine CSCs induced activation of ERK and AKT pathways as determined by increased expression of phospho-ERK and phospho-AKT. Inhibitors of ERK and AKT pathways completely reversed the effect of GHRH-R agonists on CSC proliferation. Our findings extend the observations of the expression of GHRH-R by CSCs and demonstrate that GHRH-R agonists have a direct effect on proliferation and survival of CSCs. These results support the therapeutic use of GHRH-R agonists for stimulating endogenous mechanisms for myocardial repair or for preconditioning of stem cells before transplantation.

Effects of retinoic acid on growth hormone-releasing hormone receptor, growth hormone secretagogue receptor gene expression and growth hormone secretion in rat anterior pituitary cells.

Retinoic acid (RA) is an important signaling molecule in embryonic development and adult tissue. The actions of RA are mediated by the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR), which regulate gene expression. RAR and RXR are widely expressed in the anterior pituitary gland. RA was reported to stimulate growth hormone (GH) gene expression in the anterior pituitary cells. However, current evidence is unclear on the role of RA in gene expression of growth hormone-releasing hormone receptor (Ghrh-r), growth hormone secretagogue receptor (Ghs-r) and somatostatin receptors (Sst-rs). Using isolated anterior pituitary cells of rats, we examined the effects of RA on gene expression of these receptors and GH release. Quantitative real-time PCR revealed that treatment with all-trans retinoic acid (ATRA; 10(-6) M) for 24 h increased gene expression levels of Ghrh-r and Ghs-r; however, expressions of Sst-r2 and Sst-r5 were unchanged. Combination treatment with the RAR-agonist Am80 and RXR-agonist PA024 mimicked the effects of ATRA on Ghrh-r and Ghs-r gene expressions. Exposure of isolated pituitary cells to ATRA had no effect on basal GH release. In contrast, ATRA increased growth hormone-releasing hormone (GHRH)- and ghrelin-stimulated GH release from cultured anterior pituitary cells. Our results suggest that expressions of Ghrh-r and Ghs-r are regulated by RA through the RAR-RXR receptor complex and that RA enhances the effects of GHRH and ghrelin on GH release from the anterior pituitary gland.

Transplantation of pancreatic islets to adrenal gland is promoted by agonists of growth-hormone-releasing hormone.

Here, we evaluate an alternative approach of preconditioning pancreatic islets before transplantation using a potent agonist of growth-hormone-releasing hormone (GHRH) to promote islet viability and function, and we explore the adrenal gland as an alternative transplantation site for islet engraftment. The endocrine microenvironment of the adrenal represents a promising niche with the unique advantages of exceptional high oxygen tension and local anti-inflammatory and immunosuppressive properties. GHRH agonists have been shown to promote islet graft survival and function, which may help to reduce the islet mass necessary to reverse diabetes. In the present study, the most potent GHRH agonist MR403 was tested on insulinoma cells, isolated rat islets, and adrenal ß-cell cocultures in vitro. GHRH receptor is expressed on both adrenal cells and islets. MR403 caused a significant increase in cell viability and proliferation and revealed an antiapoptotic effect on insulinoma cells. Viability of rat islets was increased after treatment with the agonist and in coculture with adrenal cells. Rat islets were transplanted into diabetic mice to the intraadrenal transplant site and compared with the classical transplants underneath the kidney capsule. Graft function and integration were tested by metabolic follow-up and immunohistochemical staining of intraadrenal grafts. A rapid decrease occurred in blood glucose levels in both models, and all animals reached normoglycemia within the first days after transplantation. Our studies demonstrated that the adrenal may be an attractive site for islet transplantation and that GHRH analogs might allow reduction of the islet mass needed to reverse a diabetic status.

Growth hormone releasing hormone receptor codon 72 mutation in a cohort of Sri Lankan patients with growth hormone deficiency.

INTRODUCTION: Growth hormone releasing hormone receptor (GHRH-R) codon 72 mutation is recognised as a common genetic cause of growth hormone deficiency (GHD) in the Indian subcontinent resulting in a characteristic lean phenotype. Genetic studies have not been previously carried out in Sri Lankans with GHD. METHODS: Patients with GHD presenting to a tertiary care referral centre were studied for GHRH-R codon 72 mutation by PCR amplification and sequencing. The phenotype of the cohort was described as the BMI SDS (Body mass index standard deviation score) based on the anthropometric data at the time of diagnosis. RESULTS: Among 91 patients from 88 families studied, eight (6 boys) carried the codon 72 mutation. The presence of this mutation was low among the Sinhalese ethnicity (3 out of 68) than among Tamil and Moor ethnicities. BMI SDS of <-2 was seen in 71% of mutation positive and 45.8% of mutation negative patients. CONCLUSIONS: Prevalence of GHRH-R codon 72 mutation in this group of GH deficient patients was 8.8%. The lean phenotype observed in 71% of the mutation positive patients was not a significant association when compared to a similar phenotype in 45.8% of the mutation negative patients.