Downloaded from Bioscientifica.Com at 09/29/2021 09:54:05PM Via Free Access 618 E FRO} HLICH and Others · Effects of Retinol on Cultured Thyrocytes

617 Retinol has specific effects on binding of thyrotrophin to cultured porcine thyrocytes Eleonore Fröhlich1, Anja Witke1, Barbara Czarnocka2 and Richard Wahl1 1Medical Clinic, Department of Endocrinology, Metabolism and Pathobiochemistry, Otfried-Muellerstrasse 10, D-72076 Tuebingen, Germany 2Medical Centre of Postgraduate Education, Department of Biochemistry, Marymoncka 99, Pl-01-813 Warsaw, Poland (Requests for offprints should be addressed to R Wahl; Email: [email protected]) Abstract Retinoids are potential candidates for the treatment of first 12 h but decreased it after 30 h and longer. After thyroid cancer. However, one of the disadvantages of d30 h incubation times with d13 µM retinol, the frac- these substances is their dedifferentiating effect on normal tion of apoptotic cells was enhanced and proliferation non-transformed thyrocytes. To identify conditions under decreased. The incubation with retinol enhanced the which no dedifferentiating effect of retinol on normal binding of [125I]bTSH to thyrocytes, but did not influence thyrocytes can be observed, we determined iodide uptake, expression of the NIS. With low retinol concentrations, protein iodination, expression of sodium–iodide symporter the effect on the binding of TSH apparently predominated (NIS) mRNA and protein, and the binding of iodine-125- and retinol increased thyroid function; with higher con- labelled bTSH in cultured porcine thyrocytes. Combina- centrations the pro-apoptotic effect of retinol overlapped tion of TSH and c6·5 µM retinol increased iodide uptake and a two-phased time course resulted. It can be con- and protein iodination compared with TSH alone over cluded that low concentrations of retinol also exert differ- the entire incubation time, whereas TSH plus d13 µM entiating effects in normal thyrocytes. retinol increased the uptake of iodine-125 only during the Journal of Endocrinology (2004) 183, 617–626 Introduction expected to occur within a range of hours. In keeping with this idea, the decrease in the expression of NIS protein has Retinoids influence proliferation, differentiation and ap- been observed after 24–36 h (Kogai et al. 1997). However, optosis in many cell types in vivo and in vitro. Their main this might not be the only action of these substances: differentiating effects on transformed cells are the induc- within 1 h, retinoic acid modulates gap junction per- tion of iodothyronine 5-deiodinase (Schreck et al. 1994, meability in neurones and in numerous mammalian cells Schmutzler & Kohrle 2000) and of the sodium–iodide (Brummer et al. 1991, Weiler et al. 1999) and markedly symporter (NIS) (Schmutzler et al. 1997, Schmutzler & reduces the concentrations of inositol phosphate and diacyl Kohrle 1998, Filetti et al. 1999, Schmutzler & Kohrle glycerol in HL-60 cells (Geny et al. 1991). 2000, Schmutzler 2001). In contrast to these positive Using normal porcine thyrocytes as a culture model, we effects on transformed cells, retinoid acid decreases the aimed to detect conditions under which no dediffer- expression of the NIS in normal rat thyrocytes (Schmutzler entiating effects on normal thyrocytes were observed and et al. 1997, Schmutzler & Kohrle 1998, Filetti et al. 1999, to detect effects that may be initiated after short periods Schmutzler & Kohrle 2000) and both the thyroid- of incubation. We determined iodide uptake, iodination stimulating hormone (TSH)-induced uptake of iodide and of protein, NIS mRNA, NIS protein expression and the the release of organified iodine in normal human thyro- number of NIS-immunoreactive cells after different cytes (Namba et al. 1993). High concentrations of retinol periods of incubation with retinoids, retinoids plus TSH also exert dedifferentiating effects on normal thyrocytes or TSH alone. The effects of retinol, other alcohols and (Fröhlich et al. 2001). These effects limit the value of retinoic acid on the binding of TSH to thyrocytes were retinoids for the treatment of thyroid carcinomas. also investigated. We confined our study mainly to the Retinoids are believed to act via nuclear receptors lower range of retinol concentrations under which differ- (Chambon et al. 1991, Chertow et al. 1996), which entiating effects on the human follicular carcinoma cells suggests that the effects on protein concentrations are line FTC 238 had been detected (unpublished results). Journal of Endocrinology (2004) 183, 617–626 DOI: 10.1677/joe.1.05693 0022–0795/04/0183–617 2004 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/29/2021 09:54:05PM via free access 618 E FRO} HLICH and others · Effects of retinol on cultured thyrocytes Materials and Methods 24–30 h, and then again stimulated with retinol (13 µM). The results from these experiments were compared with Isolation of thyroid cells and culture conditions those from cultures stimulated with TSH only. To estimate whether synthesis of new proteins influ- Porcine thyroid glands were obtained from the local ences the uptake of [125I]iodide, thyrocytes were cultured slaughterhouse, and cells were isolated as described pre- in the presence of cycloheximide (Sigma), an inhibitor of viously (Wahl et al. 1992). For the determinations of NIS 6 translation. Cycloheximide was diluted in ethanol and protein and NIS mRNA, 1·510 cells per well (12-well concentrations of 1, 10 and 100 µg/ml were used. Cyclo- Costar No. 3512 culture plates) were seeded. For the 125 6 I]NaI/well was measurement of iodide uptake, 0·1310 cells were heximide in combination with 4 kBq [ seeded per well (48-well culture plate, Costar No. 3548). added to the culture medium together with TSH alone, In all experiments, the number of cells was sufficient for TSH plus ethanol (solvent control) or TSH plus retinol. growing to confluence. NCTC (National Culture and Tissue Collection)-135 medium (standard and special Uptake of [125I]iodide retinol-free preparation, Gibco) supplemented with 2% of Uptake of [125I]iodide was determined with and without the serum substitute, Ultroser G (BioSepra, Villeneuve- stimulation with different concentrations of TSH or retin- la-Garenne, France), penicillin (200 IU/ml), streptomycin oids according to the stimulation procedures. NaI (1 µM (0·2 mg/ml) and ciprofloxacin (Ciprobay, Bayer; 60 µg/ final concentration) traced with 4 kBq/well carrier-free ml) was used for the experiments. Standard medium [125I]NaI (Amersham Pharmacia Biotech) was added to contained about 0·6 µM retinol; these trace amounts of each culture 6 h before measurements were made. The retinol are important because they decrease apoptosis and cells were collected and washed with a 48-well cell har- increase the rate of proliferation in porcine thyrocytes vester (Inotech IH 280, Inotech, Dottikon, Switzerland). compared with retinol-free medium (Fröhlich et al. 2001). Filtermats (Skatron 11731, Skatron, Lier, Norway) were In Ultroser G, no retinol was detectable by HPLC. transferred to counting tubes and their radioactivity Ultroser G contains about 130 µg/l selenium, detected by measured. To test the specificity of NIS-mediated iodide HPLC, which is essential for thyroid function (Kohrle uptake, controls were performed by the addition of 1 mM 1996, 1999). It also contains insulin (250 mg/l). The sodium perchlorate, a competing inhibitor of thyroid culture conditions were: 37 C at 100% humidity in 5% iodide transport. CO2 and 95% air. Iodination of protein Stimulation procedures 125 Cells incubated with [ I]NaI in six-well culture plates All-trans retinol and all-trans retinoic acid (Sigma), diluted were detached from the support, rinsed with 2 ml ice- in methanol, were added in concentrations from 0 to cold Earle’s balanced salt solution (EBSS) buffer and 30 nmol/well (0–40 µM). Concentrations less than 27 µM centrifuged at 500 g for 4 min. The cells were rinsed again do not induce apoptosis or necrosis to a significant degree in 2 ml ice-cold EBSS and radioactivity counted in a after incubations for up to 48 h (Fröhlich & Wahl 1999). multi-gamma counter. Thereafter, 0·5 ml 10 µM non- Control cultures contained only methanol. radioactive NaI and 0·5 ml fetal calf serum (Sigma) were Routinely, before the stimulation with porcine TSH, added, followed by 2 ml ice-cold 20% (v/v) trichloroacetic with retinoids or with both together, the thyroid cells were acid (TCA). The mixture was centrifuged at 500 g for cultured in standard medium or retinol-free standard 5 min and the resulting pellet was resuspended, washed medium with or without TSH (Sigma) for 18 h, to make twice with 10% cold TCA and the radioactivity counted as certain that follicle and monolayer cultures were investi- protein-bound iodine. gated; this time is not included in the times of incubation that are given. Unless otherwise indicated, the TSH Quantification of NIS mRNA by PCR concentration was 1 mU/ml. To investigate the influence of the retinol stimulation on the uptake of iodide, the Cells from 12-well culture plates were removed by following stimulation procedure was also used: firstly, the pipetting from the support and collected in PBS. After cells were cultured without TSH for 18 h, then stimulated centrifugation at 400 g for 4 min, the pellets were deep- with 13 µM retinol for 6 h, and subsequently 1 mU/ml frozen at 80 C. Total RNA was isolated using the TSH (without retinol) was added. Uptake of iodine-125- NucleoSpin RNA II (Macherey Nagel) kit according to labelled iodide was measured 6, 12, 30 and 48 h after the manufacturer’s recommendations. The concentrations stimulation with TSH. Secondly, to evaluate the effect of of RNA preparations were calculated by measuring the repeated stimulation with retinol, thyrocytes were stimu- absorbance at 260 nm. One microgram total RNA was lated with TSH plus retinol for 18 h, thereafter kept in used for cDNA synthesis using the 1st Strand cDNA TSH-containing medium without retinol stimulation for Synthesis Kit for RT-PCR (AMV, Roche Molecular Journal of Endocrinology (2004) 183, 617–626 www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/29/2021 09:54:05PM via free access Effects of retinol on cultured thyrocytes · E FRO} HLICH and others 619 Biochemicals).

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