scholarly journals MAPK Inhibition Requires Active RAC1 Signaling to Effectively Improve Iodide Uptake by Thyroid Follicular Cells

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5861
Author(s):  
Márcia Faria ◽  
Rita Domingues ◽  
Maria João Bugalho ◽  
Paulo Matos ◽  
Ana Luísa Silva
Keyword(s):  
Positive Impact ◽  
Mapk Pathway ◽  
Functional Expression ◽  
Braf V600e ◽  
Thyroid Cell ◽  
Follicular Cells ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Mek Inhibitors ◽  
Thyroid Follicular Cells

The Sodium/Iodide Symporter (NIS) is responsible for the active transport of iodide into thyroid follicular cells. Differentiated thyroid carcinomas (DTCs) usually preserve the functional expression of NIS, allowing the use of radioactive iodine (RAI) as the treatment of choice for metastatic disease. However, a significant proportion of patients with advanced forms of TC become refractory to RAI therapy and no effective therapeutic alternatives are available. Impaired iodide uptake is mainly caused by the defective functional expression of NIS, and this has been associated with several pathways linked to malignant transformation. MAPK signaling has emerged as one of the main pathways implicated in thyroid tumorigenesis, and its overactivation has been associated with the downregulation of NIS expression. Thus, several strategies have been developed to target the MAPK pathway attempting to increase iodide uptake in refractory DTC. However, MAPK inhibitors have had only partial success in restoring NIS expression and, in most cases, it remained insufficient to allow effective treatment with RAI. In a previous work, we have shown that the activity of the small GTPase RAC1 has a positive impact on TSH-induced NIS expression and iodide uptake in thyroid cells. RAC1 is a downstream effector of NRAS, but not of BRAF. Therefore, we hypothesized that the positive regulation induced by RAC1 on NIS could be a relevant signaling cue in the mechanism underlying the differential response to MEK inhibitors, observed between NRAS- and BRAF-mutant tumors. In the present study, we found that the recovery of NIS expression induced through MAPK pathway inhibition can be enhanced by potentiating RAC1 activity in thyroid cell systems. The negative impact on NIS expression induced by the MAPK-activating alterations, NRAS Q61R and BRAF V600E, was partially reversed by the presence of the MEK 1/2 inhibitors AZD6244 and CH5126766. Notably, the inhibition of RAC1 signaling partially blocked the positive impact of MEK inhibition on NIS expression in NRAS Q61R cells. Conversely, the presence of active RAC1 considerably improved the rescue of NIS expression in BRAF V600E thyroid cells treated with MEK inhibitors. Overall, our data support an important role for RAC1 signaling in enhancing MAPK inhibition in the context of RAI therapy in DTC, opening new opportunities for therapeutic intervention.

Effects of 4,4′-di-isothiocyano-2,2′-stilbene disulphonate on iodide uptake by primary cultures of turtle thyroid follicular cells

1987 ◽  
Vol 113 (3) ◽  
pp. 403-412 ◽  
Author(s):  
S. Y. Chow ◽  
Y. C. Yen-Chow ◽  
H. S. White ◽  
D. M. Woodbury
Keyword(s):  
Cultured Cells ◽  
Primary Cultures ◽  
Thyroid Tissue ◽  
Concentration Addition ◽  
Thyroid Cell ◽  
Follicular Cells ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Thyroid Follicular Cells ◽  
Cell Medium

ABSTRACT Iodide uptake by primary cultures of turtle thyroid follicular cells is directly proportional to the Na + concentration and is inversely proportional to the HCO3− concentration in culture medium, but is not affected by the Cl− concentration. Addition of 4,4′-di-isothiocyano-2,2′-stilbene disulphonate (DIDS; 10 μmol/l and higher doses) to medium containing different concentrations of Na+ (5–140 mmol/l), HCO3− (0–40 mmol/l) and Cl − (120 mmol/l) generally enhanced iodide uptake by the cultured cells; however, there was no significant effect in Na+-free and in low Cl− (90 mmol/l and less) medium. The inhibitory effects on iodide uptake of ouabain, frusemide and perchlorate were attenuated by DIDS which also antagonized the stimulatory effects on iodide uptake of TSH, although both DIDS and TSH increased the 125I− cell/medium ratio when they were given alone. At doses of 100 μmol/l and higher, DIDS lowered the intracellular pH of cultured cells when the pH of the medium was maintained at a constant level. It also increased the intracellular Cl − concentration, but had no effect on intracellular Na+ or K +. The input and specific resistances of cell membranes in cultured thyroid cells and in isolated thyroid slices increased (decreased conductance) after adding DIDS to the perfusion fluids. Both Na+/K+- and HCO3−-ATPase activities in homogenates of turtle thyroid tissue were inhibited by DIDS. Results from this investigation demonstrate (1) that in addition to preventing the leak of iodide from thyroid cells, DIDS may act to increase the sensitivity of the Na + -anion carrier to I− and thereby increases iodide uptake, and (2) that a HCO3−–Cl− exchange system is present in the thyroid cell membrane and appears to be linked to the transport of iodide into thyroid cells. J. Endocr. (1987) 113, 403–412

scholarly journals Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Ana Paula Santin Bertoni ◽  
Ilma Simoni Brum ◽  
Ana Caroline Hillebrand ◽  
Tania Weber Furlanetto
Keyword(s):  
Mrna Expression ◽  
Cell Function ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Sodium Iodide Symporter ◽  
Follicular Cells ◽  
Ki 67 ◽  
Thyroid Cells ◽  
Female Sex Hormones ◽  
Thyroid Follicular Cells

Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporterNIS, thyroglobulinTG, thyroperoxidaseTPO, andKI-67genes expression, in normal thyroid follicular cells, derived from human tissue.NIS,TG,TPO, andKI-67mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times,P<0.0001; 2.39 times,P=0.01; 1.58 times,P=0.0003; and 1.87 times,P<0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression ofNIS,TG, andKI-67genes increased, respectively: 1.78 times,P<0.0001; 1.75 times,P=0.037; and 1.95 times,P<0.0001, andTPOmRNA expression also increased, though not significantly (1.77 times,P=0.069). These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.

scholarly journals Detection of SARS-COV-2 receptor ACE-2 mRNA in thyroid cells: a clue for COVID-19-related subacute thyroiditis

2020 ◽  
Author(s):  
M. Rotondi ◽  
F. Coperchini ◽  
G. Ricci ◽  
M. Denegri ◽  
L. Croce ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Thyroid Tissue ◽  
Subacute Thyroiditis ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Type I ◽  
Rt Pcr ◽  
Follicular Cells ◽  
Thyroid Cells ◽  
Tissue Samples

Abstract Purpose SARS-COV-2 is a pathogenic agent belonging to the coronavirus family, responsible for the current global world pandemic. Angiotensin-converting enzyme 2 (ACE-2) is the receptor for cellular entry of SARS-CoV-2. ACE-2 is a type I transmembrane metallo-carboxypeptidase involved in the Renin-Angiotensin pathway. By analyzing two independent databases, ACE-2 was identified in several human tissues including the thyroid. Although some cases of COVID-19-related subacute thyroiditis were recently described, direct proof for the expression of the ACE-2 mRNA in thyroid cells is still lacking. Aim of the present study was to investigate by RT-PCR whether the mRNA encoding for ACE-2 is present in human thyroid cells. Methods RT-PCR was performed on in vitro ex vivo study on thyroid tissue samples (15 patients undergoing thyroidectomy for benign thyroid nodules) and primary thyroid cell cultures. Results The ACE-2 mRNA was detected in all surgical thyroid tissue samples (n = 15). Compared with two reporter genes (GAPDH: 0.052 ± 0.0026 Cycles−1; β-actin: 0.044 ± 0.0025 Cycles−1; ACE-2: 0.035 ± 0.0024 Cycles−1), the mean level of transcript expression for ACE-2 mRNA was abundant. The expression of ACE-2 mRNA in follicular cells was confirmed by analyzing primary cultures of thyroid cells, which expressed the ACE-2 mRNA at levels similar to tissues. Conclusions The results of the present study demonstrate that the mRNA encoding for the ACE-2 receptor is expressed in thyroid follicular cells, making them a potential target for SARS-COV-2 entry. Future clinical studies in patients with COVID-19 will be required for increase our understanding of the thyroid repercussions of SARS-CoV-2 infection.

Expression of an intercellular adhesion molecule, ICAM-1, by human thyroid cells

1989 ◽  
Vol 122 (1) ◽  
pp. 185-NP ◽  
Author(s):  
A. P. Weetman ◽  
S. Cohen ◽  
M. W. Makgoba ◽  
L. K. Borysiewicz
Keyword(s):  
Adhesion Molecule ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Human Thyroid ◽  
Accessory Cell ◽  
Graves Disease ◽  
Follicular Cells ◽  
Thyroid Cells ◽  
Hla Dr ◽  
Thyroid Follicular Cells

ABSTRACT Intercellular adhesion molecule-1 (ICAM-1), hitherto identified on activated B cells, macrophages, dendritic cells, endothelia and certain epithelial cells, serves as a ligand for the lymphocyte function-associated antigen-1 (LFA-1). ICAM-1 binding by LFA-1 enhances the efficiency of lymphocyte-target cell and lymphocyte-accessory cell interactions. We have investigated the in-vitro expression of ICAM-1 by cultured thyroid cells from five patients with Graves' disease using indirect immunofluorescence analysis, and found that 30 ± 11% (mean ± s.d.) of cells were ICAM-1 positive under basal conditions. The proportion of cells which were ICAM-1 positive and the amount of ICAM-1 per cell (assessed by fluorescence intensity) were both increased in all cases by the cytokines γ-interferon, interleukin-1 and tumour necrosis factor. Immunohistochemical analysis of frozen sections from thyroidectomy specimens demonstrated ICAM-1 on thyroid follicular cells in areas of lymphocytic infiltration in patients with Graves' disease (n = 2) or Hashimoto's thyroiditis (n = 2). ICAM-1 was not found in specimens from a patient with a toxic multinodular goitre or a patient with Graves' disease without focal lymphocytic accumulation. These results suggest that the thyroid epithelium may express ICAM-1 as well as major histocompatibility complex class II antigens, such as HLA-DR, in response to locally synthesized cytokines. The enhanced expression of ICAM-1 may render these cells more susceptible as targets for lymphocytemediated cytotoxicity, and together with HLA-DR antigen expression may increase the accessory cell capability of the thyroid follicular cells. Journal of Endocrinology (1989) 122, 185–191

Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 481-481
Author(s):  
Benjamin Heath Durham ◽  
Eli L. Diamond ◽  
Julien Haroche ◽  
Zhan Yao ◽  
Jing Ma ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Single Agent ◽  
Braf V600e ◽  
Rna Seq ◽  
Wild Type ◽  
Employment Equity ◽  
Mek Inhibitors ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Equity Ownership

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of the BRAF V600E mutation in ~50% of patients with LCH and the non-LCH Erdheim-Chester Disease (ECD) provided the first molecular target in these patients and novel insights into the pathogenesis of these disorders. However, recurrent mutations in the majority of the ~50% of BRAF V600E-wild type patients with non-LCH are unknown. Moreover, recurrent mutations outside of the MAP kinase pathway are undefined throughout histiocytic neoplasms. To address these issues, we performed whole exome sequencing (WES) of frozen biopsies from 24 patients with LCH (n=10) or ECD (n=14) paired with peripheral blood mononuclear cells. 13/24 patients also underwent RNA sequencing (RNA-seq). All mutations in activating kinases were validated by droplet-digital PCR, while targeted-capture next-generation sequencing validated all others. Both adult (n=18; n=2 with LCH) and pediatric cases (n=9; n=8 with LCH) were included. Using combined WES/RNA-seq, activating kinase alterations were identified in 100% of patients. In LCH, 60% and 40% had BRAF V600E and MAP2K1 mutations, respectively. In non-LCH 51%, 14%, 14%, and 7% were BRAFV600E, ARAF, MAP2K1, and NRAS mutant (Fig1A). Overall, a mean of 7 non-synonymous mutations per adult patient was identified (range 1-22) compared with 5 mutations per pediatric patient (range 4-9; p =ns). Mutations affecting diverse cellular processes were found to co-exist with kinase mutations including mutations in epigenetic modifiers and the p38/MAPK pathway. In addition to kinase point mutations, RNA-seq identified recurrent, in-frame kinase fusions-a first for these disorders. All identified fusions were validated using FISH and RT-PCR. This includes novel fusions in BRAF (RNF11-BRAF and CLIP2-BRAF), as well as therapeutically important fusions in ALK (2 separate KIF5B-ALK fusions) and NTRK1 (LMNA-NTRK1;Fig1B). Expression of each fusion in Ba/F3 cells conferred cytokine-independent growth. Importantly, the BRAF fusions were found to be sensitive to MEK inhibition but resistant to vemurafenib while the ALK fusions conferred sensitivity to the ALK inhibitors crizotinib or alectinib. We next interrogated a validation cohort of 37 BRAF V600E-wild type, non-LCH, formalin-fixed, paraffin-embedded tissue samples using targeted mutational profiling for MAP2K1, ARAF, NRAS, KRAS, and PIK3CA. This revealed activating mutations in MAP2K1 (32%; n=12), NRAS (16%; n=6), KRAS (11%; n=4), PIK3CA (8%; n=3), and ARAF (3%; n=1). Three of the investigated non-LCH patients with refractory disease and progressive organ dysfunction were treated with targeted therapies based on the discovery of novel kinase alterations described above. Treatment of 2 refractory MAP2K1- mutant, non-LCH patients with MEK inhibitors (trametinib or cobimetinib) resulted in dramatic clinical improvement (Fig1C). Both patients have been maintained on MEK inhibitor single-agent therapy with a sustained clinical response for >100 days. Further evidence of effective targeted inhibition was found in a refractory ECD patient carrying an ARAF S214A mutation. This patient failed to respond to 3 lines of prior therapies and suffered near blindness due to disease infiltration in the retina and optic nerves. Given a recent report of complete response to sorafenib in a lung cancer patient with an ARAF S214C mutation, we initiated sorafenib. Within 12 weeks, there was improvement in the patientÕs eyesight and decreased infiltrative disease, coinciding with >50% decrease in mutant ARAF DNA in plasma cell-free DNA. Whole exome and transcriptome sequencing identified activating kinase mutations or translocations in all patients with the common downstream effect of activating the MAPK pathway. The preliminary, dramatic, clinical efficacy observed with use of MEK and RAF inhibitors in MAP2K1 - and ARAF-mutated, non-LCH patients further supports the central role of targeting the MAPK pathway in these tumors. The discovery of the discussed mutations and fusions in diverse kinases provides critical new insights into the genetic events central to a spectrum of adult and pediatric histiocytic neoplasms. Figure 1. Figure 1. Disclosures Off Label Use: This abstract describes use of MEK inhibitors (both tremetinib and cobimetinib) as well as sorafenib for MEK1 and ARAF mutant histiocytosis. . Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc.: Employment. Ali:Foundation Medicine Inc.: Employment. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria.

Interaction of endothelin-1 with porcine thyroid cells in culture: a possible autocrine factor regulating iodine metabolism

1994 ◽  
Vol 142 (3) ◽  
pp. 463-470 ◽  
Author(s):  
T Tsushima ◽  
M Arai ◽  
O Isozaki ◽  
Y Nozoe ◽  
K Shizume ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Thyroid Cell ◽  
Scatchard Analysis ◽  
Camp Production ◽  
Iodide Uptake ◽  
Single Class ◽  
Thyroid Cells ◽  
Cells In Culture ◽  
Iodine Metabolism

Abstract Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET)-induced hormonal functions in various tissues. We have studied the interaction of endothelins with porcine thyroid cells in culture. Specific binding of 125I-labelled ET-1 was demonstrated in porcine thyroid cells. The binding was displaced equally by unlabelled ET-1 and ET-2, but receptor affinity for ET-3 was lower than that for ET-1 and -2. Scatchard analysis of the data revealed a single class of high-affinity ET-1 receptors with a Kd of 0·45 nmol/l and a binding capacity of 2100 sites/cell. SDS-PAGE and autoradiography of 125I-labelled ET-1 cross-linked with thyroid cell membranes demonstrated ET-1 binding sites with an apparent molecular weight of 50 kDa. These results indicated that ET-1 receptors in thyroid cells are type A ET receptors. In association with the presence of ET-1 receptors, porcine thyroid cells responded to ET-1 and ET-2 with an increase in c-fos mRNA expression. Although ET-1 did not affect DNA synthesis stimulated by either EGF or IGF-I, it dose-dependently inhibited TSH-induced iodide uptake and also inhibited iodide uptake stimulated by forskolin and 8-bromo-cAMP. ET-1 had no effect on TSH-stimulated cAMP production. Thus, ET-1 inhibited TSH-induced iodine metabolism by acting at the steps distal to cAMP production. In agreement with a recent report, immunoreactive ET-1 was detected in medium conditioned by porcine thyroid cells. Antibody to ET-1 was found to increase TSH-induced iodide uptake. These results are compatible with the notion that ET-1 negatively regulates TSH-induced iodide uptake in an autocrine manner. Journal of Endocrinology (1994) 142, 463–470

Water and electrolyte contents, cell pH and membrane potentials of cultured turtle thyroid cells

1985 ◽  
Vol 104 (1) ◽  
pp. 45-52 ◽  
Author(s):  
S. Y. Chow ◽  
Y. C. Yen-Chow ◽  
D. M. Woodbury
Keyword(s):  
Membrane Potential ◽  
Cell Membrane ◽  
Culture Medium ◽  
Resting Membrane Potential ◽  
Follicular Cells ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Cell Ph ◽  
Cells Cultured ◽  
In Cells

ABSTRACT Water and electrolyte contents, cell pH, membrane potential and 125I− uptake were determined in cultured follicular cells of turtle thyroid. The Na+, K+ and Cl− concentrations in the cultured thyroid cells were 59·2, 119·0 and 50·9 mmol/l cell water respectively. Treatment with TSH (10 mu./ml for 24 h) increased the K+ and Cl− and decreased the Na+ concentrations in cells. The water and protein contents of these cells were 81·6 and 8·7 g/100 g cells respectively. The cell pH was 6·91. With glass microelectrodes, the resting membrane potential of thyroid cells cultured in Medium 199 averaged 33·9 ± 0·63 mV which is slightly higher than 29·8 ± 1·6 mV as calculated from the data on the uptakes of [14C]methyltriphenylphosphonium and 3H2O by the cells. The potential varied linearly with the log of external K + concentration (between 15 and 120 mmol/l) with a slope of about 24 mV per tenfold change in K+ concentration. Both TSH and cyclic AMP depolarized the cell membrane. Calculations based on the values for the electrolyte concentrations in cells and in culture medium indicated that Na+, K+ and Cl− were not distributed according to their electrochemical gradients across the cell membrane. Na+ was actively transported out of the cells and K+ and Cl− into the cells. Follicular cells of turtle thyroid cultured in the medium without addition of TSH formed a monolayer. Their iodide-concentrating ability was low and they did not respond to TSH with an increase in iodide uptake. In contrast, cells cultured in medium containing TSH tended to aggregate and organize to form follicles. They had higher ability to concentrate iodide and respond to TSH. J. Endocr. (1985) 104, 45–52

Validation of Immunohistochemistry for Canine Proteins Involved in Thyroid Iodine Uptake and Their Expression in Canine Follicular Cell Thyroid Carcinomas (FTCs) and FTC-Derived Organoids

2021 ◽  
pp. 030098582110188
Author(s):  
Jana Jankovic ◽  
Martina Dettwiler ◽  
Martin González Fernández ◽  
Eve Tièche ◽  
Kerstin Hahn ◽  
...  
Keyword(s):  
Thyroid Gland ◽  
Western Blot ◽  
Follicular Cell ◽  
Iodine Uptake ◽  
Human Thyroid ◽  
Thyroid Peroxidase ◽  
Thyroid Cell ◽  
Follicular Cells ◽  
Thyroid Cells ◽  
Primary Tumors

Thyrotropin receptor (TSHR), sodium iodide symporter (NIS), pendrin, and thyroid peroxidase (TPO) are essential for the uptake of iodine by follicular thyroid cells. The aim of this study was to establish immunohistochemistry (IHC) protocols for TSHR, NIS, pendrin, and TPO in canine tissues and characterize their expression in organoids derived from canine follicular cell thyroid carcinoma (FTC) and in the respective primary tumors. This constitutes a fundamental step to establish organoids as a model to study the uptake of iodine in canine FTC. Commercially available antibodies directed against human proteins were selected. Antibody specificity was confirmed by western blot using lysates of the HTori-3 human thyroid cell line and healthy canine thyroid gland. IHC was validated using HTori-3 cells and a set of canine normal tissues including healthy thyroid gland. The expression of TSHR, NIS, pendrin, and TPO was evaluated in 3 organoid lines derived from FTC and respective primary tumors. All 4 antibodies produced specific bands by western blot and cytoplasmic labeling in follicular cells by IHC in both human HTori-3 cells and canine thyroid gland. NIS also showed basolateral membrane immunolabeling in follicular cells. All 4 proteins were highly expressed in organoids derived from FTC. The expression was similar or higher compared to the primary tumors. The results of this study characterize organoids derived from canine FTC as a suitable in vitro model to investigate iodine uptake, opening new research possibilities in the field of canine thyroid cancer therapy.

Role of 3′, 5′ cyclic adenosine monophosphate and protein kinase C in the regulation of insulin-like growth factor-binding protein secretion by thyroid-stimulating hormone in isolated ovine thyroid cells

1994 ◽  
Vol 141 (2) ◽  
pp. 231-242 ◽  
Author(s):  
J F Wang ◽  
D J Hill ◽  
G P Becks
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Primary Cultures ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Thyroid Cell ◽  
Dibutyryl Camp ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Kinase C

Abstract Isolated sheep thyroid follicles release insulin-like growth factors (IGF)-I and -II together with IGF-binding proteins (IGFBPs). We previously showed that TSH suppresses the biosynthesis and release of IGFBPs in vitro which may increase the tissue availability of IGFs, allowing a synergy with TSH which potentiates both thyroid growth and function. Many of the actions of TSH on thyroid cell function are dependent upon activation of adenylate cyclase, although increased synthesis of inositol trisphosphate and activation of protein kinase C (PKC) have also been implicated. We have now examined whether probable changes in intracellular cyclic adenosine monophosphate (cAMP) or PKC are involved in TSH-mediated suppression of IGFBP release. Confluent primary cultures of ovine thyroid cells were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin and glycyl-histidyl-lysine (designated 3H), and further supplemented with sodium iodide (10−8–10−3 mol/l), dibutyryl cAMP (0·25–1 mmol/l), forskolin (5–20 μmol/l) or 12-0-tetradecanoylphorbol-13-acetate (TPA; 10−11–10−6 mol/l), with or without exposure to TSH (200 μU/ml). The uptake and organification of Na [125I] by cells was examined after test incubations of up to 48 h, and IGFBPs in conditioned media were analysed by ligand blot using 125I-labelled IGF-II. The PKC activity in the cytosol and plasma membrane fractions of cells was measured by phosphorylation of histone using [γ-32P]ATP, and PKC immunoreactivity was visualized by Western immunoblot analysis. While dibutyryl cAMP or forskolin largely reproduced the stimulatory effect of TSH on iodine organification, they did not mimic the inhibitory effect of TSH on the secretion of IGFBPs of 43, 34, 28 and 19 kDa. Incubation with physiological or pharmacological concentrations of iodide (10−6–10−3 mol/l) for up to 48 h significantly decreased TSH action on iodide uptake and organification but did not alter the inhibitory action of TSH on IGFBP release. Incubation of cells with 10−11–10−6 mol TPA/l for 24 h inhibited the subsequent ability of TSH both to potentiate iodine organification and to suppress IGFBP release. In 3H medium, PKC activity was predominantly recovered from the membrane fraction but, following incubation for 48 h with TSH, the enzyme was no longer translocated to the membrane and was recovered predominantly from the cytosol. An 80 kDa species of immunoreactive PKC was recovered from the membranes of cells cultured in 3H medium, but its presence in membrane was decreased following incubation with TSH. The actions of TSH on intracellular PKC distribution were reversed by prior incubation with TPA, which itself stimulated the appearance of membrane PKC immunoreactivity. These results suggest that the ability of TSH to suppress IGFBP release does not depend primarily on cAMP stimulation, but may involve changes in the activation of PKC, possibly inhibition or down-regulation. Journal of Endocrinology (1994) 141, 231–242

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