ABSTRACT
PRL is mainly expressed in the pituitary and its gene expression is regulated by a variety of transcription factors including Pit-1. Brn-5 is a transcription factor that binds to Pit-1 binding elements and stimulates PRL reporter gene expression. In this study, the role of Brn-5 was examined. RNA interference (RNAi) against Brn-5 leaded to reduction in PRL content of GH3 cells, indicating endogenous Brn-5 may play a role in PRL gene expression. Furthermore Brn-5 RNAi decreased Pit-1 mRNA. Transfection of expression vectors for mPOU (human ortholog of Brn-5) modestly but significantly stimulated activities of PRL-Luc and Pit-1-Luc reporter genes in GH3 and HEK 293 cells. In addition, mPOU showed synergistic action with Pit-1 and CBP on PRL-Luc expression. mPOU-FL, a splicing variant of mPOU, showed weaker activity than mPOU. Chip assay suggested binding of mPOU to PRL and Pit-1 promoters of genomic DNA. Taken together, these results suggest that mPOU (Brn-5) enhances PRL gene expression directly in association with Pit-1 and CBP, and indirectly via the activation of Pit-1 gene expression.
Subject(s)
Gene Expression Regulation , Lactotrophs/metabolism , POU Domain Factors/metabolism , Pituitary Gland/metabolism , Prolactin/metabolism , Transcription Factor Pit-1/metabolism , Alternative Splicing , Animals , CREB-Binding Protein/metabolism , Cell Line , Genes, Reporter , Humans , POU Domain Factors/genetics , Pituitary Gland/cytology , Prolactin/genetics , Promoter Regions, Genetic , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rats , Transcription Factor Pit-1/genetics , TransfectionABSTRACT
A human pituitary cDNA library was screened using a yeast one-hybrid system to find a factor binding Pit-1 binding elements in the PRL gene other than Pit-1. Beside colonies containing Pit-1 or Oct-1 cDNA, three colonies contained mPOU cDNA, a member of the POU protein family. Immunohistochemical analysis showed mPOU-like immunoreactivity was present in human PRL-producing pituitary tumors but not in non-functioning pituitary tumors. Mobility shift analysis revealed that mPOU bound to Pit-1 binding elements of the PRL gene, 1P and 3P. mPOU activated the expression of 0.6 k PRL and 7x1P reporter genes in the presence of Pit-1 and cAMP, although it did not enhance Pit-1-induced expression of 7x3P reporter gene. These findings suggest that mPOU is involved in the activation of the PRL gene by cAMP through 1P in the presence of Pit-1.
Subject(s)
Cyclic AMP/analogs & derivatives , DNA-Binding Proteins/metabolism , Pituitary Gland/metabolism , Prolactin/genetics , Transcription Factors/metabolism , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Animals , Binding Sites , COS Cells , Calcium/metabolism , Chlorocebus aethiops , Cloning, Molecular , Cyclic AMP/pharmacology , DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , DNA-Binding Proteins/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation/drug effects , Gene Library , Genes, Reporter/genetics , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Host Cell Factor C1 , Humans , Immunohistochemistry , Mutation , Octamer Transcription Factor-1 , POU Domain Factors , Promoter Regions, Genetic/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Nucleic Acid/genetics , Sequence Alignment , Sequence Analysis, DNA , Thionucleotides/pharmacology , Transcription Factor Pit-1 , Transcription Factors/genetics , Transfection , Two-Hybrid System Techniques , Yeasts/geneticsABSTRACT
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. CONCLUSION: 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.
Subject(s)
DNA-Binding Proteins/genetics , Human Growth Hormone/genetics , Point Mutation , Prolactin/genetics , Transcription Factors/genetics , Animals , CHO Cells , COS Cells , Calcium Phosphates , Cell Nucleus/metabolism , Cricetinae , DNA-Binding Proteins/metabolism , Genes, Reporter , Humans , Luciferases/genetics , Mutagenesis , Pituitary Diseases/genetics , Promoter Regions, Genetic/genetics , Transcription Factor Pit-1 , Transcription Factors/metabolism , TransfectionABSTRACT
OBJECTIVE: mPOU is a POU protein classified as class VI. It is present in the pituitary gland as well as the brain, heart muscle, skeletal muscle, lung, and lymphocytes. In our previous investigation, mPOU bound to the Pit-1-binding DNA elements of the rat PRL gene, and promoted transcription of the GH and PRL genes. In this study, we immunohistologically investigated the expression of mPOU in pituitary adenomas. METHODS: 17 patients with pituitary adenoma underwent tumor excision by transsphenoidal approach at our hospital (PRL: 5, GH: 4, FSH: 1, non-functioning: 7). The expression in the tissue sections was investigated using immunostaining (ABC method). RESULTS: In all GH-producing and PRL-producing adenomas, mPOU protein was specifically expressed, particularly in the nuclei. DISCUSSION: Pit-1 has been considered to be a factor determining the expression of GH and PRL genes, but mPOU may also be involved in the expression.
Subject(s)
Adenoma/genetics , Adenoma/pathology , DNA-Binding Proteins/genetics , Pituitary Neoplasms/genetics , Pituitary Neoplasms/pathology , Adenoma/surgery , Adult , Aged , Biopsy, Needle , Female , Gene Expression Regulation, Neoplastic , Genetic Markers , Humans , Immunohistochemistry , Male , Middle Aged , Pituitary Neoplasms/surgery , Prognosis , Sampling Studies , Sensitivity and SpecificityABSTRACT
Pit-1 stimulates the expression of growth hormone, prolactin, and thyrotropin beta subunit genes. Consequently, abnormality of the Pit-1 gene results in combined pituitary hormone deficiency (CPHD). In this study, we analyzed the function of Pit-1 with a mutation (proline to leucine at codon 24) in the transactivation domain, P24L, which has a normal POU domain important for binding to DNA, because this mutation had been reported in a patient with CPHD. We found that codon 24 proline in the transactivation domain as well as the POU domain of Pit-1 was crucial to recruit coactivator CREB-binding protein (CBP) in the cultured cells. P24L completely lost the responsiveness to cAMP to stimulate the expression of the Pit-1-targeted genes. Furthermore, CBP and Pit-1, but not P24L, markedly enhanced the expression of the Pit-1-targeted gene to cAMP, and adenovirus E1a that binds to CBP and abrogates its function blocked the induction by cAMP of Pit-1-stimulated gene transcription in the pituitary-derived GH3 cells. These results suggest that CBP and proline at codon 24 in the transactivation domain of Pit-1 are important for the cAMP-induced activation of Pit-1-targeted genes. However, P24L maintained basal transcriptional activity, suggesting that CBP is unlikely to be an essential coactivator for Pit-1.
