Differentiation expression during proliferative activity induced through different pathways: in situ hybridization...

In canine thyrocytes in primary culture, our previous studies have identified three mitogenic agents and pathways: thyrotropin (TSH) acting through cyclic AMP (cAMP), EGF and its receptor tyrosine protein kinase, and the phorbol esters that stimulate protein kinase C. TSH enhances, while EGF and phorbol esters inhibit, the expression of differentiation. Given that growth and differentiation expression are often considered as mutually exclusive activities of the cells, it was conceivable that the differentiating action of TSH was restricted to noncycling (Go) cells, while the inhibition of the differentiation expression by EGF and phorbol esters only concerned proliferating cells. Therefore, the capacity to express the thyroglobulin (Tg) gene, the most prominent marker of differentiation in thyrocytes, was studied in proliferative cells (with insulin) and in quiescent cells (without insulin). Using cRNA in situ hybridization, we observed that TSH (and, to a lesser extent, insulin and insulin-like growth factor I) restored or maintained the expression of the Tg gene. Without these hormones, the Tg mRNA content became undetectable in most of the cells. EGF and 12-0-tetradecanoyl phorbol-13-acetate (TPA) inhibited the Tg mRNA accumulation induced by TSH (and/or insulin). Most of the cells (up to 90%) responded to both TSH and EGF. Nevertheless, the range of individual response was quite variable. The effects of TSH and EGF on differentiation expression were not dependent on insulin and can therefore be dissociated from their mitogenic effects. Cell cycling did not affect the induction of Tg gene. Indeed, the same cell distribution of Tg mRNA content was observed in quiescent cells stimulated by TSH alone, or in cells approximately 50% of which had performed one mitotic cycle in response to TSH + insulin. Moreover, after proliferation in "dedifferentiating" conditions (EGF + serum + insulin), thyrocytes had acquired a fusiform fibroblast-like morphology, and responded to TSH by regaining a characteristic epithelial shape and high Tg mRNA content. 32 h after the replacement of EGF by TSH, cells in mitosis presented the same distribution of the Tg mRNA content as the rest of the cell population. This implies that cell cycling (at least 27 h, as previously shown) did not affect the induction of the Tg gene which is clearly detectable after a time lag of at least 24 h. The data unequivocally show that the reexpression of differentiation and proliferative activity are separate but fully compatible processes when induced by cAMP in thyrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)