Growth Hormone Receptor Protein Research Articles

Reporting a novel growth hormone receptor gene variant in an Iranian consanguineous pedigree with Laron syndrome: a case report

BackgroundHuman growth hormone (hGH) plays a crucial role in growth by binding to growth hormone receptor (GHR) in target cells. Binding of GH molecules to their cognate receptors triggers downstream signaling pathways leading to the transcription of several genes, including insulin-like growth factor (IGF)-1. Pathogenic variants in the GHR gene can result in structural and functional defects in the GHR protein, leading to Laron Syndrome (LS) with the primary clinical manifestation of short stature. So far, around 100 GHR variants have been reported, mostly biallelic, as causing LS.Case presentationWe report on three siblings from an Iranian consanguineous family who presented with dwarfism. Whole-exome sequencing (WES) was performed on the proband, revealing a novel homozygous missense variant in the GHR gene (NM_000163.5; c.610 T > A, p.(Trp204Arg)) classified as a likely pathogenic variant according to the recommendation of the American College of Medical Genetics (ACMG). Co-segregation analysis was investigated using Sanger sequencing.ConclusionsTo date, approximately 400–500 LS cases with GHR biallelic variants, out of them 10 patients originating from Iran, have been described in the literature. Given the high rate of consanguineous marriages in the Iranian population, the frequency of LS is expected to be higher, which might be explained by undiagnosed cases. Early diagnosis of LS is very important, as treatment is available for this condition.

Down-regulation of hepatic expression of GHR/STAT5/IGF-1 signaling pathway fosters development and aggressiveness of HCV-related hepatocellular carcinoma: Crosstalk with Snail-1 and type 2 transforming growth factor-beta receptor.

So far, few clinical trials are available concerning the role of growth hormone receptor (GHR)/signal transducer and activator of transcription 5 (STAT5)/insulin like growth factor-1 (IGF-1) axis in hepatocarcinogenesis. The aim of this study was to evaluate the hepatic expression of GHR/STAT5/IGF-1 signaling pathway in hepatocellular carcinoma (HCC) patients and to correlate the results with the clinico-pathological features and disease outcome. The interaction between this signaling pathway and some inducers of epithelial-mesenchymal transition (EMT), namely Snail-1 and type 2 transforming growth factor-beta receptor (TGFBR2) was studied too. A total of 40 patients with HCV-associated HCC were included in this study. They were compared to 40 patients with HCV-related cirrhosis without HCC, and 20 healthy controls. The hepatic expression of GHR, STAT5, IGF-1, Snail-1 and TGFBR2 proteins were assessed by immunohistochemistry. Compared with cirrhotic patients without HCC and healthy controls, cirrhotic patients with HCC had significantly lower hepatic expression of GHR, STAT5, and IGF-1proteins. They also displayed significantly lower hepatic expression of TGFBR2, but higher expression of Snail-1 versus the non-HCC cirrhotic patients and controls. Serum levels of alpha-fetoprotein (AFP) showed significant negative correlations with hepatic expression of GHR (r = -0.31; p = 0.029) and STAT5 (r = -0.29; p = 0.04). Hepatic expression of Snail-1 also showed negative correlations with GHR, STAT5, and IGF-1 expression (r = -0.55, p = 0.02; r = -0.472, p = 0.035, and r = -0.51, p = 0.009, respectively), whereas, hepatic expression of TGFBR2 was correlated positively with the expression of all these proteins (r = 0.47, p = 0.034; 0.49, p = 0.023, and r = 0.57, p<0.001, respectively). Moreover, we reported that decreased expression of GHR was significantly associated with serum AFP level>100 ng/ml (p = 0.048), increased tumor size (p = 0.02), vascular invasion (p = 0.002), and advanced pathological stage (p = 0.01). Similar significant associations were found between down-regulation of STAT5 expression and AFP level > 100 ng/ml (p = 0.006), vascular invasion (p = 0.009), and advanced tumor stage (p = 0.007). Also, attenuated expression of IGF-1 showed a significant association with vascular invasion (p < 0.001). Intriguingly, we detected that lower expression of GHR, STAT5 and IGF-1 were considered independent predictors for worse outcome in HCC. Decreased expression of GHR/STAT5/IGF-1 signaling pathway may have a role in development, aggressiveness, and worse outcome of HCV-associated HCC irrespective of the liver functional status. Snail-1 and TGFBR2 as inducers of EMT may be key players. However, large prospective multicenter studies are needed to validate these results.

Effects of zearalenone on ovarian development of prepubertal gilts through growth hormone axis.

This experiment aimed to establish the effects of zearalenone (ZEN) on ovarian development in prepubertal gilts through the growth hormone axis [growth hormone-releasing hormone (GHRH) / growth hormone (GH) / growth hormone receptor (GHR)]. In a 40-day experiment, 48 Landrace × Yorkshire crossbred prepubertal gilts were randomly allocated to four dietary treatments, including a basal diet supplemented with 0 (control), 400 (T1), 800 (T2), and 1,600 (T3) μg/kg ZEN. The ovary index of T2 (P = 0.058) and T3 (P = 0.065) increased compared to the control group. Besides, histopathological examination revealed that ZEN promoted the development of ovaries and follicles. The GHR content, relative expression levels of GHR, janus activated kinase 2 (JAK2) mRNA, and mean optical density of GHR in the ovaries of prepubertal gilts in the T2 experimental group increased significantly at P < 0.05 compared to the control group. The T3 group had significantly higher GHR content, relative JAK2 expression levels, and signal transducer and activator of transcriptions 3 (STAT3) mRNA. In conclusion, ZEN enhances the biological effect of GH, promotes the development of the ovary (follicle), and exerts reproductive toxicity by increasing the expression level of GHR, JAK2, and STAT3 mRNA ovary and immune intensity of GHR protein.

Morphological and molecular effects of overexpressed GH on mice mammary gland

Growth Hormone (GH) plays crucial roles in mammary gland development and growth, and its upregulation has been associated with breast cancer promotion and/or progression. To ascertain how high GH levels could promote mammary tissue oncogenic transformation, morphological characteristics and the expression of receptors involved in mammary growth, development and cancer, and of mitogenic mediators were analyzed in the mammary gland of virgin adult transgenic mice that overexpress GH.Whole mounting and histologic analysis evidenced that transgenic mice exhibit increased epithelial ductal elongation and enlarged ducts along with deficient branching and reduced number of alveolar structures compared to wild type mice. The number of differentiated alveolar structures was diminished in transgenic mice while the amount of terminal end buds (TEBs) did not differ between both groups of mice. GH, insulin-like growth factor 1 (IGF1) and GH receptor mRNA levels were augmented in GH-overexpressing mice breast tissue, as well as IGF1 receptor protein content. However, GH receptor protein levels were decreased in transgenic mice. Fundamental receptors for breast growth and development like progesterone receptor and epidermal growth factor receptor were also increased in mammary tissue from transgenic animals. In turn, the levels of the proliferation marker Ki67, cFOS and Cyclin D1 were increased in GH-overexpressing mice, while cJUN expression was decreased and cMYC did not vary.In conclusion, prolonged exposure to high GH levels induces morphological and molecular alterations in the mammary gland that affects its normal development. While these effects would not be tumorigenic per se, they might predispose to oncogenic transformation.

Significant down-regulation of growth hormone receptor expression revealed as a new unfavorable prognos- tic factor in hepatitis C virus-related hepatocellular carcinoma

Background/AimsGrowth hormone (GH) is the main regulator of somatic growth, metabolism, and gender dimorphism in the liver. GH receptor (GHR) signaling in cancer is derived from a large body of evidence, although the GHR signaling pathway involved in the prognosis of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV)-related HCC, remains unclear. We aimed to explore the expression of GHR and analyze its association with clinicopathologic features and prognosis of patients with chronic hepatitis C and HCC.MethodsThe expression of GHR mRNA was investigated by quantitative real-time polymerase chain reaction in paired tumors and adjacent non-tumorous (ANT) liver tissues of 200 patients with chronic hepatitis C and HCC. Western blotting and immunofluorescence assays using the HCV-infected Huh7.5.1 cell model was performed.ResultsGHR mRNA was significantly lower in HCV-HCC tissues than in corresponding ANT liver tissues. GHR mRNA and protein levels also decreased in the HCV-infected Huh7.5.1 cell model. Notably, lower GHR expression was associated with age of >60 years (P=0.0111) and worse clinicopathologic characteristics, including alpha-fetoprotein >100 ng/mL (P=0.0403), cirrhosis (P=0.0075), vascular invasion (P=0.0052), pathological stage II–IV (P=0.0002), and albumin ≤4.0 g/dL (P=0.0055), which were linked with poor prognosis of HCC. Most importantly, the high incidence of recurrence and poor survival rates in patients with a low ratio of tumor/ANT GHR (≤0.1) were observed, indicating that low expression levels of GHR had great risk for development of HCC in patients with chronic hepatitis C.ConclusionsOur study demonstrates a significant down-regulation of GHR expression as a new unfavorable independent prognostic factor in patients with chronic hepatitis C and HCC.

Molecular modeling, docking and dynamic simulations of growth hormone receptor (GHR) of Labeo rohita

Growth hormones (GH) have diverse functions like growth promotion, metabolism, appetite, reproduction and social behavior in vertebrates, which is mediated through the growth hormone receptor (GHR). This work was aimed to analyze structural features, homology modeling and molecular docking of Labeo rohita GHR protein. A physicochemical characteristic, like molecular weight was 67.2 kDa and hydropathicity was 0.336. Protein modeling and structure confirmation of L. rohita GHR protein showed 92.7% residues are in the favored region. Selection of ligands and molecular docking shown Melengestrol and Riboflavin ligand showed uppermost binding energy values −7.8 and −7.3 kcal/mol. Molecular interactions describe conventional hydrogen bonding of Melengestrol was observed with VAL94, GLU97, GLU95, TRP57, PHE33, THR34, PRO35, ASP36, PRO37, ARG49, GLY292, LYS291, ILE290, ALA287, LYS289 residues. Riboflavin hydrogen bonds interaction was at PRO37, ASP36, PRO35, THR34, ARG49, SER144, VAL443, GLN442, PRO284, ASP294, ILE285, PRO286, SER408, ALA287, GLY292, LYS291, ILE290, PRO288, LYS287. Molecular dynamics simulation outcomes revealed that complex 2 (Riboflavin and GHR protein) is better than complex1 (Melengestrol and GHR protein). Overall, the results of the present work lead identification of novel molecules that may be agonistic of growth hormone receptor protein and can be used to surge growth in fish. Communicated by Ramaswamy H. Sarma

Regulation of extracellular and intracellular prolactin on cell proliferation and survival rate through GHR/JAK2/STAT3 pathway in NSCLC

Styrene increases serum prolactin (PRL) concentration. Hyperprolactinemia is associated with poor prognosis in lung cancer patients, but the mechanism of PRL action is unclear. The aims of this study were to (i) investigate the mechanism of PRL-action receptor in NSCLC cells (ii) measure whether PRL was secreted by NSCLC cells and its stimulatory mechanism in vitro and in vivo. We found that cell proliferation was increased after treatment of a pharmacological dose of PRL in A549 cells, which through up regulation of growth hormone receptor (GHR) and downstream of JAK2/STAT3/VEGF pathway. All NSCLC cells in the present study secreted PRL and expressed GHR, but not PRLR. Inhibition of GHR protein level led to decrease the PRL-induced cell proliferation. PRL was detected in NSCLC cells culture medium. Knockdown of intracellular PRL downregulated JAK2/STAT3 protein activities and GHR and VEGF protein levels. Furthermore, knockdown of intracellular PRL reduced the cell proliferation and the ability of colony-forming. In lung cancer tissues, PRL, GHR and VEGF levels were higher in the tumor tissues than in normal tissues and the protein expressions of these three proteins are positively correlated, respectively. High expression levels of both PRL and GHR cause a poor survival rate in lung cancer patients. Taken together, our results suggested that extracellular and intracellular PRL were involved in cell proliferation through GHR. Combination of in vitro and in vivo results, GHR and PRL are important targets for suppressing NSCLC cell proliferation, which might improve the survival rate in NSCLC patients.

GH directly inhibits steatosis and liver injury in a sex-dependent and IGF1-independent manner.

A reduction in hepatocyte growth hormone (GH)-signaling promotes non-alcoholic fatty liver disease (NAFLD). However, debate remains as to the relative contribution of the direct effects of GH on hepatocyte function vs indirect effects, via alterations in insulin-like growth factor 1 (IGF1). To isolate the role of hepatocyte GH receptor (GHR) signaling, independent of changes in IGF1, mice with adult-onset, hepatocyte-specific GHR knockdown (aHepGHRkd) were treated with a vector expressing rat IGF1 targeted specifically to hepatocytes. Compared to GHR-intact mice, aHepGHRkd reduced circulating IGF1 and elevated GH. In male aHepGHRkd, the shift in IGF1/GH did not alter plasma glucose or non-esterified fatty acids (NEFA), but was associated with increased insulin, enhanced systemic lipid oxidation and reduced white adipose tissue (WAT) mass. Livers of male aHepGHRkd exhibited steatosis associated with increased de novo lipogenesis, hepatocyte ballooning and inflammation. In female aHepGHRkd, hepatic GHR protein levels were not detectable, but moderate levels of IGF1 were maintained, with minimal alterations in systemic metabolism and no evidence of steatosis. Reconstitution of hepatocyte IGF1 in male aHepGHRkd lowered GH and normalized insulin, whole body lipid utilization and WAT mass. However, IGF1 reconstitution did not reduce steatosis or eliminate liver injury. RNAseq analysis showed IGF1 reconstitution did not impact aHepGHRkd-induced changes in liver gene expression, despite changes in systemic metabolism. These results demonstrate the impact of aHepGHRkd is sexually dimorphic and the steatosis and liver injury observed in male aHepGHRkd mice is autonomous of IGF1, suggesting GH acts directly on the adult hepatocyte to control NAFLD progression.

SUN-LB52 The Protective Effects of Hepatocyte GH Receptor (GHR) Signaling Against Steatosis and Liver Injury Is Sexually Dimorphic and Autonomous of IGF1

GH dysregulation contributes to the development of non-alcoholic fatty liver disease (NAFLD), however debate remains as to the relative contribution of the direct vs indirect effects of GH, via IGF1. Mouse models with congenital, liver-specific knockout of the GHR, JAK2 or STAT5, as adults exhibit steatosis, glucose intolerance, insulin resistance and white adipose tissue (WAT) lipolysis. It is believed that fatty liver is due to the dramatic reduction in circulating IGF1 altering systemic metabolism, due to loss of the insulin-like effects of IGF1 and the loss of IGF1 negative feedback to the pituitary leading to a rise in GH that promotes systemic insulin resistance and WAT lipolysis shifting the flux of fatty acids to the liver. In addition, low IGF1/high GH alters the development of other metabolically relevant tissues, which could indirectly contribute to the liver phenotype observed with congenital loss of hepatic GH signaling. To directly test the actions of GH on adult hepatocyte function, we developed a mouse model of adult-onset, hepatocyte-specific knockdown of the GHR (aHepGHRkd; 12 week-old, GHRfl/fl mice treated with AAV8-TBGp-Cre). aHepGHRkd enhanced hepatic de novo lipogenesis (DNL), rapidly leading to steatosis in males, but not females. In males, enhanced DNL and steatosis was sustained with age and associated with hepatocyte ballooning, inflammation and mild fibrosis. These changes occurred independent of severe systemic insulin resistance and WAT lipolysis, although the aHepGHRkd mice exhibit low IGF1/high GH similar to that of congenital models. To directly test the role of hepatocyte GHR signaling, independent of changes in IGF1, aHepGHRkd mice were treated with a vector expressing rat IGF1 targeted specifically to hepatocytes (AAV8-TBGp-rIGF1). Mice were fed standard chow diet and tissues collected 8m post-AAV. IGF1 replacement elevated plasma IGF1 in aHepGHRkd mice, resulting in a reduction in plasma GH and pituitary expression of Gh, Ghrhr and Ghsr, indicating negative feedback of IGF1 was restored. In male aHepGHRkd mice, IGF1 replacement reduced insulin and whole body lipid utilization and increased WAT, however it did not reduce steatosis or alter hepatic fatty acid composition indicative of DNL and had minimal effects on liver injury markers. RNAseq analysis of liver extracts showed IGF1 replacement also had no major impact on the differentially expressed genes observed after aHepGHRkd. These results demonstrate that steatosis, DNL and liver injury observed in male aHepGHRkd mice are autonomous of IGF1. Despite the fact that hepatic GHR protein levels were not detectable in both female and male aHepGHRkd mice, females maintained moderate levels of IGF1 and were protected from steatosis. The mechanism by which female mice are protected remains to be elucidated, however is consistent with clinical data indicating pre-menopausal women are resistance to NAFLD.

Role of ghrelin on growth hormone/insulin-like growth factor-1 axis during endotoxemia

ObjectiveTo investigate the anti-inflammatory property of ghrelin treatment on the Growth Hormone (GH)/Insulin-like Growth Factor-I (IGF-1) axis in Wistar rats that have undergone endotoxemia. DesignIn this randomized animal study, lipopolysaccharide (LPS) (5 mg/kg; intraperitoneal) was administered to induce endotoxemia, and ghrelin (15 nmol/kg; endovenous) was injected simultaneously. Blood and liver samples were collected 2 h, 6 h and 12 h after LPS administration for analysis. MeasurementsTumor necrosis factor alpha (TNF-α), interleukin (IL)-1, beta (IL-1β), and IL-6 from both blood and liver were determined by ELISA assay. Serum nitrate was determined by chemiluminescense. Growth hormone receptor (GHR) and growth hormone secretagogue receptor 1a (GHSR-1a) were determined by western blotting. GHR mRNA and IGF-1 mRNA were determined by RT-PCR. ResultsLPS administration induced a decrease in IGF-1 and GH serum levels, characterizing GH/IGF-1 axis disruption. Ghrelin treatment attenuated the decrease of serum levels of IGF-1 as well as the increase of TNF-α, IL-1β, IL-6 and nitrate induced by LPS. The increase of induced GHSR-1a protein expression seen in the LPS group after 2 h remained until 6 h after ghrelin treatment. However, attenuation of the circulating IGF-1 decrease by ghrelin treatment was not accompanied by changes in GHR protein expression nor GHR and IGF-1 gene expression. ConclusionGhrelin was able to attenuate changes in the GH/IGF-1 axis observed during systemic inflammation, which may be due to the modulation of pro-inflammatory mediators release.

Novel functional role of GH/IGF-I in neonatal lung myofibroblasts and in rat lung growth after intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a risk factor for neonatal chronic lung disease (CLD) characterized by reduced alveoli and perturbed matrix remodeling. Previously, our group showed an activation of myofibroblasts and matrix remodeling in rat lungs after IUGR. Because growth hormone (GH) and insulin-like growth factor I (IGF-I) regulate development and growth, we queried 1) whether GH/IGF-I signaling is dysregulated in lungs after IUGR and 2) whether GH/IGF-I signaling is linked to neonatal lung myofibroblast function. IUGR was induced in Wistar rats by isocaloric low-protein diet during gestation. Lungs were obtained at embryonic day (E) 21, postnatal day (P) 3, P12, and P23. Murine embryonic fibroblasts (MEF) or primary neonatal myofibroblasts from rat lungs of control (pnFCo) and IUGR (pnFIUGR) were used for cell culture studies. In the intrauterine phase (E21), we found a reduction in GH receptor (GH-R), Stat5 signaling and IGF-I expression in lungs after IUGR. In the postnatal phase (P3-P23), catchup growth after IUGR was linked to increased GH mRNA, GH-R protein, activation of proliferative Stat5/Akt signaling, cyclin D1 and PCNA in rat lungs. On P23, a thickening of the alveolar septae was related to increased vimentin and matrix deposition, indicating fibrosis. In cell culture studies, nutrient deprivation blocked GH-R/IGF-IR signaling and proliferation in MEFs; this was reversed by IGF-I. Proliferation and Stat5 activation were increased in pnFIUGR. IGF-I and GH induced proliferation and migration of pnFCo; only IGF-I had these effects on pnFIUGR. Thus, we show a novel mechanism by which the GH/IGF-I axis in lung myofibroblasts could account for structural lung changes after IUGR.

Expression of growth hormone gene in the baboon eye

The human growth hormone (GH) locus is comprised by two GH (GH1 and GH2) genes and three chorionic somatomammotropin (CSH1, CSH2 and CSH-L) genes. While GH1 is expressed in the pituitary gland, the rest are expressed in the placenta. However, GH1 is also expressed in several extrapituitary tissues, including the eye. So to understand the role of this hormone in the eye we used the baboon (Papio hamadryas), that like humans has a multigenic GH locus; we set up to investigate the expression and regulation of GH locus in adult and fetal baboon ocular tissues.We searched in baboon ocular tissues the expression of GH1, GH2, CSH1/2, Pit1 (pituitary transcription factor 1), GHR (growth hormone receptor), GHRH (growth hormone releasing hormone), GHRHR (growth hormone releasing hormone receptor), SST (somatostatin), SSTR1 (somatostatin receptor 1), SSTR2 (somatostatin receptor 2), SSTR3 (somatostatin receptor 3), SSTR4 (somatostatin receptor 4), and SSTR5 (somatostatin receptor 5) mRNA transcripts and derived proteins, by qPCR and immunofluorescence assays, respectively. The transcripts found were characterized by cDNA cloning and sequencing, having found only the one belonging to GH1 gene, mainly in the retina/choroid tissues. Through immunofluorescence assays the presence of GH1 and GHR proteins was confirmed in several retinal cell layers. Among the possible neuroendocrine regulators that may control local GH1 expression are GHRH and SST, since their mRNAs and proteins were found mainly in the retina/choroid tissues, as well as their corresponding receptors (GHRH and SSTR1-SSTR5). None of the ocular tissues express Pit1, so gene expression of GH1 in baboon eye could be independent of Pit1. We conclude that to understand the regulation of GH in the human eye, the baboon offers a very good experimental model.

Growth Hormone Receptor (GHR) gene and its applicationsin livestock: A review

The Growth Hormone Receptor (GHR) gene provides instructions for making a protein called the growth hormone receptor. The GHR mediates biological actions of growth hormone on target cells by transducing the growth hormone (GH) signal across the cell membrane and inducing transcription of many genes, including insulin-like growth factor-1 (IGF1). The gene coding for bovine GHR gene consists of nine exons. In exon 8 of the bovine GHR gene, T/A nucleotide variation results in to change in tyrosine from phenylalanine in the transmembrane domain of the GHR protein, has been reported to be associated with a major effect on milk yield in cows. GHR (growth hormone receptor) gene has been shown to harbor a causal mutation of a QTL influencing milk yield and composition GHR gene is a polymorphic gene and the polymorphisms are related to different economic traits of different species. The GHR gene influences physical traits and helps to selection of animals. The lengths of the variable TG-repeats in the P1 promoter of the bovine GHR gene are associated with growth rates in young Angus cattle. Due to various functions of GHR are viewed as promising candidate markers for selection purposes in cattle. Thus GHR gene could be a candidate gene for application in marker assisted selection (MAS).

Clinical and biochemical consequences of an intragenic growth hormone receptor (GHR) deletion in a large Chinese pedigree.

Growth hormone insensitivity (GHI) may be caused by failure of GH receptor function. Some patients bearing specific GHR mutations differ from classical GHI individuals by extremely elevated GH-binding protein (GHBP) serum concentrations. We investigated clinical, genetic and biochemical characteristics of a severely growth-retarded Chinese boy with classical Laron syndrome manifestations. DNA and mRNA from blood cells of the patient and 11 family members were investigated for GHR mutations. Basal GH, GHBP, IGF-1 and IGFBP-3 concentrations were determined in serum samples. The impact of the aberrant mRNA on GHR protein expression and secretion was analysed in vitro by transfection studies in HEK293 cells. The proband and seven relatives had excessively elevated GHBP serum concentration. Basal GH in these individuals was significantly greater compared with family members with normal GHBP. The GHBP increase originated from a novel GHR intragenic deletion comprising parts of exon and intron 8 that caused exon 8 skipping from the GHR mRNA transcript. Transfection studies revealed that the predicted loss of plasma membrane anchorage results in direct secretion of the mutant GHR. The partial GHR deletion causes excessively elevated GHBP serum concentrations regardless of the state of zygosity of the mutation. The increase in GHBP is associated with significantly elevated basal GH levels. Clinically, only homozygous carriers exhibit classical GHI manifestations. The truncated GHR protein resulting from exon 8 skipping is directly secreted out of the cell.

Expression of growth hormone receptor, plakoglobin and NEDD9 protein in association with tumour progression and metastasis in human breast cancer

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths among female population worldwide. Metastases are the common cause of morbidity and mortality in breast cancer and can remain latent for several years after surgical removal of the primary tumour. Thus, the identification and functional characterisation of molecular factors that promote oncogenic signalling in mammary tumour development and progression could provide new entry points for designing targeted therapeutic strategies for metastatic breast cancer. In the present study, we investigated the expression of proteins involved in cell signalling (growth hormone receptor (GHR) and NEDD9) and cell-cell adhesion (plakoglobin) in epithelial and stromal compartments of primary ductal invasive breast carcinomas and their axillary lymph node metastases versus non-metastatic tumours. Obtained data revealed remarkable increase in the expression levels of GHR and NEDD9 proteins in both epithelial and stromal components of axillary lymph node metastases in comparison with those of non-metastatic tumours, suggesting that the expression of these two proteins may provide biomarkers for tumour aggressiveness.

Presence of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles and improvement of in vitro preantral follicle survival and development with GH

This study aimed to demonstrate the expression of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles in order to investigate the effects of GH on the survival and development of preantral follicles. The ovaries were processed for the isolation of follicles to study GH-R mRNA expression or to localization of GH-R by immunohistochemical analysis. Pieces of ovarian cortex were cultured for 7 days in minimum essential medium+ (MEM+) in the presence or absence of GH at different concentrations (1, 10, 50, 100, and 200 ng/mL). High expression levels of GH-R mRNA were observed in granulosa/theca cells from large antral follicles. However, preantral follicles do not express mRNA for GH-R. Immunohistochemistry demonstrated that the GH-R protein was expressed in the oocytes/granulosa cells of antral follicles, but any protein expression was observed in preantral follicles. The highest (P < 0.05) rate of normal follicles and intermediate follicles was observed after 7 days in MEM+ plus 10 ng/mL GH (70%). In conclusion, GH-R mRNA and protein are expressed in caprine antral follicles, but not in preantral follicles. Moreover, GH maintains the survival of goat preantral follicles and promotes the development of primordial follicles.

Impaired renal growth hormone JAK/STAT5 signaling in chronic kidney disease

Treatment with recombinant human growth hormone (GH) is the standard therapy for short stature in children with chronic kidney disease (CKD). However, concerns have been raised on the potential renal fibrogenic effects of GH. There is no information regarding the renal GH receptor (GHR)-JAK-STAT signaling pathway in CKD. Subtotal nephrectomized (CKD) and pair-fed sham-operated control (C) juvenile rats were treated with subcutaneous GH or saline for 2 weeks. A single intravenous GH bolus or vehicle was provided prior to euthanasia. Reduced body weight in CKD was improved with GH therapy. The remnant kidney showed glomerular hypertrophy and early interstitial fibrosis without inflammatory infiltration. Treatment of CKD rats with GH did not worsen renal function or fibrosis. Kidney GHR mRNA and protein levels were reduced and basal phosphorylation of JAK2 and STAT5 was significantly impaired. However, intravenous GH administration prior to sacrifice normalized STAT5 phosphorylation. Basal renal IL6 mRNA and phosphorylation of its downstream signaling molecule STAT3 were increased as was the product of its action, the suppressor of cytokine signaling 3 (SOCS3) mRNA. Despite known unaltered circulating GH levels, remnant kidneys of uremic growth retarded juvenile rats show impaired basal signaling along the GH-activated JAK2/STAT5 signaling pathway. This may well be a consequence of the reduced GHR level and the inhibitory effect of the increase in IL-6-mediated SOCS3 expression. This renal GH insensitivity, if present in humans, may protect against the potential adverse renal effects of GH administration in CKD patients.

The role of cytochrome P450‐dependent metabolism in the regulation of mouse hepatic growth hormone signaling components and target genes by 3‐methylcholanthrene

State Department of Health, Albany, NY 3‐Methylcholanthrene (MC), a readily metabolized aryl hydrocarbon receptor (AHR) agonist, disrupts mouse hepatic growth hormone (GH) signaling and suppresses cytochrome P450 2D9 (Cyp2d9), a male‐specific gene controlled by pulsatile GH via signal transducer and activator of transcription 5b (STAT5b). To determine if these effects depend on hepatic microsomal P450‐mediated activity, we examined MC responses in LCN mice with hepatocyte‐specific deletion of NADPH‐cytochrome P450 oxidoreductase (POR). MC caused mild induction of Por and a hepatic inflammatory marker in wild‐type mice whereas strong induction by MC of AHR target genes, Cyp1a1, Cyp1a2, and Cyp1b1, occurred in wild‐type and LCN mice. Two STAT5b target genes, Cyp2d9 and major urinary protein 2 (Mup2), were suppressed by MC in wild‐type mice and the CYP2D9 mRNA response was maintained in LCN mice. In wild‐type mice, MC decreased GH receptor (GHR) mRNA but increased GHR protein levels. There was a trend for impairment of STAT5 phosphorylation by MC in both mouse strains. Basal levels of MUP2 mRNA, GHR mRNA and protein, and the activation status of extracellular signal‐regulated kinase 2 and Akt were influenced by hepatic Por genetic status. The effects of MC on hepatic GH signaling components and target genes are complex, involving aspects that are both dependent and independent of hepatic microsomal P450‐mediated activity. [Support: CIHR, NIH‐NCI]

SiRNA-targeted inhibition of growth hormone receptor in human colon cancer SW480 cells

To determine the effects of RNAi-mediated inhibition of the growth hormone receptor (GHR) gene on tumors and colon cancer cells in vivo. Construction of a eukaryotic vector for human GHR expression, the pcDNA6.2-GW/EmGFP-small interfering RNAs (siRNAs)-GHR plasmid, was used to inhibit GHR expression. Thirty-six BALB/c nude mice were randomly divided into groups and treated with normal saline (NS), recombinant plasmid (G₂), growth hormone (GH), 5-fluorouracil (FU), G₂+FU or G₂+FU+GH. Each nude mouse was subcutaneously inoculated with 1 × 10(7) human colon cancer SW480 cells; the nude mice were weighed before inoculation and on the 2(nd), 5(th), 8(th), 11(th), 14(th) and 17(th) day after inoculation. All nude mice were sacrificed after 17 d. Each subcutaneous tumor was removed and studied. Tumor volume was measured on the 5(th), 8(th), 11(th), 14(th) and 17(th) day after inoculation. The expression of GHR protein in the tumor tissue was detected by Western blotting analysis, and the differences in GHR mRNA expression in the tumor tissue were detected by real-time quantitative reverse transcription-polymerase chain reaction. Compared to the control group, the weights of the inoculated nude mice on the 17(th) day after inoculation were: G₂: 21.60 ± 0.71 g, GH: 21.64 ± 0.45 g, FU: 18.94 ± 0.47 g, FU+G₂: 19.40 ± 0.60 g, G₂+FU+GH: 21.04 ± 0.78 g vs NS: 20.68 ± 0.66 g, P < 0.05; the tumor volumes after the subcutaneous inoculation were: G₂: 9.71 ± 3.82 mm(3), FU: 11.54 ± 2.42 mm(3), FU+G₂: 11.42 ± 1.11 mm(3), G₂+FU+GH: 10.47 ± 1.02 mm(3) vs NS: 116.81 ± 10.61 mm(3), P < 0.05. Compared to the GH group, the tumor volumes were significantly decreased in the experimental groups. The GHR protein expression (G₂: 0.39 ± 0.02, FU: 0.40 ± 0.02, FU+G₂: 0.38 ± 0.01, G₂+FU+GH: 0.39 ± 0.01 vs NS: 0.94 ± 0.02, P < 0.05) and the GHR mRNA expression (G₂: 14.12 ± 0.10, FU: 15.15 ± 0.44, FU+G₂: 16.46 ± 0.27, G₂+FU+GH: 15.37 ± 0.57 vs NS: 12.63 ± 0.14, P < 0.05) were significantly decreased and increased, respectively, in the experimental groups. Inhibition of GHR in human colon cancer SW480 cells resulted in anti-tumor effects in nude mice.

The Role of Cytochrome P450–Dependent Metabolism in the Regulation of Mouse Hepatic Growth Hormone Signaling Components and Target Genes by 3-Methylcholanthrene

3-Methylcholanthrene (MC) is a readily metabolized aryl hydrocarbon receptor (AHR) agonist. MC disrupts expression of mouse hepatic growth hormone (GH) signaling components and suppresses cytochrome P450 2D9 (Cyp2d9), a male-specific gene controlled by pulsatile GH via signal transducer and activator of transcription 5b (STAT5b). To determine if these effects of MC depend on hepatic microsomal P450-mediated activity, we examined biologic responses to MC treatment in liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of NADPH-cytochrome P450 oxidoreductase (POR). MC caused mild induction of Por and a hepatic inflammatory marker in wild-type mice, whereas MC caused strong induction of AHR target genes, Cyp1a1, Cyp1a2, and Cyp1b1 in wild-type and LCN mice. Two mouse hepatic STAT5b target genes, Cyp2d9 and major urinary protein 2 (Mup2), were suppressed by MC in wild-type mice, and the CYP2D9 mRNA response was maintained in LCN mice. In wild-type mice only, MC decreased hepatic GH receptor (GHR) mRNA but increased GHR protein levels. There was an apparent impairment of STAT5 phosphorylation by MC in wild-type and LCN mice, but large interanimal variation prevented achievement of statistical significance. In vehicle-treated mice, basal levels of MUP2 mRNA, GHR mRNA, GHR protein, and the activation status of extracellular signal-regulated kinase 2 and Akt were influenced by hepatic Por genetic status. These results indicate that the effects of MC on hepatic GH signaling components and target genes are complex, involving aspects that are both dependent and independent of hepatic microsomal P450-mediated activity.