ABSTRACT
Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial-to-mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer-compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/prevention & control , Chromatin/genetics , Epigenesis, Genetic/genetics , Genome/genetics , Animals , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Female , Humans , Mice , Neoplastic Stem CellsABSTRACT
Previous animal studies have demonstrated that following systemic administration phosphorothioate oligodeoxynucleotides (S-ODNs) are primarily excreted by the kidneys and that renal tissue levels of S-ODNs exceed that of other organs. Thus, the kidney may be an ideal target organ for application of antisense S-ODNs in vivo. We examined which cells within the rat kidney have uptake of radiolabeled S-ODNs following intravenous infusion. A 20-base 35S-ODN was infused into 6 adult male Wistar rats. Three animals each were sacrificed 30 min and 4 h after infusion. The kidneys were then removed, fixed, and tissue autoradiography was performed. Similar results were obtained in both groups. The highest level of radioactivity was seen within the proximal tubules. Lower levels of activity were seen within the glomerulus, the parietal epithelial cells of Bowman's space, and distal tubular cells. Very weak activity was also detected within the cells of the loop of Henle and the medullary collecting ducts. These results demonstrated that within the kidney S-ODNs were taken up primarily by proximal tubular cells, with much lower uptake by cells in other segments of the nephron.
Subject(s)
Kidney/metabolism , Oligonucleotides, Antisense/pharmacokinetics , Thionucleotides/pharmacokinetics , Animals , Autoradiography , Infusions, Intravenous , Kidney/cytology , Kidney Tubules/cytology , Kidney Tubules/metabolism , Male , Oligonucleotides, Antisense/administration & dosage , Rats , Rats, Wistar , Sulfur Radioisotopes , Thionucleotides/administration & dosageABSTRACT
Activating mutations encoding substitutions at positions Arg201 and Gln227 of the alpha-subunit of the stimulatory G protein. G10 have been found in about 40% of pituitary somatotroph tumors. Although the etiology of thyrotroph adenomas is unknown, their autonomous behavior and blunted response to stimulatory hypothalamic hormone superficially resemble those of somatotroph tumors. We hypothesized that a subset of thyrotroph tumors might be caused by dominant somatic mutations that lead to inappropriate activation of the Gq/phospholipase C beta/Ca2+/protein kinase C. pathway normally triggered by occupancy of the TRH receptor (TRHR). We, therefore, screened samples from nine thyrotroph tumors for the presence of activating mutations of the alpha q, alpha 11, and TRHR genes. Fragments of alpha q and alpha 11 complementary DNA encompassing residues (Arg183 and Gln209) that correspond to Arg201 and Gln227 of alpha q were amplified and sequenced. Temperature gradient gel electrophoresis was used to screen for heterozygous mutations in the TRHR coding sequence as well as for known alpha s mutations. No mutations were detected. We conclude that mutations in these regions of the alpha q, alpha 11, alpha s, and TRHR genes occur infrequently, if at all, in human thyrotroph tumors. Alternative mechanisms underlying thyrotroph tumorigenesis, including changes in the expression levels of G protein alpha-subunits or TRHR, dysregulation of downstream components, inappropriate activation of other stimulatory pathways, or loss of inhibitory inputs, remain to be explored.
Subject(s)
Genetic Testing , Oncogenes , Pituitary Neoplasms/genetics , Pituitary Neoplasms/metabolism , Thyrotropin/metabolism , Adult , Base Sequence , Female , GTP-Binding Proteins/genetics , Genes , Heterozygote , Humans , Male , Middle Aged , Molecular Probes/genetics , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , Receptors, Thyrotropin-Releasing Hormone/geneticsABSTRACT
The family of 70-kDa heat-shock proteins (HSP-70) is evolutionarily highly conserved and has been shown to enhance cell survival from thermal injury. This study characterized HSP-72 induction in human epidermoid A-431 cells exposed to 45 degrees C for 10 min and determined the relationship between HSP-72, intracellular pH (pHi), adenosine 3',5'-cyclic monophosphate (cAMP), G proteins, and intracellular cytosolic free Ca2+ concentration ([Ca2+]i). Heat shock induced HSP-72 production, which was dependent on both temperature and the duration of heating. This HSP-72 induction was confirmed by Western blot analysis. HSP-72 levels in cells that had been heated then returned to 37 degrees C were elevated at 2 h (1.5 +/- 0.1 x control), reached a maximum at 8 h (2.7 +/- 0.1 x control), and remained above baseline for up to 4 days. Levels of HSP-72 mRNA, determined by dot-blot analysis, reached a maximum at 2 h and returned to baseline within 8 h. Both actinomycin D and cycloheximide blocked HSP-72 induction. Because heating causes intracellular acidification and increases in cAMP and [Ca2+]i, we studied the effect of pHi, cellular cAMP, and [Ca2+]i on HSP-72 induction. The reduction of pHi to 6.9 by acid loading did not affect the basal level of HSP-72 in unheated cells. Treatment with pertussis toxin, cholera toxin, or forskolin, but not 8-bromo-cAMP, 3-isobutyl-1-methylxanthine, or N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide potentiated heat-induced HSP-72 production. Inhibition of the heat-induced increase in [Ca2+]i attenuated, but failed to completely block, heat-induced HSP-72 production, mRNA synthesis, and the heat-shock transcriptional factor-heat-shock element binding complex formation, which suggests there are Ca(2+)-dependent and -independent processes involved in HSP-72 synthesis. Our results show that an increase in [Ca2+]i or activation of G proteins, but not pHi and cAMP, enhances HSP-72 induction.
Subject(s)
Calcium/metabolism , Cyclic AMP/physiology , GTP-Binding Proteins/metabolism , Heat-Shock Proteins/biosynthesis , Hydrogen/metabolism , Intracellular Membranes/metabolism , Heat-Shock Proteins/genetics , Hot Temperature , Humans , Hydrogen-Ion Concentration , Osmolar Concentration , Protein Biosynthesis , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
RNA was isolated from fibroblasts from the retroocular area, from endomysial fibroblasts obtained from orbital lateral rectus muscle, and from abdominal skin fibroblasts. The RNA was reverse transcribed into cDNA which was then used as a template for PCR with primers encompassing a portion (nucleotides 989-1235) of the extra-cellular domain of the human TSH receptor (hTSH-R). A definite 247 BP product was detected from fibroblast RNA by ethidium bromide staining, and was confirmed by hybridization with labelled hTSH-R cDNA. The product had homology with the known TSH-R cDNA. These studies indicate that human fibroblasts can express hTSH-R, and they suggest that a cross reactive immunologic response between anti-hTSH-R and these fibroblast TSH receptors may play a role in the genesis of Graves' ophthalmopathy.
Subject(s)
Fibroblasts/cytology , Oculomotor Muscles/cytology , Receptors, Thyrotropin/genetics , Base Sequence , Blotting, Southern , Cells, Cultured , DNA/analysis , DNA/genetics , Ethidium , Fibroblasts/chemistry , Fibroblasts/ultrastructure , Gene Expression , Graves Disease/etiology , Humans , Molecular Sequence Data , Polymerase Chain Reaction , RNA/analysis , RNA/genetics , Receptors, Thyrotropin/analysis , Receptors, Thyrotropin/physiology , Sequence Homology, Nucleic Acid , Transcription, GeneticABSTRACT
Thyroid hormones may stimulate or repress transcriptional activity depending upon the specific gene. Whereas, a palindromic DNA sequence, TREpal, mediates positive regulation by thyroid hormone, the negative response element (nT3RE) remains undefined. Therefore, we have examined the DNA sequences that mediate the inhibitory effects of thyroid hormone on the transcription of the beta-subunit gene of rat thyrotropin (rTSH beta). In rat pituitary tumor cells (GH3), transient expression of plasmid constructs containing the putative nT3RE of rTSH beta mediated negative regulation by L-triiodothyronine (T3). Since this nT3RE contained sequences which resembled a half-site motif of the consensus T3RE and the idealized palindrome (TREpal), we tested a construct containing this half-site motif in the same cells. T3 decreased the activity of this plasmid. Cotransfection studies in T3-receptor (T3R)-deficient cells indicated that either alpha or beta isoforms of T3R were required for the inhibitory effects of the hormone. Both T3R isomers bind to DNA sequences containing the nT3RE from rTSH beta DNA or the half-site motif of TREpal. In summary, our results show that the repressive properties of T3 are mediated by a nT3RE from rTSH beta. Unexpectedly, this motif resembles a half-site component of TREpal which enhances promoter activity in response to T3.
Subject(s)
Gene Expression Regulation , Promoter Regions, Genetic , Receptors, Thyroid Hormone/physiology , Thyrotropin/genetics , Triiodothyronine/physiology , Animals , Base Sequence , DNA Primers/chemistry , DNA-Binding Proteins/physiology , Genes , Molecular Sequence Data , Rats , Repressor Proteins/physiologyABSTRACT
The hypothalamic neuropeptide TRH, through G-protein-coupled transmembrane pituitary receptors, rapidly stimulates intracellular signaling events that, in turn, stimulate gene transcription. Our previous studies in transfected pituitary tumor cells indicated that TRH stimulation of thyrotropin beta-subunit (TSH beta) gene expression involves both calcium mobilization and protein kinase-C activation. To characterize the gene-proximal elements of the intracellular signaling pathways involved, we examined the effects of TRH, ionomycin, and phorbol ester (TPA) on cellular protooncogenes (c-jun and c-fos) known to be responsive to calcium mobilization and protein kinase-C activation. TRH stimulated a 3-fold increase in both c-jun and c-fos mRNA levels within 1 h, followed by a rapid decline in steady state mRNA levels. A secondary response to the single administration was noted, culminating in a 5-fold stimulation at 20 h. The increase in c-jun and c-fos mRNA levels occurred before the increased steady state mRNA levels of both PRL and TSH beta chimera in transfected pituitary GH3 cells. Furthermore, we examined the role of calcium in these effects using the ionophore ionomycin to elevate and TMB-8 to decrease intracellular calcium. We used the phorbol ester TPA to investigate the effects of increased protein kinase-C activity and H7 or pretreatment with TPA to monitor the decreased kinase activity. Our data indicate that calcium mobilization and protein kinase activation represent distinct components of the signaling events initiated by TRH resulting in increased c-jun and c-fos mRNA levels. Only c-fos mRNA is increased by all three factors, suggesting that c-fos may be a key element in mediating the intracellular processes reflecting TRH action.
Subject(s)
Calcium/metabolism , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/genetics , RNA, Messenger/metabolism , Thyrotropin-Releasing Hormone/pharmacology , Biological Transport , Calcium Channel Blockers/pharmacology , Cell Line , Enzyme Activation/drug effects , Intracellular Membranes/metabolism , Pituitary Gland/cytology , Pituitary Gland/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/physiology , RNA, Messenger/antagonists & inhibitorsABSTRACT
P1 is a nuclear protein found exclusively in rat liver and binds to a motif that spans nucleotides -310 to -288 of the thyroid hormone responsive gene, S14. We expect P1 to play an important role in regulating gene expression because the binding motif for this factor is contained within a DNase I-hypersensitive site of S14 chromatin. In this report, we have attempted to define the function of P1 by correlating its DNA binding activity with levels of mRNA-S14 in response to aging and obesity. Results of all studies revealed inverse relationships between the activity of P1 and levels of mRNA-S14, thus suggesting that P1 may function as a repressor of S14 gene expression. Accordingly, we tested the repressor hypothesis using cell-free transcription and transient transfection assays to measure the activity of reporter constructs with and without the P1 binding motif. In the presence of the P1 motif, S14 promoter activity was repressed and the negative effect on gene transcription was further enhanced by thyroid hormone. These observations are consistent with P1 being a repressor of S14 gene transcription.
Subject(s)
Gene Expression , Liver/metabolism , Nuclear Proteins/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Animals , Blotting, Northern , Cells, Cultured , Chloramphenicol O-Acetyltransferase/metabolism , DNA/metabolism , Male , Nuclear Proteins/isolation & purification , Obesity/metabolism , Proteins/genetics , RNA/genetics , RNA/isolation & purification , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Receptors, Thyroid Hormone/metabolism , Recombinant Proteins/metabolism , Repressor Proteins/isolation & purification , Templates, Genetic , Thinness/metabolism , TransfectionABSTRACT
The precursor peptide of TRH (prepro-TRH) contains five copes of pro-TRH linked by other peptide sequences. These peptides are coprocessed with TRH in the median eminence of the hypothalamus and released into the portal circulation, rendering this family of peptides available to act at the level of the anterior pituitary. Therefore, we tested the potential bioactivity of one cryptic peptide, prepro-TRH amino acids 160-169 [prepro-TRH-(160-169)], in a TRH-responsive pituitary cell line (GH3). In a heterologous TSH expression assay, we found that prepro-TRH-(160-169) stimulated TSH beta gene promoter activity in a time- and dose-dependent manner; moreover, the effect of prepro-TRH-(160-169) was more rapid and of greater magnitude than that of TRH on TSH beta-directed chloramphenicol acetyltransferase synthesis. In the same cells, we found that prepro-TRH-(160-169) stimulated PRL synthesis and secretion, but the effect was similar in magnitude and duration to that of TRH. The effect of prepro-TRH-(160-169) appears to be additive to that of TRH, suggesting that prepro-TRH-(160-169) may act through a mechanism separate from that of TRH. Thus, prepro-TRH-(160-169) has potent endocrinological effects at the level of the genome.
Subject(s)
Gene Expression/drug effects , Peptide Fragments/pharmacology , Protein Precursors/pharmacology , Thyrotropin-Releasing Hormone/pharmacology , Thyrotropin/genetics , Amino Acid Sequence , Animals , Cell Line , Conserved Sequence , Hypothalamus/chemistry , Molecular Sequence Data , Peptide Fragments/analysis , Peptide Fragments/chemistry , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Prolactin/biosynthesis , Prolactin/metabolism , Promoter Regions, Genetic/drug effects , Protein Precursors/analysis , Protein Precursors/chemistry , Rats , Thyrotropin-Releasing Hormone/analysis , Thyrotropin-Releasing Hormone/chemistryABSTRACT
Clarifying the role of the TSH receptor protein in the autoimmune process may be the key to understanding the development of Graves' disease. In the present study we used a 16 amino acid peptide of the human TSH receptor (hTSHR) to immunize rabbits. A comparable, but theoretically less immunogenic, peptide was injected into other rabbits. The antibody response against these and other peptides, as well as against solubilized human thyroid membrane (TM) and guinea pig fat cell membrane (GPF) proteins, was tested using ELISA and Western blots. The GPF and TM binding pattern of rabbits' sera was compared to that of Graves' patients' sera. We have identified an area of antigenic cross-reactivity between GPF and TM; a 63 kD protein was present in both GPF and TM, and this protein uniformly bound IgG-s of the rabbits' postimmunization sera and one of eight Graves' patient's serum. We have shown that i) a theoretically immunogenic 16 amino acid peptide was indeed highly immunogenic in rabbits, ii) antibodies binding to GPF and TM were detected after immunization, and iii) the peak of thyroid stimulating immunoglobulin activity of sera was followed by a transient elevation of serum triiodothyronine levels. Further studies investigating the immunogenic epitopes of the hTSHR as well as characterizing the 63 kD protein are indicated.
Subject(s)
Antibodies/blood , Autoantibodies/blood , Cross Reactions/immunology , Graves Disease/immunology , Oligopeptides/immunology , Receptors, Thyrotropin/immunology , Amino Acid Sequence , Animals , Antibodies/genetics , Disease Models, Animal , Graves Disease/blood , Graves Disease/genetics , Guinea Pigs , Humans , Immunoglobulins, Thyroid-Stimulating , Molecular Sequence Data , Oligopeptides/administration & dosage , Rabbits , Receptors, Thyrotropin/genetics , Sequence Homology, Amino Acid , VaccinationABSTRACT
The clinical applicability of a newly described polymerase chain reaction directed protein expression system was assessed for the in vitro synthesis and partial epitope mapping of large radiolabeled human thyrotropin receptor (hTSH-R) protein segments. PCR amplification of targeted regions within the hTSH-R cDNA followed by in vitro transcription and translation permitted rapid synthesis of protein segments ranging in size from 18 to 62 kDa. Initial epitope mapping was directed at a 52 amino acid segment unique to the hTSH-R compared to otherwise homologous glycoprotein hormone receptors. Sera from Graves' disease patients known to have autoantibodies against the hTSH-R were used to immunoprecipitate two protein fragments differing only by the presence of the unique region in the larger fragment (E5) but not in the smaller fragment (E4). Dense precipitation bands were obtained using Graves' sera to immunoprecipitate E5 whereas little or no specific immunoprecipitation of E4 occurred. Normal sera gave only weak immunoprecipitation bands of E5. The technique provides significant advantages over conventional cloning methods and should have general applicability in the study of other protein targets of autoimmune disease.
Subject(s)
Autoantigens/chemistry , Epitopes/chemistry , Receptors, Thyrotropin/immunology , Amino Acid Sequence , Autoantigens/biosynthesis , Base Sequence , Humans , In Vitro Techniques , Molecular Sequence Data , Polymerase Chain Reaction , Receptors, Thyrotropin/biosynthesis , Recombinant ProteinsABSTRACT
Thyroid hormones suppress the synthesis and release of thyrotropin from thyrotropes in the anterior pituitary gland, a feature that is critical in the classic negative-feedback loop of the pituitary-thyroid endocrine axis. The major effect of thyroid hormones in this system is exerted at the transcriptional level. The molecular mechanisms by which there is negative regulation of TSH subunit gene expression by thyroid hormone have been elucidated. The TSH subunit genes have isolated and characterized. Structure-function analyses using fusion genes and DNA transfection approaches have defined the putative negative TREs among the promoters of the rat, mouse, and human alpha and TSH beta genes. These sequences are either largely overlapping direct TRE half-sites, TRE half-sites as direct repeats gapped by two nucleotides, or single TRE half-sites. These arrangements are distinct from those seen in positive TREs. Recent knowledge regarding the molecular mechanisms of thyroid action in general forces consideration of multiple TR isoforms, TR heterodimer partners (TRAPs), and thyroid hormones in the ultimate mechanisms of negative action. Several models have been proposed, but none has yet been proved. In addition, the role of thyroid hormone in the regulation of gene expression at the posttranscriptional level is beginning to be addressed. Future work should continue to illuminate these important facets of gene regulation.
Subject(s)
Gene Expression Regulation/drug effects , Thyroid Hormones/pharmacology , Thyrotropin/genetics , Animals , Base Sequence , DNA/chemistry , Humans , Molecular Sequence Data , Receptors, Thyroid Hormone/chemistry , Receptors, Thyroid Hormone/physiology , Thyrotropin/biosynthesis , Thyrotropin/chemistry , Thyrotropin/metabolismABSTRACT
Production of neuroendocrine peptides by human lymphocytes is thought to facilitate control of the immune response. The presence of neuroendocrine peptide gene expression, specifically the TSH beta subunit gene, was studied in human lymphocytes using Northern blot analysis and polymerase chain reaction (PCR) techniques. Northern blot analysis of human lymphocyte RNA probed with a TSH beta cDNA probe failed to demonstrate TSH beta subunit steady state message levels. PCR amplification of lymphocyte-derived cDNA using TSH beta subunit complementary primers resulted in amplification of a .38 Kb DNA fragment, confirming expression and initial exonic splicing of TSH beta subunit gene exons 2 and 3 in human lymphocytes. Sequence analysis of this .38 Kb DNA fragment demonstrated conservation of exon borders after splicing (exons 2 and 3) and predicted an amino acid translation product similar, if not identical, to human TSH beta peptide sequence. Hybridization with a TSH beta subunit cDNA probe of PCR-amplified reverse-transcribed lymphocyte RNA suggested that: (1) the abundance of TSH beta subunit gene transcripts in human lymphocytes is less than the relative abundance in T3-treated pituitary; and (2) this messenger RNA may be modulated by the presence of certain thyromimetic compounds (T2, T3, TRIAC).
Subject(s)
Lymphocytes/physiology , Thyrotropin/genetics , Amino Acid Sequence , Base Sequence , Blotting, Northern , Gene Expression , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Polymerase Chain Reaction , RNA, Messenger/geneticsABSTRACT
TRH is synthesized as a precursor peptide containing five copies of the sequence Gln-His-Pro-Gly, QHPG, flanked by paired basic amino acids, and linked by other peptides. We tested one cryptic peptide, PPT (160-169, SFPWMESDVT), as a possible physiological regulator of pituitary activity in vivo. Male rats were cannulated (jugular) and received a single dose of either PPT or TRH (10(-8)-10(-6) M). PPT caused no consistent effects on either TSH or PRL secretion, while TRH stimulated the secretion of both hormones. However, PPT stimulated a dose-dependent increase in both pituitary TSH beta and PRL mRNA content at 240 min similar to TRH. In primary cultures of rat pituitaries, PPT stimulated a maximum 4-fold increase in TSH beta mRNA and a 2-fold increase in PRL mRNA in 4 h, while TRH increased both TSH beta and PRL mRNA approximately 3-fold. Again, PPT had no significant effect on TSH or PRL secretion into the medium. Thus, PPT appears to be a physiological regulator of both TSH and PRL synthesis, but, unlike TRH, does not act as a secretagogue.
Subject(s)
Peptide Fragments/pharmacology , Protein Precursors/pharmacology , Thyrotropin-Releasing Hormone/pharmacology , Amino Acid Sequence , Animals , Cells, Cultured , Male , Molecular Sequence Data , Peptide Fragments/chemistry , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Prolactin/genetics , Prolactin/metabolism , Protein Precursors/chemistry , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Thyrotropin/genetics , Thyrotropin/metabolism , Thyrotropin-Releasing Hormone/chemistryABSTRACT
We have previously identified a rat hepatonuclear factor, PS-1 that binds to the thyroid hormone responsive gene, S14. To determine whether PS-1 is involved in regulating tissue-specific expression of the S14 gene, we have correlated the DNA binding activity of PS-1 with mRNA-S14 expression in a variety of tissues. Gel retardation analysis revealed a pattern of binding to the recognition site that was characteristic of tissues with high levels of mRNA-S14, a different pattern was found in tissues which do not express the gene. Competition studies using mutant oligonucleotides showed that the first 4 nucleotides and the CAAT motif contained within the PS-1 recognition sequence are essential for protein binding. C/EBP, a CCAAT-transcription factor binds to the PS-1 recognition site thus raising the possibility that both C/EBP and PS-1 may belong to the same family of proteins. Next we used a cell-free transcription assay to measure activity of a template, pS14-GFC(-72), that contained the PS-1 sequence. The pS14-GFC(-72) template was active in hepatonuclear extracts but deletion of or competition with the PS-1 binding sequence rendered the construct inactive. A template containing three PS-1 binding sequences increased S14 promoter activity by 12- to 13-fold. In nuclear extracts from spleen and testis, relative S14 promoter activity was only 2% of that in the liver, this observation mimicked closely in vivo expression of the gene. Mixing together extracts from liver and spleen in varying proportions, prior to incubation with S14 template, yielded a linear increase in S14 promoter activity that correlated with the amount of liver extract in the reaction. This finding is consistent with the absence of an essential factor or factors in spleen that is/are required for S14 promoter activity in vitro. In summary, PS-1 binds to a DNA sequence that contains a CAAT motif and appears to play a critical role in determining tissue-specific activity of the S14 promoter in vitro.
Subject(s)
DNA-Binding Proteins/metabolism , Liver/metabolism , Malate Dehydrogenase/genetics , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , Receptors, Thyroid Hormone/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Binding, Competitive , Cell-Free System , DNA Probes , Humans , Male , Molecular Sequence Data , Nuclear Proteins/metabolism , Oligonucleotide Probes , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Sequence Deletion , Templates, Genetic , Transcription, GeneticABSTRACT
We have previously identified a 57-bp DNA fragment encompassing exon 1 of the beta-subunit gene of rat thyrotropin (rTSH beta) that mediates the negative response to L-triiodothyronine (T3). To determine the specific motif that confers this negative regulation, we tested the T3 sensitivity of various segments of this 57-bp gene fragment in transiently transfected pituitary tumor cells, GH3. The suppressive effects were mediated by a 17-bp motif (+11/+27, CGCCAGTGCAAAGTAAG) located at the 3' end of exon 1. The inhibitory effects mediated by the sequence were evident when a single copy of the motif was inserted 125 bp upstream or 11 bp downstream of the transcriptional start site. These findings indicate that the suppressive effect of T3 is an intrinsic property of the T3-responsive element and not dependent on position relative to the promoter. The T3 receptor (T3R) extracted from GH3 cells or expressed in vitro bound specifically to this sequence. Specific mutations introduced into this region result in a selective loss of nuclear protein binding and a corresponding loss of T3 sensitivity. Additional studies showed that the 17-bp sequence was not responsive to T3 in COS cells which lack endogenous T3R. Cotransfection of a T3R restored the T3 responsiveness of the TSH beta motif. In summary, we have identified an element in the rTSH beta gene that mediates negative regulation by T3 and binds to the T3R.
Subject(s)
Gene Expression Regulation/drug effects , Thyroid Hormones/pharmacology , Thyrotropin/genetics , Triiodothyronine/genetics , Base Sequence , DNA/metabolism , Molecular Sequence Data , Nuclear Proteins/metabolism , Pituitary Neoplasms/metabolism , Promoter Regions, Genetic , RNA, Messenger/metabolism , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
The induction of rat hepatic mRNA S11 by L-T3 (T3) is a useful model for studying the mechanisms of thyroid hormone action. Although numerous reports have examined the response of mRNA S11 to various physiological and hormonal manipulations, the role of S11 protein in cellular metabolism remains unknown. In this study we show that mRNA S11 is abundantly expressed and regulated by T3 only in liver and small intestine. High levels of the mRNA are present at birth, but drop sharply between 30-60 days of age. These and other features of the S11 gene product were similar to those of rat apolipoprotein-A1 (Apo-A1). The sequence of S11 cDNA was identical to a portion of the Apo-A1 mRNA, thus confirming identity of the S11 mRNA. To examine whether DNA sequences immediately adjacent to the transcription start site mediate the effects of thyroid hormone, we measured the activity of an Apo-A1 gene fragment, U-1 (-474 to -7) using a transient transfection assay. The activity of the full-length U-1 DNA in HuH-7 hepatoma cells was 2- to 2.5-fold higher in the presence of thyroid hormone. This finding closely matched previous results using the in vitro nuclear run-on assay. Internal deletion of a motif that resembles a thyroid hormone response element from U-1 DNA not only abolished the induction by T3, but suppressed promoter activity by 3- to 4-fold in response to the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Apolipoprotein A-I/genetics , Gene Expression Regulation/drug effects , Liver/drug effects , Promoter Regions, Genetic/drug effects , RNA, Messenger/genetics , Triiodothyronine/pharmacology , Aging/metabolism , Animals , Apolipoprotein A-I/biosynthesis , Base Sequence , DNA, Recombinant/genetics , Hyperthyroidism/metabolism , Hypothyroidism/metabolism , Liver/growth & development , Liver/metabolism , Male , Molecular Sequence Data , Organ Specificity , RNA, Messenger/analysis , Rats , Rats, Inbred F344 , Rats, Inbred Strains , Stimulation, ChemicalABSTRACT
Cholinergic mechanisms have been implicated in the regulation of anterior pituitary hormone secretion. The present study was designed to determine the effect of a single injection of an organophosphate acetylcholinesterase inhibitor, diisopropylfluorophosphate (DFP), on anterior pituitary function in male rats. DFP increased serum ACTH (2.7-fold) and corticosterone (9.1-fold), while suppressing TSH, PRL, LH, and GH by up to 95%. The earliest response was at 1 hr, with a duration of at least 18 hr for TSH and LH. Responses were similar in adrenalectomized animals. After DFP, responses to hypothalamic releasing factors were normal for TSH, GH, and ACTH, but significantly blunted for PRL and LH. TSH suppression was partially prevented by combined therapy with a nicotinic (mecamylamine) and a muscarinic (atropine) antagonist. TSH suppression was partially reversed by immunoneutralization with somatostatin antibody, and PRL suppression was completely prevented by a dopamine antagonist (haloperidol). Atropine alone prevented the effects on corticosterone. TSH pituitary content and TSH-beta mRNA were reduced by 37 and 22%, respectively, by DFP. In contrast, PRL mRNA was unchanged but PRL content was increased 3-fold. We conclude that cholinesterase inhibition evokes a multiplicity of effects on anterior pituitary function. There is a hierarchy of responses, with corticosterone being the most and TSH the least sensitive. There is evidence for inhibition at both the hypothalamic and pituitary levels, involving both nicotinic and muscarinic receptors. Although cholinesterase inhibition is the proximate event, other neurotransmitter pathways involved in TSH and PRL suppression are somatostatin and dopamine, respectively.
Subject(s)
Adrenocorticotropic Hormone/blood , Corticosterone/blood , Growth Hormone/blood , Isoflurophate/pharmacology , Luteinizing Hormone/blood , Prolactin/blood , Thyrotropin/blood , Adrenalectomy , Animals , Carbachol/pharmacology , Cholinesterase Inhibitors/pharmacology , Dopamine/metabolism , Dose-Response Relationship, Drug , Hypothalamus/drug effects , Hypothalamus/physiology , Male , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/physiology , Pituitary Hormones/metabolism , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains , Sheep , Somatostatin/pharmacology , Synaptic Transmission/drug effects , Time FactorsABSTRACT
Our previous studies demonstrated TRH stimulation of TSH beta gene expression in rat pituitary cell cultures and GH3 tumor cells in a transient expression assay. To begin to characterize the gene-proximal elements of the pathways involved in TRH stimulation of TSH beta gene transcription, we examined the effects of factors that increase intracellular calcium concentration, [Ca2+]i, or activate protein kinase C on TSH beta promoter activity in transfected GH3 cells. TPA, a tumor-promoting phorbol ester, stimulated a dose-dependent increase in TSH beta promoter activity at 8 h similar to TRH (2-3-fold). TPA did stimulate protein kinase C activation without [Ca2+] mobilization. The calcium ionophore ionomycin increased cytoplasmic free [Ca2+] by stimulating both calcium influx and release from internal stores without affecting protein kinase C. Ionomycin also stimulated a dose-dependent increase (2-fold) in TSH beta promoter activity at 8 h. However, the voltage-dependent Ca2+ channel agonist Bay K 8644, which increased influx of extracellular calcium, had little or no effect on TSH beta gene expression until 48 h (5-fold). Similar effects on prolactin/mRNA levels were observed in these cells. Effects of these factors were not additive, suggesting a common pathway(s) to stimulate gene expression. Inhibition of intracellular calcium mobilization by treatment with 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) inhibited ionomycin effects on gene expression without affecting phorbol ester activity, and, conversely, inhibition of protein kinase C activity by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) or TPA desensitization blocked TPA effects without affecting ionomycin activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Calcium/metabolism , Gene Expression Regulation , Protein Kinase C/metabolism , Thyrotropin-Releasing Hormone/pharmacology , Thyrotropin/genetics , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Animals , Cells, Cultured , Cloning, Molecular , Enzyme Activation/drug effects , Gallic Acid/analogs & derivatives , Gallic Acid/pharmacology , Gene Expression Regulation/drug effects , Ionomycin/pharmacology , RNA, Messenger/metabolism , Rats , Substrate Specificity , Tetradecanoylphorbol Acetate/pharmacology , Thyrotropin/biosynthesisABSTRACT
An earlier report of HIV-1 gene sequences in thyroid cell genomic DNA from patients with Graves' disease prompted use of the polymerase chain reaction technique to identify such sequences in Graves' disease thyroid tissue and in white blood-cells from these patients. We were unable to confirm the existence of HIV-1-related DNA sequences in Graves' specimens.