hGH-1 Gene Research Articles

Transgenic mice expressing the human growth hormone gene provide a model system to study human growth hormone synthesis and secretion in non-tumor-derived pituitary cells: Differential effects of dexamethasone and thyroid hormone

Growth hormone (GH) is regulated by pituitary and hypothalamic factors as well as peripheral endocrine factors including glucocorticoids and thyroid hormone. Studies on human GH are limited largely to the assessment of plasma levels in endocrine disorders. Thus, insight into the regulation of synthesis versus secretion has come mainly from studies done on non-human GH and/or pituitary tumor cells. However, primate and non-primate GH gene loci have differences in their structure and, by extension, regulation. We generated transgenic (171hGH/CS-TG) mice containing the intact hGH1 gene and locus control region, including sequences required for integration-independent and preferential pituitary expression. Here, we show hGH co-localizes with mouse (m) GH in somatotrophs in situ and in primary pituitary cells. Dexamethasone treatment increased hGH and mGH, as well as GH releasing hormone (GHRH) receptor RNA levels, and hGH release was stimulated by GHRH treatment. By contrast, triiodothyronine decreased or had no effect on hGH and mGH production, respectively, and the negative effect on hGH was also seen in the presence of dexamethasone. Thus, 171hGH/CS-TG mouse pituitary cultures represent a model system to investigate hormonal control of hGH synthesis and secretion.

The Human Growth Hormone Gene Contains a Silencer Embedded within an Alu Repeat in the 3′-Flanking Region

Alu family sequences are middle repetitive short interspersed elements (SINEs) dispersed throughout vertebrate genomes that can modulate gene transcription. The human (h) GH locus contains 44 complete and four partial Alu elements. An Sx Alu repeat lies in close proximity to the hGH-1 and hGH-2 genes in the 3'-flanking region. Deletion of the Sx Alu repeat in reporter constructs containing hGH-1 3'-flanking sequences increased reporter activity in transfected pituitary GC cells, suggesting this region contained a repressor element. Analysis of multiple deletion fragments from the 3'-flanking region of the hGH-1 gene revealed a strong orientation- and position-independent silencing activity mapping between nucleotides 2158 and 2572 encompassing the Sx Alu repeat. Refined mapping revealed that the silencer was a complex element comprising four discrete entities, including a core repressor domain (CRD), an antisilencer domain (ASE) that contains elements mediating the orientation-independent silencer activity, and two domains flanking the CRD/ASE that modulate silencer activity in a CRD-dependent manner. The upstream modulator domain is also required for orientation-independent silencer function. EMSA with DNA fragments representing all of the silencer domains yielded a complex pattern of DNA-protein interactions indicating that numerous GC cell nuclear proteins bind specifically to the CRD, ASE, and modulator domains. The silencer is GH promoter dependent and, in turn, its presence decreases the rate of promoter-associated histone acetylation resulting in a significant decrease of RNA polymerase II recruitment to the promoter. The silencer may provide for complex regulatory control of hGH gene expression in pituitary cells.

Reduced human growth hormone (hGH) bioactivity without a defect of the GH-1 gene in three patients with rhGH responsive growth failure.

A GH deficiency-like phenotype with normal or high hGH secretion, pathologically low IGF-I serum levels, and catch-up growth under treatment with recombinant hGH is suggestive of the presence of biologically inactive hGH syndrome, whose presumably heterogenous molecular basis is substantially unknown. Serum samples from patients who fulfilled the above criteria and from controls with idiopathic short stature were measured by polyclonal hGH-RIA, Nb2 rat lymphoma proliferation assay, and hGH immunofunctional assay (IFA). If assays were suggestive of the presence of bioinactive GH, mutational analysis of the hGH-1 gene was performed. Three patients were selected because of clinical and biochemical evidence. At the time of diagnosis mean age was 3.4 (2.2, 3.5, 4.5) years, mean height -3.5 (-2.8, -3.6, -4.2) SD score (SDS) and mean growth rate -1.5 (-1.4, -1.5, -1.6) SDS. Mean IGF-I serum levels were -1.9 (-0.7, -2.4, -2.5) SDS and mean IGFBP-3 serum levels -1.2 (-1.1, -1.2, -1.2) SDS. Stimulated and spontaneous GH peaks measured by a polyclonal radioimmunoassay were all above 14 microg/l. GHBP serum levels were normal, and antihGH antibodies were not detected. Therapy with rhGH was effective in causing catch-up growth of the three children with an initial mean growth rate of + 2.9 (+ 1.7, + 2.1, + 5.0) SDS, and normalization of IGF-I (mean: -0.66 SDS: -1.8, - 1.2, + 1.1 SDS) and IGFBP-3 serum levels (mean: + 0.81 SDS: -0.2, + 0.8, + 1.8 SDS). In comparison to controls, the patients' serum hGH levels were much lower when measured by Nb2 rat lymphoma cell proliferation bioassay (mean: -2.3 SDS, range: -1.7- -4.1) and by the immunofunctional assay (IFA) (-1.5 SDS, range: -0.2- -3.1) than by RIA. Retesting of two of the three patients including an one year break of therapy confirmed the rhGH dependence of growth in spite of normal endogenous GH secretion. Radioactive direct sequencing of both strands of PCR-amplified genomic DNA and cDNA excluded a GH-1 gene mutation in all three children. Mutations of the GH-1 gene are probably not the main genetic defect in children with biologically inactive hGH syndrome. Posttranslational processing of hGH might reduce the biological activity of the normal translation product.

Long-Term Treatment of Growth Hormone Insensitivity Syndrome with IGF-I

A total of 33 patients (17 female, 16 male) with Laron syndrome (n = 31) or hGH-1 gene (n = 2, type IA deletion) from 22 centres in 12 countries were enrolled in a study conducted by Pharmacia & Upjohn, Stockholm, which was designed to test the efficacy, in terms of growth promotion and safety, of IGF-I (Igef^TM). The patients were treated with 40–120 μg/kg IGF-I s.c. twice daily after meals. After the study ended, the patients continued to be treated on an individual basis. The results of 17 patients, who were treated for 48 months or longer were available for the present analysis. Six patients were treated for up to 72 months. When treatment started, the mean age of these patients (8 female, 9 male) was 9.1 (3.7–13.5) years and mean height was –6.5 ± 1.3 SDS. At the end of the observation period, the mean age of the 17 patients was 14.2 (9.1–17.7) years and mean height was –4.9 ± 1.9 SDS. All patients showed a significant increase in growth during the final year on IGF-I, with two of them reaching the age-corresponding 3rd centile. The total gain in height (ΔHT) was 1.7 ± 1.2 SDS. ΔHT SDS correlated negatively with age at onset of treatment (R² = –0.78, p < 0.02). BMI was 0.6 ± 1.8 SDS at start of treatment and 1.8 ± 1.5 SDS at the end of observation. Total ΔHT SDS correlated positively with total ΔBMI SDS (R² = 0.59, p < 0.01). Long-term treatment of patients with GHIS thus proved to be effective in promoting growth. If treatment is started at an early age, there is considerable potential for achieving height normalisation. The treatment modalities need to be optimized with respect to the growth-promoting and metabolic effects of IFG-I.

An improved polymerase chain reaction (PCR) protocol for unambigous detection of growth hormone gene deletions.

hGH-1 gene deletions are detected by simultaneous PCR amplification along the two homologous DNA sequences flanking the hGH-1 gene on both sides and are differentiated by SmaI restriction enzyme digestion. We have observed that among the SmaI digested PCR products from normal homozygous subjects, from those heterozygous for the 7.6 kb deletion and from those heterozygous for a 6.7 kb deletion, along with the expected fragments there is an unexpected 1470 bp fragment. This fragment arises from the co-amplification of a third homologous sequence located downstream from the hGH-1 gene and it confuses differentiation between normal homozygous and heterozygous for 7.6 kb subjects from the 6.7 kb heterozygous subjects. To overcome this problem we have improved PCR conditions using a different reverse primer. These changes avoid the interaction of the primers with the third homologous sequence located downstream from the hGH-1 gene and prevent the appearance of this additional band that complicates the interpretation of the results. We conclude that the new reverse primer sequence avoids the amplification of the downstream hGH-1 gene sequence and the production of the 1474 bp band after SmaI endonuclease enzyme digestion and makes it possible to differentiate homozygous normal subjects and those who are heterozygous for a 7.6 kb deletion from those who are heterozygous for a 6.7 kb deletion.

Allelic variations in the human growth hormone-1 gene promoter of growth hormone-deficient patients and normal controls.

Isolated growth hormone deficiency (IGHD) type IB is suggested to be more probably due to alterations in the genes directly involved in the hypothalamo-pituitary axis and/or in the specific transcriptional regulation (cis-trans coupling) of the hGH-1 gene than to alterations in the gene itself. In this study we analyzed the hGH-1 gene promoter region for structural alterations and allelic variations. The hGH-1 gene promoter region was analyzed by PCR, cycle sequencing and direct-blotting electrophoresis in a total of 212 individuals including 113 patients with IGHD type IB, 21 unaffected family members and 78 normal controls. Twenty-two sequence variation sites were identified. Of these, 14% were located around the region of -1075bp, 77% between -550bp and the translational start site (+1bp) and 9% within the first intron. Only one variation site affected a characterized cis-acting element, namely that of NF-1. Importantly, all the variations found in patients were also observed in non-affected family members as well as in normal unrelated controls. These findings imply that it is not a single variation within the GH-1 gene promoter, and therefore in the cis-acting elements, which causes IGHD. However, we can not exclude the possibility that combinations of variations might perturb expression. Furthermore, these data illustrate the normal heterogeneity of the GH-1 gene promoter region, a fact that has to be borne in mind whenever transcriptional studies are performed.

Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells.

We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.

The use of the polymerase chain reaction in prenatal diagnosis of growth hormone gene deletions.

Familial isolated growth hormone deficiency (IGHD) type IA is characterized by a complete absence of human growth hormone (hGH) resulting in most cases from either a 6.7 or 7.7 kb deletion of DNA containing the hGH-1 gene. These patients have a strong initial anabolic response to exogenous recombinant hGH (r-hGH) therapy, frequently associated with the development of immune intolerance to r-hGH which causes an arrest of response to r-hGH replacement. This disorder is inherited as an autosomal recessive trait. In two pregnancies at risk, the polymerase chain reaction (PCR) was applied as a method for identifying hGH-1 gene deletions in DNA obtained by chorionic villus sampling (CVS) in the first trimester. Homozygotes for the 6.7kb deletion of DNA containing the hGH-1 gene were easily and conclusively detected by the absence of 1900, 761 and 712bp fragments after SmaI digestion of the polymerase chain reaction products. In contrast, the pattern found in heterozygotes for the hGH-1 gene deletion was difficult to distinguish from the pattern found in normal homozygotes. We conclude that the polymerase chain reaction method is valuable for diagnosing individuals who are homozygous for hGH-1 gene deletions, while heterozygotes and normal individuals may be difficult to distinguish from each other. We suggest that, in these cases, Southern blotting remains the analysis to perform.

Prevalence of human growth hormone-1 gene deletions among patients with isolated growth hormone deficiency from different populations.

Familial isolated growth hormone deficiency type IA results from homozygosity for either a 6.7-kb or a 7.6-kb hGH-1 gene deletion. Genomic DNA was extracted from circulating lymphocytes of 78 subjects with severe isolated growth hormone deficiency (height less than -4.5 SD score) and studied by polymerase chain amplification and by restriction endonuclease analysis looking for gene deletions within the hGH-gene cluster. The individuals analyzed were broadly grouped into three different populations (North-European, n = 32; Mediterranean, n = 22; and Turkish, n = 24). Ten out of 78 patients studied presented with an hGH-1 gene deletion; eight out of these 10 showed a 6.7-kb gene deletion, the remaining two a 7.6-kb hGH-1 gene deletion. Five of the 10 subjects developed anti-hGH antibodies to hGH replacement followed by a stunted growth response. Family studies of the affected patients were performed, revealing consanguinity in all the families, and the corresponding heterozygosity for the deletion was present in each of the parents. The results of our study revealed a prevalence for an hGH-1 gene deletion in three out of 32 North-European, three out of 22 Mediterranean, and four out of 24 Turkish patients with growth hormone deficiency (height less than 4.5 SD score). These data are important for prenatal diagnosis of at-risk pregnancies and for families at risk for recurrence and underline clearly the fact that the hGH-I gene deletion represents a common cause for growth hormone deficiency associated with severe growth retardation (height less than -4.5 SD score).

Short Stature in a Patient with Cystic Fibrosis Caused by a 6.7-kb Human Growth Hormone Gene Deletion

A recent longitudinal study in children with cystic fibrosis (CF) challenged the common idea that CF is causing short stature. The data, however, showed clearly that short stature cannot be explained by CF alone after the first year of life. We report on a girl suffering from CF and short stature in whom DNA analysis using polymerase chain reaction and Southern blot techniques of the human growth hormone (hGH) gene cluster revealed a 6.7-kb gene deletion encompassing the hGH-1 gene. Anti-hGH antibodies of polyclonal origin developed, leading to a growth arrest after only 2 months of hGH replacement. In addition, a family study was performed, and the haplotypes of the CF gene and hGH gene cluster were analyzed.

Isolated Growth Hormone Deficiency: Analysis of the Growth Hormone (GH)-Releasing Hormone Gene and the GH Gene Cluster

Isolated GH deficiency (IGHD) cannot be distinguished on the grounds of anti-human (h) GH antibodies and stunted growth response to exogenous hGH. DNA analysis was proposed to classify children with IGHD. Genomic DNA was extracted and studied by restriction endonuclease analysis after extraction from the circulating lymphocytes of 53 children with IGHD. These children included 5 pairs of siblings and 5 individuals from 10 families, whose parents (n = 20) and brothers and sisters (n = 5) were also analyzed. Twenty-five adults, including individuals from 3 families of normal height, were studied as controls. No deletion within the hGH gene cluster was identified using a [32P]hGH cDNA clone as a probe. A compound heterozygosity for a hGH-1 deletion or a mutation have not been found. The allelic frequencies for 5 common restriction fragment length polymorphisms were similar in patients and controls. The distribution and frequency of the distinct haplotypes in the hGH gene family revealed no differences between IGHD (n = 30 chromosomes) and controls (n = 48 chromosomes). No deletion or restriction fragment length polymorphisms could be found using a hGH-releasing hormone cDNA clone as a probe in patients or controls. This large volume of data gathered from a caucasian population indicates that the great majority of patients with IGHD has no structural abnormalities of the hGH gene cluster, particularly no hGH-1 gene deletion. In addition, they have no gross deletions within the hGH-releasing hormone gene.

Regulated expression of chimaeric genes containing the 5'-flanking regions of human growth hormone-related genes in transiently transfected rat anterior pituitary tumor cells.

The expression and hormonal regulation of chimaeric genes containing the 5'-flanking regions of the normal human growth hormone (hGH-1), the variant hGH (hGH-2) and chorionic somatomammotropin (hCS-1) genes fused to the chloramphenicol acetyl transferase (CAT) gene has been examined after transient transfection into cultured rat pituitary (GC), and non-pituitary (HeLa and Rat 2) tumor cells. As assessed by levels of CAT activity, the hGH-1 and hCS-1 gene hybrids were expressed at 5- to 25-fold higher levels in GC cells than in HeLa or Rat 2 cells. The hGH-2 gene hybrid was expressed at very low levels in all 3 cell types. Triiodothyronine treatment of transiently transfected GC cells had little effect on CAT activity from the hGH-1 gene hybrid but increased CAT activity from the hCS-1 gene hybrid. A slight but significant increase in CAT expression was detected with both genes after dexamethasone treatment. The data indicate that elements present on the hGH-1 and hCS-1 genes' 5'-flanking DNA are required for the efficient expression of these genes in GC cells.