DIFFERENTIAL EXPRESSION OF THROMBOSPONDIN, COLLAGEN, AND THYROGLOBULIN BY THYROID-STIMULATING HORMONE AND TUMOR-PROMOTING PHORBOL ESTER IN CULTURED PORCINE THYROID-CELLS

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by t hyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing mediu m on poly (L-lysine)-coated dishes. Cells differentiated into follicul ar or vesicular-like structures were distinguished by their ability to organify Na[I-125] and to respond to TSH stimulation. After an incuba tion of the cells with radiolabeled proline or methionine, two major p roteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthes is of [I-131]-labeled polypeptides with electrophoretic properties ide ntical to those of authentic Tg molecules. P450-480 resolved to Mr 190 ,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrop horesis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by pept ide sequencing of some of its tryptic fragments that displayed identit y to thrombospondin I. Collagen synthesis was demonstrated by the form ation of radioactive hydroxyproline and by the synthesis of pepsin-res istant polypeptides ranging from Mrs 120,000 to 200,000. When the cell s were cultured in the presence of 100 nM TPA, the culture medium cont ents of thrombospondin and collagen were increased by 2.7 and 1.6-fold , respectively, whereas Tg content was decreased by a factor 3.9. In c ontrast, the acute treatment of control cells with TPA induced a decre ase in both Tg and collagen content by factors 3.0 and 1.5, respective ly, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulati ng effect of TPA on extracellular matrix components synthesis. In cont rast, when cells were cultured in the presence of TSH alone at concent rations higher than 0.1 mU/ml, collagen and thrombospondin in the medi um were decreased by a factor 2.0 and 1.9, respectively, and TSH prefe rentially activated Tg synthesis. However, no acute response to TSH wa s observed in cells incubated for 2 days without effecters (control ce lls). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factors 1.2 and 1.8, respectively, whereas it increase d the one of thrombospondin by a factor 2. These results, together wit h the stimulating effect of TPA on TSH mediated cell proliferation, ar gue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium. (C) 1994 Wiley-Liss, Inc.