Cultured Rat Thyroid Cells Research Articles

Poly-L-lactic acid and poly-ε-caprolactone as biomaterials for ex-situ bioengineering of the rat thyroid tissue

We are currently developing innovative methodologies for rapid prototyping of threedimensional (3D) replicas of the vascular matrix of soft and hard tissue organs, primarily the thyroid and bones, with the intent of obtaining organomorphic scaffolds for their ex situ reconstruction (1, 2). To identify biocompatible materials suitable for this application, we have studied the effect of poly-L-lactic acid (PLLA) and poly-e-caprolactone (PCL) on survival, adhesion and hormonal secretory activity of primary rat thyroid cells. Sprague- Dawley male rats (225-250 g) were used as thyroid donors. After penthobarbital anesthesia rats were thyroidectomised, thyroids surgically excised, and primary cells prepared using enzymatic breaking. After 72 hs in standard monolayer culture, cells were trypsinized and seeded (20 x103 / cm2) onto microporous scaffold sheets. Biomaterial scaffolds were prepared using either 3.5-5% PLLA (Lacea H100E, Mistui Chemicals) in anydrous dichloromethane / amylene (Sigma) or 3% PCL (Aldrich) in anydrous tetrahydrofurane / BHT (Aldrich). Solutions were dropped onto a wet glass support, and let polymerize as a thin layer at 25°C in the absence of forced air flow. Pattern of polymerization was assessed with thermogravimetry. Cells were let grow on biomaterials up to 8 days. Single biomaterial sheets without cells, and standard multiwell cultures were used as controls. Every 2 days, percentage of viable cells was assessed using a count chamber and Trypan blue exclusion, whereas morphology of growing cells was analyzed using a scanning electron microscopy (SEM) technique without critical point drying. Culture supernatants were collected every 48 hs, and free forms of thyroid hormone (FT3 and FT4) assessed with chemiluminescent immunoassays. PLLA and PCL scaffold sheets exhibited a quite homogeneous thickness (10-12 μm), a repetitive and regular pore geometry, and a crystalline pattern of polymerization. Both biomaterials promoted survival, adhesion and proliferation of primary thyroid elements. Secretion of FT3 and FT4 was variably maintained during the culture period, and resulted statistically higher with respect to monolayer cultures on standard multiwells. Our results indicate that PLLA and PCL are suitable for growth and differentiation of adult rat thyroid cells in culture, and suggests that they might be exploited as bioerodible materials for rapid prototyping of vascular-like, organomorphic scaffolds.

Transcriptional regulation of major histocompatibility complex class I gene by insulin and IGF-I in FRTL-5 thyroid cells

Increased major histocompatibility complex (MHC) class I gene expression in nonimmune cell 'target tissues' involved in organ-specific diseases may be important in the pathogenesis of autoimmune diseases. This possibility in part evolves from studies of cultured thyrocytes where properties appear relevant to the development of thyroid autoimmune disease. In FRTL-5 rat thyroid cells in continuous culture, hormones and growth factors that regulate cell growth and function specifically decrease MHC class I gene expression. We hypothesized that this could reflect a mechanism to preserve self-tolerance and prevent autoimmune disease. The mechanisms of action of some of these hormones, namely TSH and hydrocortisone, have been already characterized. In this report, we show that IGF-I transcriptionally downregulates MHC class I gene expression and that its action is similar to that of insulin. The two hormones have a complex effect on the promoter of the MHC class I gene, PD1. In fact, they decrease the full promoter activity, but upregulate the activity of deleted mutants that have lost an upstream, tissue-specific regulatory region but still retain the enhancer A region. We show that insulin/IGF-I promotes the interactions of the p50/p65 subunits of NF-kappaB and AP-1 family members with these two regions, and that the tissue-specific region acts as a dominant silencer element on insulin/IGF-I regulation of promoter activity. These observations may be important to understand how MHC class I gene transcription is regulated in the cells.

Effects of kojic acid on thyroidal functions in rats by single-dose administration and in cultured rat thyroid cells (FRTL-5 cells).

The effects of kojic acid (KA) on thyroidal function were studied by single-dose administration in rats and in cultured rat thyroid cells (FRTL-5 cells). In rats receiving a single dose of 1,000 mg/kg KA orally, the 125I uptake from blood into the thyroid gland was significantly lower than that of the control group from 30 min to 24 hr after administration. The 125I organification activity of the KA groups was significantly lower than control from 30 min to 6 hr after administration. However, the 125I organification activity at 24 hr or 48 hr after administration recovered enough to be nearly comparable with the control group. In the study in FRTL-5 cells, KA inhibited iodine organification dose-dependently, but did not inhibit iodine uptake. These results suggest that the observed lower iodine uptake activity in the single-dose administration study in rats was due to the inhibition of iodine organification caused by the oral administration of KA, consequently decreasing iodine in the entire thyroid gland. Although serum T4 showed a tendency to decrease from 2 hr to 48 hr after administration of KA, serum TSH did not show any evident change associated with KA in the single-dose administration study in rats. Based on these results, it is presumed that a massive dose or long administration period might be needed to decrease serum T4 and increase serum TSH. From these results, it is presumed that KA affected thyroidal function when given at a massive dose or in a long administration period by inhibiting iodine organification in the thyroid.

Heregulin-dependent autocrine loop regulates growth of K-ras but not erbB-2 transformed rat thyroid epithelial cells.

The EGF-like family of proteins, such as epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), amphiregulin (AR), betacellulin (BTC), cripto-1 (CR-1), and heregulin (HRG), plays an important role in the pathogenesis of several human carcinomas as autocrine growth factors. Differentiation and proliferation of rat thyroid cells in culture (FRTL-5 cells) are regulated by thyrotropin (TSH); withdrawal of TSH from culture medium produces growth arrest, whereas its addition to quiescent cells stimulates cell entry into S phase. Instead, transformed thyroid cell lines as FRTL-5H2 cell line, overexpressing erbB-2, Kimol cells, transformed by the wild-type K-ras and A6 clone, transformed by a temperature sensitive K-ras mutant, can grow without addition of TSH to the culture medium. In order to identify whether EGF-like growth factors and corresponding receptors (erbB-2, erbB-3, and erbB-4) could be involved in the autonomous growth of these transformed rat thyroid epithelial cells, Northern blot for mRNA analysis and Western blot for protein expression were performed. In contrast to normal control FRTL-5 cells, both K-ras and erbB-2-transformed cells expressed elevated levels of erbB-2 receptor. Moreover, both K-ras transformed cells, Kimol and A6 cells, but no FRTL-5H2 cells, were found able to express also high levels of erbB-4 receptor and HRG/NDF ligand. Treatment of K-ras transformed thyroid cells with neutralizing antibody against HRG/NDF reduced by 50% cell proliferation. These data indicate that unlike the erbB-2 overexpressing FRTL-5 cells, in K-ras rat thyroid epithelial cells, the growth factor heregulin signals through the heterodimer erbB-2/erbB-4 receptors in an autocrine fashion.

Cyclin D1 and Cyclin E expression in malignant thyroid cells and in human thyroid carcinomas.

Evidence of the involvement of cyclin gene alterations in human cancer is growing. In this study, we sought to determine the pattern of expression of cyclin D1 and cyclin E in normal and malignant thyroid cells. Quiescent rat thyroid cells in culture, induced to synthesize DNA by thyrotropin (TSH), expressed cyclin D1 gene after 6 hr and cyclin E gene with a peak at 18 hr from the stimulus; K-ras-transformed rat thyroid cells, which grew without addition of hormones necessary for normal cell proliferation, expressed elevated levels of cyclin D1 and cyclin E, compared with normal differentiated thyroid cells. Human benign and malignant thyroid tumors and their relative normal tissues were then analyzed. Neither major genetic alterations nor amplifications for cyclin D1 and cyclin E genes were found by Southern blot analysis in genomic DNAs extracted from all types of thyroid tumors. Moreover, statistical analyses of densitometric values from Northern blots did not show increased levels of cyclin D1 and E mRNAs in the tumor samples, compared with normal thyroid. Immunohistochemical analyses of formalin-fixed paraffin-embedded sections of tissues with specific antibodies revealed a prevalent cytoplasmic cyclin E staining in the thyroid tissues analyzed. Cyclin D1, instead, was present in the cytoplasm of normal thyroids and adenomas, but in 31% of thyroid papillary carcinomas analysed, it was overexpressed, with a localization in the nucleus. Our in vivo observations suggest that unlike cyclin E, elevated nuclear cyclin D1 expression defines a subset of thyroid papillary carcinomas, and might be a contributory factor to thyroid tumorigenesis.

Quantitative analysis of rat thyroglobulin messenger rna in FRTL-5 cells by competitive polymerase chain reaction with human thyroglobulin messenger RNA

To measure relative expression level of mRNA in a small number of cultured rat thyroid cells (FRTL-5), we developed a system of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Human thyroglobulin mRNA in total RNA extracted from a human thyroid tissue was used as an internal control. FRTL-5 cells in a 24 well dish were lysed with denaturing solution containing human RNA. Total RNA was extracted followed by reverse transcription and polymerase chain reaction. After digestion with a restriction enzyme, PCR products were separated by electrophoresis and stained with Sybr Green ITM, then their fluorescence was measured with fluorescent image analyser. Increase of thyroglobulin mRNA in FRTL-5 cells stimulated by thyroid stimulating hormone (TSH) was observed by this technique. Because this method does not require a large number of cells or radioactive isotopes, it is as useful for the analysis of the relative expression level of mRNAs in the cells as the conservative methods such as Northern Blot.

Negative correlation between the conversion of thyrotropin receptor-bound blocking type thyrotropin receptor antibody to the stimulating type by anti-human IgG antibodies and the biological activity of blocking type thyrotropin receptor antibody.

It has been reported that receptor-bound blocking type TSH receptor antibody (TRAb) can be converted to the stimulating type by anti-human IgG antibodies. To evaluate the relationship between the conversion of receptor-bound blocking type TRAb to the stimulating type and the biological activity of blocking type TRAb, we compared converting activities of blocking type TRAb from 10 patients with primary nongoitrous hypothyroidism with both the doses of blocking type TRAb which show 50% inhibition of 125I-bTSH binding to the TSH receptor and those which show 50% inhibition of TSH-stimulated cAMP production in cultured rat thyroid cells (FRTL-5). The additions of anti-human IgG antibody to FRTL-5 cell-bound blocking IgGs resulted in the increase in cAMP production in a dose-dependent manner and the converting activities (percent increase of cAMP production) also depended on the doses of blocking IgGs. The converting activities were significantly correlated with the doses of blocking IgGs which showed 50% inhibition of 125I-bTSH binding to the TSH receptor (r = 0.71, p = 0.011). And these converting activities were also significantly correlated with the doses of blocking IgGs which showed 50% inhibition of TSH-stimulated cAMP increase (r = 0.81, p = 0.002), and were negatively correlated with thyroid stimulation blocking antibody activities (r = 0.58, p = 0.02). We have demonstrated that all cell-bound blocking type TRAb were converted to the stimulating type by anti-human IgG antibody and the degree of conversion was negatively correlated with the biological activity of blocking type TRAb.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin.

We investigated the glucose transporter mRNAs expressed in FRTL5, a rat thyroid cell line, and their regulation by TSH by means of the polymerase chain reaction. FRTL5 cells as well as rat thyroid tissue expressed three types of glucose transporter mRNAs: GLUT1 or erythrocyte/HepG2/brain isoform, GLUT2 or pancreatic beta-cell/liver isoform, and GLUT4 or muscle/fat isoform. GLUT1 mRNA predominated, GLUT4 mRNA was minor, and GLUT2 mRNA expression was faint. Incubation of FRTL5 cells with TSH induced a time- and concentration-dependent increase in GLUT1 mRNA levels, while GLUT4 mRNA levels were decreased. The response of GLUT1 mRNA to TSH was evident at 3 h, and the maximal response was achieved at 12 h. TSH at a dose of 1 mU/ml elicited an approximately 3-fold increase in GLUT1 mRNA levels. (Bu)2cAMP (1 mM), 8-bromo-cAMP (1 mM), and forskolin (50 microM) mimicked the effect of TSH on GLUT1 and GLUT4 mRNA levels. The increase in GLUT1 mRNA by TSH was correlated with the increase in GLUT1 protein and the increase in 2-deoxyglucose transport activity. These observations suggest that in thyroid cells, TSH stimulates glucose transport at least in part by enhancing GLUT1 gene expression, and that the effect of TSH on GLUT1 and GLUT4 mRNA levels is mediated by a cAMP-dependent pathway.

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin

TSH is able to induce cell cycle-related gene expression in rat thyroid cell

Rat thyroid cells in culture (FRTL-5 strain) require thyrotropic hormone (TSH) for growth. TSH alone in serum free medium is able to induce DNA synthesis of FRTL-5 cells. DNA synthesis occurs 18–20 hours following TSH stimulation of quiescent cells. Here we demonstrate that two sets of genes, related to the entry of cells in the S phase, are induced by TSH: 1) immediate early genes, such as c- jun and a gene coding for a zinc-finger protein Xrox 20 Egr2 , both having a pattern of expression similar to the c- fos oncogene; 2) early delayed genes such as ornithine decarboxylase (ODC), 2F-1, a gene that shows a strong similarity in aminoacid sequence to a mitochondrial ADP ATP carrier, and the asparagine synthetase gene (TS11). Furthermore, an increased expression of the histone H3 gene, a typical marker of S phase, has been observed in TSH-treated FRTL-5 cells.

Heterogeneity of thyrotropin binding inhibitor immunoglobulins in serum from untreated patients with hyperthyroid Graves' disease

We have previously established an assay for the simultaneous assessment of thyrotropin (TSH) binding inhibitor immunoglobulin (TBII) and thyroid stimulating autoantibody activities in cultured rat thyroid cells (FRTL-5 cell), and found a discrepancy in some patients with untreated Graves' disease between the activities of TBII measured in FRTL-5 cells (TBII-rc) and in solubilized thyroid membranes (TBII-pm). In three selected patients with untreated Graves' disease, the different dose-response relationship between TBII-rc and TBII-pm clearly indicated the heterogeneous populations of TBII-pm in patients' sera, with different binding affinities for TSH receptor in intact cells.

Cycloheximide Inhibits Interferon-γ-Induced Class II Major Histocompatibility Complex Antigen Expression in Cultured Rat Thyroid Cells

The effects of the agents that are related to intracellular events on interferon-gamma-induced class II major histocompatibility complex antigen expression were studied using the technique of immunocytochemistry. Rat class II major histocompatibility complex antigen (RT1.B) was expressed in 88.3 +/- 3.3% (n = 3) of the functioning rat thyroid cells (FRTL-5) cultured in a medium containing 100 U/ml recombinant rat interferon-gamma (IFN gamma). Deprivation of bovine TSH had no effect on the expression of RT1.B antigen by IFN gamma. A23187 (1 nM to 2 microM) and/or 10 nM to 10 microM phorbol 12-myristate 13-acetate did not induce the expression of RT1.B antigen. IFN gamma-induced RT1.B expression was not inhibited by either 10 nM to 100 microM 1-(5-isoquinolysulfonyl)-2-methylpiperazine or 200 nM to 200 microM 8-(N,N-dimethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride. It was also not inhibited by either 5-200 microM verapamil or 500 nM to 20 microM trifluoperazine. However, 0.01-10 micrograms/ml cycloheximide inhibited IFN gamma-induced RT1.B antigen expression in a dose-dependent manner. These results suggest that IFN gamma induces RT1.B antigen expression in FRTL-5 cells via de novo protein synthesis independent of the cAMP system, phosphatidylinositide system, and voltage-dependent calcium channel.

Rosmarinic Acid and Freeze-Dried Extract (FDE) ofLycopus virginicusare able to Inhibit Forskolin-Induced Activation of Adenylate Cyclase in Cultured Rat Thyroid Cells

Rosmarinic Acid and Freeze-Dried Extract (FDE) of<i>Lycopus virginicus</i>are able to Inhibit Forskolin-Induced Activation of Adenylate Cyclase in Cultured Rat Thyroid Cells

Organization of the thyroglobulin polysome, studied in Endoplasmic Reticulum surface views of rough endoplasmic reticulum in cultured rat thyroid cells

Thyroglobulin is synthesized in the thyroid gland and is subsequently degraded to provide thyroid hormones. Rat thyroglobulin is made up of two identical 330 kD subunits, and the mRNA for each subunit contains about 8,500 nucleotides. Since polysomes have approximately one ribosome for each 90-100 nucleotides of mRNA, a polysome of about 85-95 ribosomes would be expected for thyroglobulin. We have been interested in how this very large polysomes is organized on the membranes of the rough endoplasmic reticulum (RER).It is well known that bound polysomes assume characteristic shapes on the surface of the RER, resembling beads on a string arranged in circles, spirals, loops, hairpins or other forms. These polysomal shapes can be observed in conventional electron micrographs when the membranes of the RER are seen in surface or en face view, rather than in the usual cross section. Clearcut surface views are infrequent, but the likelihood of seeing them is greatly improved when flattened cells in culture are sectioned in the plane of the cell, since the RER in a flattened cell tends to be oriented in that plane.

Incidence of Antibodies Blocking Thyrotropin EffectIn Vitroin Patients with Euthyroid or Hypothyroid Autoimmune Thyroiditis*

Autoantibodies blocking the TSH-dependent production of cAMP in thyroid cells (TSH-BAb) have been described in atrophic thyroiditis (AT; idiopathic myxedema) and in neonates with transient hypothyroidism, but their incidence in autoimmune thyroiditis in relation to thyroid status remains to be completely established. To this purpose TSH-BAb were evaluated in a group of 140 consecutive patients with autoimmune thyroiditis, which included 26 cases of AT and 114 subjects with goitrous Hashimoto's thyroiditis (HT); among the goitrous group 27 were euthyroid (HT-E), 32 had subclinical hypothyroidism (HT-SH), and 55 had clinical hypothyroidism (HT-H). TSH-BAb were measured in immunoglobulin G prepared by DEAE-Sephadex A-50 by determining their ability to inhibit TSH-dependent cAMP production in a differentiated strain of cultured rat thyroid cells (FRTL-5). Using this sensitive and reproducible method, TSH-BAb were detected in 12 of 26 (46%) patients with AT, in 1 of 27 (3.7%) subjects with HT-E, in 3 of 32 (9.4%) with HT-SH, and in 20 of 55 (36%) with HT-H. The prevalence of TSH-BAb was higher in AT vs. HT-H (P less than 0.001), HT-SH (P less than 0.001), or HT-E (P less than 0.001), and in HT-H vs. HT-SH (P less than 0.001) or HT-E (P less than 0.001). Mean TSH-BAb levels in AT were higher than those in HT-H (P less than 0.005) and HT-SH (P less than 0.025); the difference was not significant between HT-H and HT-SH. An inverse correlation was found between TSH-BAb levels and estimated goiter weight (P less than 0.005). The results of the present study indicate that 1) in autoimmune thyroiditis TSH-BAb are detectable almost exclusively in hypothyroid patients, their prevalence being higher in overt hypothyroidism than in subclinical thyroid failure; 2) the prevalence of TSH-BAb and their mean levels are higher in hypothyroid patients with AT than in those with HT; and 3) therefore, the presence of circulating TSH-BAb appears to be related to the development of hypothyroidism and thyroid atrophy.

Chromatographically Purified Immunoglobulin G of Endemic and Sporadic Goiter Patients Stimulates FRTL5 Cell Growth in a Mitotic Arrest Assay*

A strain of differentiated rat thyroid cells (FRTL5) in continuous culture was used to study the presence of thyroid growth-promoting immunoglobulins (TGI) in the serum of patients with endemic and sporadic euthyroid goiters. To identify true in vitro cell proliferation a microscopic mitotic arrest assay was used. Immunoglobulins G (IgGs) were prepared with QAE-Sephadex A-50 or protein-A-Sepharose. A positive growth stimulation index was found in IgG preparations of 65 of 71 patients with endemic goiter and in 9 of 14 IgG preparations of patients with sporadic goiter. IgG preparations of 15 control subjects from an area where endemic goiter due to iodine deficiency does not occur and of 18 subjects without iodine deficiency and without thyroid enlargement living in the endemic area did not stimulate FRTL5 cell growth. FRTL5 cell growth stimulation with IgGs of these euthyroid goiter patients could only be detected when IgG was tested in combination with a small dose of TSH. Immunoprecipitation with polyclonal and monoclonal antihuman IgG was able to abolish the growth-promoting effects. In 32 blinded samples the Feulgen cytobiochemical assay, formerly used to detect TGI, was compared with the FRTL5 mitotic arrest assay. The two methods showed similar results. Our observations of chromatographically purified IgG promoting thyroid cell proliferation in vitro provide good evidence that IgG was responsible for thyroid cell growth in vitro and suggest that autoimmune growth mechanisms may be involved in the pathogenesis of both endemic and sporadic goiters.

Studies on the thyroid stimulating hormone-activated Na +K +-ATPase and iodide transporter in cultured rat thyroid cells and the effects of drugs

Exposure of rat thyroid FRTL-5 cells to high concentrations of thyroid stimulating hormone (TSH) for 24 hr in vitro activated 125iodide but not ouabain-sensitive 86Rb transport in the cells, whereas after 27 hr and above there were significant increases in the activity of both transporters. This was similar to results obtained in propylthiouracil-treated rats in vivo where, unlike the early reported increases in iodide transport, activation of thyroidal Na +K +-ATPase activity in thyroid membranes occurred around 24–48 hr following rises in serum TSH. These data confirm the presence of a Na +K +-ATPase in cultured rat FRTL-5 thyroid cells, which is activated several hours later than iodide accumulation by TSH stimulation. This pump is sensitive to the cardiac glycoside ouabain as well as other agents affecting intracellular Na + ion concentrations such as monensin and amiloride.

Sequence and chromosomal assignment of a novel cDNA identified by immunoscreening of a thyroid expression library: similarity to a family of mitochondrial solute carrier proteins.

An immunoglobulin G (IgG) preparation of the serum from a patient with active Graves' disease was used to isolate cDNA clones from a lambda gt11 cDNA library of human thyroid follicular carcinoma tissue by immunoscreening. One of these clones, hML-7, is further characterized herein by sequencing, Northern analysis, and chromosomal mapping. The clone reacted with IgG preparations from the sera of 14 of 19 patients with active Graves' disease but not with IgG preparations from 11 normal individuals, three patients with toxic thyroid adenoma, and three with rheumatoid arthritis. The hML-7 cDNA hybridized to a 3.6 kilobase (kb) mRNA transcript in poly(A+) RNA preparations from human thyroid tissue and continuously cultured rat thyroid cells; expression of this transcript in rat FRTL-5 thyroid cells was positively regulated by TSH. The 3.6 kb transcript was less abundant in rat liver (BRL3A) cells or differentiated rat (L6) myoblasts than in cultured rat thyroid cells and was not detectable in mouse L-M fibroblasts, human IM-9 lymphocytes, Chinese hamster ovary cells, or human cervical carcinoma cells. The cDNA from hML-7 was sequenced and compared with the sequence of cross-hybridizing cDNA clones isolated from human Graves' thyroid and rat FRTL-5 thyroid cell lambda gt11 expression libraries. A 1.05 kb open reading frame, which is highly conserved between human and rat, was defined. The predicted amino acid sequence of 348 residues exhibited a strong homology with the mitochondrial ADP/ATP carrier protein (adenine nucleotide translocase; ADP/ATP translocator) and with two other members of the same mitochondrial protein family, the phosphate carrier and the hydrogen ion uncoupling protein. The gene represented by the hML-7 cDNA has been assigned to human chromosome 10.

Iodine Inhibits the Proliferation of Rat Thyroid Cells in Culture*

We have explored the mechanisms whereby iodine inhibits thyroid growth using as models both the FRTL5 line of rat thyroid follicular cells that require TSH for growth and the M12 line of mutant cells that grow in the absence of TSH. Between 0.01-1.0 mM, NaI produced a dose-dependent inhibition of TSH stimulation of [3H]thymidine incorporation and replication in FRTL5 cells as well as spontaneous growth in M12 cells. Iodide also inhibited the cAMP-dependent growth of FRTL5 cells induced by forskolin and (Bu)2cAMP, as well as the cAMP-independent mitogenesis induced by insulin-like growth factor-I. The effect of iodide to inhibit both TSH- and insulin-like growth factor-I-stimulated growth in FRTL5 cells was abolished by concomitant culture with methimazole, and no iodide inhibition of growth was observed in L6 myoblasts and BRL 30E hepatocytes. Exposure of cells to iodide under conditions that resulted in inhibition of TSH-stimulated growth did not significantly alter the ability of TSH to increase the intracellular cAMP concentration, nor did iodide alter two responses to TSH in FRTL5 cells that depend upon an increase in cAMP concentration: down-regulation of TSH receptor and cytoskeletal reorganization. We conclude that iodide exerts its inhibitory action on the growth of thyroid cells at multiple loci related to both the cAMP-dependent and cAMP-independent pathways of mitogenic regulation.

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.