Human Pituitary Growth Hormone Research Articles

Production and engineering of nanobody-based quenchbody sensors for detecting recombinant human growth hormone and its isoforms.

Due to athletes' misuse of recombinant human growth hormone (rhGH) for performance improvement, the World Anti-Doping Agency has designated rhGH as a prohibited substance. This study focuses on the development and improvement of a simple and fast rhGH detection method using a fluorescence-incorporated antibody sensor "Quenchbody (Q-body)" that activates upon antigen binding. Camelid-derived nanobodies were used to produce stable Q-bodies that withstand high temperatures and pH levels. Notably, pituitary human growth hormone (phGH) comprises two major isoforms, namely 22 and 20 kDa GH, which exist in a specific ratio, and the rhGH variant shares the same sequence as the 22 kDa GH isoform. Therefore, we aimed to discriminate rhGH abuse by analyzing its specific isoform ratio. Two nanobodies, NbPit (recognizing phGH) and NbRec (preferentially recognizing 22 kDa rhGH), were used to develop the Q-bodies. Nanobody production in Escherichia coli involved the utilization of a vector containing 6xHis-tag, and Q-bodies were obtained using a maleimide-thiol reaction between the N-terminal of the cysteine tag and a fluorescent dye. The addition of tryptophan residue through antibody engineering resulted in increased fluorescence intensity (FI) (from 2.58-fold to 3.04-fold). The limit of detection (LOD) was determined using a fluorescence response, with TAMRA-labeled NbRec successfully detecting 6.38 ng/ml of 22 kDa rhGH while unable to detect 20 kDa GH. However, ATTO520-labeled NbPit detected 7.00 ng/ml of 20 kDa GH and 2.20 ng/ml 22 kDa rhGH. Q-bodies successfully detected changes in the GH concentration ratio from 10 to 40 ng/ml in human serum within 10 min without requiring specialized equipment and kits. Overall, these findings have potential applications in the field of anti-doping measures and can contribute to improved monitoring and enforcement of rhGH misuse, ultimately enhancing fairness and integrity in competitive sports.

Obesity and regulation of human placental lactogen production in pregnancy.

The four genes coding for placental members of the human (h) growth hormone (GH) family include two that code independently for placental lactogen (PL), also known as chorionic somatomammotrophin hormone, one that codes for placental growth hormone (PGH) and a pseudogene for which RNA but no protein product is reported. These genes are expressed preferentially in the villus syncytiotrophoblast of the placenta in pregnancy. In higher primates, the placental members, including hPL and PGH, are the result of multiple duplication events of the GH gene. This contrasts with rodents and ruminants, where PLs result from duplication of the prolactin (PRL) gene. Thus, unlike their mouse counterparts, the hPL and PGH hormones bind both lactogenic and somatogenic receptors with varying affinity. Roles influenced by nutrient availability in both metabolic control in pregnancy and maternal behaviour are supported. However, the effect maternal obesity has on the activation of placental members of the hGH gene family, particularly the expression and function of those genes, is poorly understood. Evidence from partially humanised hGH/PL transgenic mice indicates that both the remote upstream hPL locus control region (LCR) and more gene-related regulatory regions are required for placental expression in vivo. Furthermore, a specific pattern of interactions between the LCR and hPL gene promoter regions is detected in term placenta chromatin from women with a normal body mass index (BMI) in the range 18.5-25kgm-2 by chromosome conformation capture assay. This pattern is disrupted with maternal obesity (class II BMI>35kgm-2 ) and associated with a >40% decrease in term hPL RNA levels, as well as serum hPL but not PRL levels, during pregnancy. The relative importance of the chromosomal architecture and predicted properties for transcription factor participation in terms of hPL production and response to obesity are considered, based on comparison with components required for efficient human pituitary GH gene expression.

In Alzheimer's Mice, Decades-Old Human Cadaveric Pituitary Growth Hormone Samples Can Transmit and Seed Amyloid-Beta Pathology

In Alzheimer's Mice, Decades-Old Human Cadaveric Pituitary Growth Hormone Samples Can Transmit and Seed Amyloid-Beta Pathology

Application of artificial gel antibodies for investigating molecular polymorphisms of human pituitary growth hormone

Artificial gel antibodies were used to investigate human growth hormone (GH) activity in preparations purified from human pituitary glands. A partially purified fraction containing differently sized structural variants of GH was processed to yield monomeric and dimeric forms suitable for synthesizing artificial polyacrylamide gel antibodies. These two types of GH antibodies were used for investigating GH activity in experiments using HPLC gel-permeation and ion-exchange chromatography. In the size-exclusion experiments, both hormone fractions eluted as homogeneous peaks, whereas the ion exchanger resolved the hormones into several active components. The GH monomer antibodies exhibited a much higher affinity for monomeric GH than for dimeric GH, and the GH dimer antibodies exhibited a much higher affinity for dimeric GH than for monomeric GH. It was concluded that these two sets of antibodies might be useful for discriminating between dimeric and monomeric GH in clinical samples.

Identification of Human Pituitary Growth Hormone Variants by Mass Spectrometry

The heterogeneity of human endogenous growth hormone (GH) is used in doping control analysis to distinguish it from the homogeneous recombinant analogue in plasma samples. Pituitary GH variants were characterized by gel electrophoresis and mass spectrometry. Besides 22 and 20 kDa isoforms, fragments of 9 and 12 kDa were identified and a glycosylated 23 kDa GH variant was elucidated to bear a HexHexNac 2 NeuAc modification presumably located at Thr 60.

Preparative alkaline urea gradient PAGE: Application to purification of extraordinarily‐stable disulfide‐linked homodimer of human growth hormone

The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.

Epigenetic Activation of the Human Growth Hormone Gene Cluster during Placental Cytotrophoblast Differentiation

The hGH cluster contains a single human pituitary growth hormone gene (hGH-N) and four placenta-specific paralogs. Activation of the cluster in both tissues depends on 5' remote regulatory elements. The pituitary-specific locus control elements DNase I-hypersensitive site I (HSI) and HSII, located 14.5 kb 5' of the cluster (position -14.5), establish a continuous domain of histone acetylation that extends to and activates hGH-N in the pituitary gland. In contrast, histone modifications in placental chromatin are restricted to the more 5'-remote HSV-HSIII region (kb -28 to -32) and to the placentally expressed genes in the cluster, with minimal modification between these two regions. These data predict distinct modes of hGH cluster gene activation in the pituitary and placenta. Here we used cell culture models to track structural changes at the hGH locus through placental-gene activation. The data revealed that this process was initiated in primary cytotrophoblasts by histone H3K4 di- and trimethylation and H4 acetylation restricted to HSV and to the individual placental-gene repeat (PGR) units within the cluster. Later stages of transcriptional induction were accompanied by enhancement and extension of these modifications and by robust H3 acetylation at HSV, at HSIII, and throughout the placental-gene regions. These data suggested that elements restricted to HSIII-HSV regions and each individual PGR might be sufficient for activation of the hCS genes. This model was tested by comparing hCS transgene expression in the placentas of mouse embryos carrying a full hGH cluster to that in placentas in which the HSIII-HSV region was directly linked to the individual hCS-A PGR unit. The findings indicate that the HSIII-HSV region and the PGR units, although targeted for initial chromatin structural modifications, are insufficient to activate gene expression and that this process is dependent on additional, as-yet-unidentified chromatin determinants.

Both pituitary and placental growth hormone transcripts are expressed in human peripheral blood mononuclear cells (PBMC).

The hGH-V gene codes for a variant of human pituitary growth hormone (hGH-N) named placental growth hormone (hPGH). hPGH shares 93% amino acid identity with hGH-N. Until now the hGH-V gene was considered to be exclusively expressed in human placenta, where it replaces maternal circulating hGH-N at the end of pregnancy. In this study we investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis hGH-N, and hGH-V, gene expression in PBMC in men, women and pregnant women. We have demonstrated that hGH-N and hGH-V transcripts are simultaneously produced by PBMC in both men and women as well as pregnant women. The PBMC of a PIT-1-negative woman expressed only the hGH-V transcript, but not the hGH-N one as expected. In conclusion, hGH-V mRNA is expressed by cells other than the syncytiotrophoblast, is not regulated by PIT-1, and may be involved in immune regulation, as is pituitary GH.

Large-scale preparation and in vitro characterization of biologically active human placental (20 and 22K) and pituitary (20K) growth hormones: Placental growth hormones have no lactogenic activity in humans

Expression plasmids containing DNA sequences optimized for expression in Escherichia coli were prepared encoding human pituitary (hGH-N 20K) and placental (hGH-V 20 and 22K) growth hormones. The proteins were expressed in bacteria, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose according to a unique protocol developed for each protein. The yields from 5 l of fermentation culture varied between 400 and 700 mg of electrophoretically pure, over 95% monomeric protein. Circular dichroism (CD) analysis revealed similarity of the purified hGHs’ secondary structure to that of the pituitary hGH-N 22K, except for hGH-V 20K, in which the α-helix content was lower. The purified proteins were stable as a 0.1% sterile solution held at pH 10–11 at 4 °C for at least one month. All three purified hGH molecules formed a 1:2 complex with hGH receptor extracellular domain (hGHR-ECD), similar to hGH-N 22K. Binding experiments using hGHR-ECD revealed that the differences between the two 22K variants or between the two 20K variants were not significant, except that hGH-V 20K exhibited slightly lower affinity. Somatogenic activity was tested in vitro using FDC-P1 cell lines. Whereas the bioactivity of 22K hGHs and hGH-N 20K in FDC-P1-9D11 cells stably transfected with hGHR was almost equal and two to threefold higher than that of hGH-V 20K, in FDC-P1 3B9 cells stably transfected with rabbit (rb) GHR, the bioactivity of both 20K analogues was significantly (five to ninefold) lower than that of the 22K hormones. The lactogenic activity measured in heterologous assays (Nb2-11C cells and Baf/3 cells stably transfected with the long form of rabbit prolactin receptor) revealed that the activity of hGH-N 20K was close to that of hGH-N 22K in the Baf/3 cells, but 4.5-fold lower in the Nb2 cells. The activity of hGH-V 22K was ninefold less in Nb2 cells and 55-fold less in Baf/3 cells, whereas hGH-V 20K had no lactogenic activity in either bioassay. In contrast, in a homologous lactogenic assay using Baf/3 LP cells stably transfected with hPRLR, the activity of both placental hGHs was nil and the activity of hGH-N 20K was 4.3-fold lower than that of hGH-N 22K. The latter finding raises the question of whether the lack of intrinsic lactogenic activity in the placental hGHs that dominate during pregnancy has any physiological relevance.

Effects of Soy Protein and Soy Phytochemicals on Mammary Tumor Development in Female Transgenic Mice Overexpressing Human Pituitary Growth Hormone

The objective of this study was to determine the effect of soy-based diets on mammary tumors in female cancer- prone mice. Transgenic virgin female mice expressing human pituitary growth hormone and their respective phenotypically normal littermates were fed a diet containing either casein (C), low-isoflavone soy protein (LIS), or high-isoflavone soy protein (HIS). Indices of tumor development were measured throughout the study. Both days from birth until death and days on diet until death were increased [by 20% (P = .01) and 19% (P = .02), respectively] in the LIS group when compared with the C group. Both intervals were increased also (by 16% and 17%, respectively; P < .05) in the HIS group when compared with the C group. Days from birth to first tumor were increased by 7% (P < .05), as was days on diet to first tumor by 5% (P < .05), in the LIS group when compared with the C group. First-onset number of tumors was decreased (P = .02) by 41% and 34% in the LIS and C groups, respectively, when compared with the HIS group. Final onset of tumors was decreased (P < .05) by 44% and 9% in the LIS and HIS groups, respectively, when compared with the C group. Total area of final tumors was decreased (P < .05) by 30% in the LIS group when compared with the C group. Taken cumulatively, these data suggest that a diet rich in soy protein may provide protective benefits regarding tumor development in female cancer-prone mice. Furthermore, some bioactive compounds in the HIS diet appeared to confound the soy protein-induced beneficial effects.

Receptors and Sites of Synthesis and Storage of γ-Aminobutyric Acid in Human Pituitary Glands and in Growth Hormone Adenomas

gamma-Aminobutyric acid (GABA) is an important regulatory factor of pituitary gland function, which in addition to hypothalamic neurons, can be derived from intrapituitary sources, ie, growth hormone (GH) cells of rat and monkey. We report that human pituitary glands also express 2 isoforms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD 65; GAD 67), the vesicular GABA transporter (VGAT), and multiple subunits of GABA (A, B, and C) receptors. GABA production and storage occurs in GH cells, as demonstrated by cellular colocalization of immunoreactive GAD and VGAT in GH cells and by reverse transcription-polymerase chain reaction analysis of laser capture-microdissected immunostained GH cells. It is interesting that human pituitary GH adenomas share expression of VGAT and GABA receptors with normal pituitary glands but lack GAD 65. We propose that GABA, synthesized by GH cells, might act as a paracrine or autocrine regulating factor in the human pituitary gland and in human GH adenoma. Because many drugs interfere with GABA function, the identification of GABA system components might have clinical implications.

100 years of hormonology: a view from No. 1 Wimpole Street

100 years of hormonology: a view from No. 1 Wimpole Street

Creutzfeldt-Jakob disease in United Kingdom patients treated with human pituitary growth hormone.

To investigate risk factors for Creutzfeldt-Jakob disease (CJD) in patients in the United Kingdom treated with human pituitary growth hormone (hGH). Incidence rates of CJD, based on person-year denominators, were assessed in a cohort of 1,848 patients treated with hGH in the United Kingdom from 1959 through 1985 and followed to the end of 2000. CJD developed in 38 patients. Risk of CJD was significantly increased by treatment with hGH prepared by the Wilhelmi method of extraction from human pituitaries. Risk was further raised if this treatment was administered at ages 8 to 10 years. The peak risk of CJD was estimated to occur 20 years after first exposure, and the estimated lifetime cumulative risk of CJD in Wilhelmi-treated patients was 4.5%. Size-exclusion chromatography, used in non-Wilhelmi preparation methods, may prevent CJD infection. Susceptibility to CJD may vary with age, and susceptibility may be present in only a few percent of the population.

Risk of cancer in patients treated with human pituitary growth hormone in the UK, 1959-85: a cohort study.

Growth hormone raises serum concentrations of insulin-like growth factor IGF-I, which is mitogenic and antiapoptotic. There is evidence that raised endogenous levels of growth hormone and IGF-I might be associated with increased risk of certain solid cancers, but there have been no data on long-term risks of solid cancers after growth hormone treatment. We did a cohort study to investigate cancer incidence and mortality in 1848 patients in the UK who were treated during childhood and early adulthood with human pituitary growth hormone during the period from 1959 to 1985. Patients were followed up for cancer incidence to December, 1995 and for mortality to December, 2000. Risk of cancer in the cohort was compared with that in the general population, controlling for age, sex, and calendar period. Patients treated with human pituitary growth hormone had significantly raised risks of mortality from cancer overall (standardised mortality ratio 2.8, 95% CI 1.3-5.1; ten cases), colorectal cancer (10.8, 1.3-38.8; two cases), and Hodgkin's disease (11.4, 1.4-41.3; two cases). Incidence of colorectal cancer was also greatly raised (7.9, 1.0-28.7). After exclusion of patients whose original diagnosis rendered them at high risk of cancer, the significance and size of the risks of colorectal cancer incidence and mortality, and of Hodgkin's disease mortality were increased. Although based on small numbers, the risk of colorectal cancer is of some concern and further investigation in other cohorts is needed. We have no evidence as to whether growth hormone in modern dosage regimens is associated with an increased risk of colorectal cancer.

Prolactin (PRL)-Releasing Peptide Stimulates PRL Secretion from Human Fetal Pituitary Cultures and Growth Hormone Release from Cultured Pituitary Adenomas

Prolactin (PRL)-Releasing Peptide Stimulates PRL Secretion from Human Fetal Pituitary Cultures and Growth Hormone Release from Cultured Pituitary Adenomas

Prolactin (PRL)-releasing peptide stimulates PRL secretion from human fetal pituitary cultures and growth hormone release from cultured pituitary adenomas.

The hypothalamic peptide PRL-releasing peptide (PrRP) has recently been cloned and identified as a ligand of an orphan pituitary receptor that stimulates in vitro PRL secretion. PrRP also induces PRL release in rats in vivo, especially in normal cycling females. However, no information on the effects of PrRP in the human is available. To elucidate the role of PrRP in regulating human anterior pituitary hormones, we used human PrRP-31 in primary cultures of human pituitary tissues, including fetal (20--27 weeks gestation) and normal adult pituitaries, as well as PRL- and GH-secreting adenomas. PrRP increased PRL secretion from human fetal pituitary cultures in a dose-dependent manner by up to 35% (maximal effect achieved with 10 nM), whereas TRH was slightly more potent for PRL release. Coincubation with estradiol resulted in enhanced fetal PRL response to PrRP, and GH release was only increased in the presence of estradiol. Although PRL secretion from PRL-cell adenomas was not affected by PrRP, PrRP induced PRL release from cultures of a GH-cell adenoma that cosecreted PRL. PrRP enhanced GH release in several GH-secreting adenomas studied by 25--27%, including GH stimulation in a mixed PRL-GH-cell tumor. These results show for the first time direct in vitro effects of PrRP-31 on human pituitary cells. PrRP is less potent than TRH in releasing PRL from human fetal lactotrophs and is unable to release PRL from PRL-cell adenomas in culture, but stimulated GH from several somatotroph adenomas. Thus, PrRP may participate in regulating GH, in addition to PRL, in the human pituitary.

Intact leptin receptor is selectively expressed in human fetal pituitary and pituitary adenomas and signals human fetal pituitary growth hormone secretion.

Leptin, a circulating hormone secreted by adipocytes, communicates peripheral nutritional status to hypothalamic centers affecting satiety, energy expenditure, and body weight. The intact leptin receptor (OB-R), a single membrane-spanning peptide containing an approximately 300-amino acid intracellular domain, is highly expressed in the hypothalamus, whereas shorter OB-R isoforms with truncated cytoplasmic regions resulting from alternative splicing have also been identified. We studied expression of OB-R isoforms in human fetal pituitaries, adult anterior pituitaries, and human pituitary adenomas. Using RT-PCR, messenger ribonucleic acid expression of the OB-R intact isoform was detected in fetal anterior pituitary tissues, but not in adult anterior pituitary glands, whereas both fetal and adult tissues expressed the short forms. Messenger ribonucleic acid of both intact and short OB-R isoforms were expressed in 4 of 5 GH-secreting, all 9 PRL-secreting, and 26 of 29 nonfunctioning pituitary adenomas. Recombinant human leptin (3-6 nmol/L) specifically stimulated GH secretion from primary human fetal pituitary cultures by 40-90% (P < 0.05) without altering fetal ACTH, PRL, or gonadotropin secretion. Thus, the intact OB-R is selectively expressed in human fetal and adult pituitary tumor tissues, but not in normal adult pituitary. Leptin specifically stimulates GH release from normal fetal somatotrophs, substantiating the functionality of its intact receptor in the fetal pituitary. Thus, pituitary adenomas appear to revert to a fetal phenotype of leptin receptor expression.

Intact Leptin Receptor Is Selectively Expressed in Human Fetal Pituitary and Pituitary Adenomas and Signals Human Fetal Pituitary Growth Hormone Secretion

Intact Leptin Receptor Is Selectively Expressed in Human Fetal Pituitary and Pituitary Adenomas and Signals Human Fetal Pituitary Growth Hormone Secretion

Age-related variations in the growth hormone response to growth hormone secretagogues

Growth hormone (GH) secretagogues are synthetic peptidyl and non-peptidyl molecules which possess a strong, dose-dependent and reproducible GH-releasing effect after intravenous and even oral administration in humans. This effect is probably mediated via the activation of specific receptors, mainly present at the pituitary and hypothalamic level; a human pituitary GH secretogogue receptor has already been cloned, pointing to the existence of an endogenous GH secretagogue-like ligand. The GH-releasing effect of GH secretagogues is gender-independent but undergoes marked age-related variations. In fact, the effect is low at birth, increases markedly at puberty, persists at a similar level in adulthood and decreases thereafter, being already similar in middle age to that in elderly subjects. It is likely that the reduced activity of GH secretagogues in aging reflects the age-related changes in the neural control of somatotrope function. These could include the hypothetical impairment in the activity of the putative natural GH secretagogue-like ligand. Prolonged treatment with non-peptidyl GH secretagogues has been shown to restore spontaneous GH pulsatility and insulin-like growth factor I (IGF-I) levels in aged humans as well as in animals. The possibility that chronic treatment with GH secretagogues in aging rejuvenates the activity of the GH/IGF-I axis and counteracts the age-related changes in body composition, structure functions and metabolism seems very attractive.

The not-so-good old days: Working with pituitary growth hormone in North America, 1956 to 1985

Before 1985 the use of growth hormone (GH) was governed by a philosophy of scarcity and conservation of resources. Between 1956 and 1959 human pituitary GH was shown to be effective. The competition for gland collection and extraction that followed benefited only certain patients with motivated parents and only a few investigators. To maximize gland collection, the distribution of GH for clinical investigation, and the number of patients who could be treated, the National Institutes of Health and the College of American Pathologists formed the National Pituitary Agency (NPA). In Canada a similar program was developed by the Canadian Medical Research Council. For more than 20 years the NPA supervised most of the GH treatment in the United States. Commercial pituitary GH entered the U.S. market in 1976, and competition soon appeared. Patients treated through the NPA were subjects in clinical studies for part of the first year of treatment, after which the limited availability of GH dictated treatment for only part of the year and caps on final heights. By 1984 treatment was year round and the height caps largely unenforced. In the last year of its distribution NPA GH was used in 2450 patients in the United States and commercial pituitary GH was used in 600 to 800; slightly more than 300 patients were being treated in Canada. And then, in 1985, came Creutzfeldt-Jakob disease. While the not-so-good old days are gone and need not be lamented, there remains virtue in a conservative therapeutic philosophy. If anything can be learned from the use of pituitary GH in children, it is a healthy respect for the law of unintended consequences. (J Pediatr 1997;131:S1-4)