Regulation of Epidermal Growth Factor Receptors by TSH, and Effects of EGF, TSH and Phorbol Ester on DNA Synthesis of Cultured Porcine Thyroid Cells
1986 Volume 62 Issue 12 Pages 1362-1375
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1986 Volume 62 Issue 12 Pages 1362-1375
Using porcine thyroid cells of primary monolayer culture, this study was conducted to clarify the nature and characteristics of epidermal growth factor (EGF) receptors on porcine thyroid cells and also to investigate the effects of EGF, TSH and phorbol ester on DNA synthesis. Receptors for EGF on porcine thyroid cells exist in two forms which differ in both affinity and capacity. Scatchard analysis of saturation binding assay performed at 4°C for 6h indicates that there is a high affinity class of receptors with low capacity (K1 = 4.70 ± 0.90 × 10-9M and 8,600 ± 1,200 sites/cell) and low affinity receptors with high capacity (K2 = 2.24 ± 1.17 × 10-7M and 65,500 ± 18,000 sites/cell) on the cells cultured for 4 days in the absence of TSH. When thyroid cells were cultured in the presence of various concentrations of TSH (0-50 mU/ml) and for various times (0-96h) with TSH (10mU/ml), specific EGF binding to the cells increased dose- and time-dependently. On TSH (10mU/ml) -treated cells for 4 days, two kinds of EGF receptors, i.e. high affinity and low capacity (K1 = 5.39 ± 1.75 × 10-9M and 17,200 ± 2,500 sites/cell) and low affinity and high capacity (K2 = 1.70 ± 1.40 × 10-7M and 76,300 ± 17,900 sites/cells), were resolved. The results indicate that TSH can modulate EGF receptors by increasing the number of high affinity sites on porcine thyroid cells. Next, using [Me-3 H] thymidine incorporation into TCA precipitable materials for 48h, we studied the biological effects of EGF, TSH and phorbol myristate acetate (one of the potent phorbol esters) on DNA synthesis. Both EGF and PMA can promote [Me-3 H] thymidine incorporation. Maximal responses were obtained with EGF ranging from 10-9 to 10-7M and with PMA from 10-9 to 10-7M. In contrast, TSH inhibits [Me-3H] thymidine incorporation dose-dependently. Almost the same results were obtained by these agents on TSH (10mU/ml) -treated cells for 4 days, but EGF stimulated cell growth at a lower concentration of 10-11 M, which was.possibly related to an increase of high affinity receptor numbers. The growth promoting effect of EGF and PMA in combination was additive, and TSH suppressed the cell growth in the concomitant presence of EGF and PMA.
From the present study, it is indicated that TSH can modulate EGF receptors on porcine thyroid cells, and also that both EGF and PMA can stimulate DNA synthesis of porcine thyroid cells at the concentrations of 10-11-10-7M. However, TSH, itself, may inhibit DNA synthesis.
