Thyroid Peroxidase mRNA Research Articles

An Abnormal Splice Donor Site in One Allele of the Thyroid Peroxidase Gene in FRTL5 Rat Thyroid Cells Introduces a Premature Stop Codon: Association with the Absence of Functional Enzymatic Activity

Low stringency screening of an FRTL5 cDNA library with a human thyroid peroxidase (TPO) cDNA probe yielded two different types of TPO cDNA clones. One type contained the full-length structural gene, but there was an A at the first nucleotide of an intronic splice donor site that leads to alternate splicing, with the retention of part of an intron. This 54-basepair retained intron fragment contains a premature inframe stop codon that would truncate the protein at its carboxyl-terminus by 71% with the loss of enzymatic activity. The second type of TPO cDNA does not contain the retained intron fragment and premature stop codon, but it is not full-length and lacks 580-680 basepairs at its 5' end. However, Northern blot analysis reveals only full-length copies (3.2 kilobases) of TPO mRNA in FRTL5 cells, and this 5' truncation is, therefore, an artifact of library construction. The relative proportions of the two types of TPO mRNA in FRTL5 cells was determined by the polymerase chain reaction, using as template single stranded cDNA generated by reverse transcription of FRTL5 mRNA. Slightly more than half of the TPO mRNA in the FRTL5 cells represented the form with the abnormal splice donor site. The relative proportions of the two TPO mRNA forms was not influenced by TSH stimulation of the FRTL5 cells, and the proportion remained unaltered even after the FRTL5 cells were subcloned by limiting dilution. At the genomic level, we used allele-specific oligonucleotides for the mutant and normal forms of TPO, and found FRTL5 cells to have both normal and abnormal TPO alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal Regulation of Thyroid Peroxidase In Normal And Transformed Rat Thyroid Cells

The hormonal induction of thyroid peroxidase (TPO) mRNA is studied in the functional rat thyroid cell line FRTL-5 and compared to the induction of thyroglobulin (TG) mRNA and I- uptake. TPO and TG mRNAs are regulated by TSH and by insulin-like growth factor I (IGF-I) and/or insulin. However, while TPO is more sensitive to TSH regulation (5- to 6-fold increase vs. 2- to 3-fold increase by IGF-I), TSH and IGF-I are equally potent in increasing TG mRNA levels (3- to 4-fold). Regulation of I- uptake appears to be different: thus TSH greatly (15-fold) increases I- uptake, while IGF-I or insulin are completely ineffective. TPO and TG mRNAs and I- transport display different sensitivity to transformation of rat thyroid cells. Thus, when another differentiated rat thyroid cell line, the PC cells, are transformed by human c-myc (PC myc), TPO and TG mRNAs are both present at normal levels, while I- uptake is slightly decreased; in the PC cells transformed by polyomavirus middle-T-antigen (PC PyMLV) TPO mRNA is undetectable and I- uptake is greatly decreased, while TG mRNA is present at normal levels. All three differentiated functions are switched off in PC cells transformed by the cooperation of c-myc and polyomavirus middle-T-antigen (PC myc + PyMLV).

Inhibition of human thyroid peroxidase gene expression by interleukin 1.

We have already demonstrated the inhibitory effect of interleukin 1 on thyroglobulin gene expression. Recent availability of thyroid peroxidase cDNA has allowed us to investigate the regulation of thyroid peroxidase gene. Therefore, the regulation of thyroid peroxidase mRNA by interleukin 1 in cultured human thyrocytes was investigated. Thyrocytes dispersed from thyroid tissues from patients with Graves' disease were incubated with TSH with or without recombinant human interleukin 1. Unstimulated human thyrocytes did not contain any detectable thyroid peroxidase mRNA, however, TSH-stimulated thyrocytes expressed four thyroid peroxidase mRNA transcripts (4.0, 3.2, 2.1 and 1.7 kb, respectively). Both interleukin 1 alpha and beta inhibited TSH-induced thyroid peroxidase mRNA in a dose responsive manner; 10(3) U/1 interleukin 1 caused maximal suppression of TSH-induced thyroid peroxidase mRNA level to nearly basal levels. Interleukin 1 also inhibited cAMP analogue 8-bromo-cyclic AMP induced thyroid peroxidase mRNA level. In contrast the gamma-actin mRNA hybridization signal was not altered in control or treated cells. These results demonstrate that interleukin 1 directly inhibits TSH-induced thyroid peroxidase gene expression and provide further evidence for a paracrine role of interleukin 1 as a local inhibitor of thyroid hormone synthesis.

INTERFERON-yINHIBITS THYROTROPIN-INDUCED THYROIDAL PEROXIDASE GENE EXPRESSION IN CULTURED HUMAN THYROCYTES

We have previously demonstrated that interferon-gamma (IFN-gamma) induced HLA-DR antigen and also inhibited thyrotropin (TSH)-induced triiodothyronine (T3) and thyroglobulin (Tg) secretion from cultured human thyrocytes. In order to further clarify the inhibitory effect of IFN-gamma on TSH-stimulated thyroid hormone secretion, we have examined human thyroid peroxidase (TPO) gene expression. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without IFN-gamma. Total RNA was extracted, separated and hybridized with 32P-labelled human TPO cDNA. Thyrocytes expressed four TPO mRNA transcripts (4.0, 3.2, 2.1 and 1.7kb, respectively), all of which were stimulated by TSH. IFN-gamma inhibited TSH-stimulated TPO mRNA in a dose dependent manner (0.01-10U/mL). 1 U/mL IFN-gamma caused maximal suppression of TSH-stimulated TPO mRNA levels to basal levels. IFN-gamma also inhibited 8-bromo-cyclic AMP-stimulated TPO mRNA levels. In contrast, the gamma-actin mRNA hybridization signal was not altered in control or treated cells. These results demonstrate that IFN-gamma directly inhibits TSH-stimulated TPO gene expression and provide further support for a role of IFN-gamma as a local modulator of thyroid hormone synthesis.

Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen.

Activation of the thyroid peroxidase gene in human thyroid cells: effect of thyrotrophin, forskolin and phorbol ester.

The enzyme thyroid peroxidase (TPO) plays a central role in thyroid hormone synthesis and is the target for the autoimmune attack in lymphocytic thyroiditis. We have examined the activation of the TPO gene in cultured human thyrocytes using slot-blot hybridization with a synthetic 40 mer oligonucleotide probe derived from the nucleotide sequence of the human TPO gene. The oligonucleotide probe was shown by Northern blotting to hybridize specifically to an approximately 3 kb RNA species from thyroid tissue of patients with Graves' disease, but not to RNA preparations from human or bovine retinal tissue, providing compelling evidence for the specificity of the probe for TPO mRNA. Addition of TSH (10 mU/ml) to primary thyroid cultures for 4 h led to increased TPO mRNA levels which were maximal after 48 h and significantly higher than basal even after 7 days of co-culture. Activation of TPO mRNA by TSH showed dose dependency over a wide range (0.01-100 mU/ml), with a maximal effect at 10 mU TSH/ml in cells cultured for a period of 72 h. Comparison of TPO mRNA levels with the accumulation of thyroglobulin mRNA levels following stimulation by TSH indicated that the induction of the gene encoding thyroglobulin precedes transcription of the TPO gene. The adenylate cyclase activator forskolin (1-100 microM) mimicked TSH in increasing TPO mRNA levels whilst, in contrast, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA; 0.01-1 microM) led to levels of TPO mRNA that were lower than basal. Thus TSH induces a specific dose-dependent activation of TPO mRNA which is mimicked by agents which increase cyclic AMP. In contrast, TPA-induced activation of protein kinase C inhibits this response.

Thyrotropin Regulation of Thyroid Peroxidase Messenger Ribonucleic Acid Levels in Cultured Rat Thyroid Cells: Evidence for the Involvement of a Nontranscriptional Mechanism*

The influence of TSH on thyroid peroxidase (TPO) gene expression was investigated in FRTL5 rat thyroid cells. Cultured in the presence of TSH, these cells express a TPO mRNA species of 3.1 kilobases. TSH withdrawal from the culture medium led to a decline in TPO mRNA levels over 24 h. In contrast, no decline in beta-actin mRNA levels occurred after 24 h of incubation in TSH-free medium. TSH (1 mU/ml) added to FRTL5 cells cultured in the absence of TSH increased TPO mRNA levels 7- to 9-fold compared to levels in control cells. This effect of TSH on TPO mRNA accumulation in FRTL5 cells was time related (it was already present after 4 h and was maximal after 24 h of cell exposure to TSH), dose related (0.01 and 1 mU/ml were, respectively, the lowest and the maximally effective doses), and independent of new protein synthesis, in that it was not prevented by cycloheximide (100 microM). cAMP analogues [8-bromo-cAMP and (Bu)2cAMP] also increased TPO mRNA levels, although to a lesser degree than TSH. Run-on transcription analysis in nuclei prepared from FRTL5 cells previously cultured in the presence or absence of TSH did not reveal any difference in TPO mRNA transcripts. These results suggest that TSH regulates the level of TPO mRNA in FRTL5 cells, in part via the second messenger cAMP and by a nontranscriptional mechanism. This TSH effect may represent a primary site of TSH action in regulating TPO bioactivity.

Regulation of thyroid peroxidase and thyroglobulin gene expression by thyrotropin in cultured human thyroid cells.

cDNAs for thyroid peroxidase (TPO) and thyroglobulin (Tg) have been cloned and sequenced. Using such cDNAs, we investigated the regulation of TPO and Tg gene expression by various agents in cultured human thyroid cells. Unstimulated human thyroid cells contained a major RNA species [3.2 kilobases (kb) in length] and several minor RNA species (less than 3.2 kb mRNA) of TPO, but no detectable Tg transcripts. However, TSH-stimulated thyroid cells contained four distinct TPO mRNA species (4.0, 3.2, 2.1, and 1.7 kb) and a single Tg mRNA species (8.5 kb). TSH stimulated the TPO and Tg mRNA levels in a dose- and time-dependent manner. The same results were obtained with 8-bromo-cAMP, a cAMP analog, but not with insulin or insulin-like growth factor I. Furthermore, inductions of these mRNAs by TSH and 8-bromo-cAMP were almost completely blocked by cycloheximide, a protein synthesis inhibitor. These results suggest that human thyroid cells contain four distinct TPO mRNAs and a single species of Tg mRNA, and the levels of all mRNAs are increased by TSH/cAMP stimulation. These increases are blocked by inhibiting protein synthesis, indicating that TSH stimulation of TPO and Tg mRNA levels may be mediated by newly synthesized protein(s).

Regulation of thyroperoxidase, thyroglobulin and iodide levels in sheep thyroid cells by TSH, tumor promoters and epidermal growth factor

Using sheep thyroid cells in culture, we have studied the effects of thyroid stimulating hormone (TSH), epidermal growth factor (EGF) and the tumor promoter, 12- O-tetradecanoylphorbol-13-acetate (TPA) on the activity and expression of both thyroglobulin (Tg) and thyroid peroxidase (TPO) and on the ability of cells to trap and organify iodide. Using Western blotting techniques, we found that TSH increased the absolute cellular levels of Tg. The optimum TSH concentration for Tg mRNA production was between 0.1–1.0 mU/ml. Thyroglobulin mRNA levels were stimulated by TSH but detectable levels were also present in cultures grown in its absence containing cortisol, insulin, transferrin, somatostatin and glycyl-lysyl-histidyl acetate. Unlike Tg, TPO protein levels were found to be completely dependent upon TSH. A time course of TSH stimulation of TPO mRNA showed increases after 8 h of TSH stimulation, whereas induction of Tg mRNA by TSH was seen at 24 h. Iodide trapping and organification were also TSH-dependent processes, showing maximum activities at 300–500 μU/ml of TSH. The addition of 10 nM TPA caused a biphasic decrease in radiolabeled pertechnetate uptake, with complete inhibition being seen at 14 h. Inhibition of iodide organification occurred more rapidly. TPA and EGF (1 nM) reduced the amount of newly synthesized Tg in TSH-stimulated cells by 50% but the absolute amount of Tg within the cells was not markedly inhibited at these early times. The amount of TPO protein, however, was markedly decreased by TPA and EGF. The results show that 2 agents which inhibit differentiated function do so, at least in part, by reduction of the protein levels of both TPO and Tg. Regulation of the amount of TPO protein appears to be more sensitively regulated than that of Tg by TSH, TPA and EGF.

Thyrotropin Stimulation of Cultured Thyroid Cells Increases Steady State Levels of the Messenger Ribonucleic Acid for Thyroid Peroxidase

We have examined the effect of TSH on thyroid peroxidase (TPO) mRNA levels in dog thyroid cell primary cultures. Freshly dispersed dog thyroid cells were cultured for up to 5 days in the absence or presence of 5 mU/ml bovine TSH. At the outset of culture, and at daily intervals thereafter, total cytoplasmic RNA was extracted and applied to Nytran paper using a slot-blot apparatus. A nick-translated cDNA fragment of the porcine TPO gene was used to probe these filters. Autoradiographs were quantified by densitometry. Nonspecific binding was negligible as determined using a pUC18 probe. During the first 2 days of culture, TPO mRNA levels declined irrespective of whether or not TSH was present in the medium. TSH did not affect this decline. Between 3 and 5 days of culture, TPO mRNA levels in control (no TSH) cells increased to 3 times the initial level (expressed relative to cellular DNA). However, during the same period TSH stimulated TPO mRNA levels 8-fold above the initial level. To confirm that the signal with the cDNA probe was actually that of dog TPO mRNA, cellular RNA (day 4 of culture) was subjected to Northern blot analysis using the same cDNA probe. Specific bands of 2.9 kilobases were detected corresponding to the known size of TPO mRNA in pig thyroid tissue. The signal of this 2.9 kilobase species was enhanced by TSH. In conclusion, the data indicate that chronic TSH stimulation raises steady state levels of TPO mRNA and provide an explanation, at least in part, for the mechanism by which TSH enhances TPO bioactivity in thyroid tissue.