Description of the SAGhE Cohort: A Large European Study of Mortality and Cancer Incidence Risks after Childhood Treatment with Recombinant Growth Hormone.

BACKGROUND The long-term safety of growth hormone treatment is uncertain. Raised risks of death and certain cancers have been reported inconsistently, based on limited data or short-term follow-up by pharmaceutical companies. PATIENTS AND METHODS The SAGhE (Safety and Appropriateness of Growth Hormone Treatments in Europe) study assembled cohorts of patients treated in childhood with recombinant human growth hormone (r-hGH) in 8 European countries since the first use of this treatment in 1984 and followed them for cause-specific mortality and cancer incidence. Expected rates were obtained from national and local general population data. The cohort consisted of 24,232 patients, most commonly treated for isolated growth failure (53%), Turner syndrome (13%) and growth hormone deficiency linked to neoplasia (12%). This paper describes in detail the study design, methods and data collection and discusses the strengths, biases and weaknesses consequent on this. CONCLUSION The SAGhE cohort is the largest and longest follow-up cohort study of growth hormone-treated patients with follow-up and analysis independent of industry. It forms a major resource for investigating cancer and mortality risks in r-hGH patients. The interpretation of SAGhE results, however, will need to take account of the methods of cohort assembly and follow-up in each country.

Effects of aerobic exercise on ectopic lipids in patients with growth hormone deficiency before and after growth hormone replacement therapy

Growth hormone replacement therapy (GHRT) increases exercise capacity and insulin resistance while it decreases fat mass in growth hormone-deficient patients (GHD). Ectopic lipids (intramyocellular (IMCL) and intrahepatocellular lipids (IHCL) are related to insulin resistance. The effect of GHRT on ectopic lipids is unknown. It is hypothesized that exercise-induced utilization of ectopic lipids is significantly decreased in GHD patients and normalized by GHRT. GHD (4 females, 6 males) and age/gender/waist-matched control subjects (CS) were studied. VO2max was assessed on a treadmill and insulin sensitivity determined by a two-step hyperinsulinaemic-euglycaemic clamp. Visceral (VAT) and subcutaneous (SAT) fat were quantified by MR-imaging. IHCL and IMCL were measured before and after a 2 h exercise at 50-60% of VO2max using MR-spectroscopy (∆IMCL, ∆IHCL). Identical investigations were performed after 6 months of GHRT. VO2max was similar in GHD and CS and significantly increased after GHRT; GHRT significantly decreased SAT and VAT. 2 h-exercise resulted in a decrease in IMCL (significant in CS and GHRT) and a significant increase in IHCL in CS and GHD pre and post GHRT. GHRT didn't significantly impact on ∆IMCL and ∆IHCL. We conclude that aerobic exercise affects ectopic lipids in patients and controls. GHRT increases exercise capacity without influencing ectopic lipids.

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against l-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.

Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression

The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile but not continuous GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, which is proposed to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. We have evaluated the importance of STAT5b for the physiological effects of GH pulses using a mouse gene knockout model. STAT5b gene disruption led to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression were decreased to wild-type female levels in STAT5b−/− males, while female-predominant liver gene products were increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient Little mice, STAT5b−/− mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in STAT5b−/− mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute.

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in Cpefat mice associated with a carboxypeptidase E mutation

Cpefat mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia–diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in Cpefat mice. In Cpefat mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K+ stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of Cpefat mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of Cpefat mice.

Growth hormone-releasing hormone: An autocrine growth factor for small cell lung carcinoma

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers in vivo. This effect is thought to be exerted through suppression of the pituitary growth hormone–hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured in vitro express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1–29)NH2 stimulated the proliferation of H-69 and H-510A SCLCs in vitro, and GHRH antagonist JV-1–36 inhibited it. JV-1–36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (P < 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.

Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancers in vitro and in vivo by mechanisms that include apparent direct effects through specific binding sites expressed on tumors and that differ from pituitary human GHRH (hGHRH) receptors. In this study, GHRH antagonist JV-1–38 (20 μg/day per animal s.c.) inhibited the growth of orthotopic CAKI-1 human renal cell carcinoma (RCC) by 83% and inhibited the development of metastases to lung and lymph nodes. Using ligand competition assays with 125I-labeled GHRH antagonist JV-1–42, we demonstrated the presence of specific high-affinity (Kd = 0.25 ± 0.03 nM) binding sites for GHRH with a maximal binding capacity (Bmax) of 70.2 ± 4.1 fmol/mg of membrane protein in CAKI-1 tumors. These receptors bind GHRH antagonists preferentially and display a lower affinity for hGHRH. The binding of 125I-JV-1–42 is not inhibited by vasoactive intestinal peptide (VIP)-related peptides sharing structural homology with hGHRH. The receptors for GHRH antagonists on CAKI-1 tumors are distinct from binding sites detected with 125I-VIP (Kd = 0.89 ± 0.14 nM; Bmax = 183.5 ± 2.6 fmol/mg of protein) and also have different characteristics from GHRH receptors on rat pituitary as documented by the insignificant binding of [His1,125I-Tyr10,Nle27]hGHRH(1–32)NH2. Reverse transcription-PCR revealed the expression of splice variants of hGHRH receptor in CAKI-1 RCC. Biodistribution studies demonstrate an in vivo uptake of 125I-JV-1–42 by the RCC tumor tissue. The presence of specific receptor proteins that bind GHRH antagonists in CAKI-1 RCC supports the view that distinct binding sites that mediate the inhibitory effect of GHRH antagonists are present on various human cancers.

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV1 and SV2 are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV1 is identical to that of human pituitary GHRH-R, whereas in SV2 exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV1 is identical to that of pituitary GHRH-R. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between −14.6 kb and −32 kb 5′ to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (−14.6 to −16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen in vivo. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.

Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ × DW/J)F1 background. Mutant dwJ/dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhrlit mutation, which like the Pit1dw mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1dw mutant, and the closely related, long-lived Prop-1df (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.

Alternative splicing of exon 3 of the human growth hormone receptor is the result of an unusual genetic polymorphism.

Two isoforms of the human growth hormone receptor (hGHR), which differ in the presence (hGHRwt) or absence (hGHRd3) of exon 3, are expressed in the placenta. Specifically, three expression patterns are observed: only hGHRwt, only hGHRd3, or an approximately 1:1 combination of both isoforms. We investigated several potential regulatory mechanisms which might account for the expression of the hGHR isoforms. The frequency of hGHRd3 expression did not change when placentas from differing stages of gestation were examined, suggesting splicing was not developmentally regulated. However, when hGHR isoform expression patterns were examined in each component of a given placenta, it was evident that alternative splicing of exon 3 is individual-specific. Surprisingly, the individual-specific regulation of hGHR isoforms appears to be the result of a polymorphism in the hGHR gene. We analyzed hGHRwt and hGHRd3 expression in Hutterite pedigrees, and our results are consistent with a simple Mendelian inheritance of two differing alleles in which exon 3 is spliced in an "all-or-none" fashion. We conclude the alternative splicing of exon 3 in hGHR transcripts is the result of an unusual polymorphism which significantly alters splicing of the hGHR transcript and that the relatively high frequency (approximately 10%) of homozygous hGHRd3 expression suggests the possibility it may play a role in polygenic determined events.

Binding in the growth hormone receptor complex.

Binding reactions between human growth hormone (hGH) and its receptor provide a detailed account of how a polypeptide hormone activates its receptor and more generally how proteins interact. Through high-resolution structural and functional studies it is seen that hGH uses two different sites (site 1 and site 2) to bind two identical receptor molecules. This sequential dimerization reaction activates the receptor, presumably by bringing the intracellular domains into close proximity so they may activate cytosolic components. As a consequence of this mechanism it is possible to build antagonists to the receptor by introducing mutations in hGH that block binding at site 2 and to build even more potent antagonists by combining these with mutants that enhance binding at site 1. Alanine-scanning mutagenesis of all contact residues at the site 1 interface shows that only a small and complementary set of side chains clustered near the center of the interface affects binding. The most important contacts are hydrophobic, and these are surrounded by polar and charged interactions of lesser importance. Kinetic analysis shows for the most part that the important side chains function to maintain the complex, not to guide the hormone to the receptor. Hormone-induced homodimerization or heterodimerization reactions are turning out to be pervasive mechanisms for signal transduction. Moreover, the molecular recognition principles seen in the hGH-receptor complex are likely to generalize to other protein-protein complexes.