ABSTRACT
The novel heterodimeric glycoprotein hormone thyrostimulin consists of two unique subunits, A2 and B5. To understand its yet unknown transcriptional regulation, we characterised the 3.1-kb immediate 5'-flanking region of the human A2 gene localised on chromosome 11q13. In transient transfection assays this sequence exhibited promoter activity, which could be confined to nucleotides -506 to -347 relative to the ATG start codon. Interestingly, this minimal promoter appeared to be non-tissue-specific. Deletional, mutational and gel shift analyses revealed regulatory elements that are essential for the regulation of the A2 gene expression. Another noteworthy feature of this gene is the presence of silencer elements upstream and downstream of the promoter. To surmise, our results provide an initial step toward a detailed analysis of the underlying molecular mechanisms of the human thyrostimulin gene expression.
Subject(s)
Cloning, Molecular , Glycoproteins/genetics , Peptide Hormones/genetics , Promoter Regions, Genetic , Regulatory Elements, Transcriptional , Transcription, Genetic , Animals , Cell Line , Codon/genetics , Codon/physiology , Electrophoretic Mobility Shift Assay , Exons/genetics , Exons/physiology , Gene Deletion , Gene Expression Regulation , Glycoproteins/physiology , Humans , Mice , Mutation , Peptide Hormones/physiology , Silencer Elements, Transcriptional , Transcription Factors/genetics , Transcription Factors/physiology , TransfectionABSTRACT
The diesters of benzene-1,2-dicarboxylic (phthalic) acid, the phthalates, are used to make plastics flexible and can comprise 40% of the weight of plastic. Human exposure to phthalates can occur via ingestion, inhalation and dermal routes, as well as through parenteral exposure from medical devices containing phthalates. Since earlier morphological studies showed that some phthalates induced thyroid hyperactivity, we thought it important to investigate possible effects of six major phthalates on the transcriptional activity of sodium/iodide symporter (NIS). Di-isodecyl phthalate (DIDP), benzyl butyl phthalate (BBP) and di-octyl phthalate (DOP) increased the activity of the human NIS promoter construct 2.5-, 2.6- and 2.4-fold, respectively. Likewise, these phthalates also enhanced the rat NIS endogenous mRNA expression ca. 2-fold. No effect was observed for bis-(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DINP), whereas dibutyl phthalate (DBP) appeared to down-regulate hNIS promoter. Although the demonstrated stimulation of NIS gene transcription by DIDP, BBP and DOP is not very strong, this finding is of great importance as humans are routinely exposed for long periods to phthalate plasticisers, the accumulation of which may contribute to thyroid hyperfunction.
Subject(s)
Phthalic Acids/pharmacology , Plasticizers/pharmacology , Symporters/drug effects , Transcription, Genetic/drug effects , Cells, Cultured , Humans , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Symporters/genetics , Symporters/metabolismABSTRACT
This study shows that organification of radioiodide into proteins of thyroid cancer cells exogenously co-expressing the thyroid peroxidase (TPO) and the sodium/iodide symporter (NIS) is independent of NIS function. When administering (125) I to cells constitutively expressing either NIS, or TPO or NIS/TPO, next to iodide accumulation due to NIS activity, organification was exclusively observed in TPO expressing/co-expressing cells. The use of specific inhibitors for TPO and NIS showed that organification is strictly dependent of TPO and not of NIS. An identical pattern of iodoproteins migrating between approximately 75 and 200 kDa in all cell lines tested was observed. Among the five major iodoproteins, two polypeptides appear to be related and three are most probably unrelated, according to their peptide pattern. Our results significantly indicate that co-expression of TPO in NIS transfected cells mediates iodination on the one hand but on the other hand does not contribute to augmentation of a putative NIS-based radioiodide concentrator gene therapy.
Subject(s)
Autoantigens/metabolism , Iodide Peroxidase/metabolism , Iodine/metabolism , Iodoproteins/biosynthesis , Iron-Binding Proteins/metabolism , Proteins/metabolism , Symporters/metabolism , Autoantigens/genetics , Autoradiography , Cell Line, Tumor , Humans , Hydrogen Peroxide/pharmacology , Iodide Peroxidase/genetics , Iodine Radioisotopes , Iodoproteins/analysis , Iron-Binding Proteins/genetics , Symporters/genetics , Thyroid Neoplasms/pathology , TransfectionABSTRACT
Graves' thyroid tissue has been shown to express elevated levels of human sodium/iodide symporter (hNIS) mRNA and protein. In the present work, we demonstrate for the first time that hNIS gene expression in Graves' disease (GD) is up-regulated by Graves' IgG. Here, in transient transfection experiments using FRTL-5 cells, hNIS promoter and enhancer/luciferase construct showed an up to six-fold increase in transcriptional activity after incubation with purified Graves' IgG. Mutation of a CRE site in hNIS enhancer inhibited Graves' IgG response. In addition, mutation of a novel putative regulatory region in hNIS promoter reduced the stimulation three-fold. This discovered putative regulatory sequence might play a role in hNIS up-regulation by Graves' IgG and TSH. The data presented here complement our current knowledge of the pathogenesis of GD and will contribute to a better understanding of mechanisms regulating the thyroid iodide concentrating activity.
