Sophie Reuse

Multiple facets of the modulation of growth by cAMP.

Publisher Summary This chapter describes multiple facets of the modulation of growth by cyclic AMP (cAMP). The successes in elucidating the functions of cell-transforming proteins encoded by various oncogenes and the demonstration of antioncogenes have shed light on the assumption that cancers mostly develop from alterations of growth control mechanisms, normally involved in homeostasis, tissue repair, and development. cAMP is the first identified intracellular second messenger of hormone action. Pharmacological and genetic tools used to manipulate the CAMP-PKA cascade are summarized in the chapter. Forskolin, a plant diterpene, activates vertebrate adenylate cyclase directly and enhances its response to activated GS, thus to the receptors that regulate GS. Inhibitors of cyclic nucleotide phosphodiesterases by inhibiting the catabolism of cAMP also raise cAMP levels. It is found that due to the negative cooperativity of the phosphodiesterase system, these inhibitors are much more efficient in increasing the cAMP response to activators of adenylate cyclase than in increasing basal levels of cAMP.

Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathways in dog primary thyrocytes: a positive and negative control by cyclic AMP on c-myc expression.

The proliferation of dog thyrocytes in primary culture is stimulated by three distinct intracellular signaling pathways: (1) the thyrotropin or forskolin-cyclic AMP-mediated cascade which is compatible with the differentiated state of the cell; (2) the protein kinase C pathway activated by diacylglycerol and phorbol esters; and (3) a protein tyrosine kinase system activated by epidermal growth factor. The two latter pathways also induce dedifferentiation. The activation of the three cascades induced the expression of the protooncogenes c-fos and c-myc with dose-response curves similar to those for DNA synthesis. After TPA and EGF, the time courses of stimulation of c-fos and c-myc were the same as those for mitogenically stimulated fibroblasts. However, after the cyclic AMP stimulation, c-myc expression was biphasic with an enhancement at 1 h followed by a down-regulation. A similar inhibition by cyclic AMP was also observed on the increased c-myc expression induced by EGF. This down-regulation is suppressed by cycloheximide, which suggests the involvement of a neosynthesized or a labile protein intermediate. The action of cyclic AMP on c-myc mRNA levels could be related to the opposite requirements of the stimulation of both proliferation and differentiation expression by the cyclic AMP pathway in the differentiated thyrocytes.

Differential regulation of protooncogenes c-jun and jun D expressions by protein tyrosine kinase, protein kinase C, and cyclic-AMP mitogenic pathways in dog primary thyrocytes: TSH and cyclic-AMP induce proliferation but downregulate C-jun expression.

The expressions of the protooncogenes c-jun and jun D have been investigated in dog thyrocytes in a primary culture whose proliferation is stimulated by three distinct intracellular signaling pathways (1) the thyrotropin (TSH) or forskolin-cyclic-AMP-mediated cascade; (2) the protein kinase C pathway activated by diacylglycerol (DAG) and phorbol esters (TPA); (3) a protein tyrosine kinase system activated by epidermal growth factor (EGF). While the first cascade is compatible with the differentiated state of the cell, the two latter pathways induce dedifferentiation. Following the stimulation by TPA or EGF, the expression of c-jun was increased and the expression of jun D was faintly increased. Both expressions are superinduced in the presence of cycloheximide as in mitogenically stimulated fibroblasts but, in the presence of cycloheximide alone, the expressions of c-jun and jun D are clearly unstable with time. This indicates that cycloheximide controls should be included at all time points examined in such experiments. Increasing intracellular concentrations of cyclic-AMP by forskolin or TSH was followed by an inhibition of the expression of c-jun. This inhibition was independent of protein synthesis. Similarly, the TPA or EGF stimulation of c-jun expression was also inhibited by TSH or forskolin, as in fibroblasts in which cyclic-AMP inhibits proliferation. Our results show that the expression of c-jun is not universally correlated with the stimulation of cell proliferation. The stimulation of c-jun expression is not common between the three mitogenic pathways. It thus represents another of the very different responses elicited by the cyclic-AMP cascade as compared to the more studied tyrosine kinase and protein kinase C mitogenic pathways.

Activation of the cyclic AMP cascade as an oncogenic mechanism: the thyroid example.

Three cascades activate thyroid cell proliferation: the EGF-protein tyrosine kinase pathway, the phorbol ester-protein kinase C pathway and the thyrotropin-cyclic AMP pathway. While the first 2 cascades converge early, they remain distinct from the cyclic AMP cascade until very late in G1. The cyclic AMP cascade is characterized by an early and transient expression of c-myc, which may explain why it induces proliferation and differentiation expression. Constitutive activation of this cascade causes growth and hyperfunction, ie, hyperfunctioning adenomas. The various possible defects that could lead to such a constitutive activation are discussed.

Lack of correlation between the activation of the Ca2+ phosphatidylinositol cascade and the regulation of DNA synthesis in the dog thyrocyte

Changes in the [Ca2+]i and/or activation of phospholipase C are thought to participate in the control by several growth factors of the mammalian cell proliferation. It has even been claimed that activation of the Ca(2+)-phosphatidylinositol cascade is sufficient to elicit cell proliferation [Jackson et al. (1988) Nature 335, 437-440; Julius et al. (1989) Science 244, 1057-1062]. In this work, we have evaluated the control of DNA synthesis by this cascade in a differentiated epithelial cell model: the dog thyrocyte in primary culture. We first observed that potent activators of the dog thyrocyte (2+)-phosphatidylinositol cascade such as carbachol or bradykinin failed to promote the onset of DNA synthesis in these cells. Moreover, carbachol inhibited the mitogenic effect of thyroid stimulating hormone (TSH) and of epidermal growth factor (EGF). The mitogenic effect of EGF was also reduced by bradykinin. Nevertheless, carbachol enhanced the expression of the protooncogenes c-fos and c-myc mRNAs. The time course of this enhancement was identical to the time course for the induction of c-fos and c-myc mRNAs by phorbol esters or EGF. On the other hand, in most experiments, TSH and EGF were able to trigger the onset of dog thyrocyte DNA synthesis without affecting their intracellular free Ca2+ concentration [Ca2+]i, 45Ca2+ efflux, or inositol phosphate generation. In several experiments, TSH increased the dog thyrocyte 45Ca2+ release and promoted a rise in the [Ca2+]i or the inositol phosphate accumulation but these effects were weak. In contrast to the effect of carbachol, the TSH effects on the [Ca2+]i and the 45Ca2+ efflux appeared slowly, were sustained, and were extremely sensitive to extracellular Ca2+ depletion. They were observed at hormone concentrations higher than the concentration achieving maximal stimulation of DNA synthesis. Similarly, in a few experiments, a slight increase in the [Ca2+]i or in the inositol trisphosphate generation were provoked by EGF. However, these modifications were not associated with an increased mitogenic potency of EGF. Finally, in all experiments, fetal calf serum slightly accelerated the dog thyrocyte 45Ca2+ efflux and increased their inositol phosphate generation.(ABSTRACT TRUNCATED AT 400 WORDS)

Transducing Systems in the Control of Human Thyroid Cell Function, Proliferation and Differentiation

Our laboratory has been involved in the study of thyroid regulation at the cellular level for many years. The complex picture emerging from these studies leads to conclusions of general relevance. The regulation of the thyroid cell was once a classical example of the concept one hormone — one cell type — one intracellular secondary messenger with its pleiotypic effects. It should now rather be considered as a network of crosslinked regulatory steps where the extracellular and intracellular signal-molecules act on their receptors as bits of information in an electronic circuit, i.e., express on/off regulations with no definite general physiological meaning per se. Such networks differ from one cell type to another and for a given cell type from one species to another. In the case of the thyroid many apparent discrepancies in the literature are explained if this is taken into account. In this presentation, we wish to draw mainly on the results of our group to illustrate this point with regard to the regulation of function, proliferation and differentiation of the thyroid cell.

The Control of Human Thyroid Cell Function, Proliferation and Differentiation

The study of thyroid regulation at the cellular level is the main interest of our laboratory since many years. The complex picture emerging from these studies leads to conclusions of general relevance. The regulation of the thyroid cell was once a classical example of the concept one hormone — one cell type — one intracellular secondary messenger with its pleiotypic effects. It should now rather be considered as a network of crosslinked regulatory steps where the extracellular and intracellular signal-molecules act on their receptors as bits of information in an electronic circuit, i.e., express on/off regulations with no definite general physiological meaning per se. Such networks differ from one cell type to another and for a given cell type from one species to another. In the case of the thyroid, many apparent discrepancies in the literature are explained if this is taken into account. In this presentation, we wish to draw mainly on the results of our group to illustrate this point with regard to the regulation of function, proliferation and differentiation of the thyroid cell.