The R271W mutant form of Pit-1 does not act as a dominant inhibitor of Pit-1 action to activate the promoters of GH and prolactin genes.
OBJECTIVE
Genetic abnormalities of the pituitary specific transcription factor, Pit-1, have been reported in several patients with GH, prolactin (PRL) and TSH deficiencies. The most common is a mutation altering an arginine to a tryptophan in codon 271 (R271W) in one allele of the Pit-1 gene. According to the previous in vitro expression study, R271W acted as a dominant negative inhibitor of the wild type to activate the GH promoter. However, healthy carriers with this mutation, who should be affected by the dominant negative effect of R271W, have also been reported. The aim of this study was to clarify in more detail the function of this mutant form of Pit-1.


METHODS
Transcriptional activity of R271W for the expression of Pit-1-associated genes was investigated in COS7 cells with the aid of transient transfection assays. The 1.8 kb rat GH, 0.6 kb rat PRL or 1.9 kb rat PRL 5'-flanking regions were inserted upstream of the luciferase reporter gene and were used for functional analysis of R271W. Another reporter gene containing seven Pit-1 responsive elements was also used. The same experiments were also performed using JEG3 and CHO cells.


RESULTS
We could not confirm the dominant negative effect of R271W on wild type Pit-1. Furthermore, our expression study revealed that R271W could activate the promoters of GH and PRL genes to levels similar to the wild type.


CONCLUSIONS
Taken together with the evidence that phenotypically normal cases have been reported with this mutation, our results deny the relationship between R271W and combined pituitary hormone deficiency.
Kishimoto
Masahiko
M
Division of Endocrinology/Metabolism, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-10-1 Tomogaoka, Suma-ku, Kobe 654-0142, Japan.
Okimura
Yasuhiko
Y
Fumoto
Mariko
M
Iguchi
Genzo
G
Iida
Keiji
K
Kaji
Hidesuke
H
Chihara
Kazuo
K
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002130:Calcium Phosphates; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D016296:Mutagenesis; D010900:Pituitary Diseases / Q000235:genetics; D017354:Point Mutation; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D002130:Calcium Phosphates; D004268:DNA-Binding Proteins; C494187:POU1F1 protein, human; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D019382:Human Growth Hormone; C485838:calcium phosphate, monobasic, anhydrous; D011388:Prolactin; C020243:calcium phosphate; D008156:Luciferases; C485829:calcium phosphate, dibasic, anhydrous