Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells.

Abstract

Abstract. 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 ceils. 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 EGE 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 dissoc.iated 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 ,'050 % 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. The inhibition of differentiation could be a side effect of the activation of the EGF and phorbol ester pathways, rather than a consequence of cell cycling itself.ULT thyroid epithelial follicular cells (thyrocytes) in primary culture keep many thyroid functions and responsiveness to various hormones, neurotransmitters, and growth factors (Lissitzky et al., 1971;Westermark et al., 1983;Roger and Dumont, 1984;Roger et al., 1982Roger et al., , 1985Errick et al., 1985; Rasp6 et al., 1986; G...

Signal transduction in the human thyrocyte and its perversion in thyroid tumors

“…1B). TSH and cAMP induce differentiation expression, whereas growth factors and more potently EGF reversibly inhibit the expression of all differentiation markers while inducing a morphological transformation analogous to an epithelium/mesenchyme transition (EMT) (Roger and Dumont, 1984;Pohl et al, 1990;Roger et al, 1985;Coclet et al, 1991;Hebrant et al, 2007). Both TSH and EGF mitogenic stimulations are permitted or facilitated by the comitogenic activity of IGF -I (reproduced in vitro by high insulin concentrations) acting through IGF-I tyrosine kinase receptors (Roger et al, 1987;Burikhanov et al, 1996).…”

“…In the thyroid, the main physiological feedbacks involve the thyroid hormone inhibition of TSH synthesis in, and release by, the thyrotrophs of the pituitary and the dampening by M a n u s c r i p t 14 induce the proliferation of dog thyrocytes while maintaining differentiation expression; both proliferation and differentiation programs can be triggered by TSH in the same cells at the same time (Pohl et al, 1990;Pohl et al, 1993). It is tempting to relate this apparent paradox to the unique characteristics of the cAMP-dependent mitogenic pathway, such as the lack of activation (or even the inhibition) of the Ras-ERK-c-jun-cyclin D1 cascade, as demonstrated in dog and human thyrocytes.…”

The dog thyroid primary culture system: a model of the regulation of function, growth and differentiation expression by cAMP and other well-defined signaling cascades

“…52,55,65,68,71). The inhibitory effect of EGF on the expression of these differentiation markers is independent of its mitogenic activity (see below).…”

“…The basal expression of Tg gene (but not its stimulation by TSH) is strictly dependent on the presence of insulin in the culture medium (68,69), but insulin does not affect TPO gene expression (69,71) and iodide transport (72). Hydrocortisone potentiates the TSH-stimulation of iodide transport capacity (14) but does not modify Tg mRNA accumulation (the opposite is found in calf thyroid primary cultures (73)).…”

“…in the terminology of embyologists they remain determined. Indeed, after elimination of EGF and addition of TSH, high iodide trapping capacity and Tg and TPO mRNA are restored (18,23,68). In the other thyroid primary culture systems this redifferentiation activity of TSH has not been demonstrated and the maintenance of differentiated functions requires the continuous presence of TSH.…”