title: Effect of different growth hormone (GH) mutants on the regulation of GH-receptor gene transcription in a human hepatoma cell line.
abstract: OBJECTIVE
  G to A transition at position 6664 of the growth hormone (GH-1) gene results in the substitution of Arg183 by His (R183H) in the GH protein and causes a new form of autosomal dominant isolated GH deficiency (IGHD type II). The aim of this study was to assess the bioactivity of this R183H mutant GH in comparison with both other GH variants and the 22-kDa GH in terms of GH-receptor gene regulation.
  
  
  METHODS
  The regulation of the GH-receptor gene (GH-receptor/GH binding protein, GHR/GHBP) transcription following the addition of variable concentrations (0, 12.5, 25, 50 and 500 ng/ml) of R183H mutant GH was studied in a human hepatoma cell line (HuH7) cultured in a serum-free hormonally defined medium. In addition, identical experiments were performed using either recombinant human GH (22-kDa GH) as a positive control or two GH-receptor antagonists (R77C mutant GH and pegvisomant (B-2036-PEG)) as negative controls. GHR/GHBP mRNA expression was quantitatively assessed by RT-PCR amplification after 0, 1, 3 and 6 h incubation.
  
  
  RESULTS
  Following the addition of R183H mutant GH, GHR/GHBP mRNA changed at a similar rate to that seen in experiments where 22-kDa GH was added, indicating equal bioactivity. At all times and concentrations studied, the addition of R77C mutant GH, however, resulted in a significantly lower increase (P<0.001) of GHR/GHBP mRNA concentration compared with that caused by the addition of either 22-kDa GH or R183H mutant GH. Furthermore, in additional experiments, pegvisomant resulted in an absolute block of GHR/GHBP mRNA expression identical to that seen in control experiments where no 22-kDa GH was added at all.
  
  
  CONCLUSIONS
  These data indicate that the R183H mutant GH, although causing an autosomal dominant form of IGHD has an identical effect on GHR/GHBP transcription as its wild-type, the 22-kDa GH. This implies that the IGHD caused by the R183H heterozygous mutation of the GH-1 gene is mainly due to a block of its regulated GH secretion. In addition, the R77C-GH variant and pegvisomant have an antagonistic effect at the level of GHR/GHBP transcription. All these data were confirmed by run-on experiments. In addition, these data highlight, as far as the GH variants are concerned, that a mutational alteration within the GH-1 gene might cause short stature also on the basis of an altered secretory pathway. This fact has to be taken into consideration when growth retardation is clinically diagnosed and studied at the molecular level. Secretory pathways and, therefore, cell-biological mechanisms are of importance and have to be considered in future not only at the scientific but also at the clinical level.
authors.affiliation: Department of Paediatrics, Division of Paediatric Endocrinology, Inselspital, CH-3010 Bern, Switzerland.
authors.forename: Johnny
authors.initials: J
authors.lastname: Deladoëy
authors.forename: Grégoire
authors.initials: G
authors.lastname: Gex
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Vuissoz
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Strasburger
authors.forename: Michael P
authors.initials: MP
authors.lastname: Wajnrajch
authors.forename: Primus E
authors.initials: PE
authors.lastname: Mullis
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D019382:Human Growth Hormone / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D008970:Molecular Weight; D009154:Mutation / Q000502:physiology*; D011986:Receptors, Somatotropin / Q000037:antagonists & inhibitors / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D014158:Transcription, Genetic / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D019382:Human Growth Hormone; C406545:pegvisomant