Respective Roles of Carbamylcholine and Cyclic Adenosine Monophosphate in Their Synergistic Regulation of Cell Cycle in Thyroid Primary Cultures¹

Abstract

The stimulation of thyroid cell proliferation by TSH through cAMP depends on permissive comitogenic factors, generally the insulin-like growth factors and insulin. In dog thyroid primary cultures, the use of the phosphodiesterase-resistant analog of cAMP (Bu) 2 cAMP instead of TSH allowed to unveil a potent comitogenic activity of carbamylcholine, which can substitute for insulin and was shown to mimic insulin action on cell cycle regulatory proteins. Like insulin, carbamylcholine induced the accumulation of cyclin D3 and overcame the repression by cAMP of this protein, which was shown 1) to be essential for cell cycle progression by means of microinjections of a neutralizing antibody; and 2) to be rate limiting for the cAMPdependent assembly of cyclin D3-cdk4 complexes, their nuclear translocation and the phosphorylation of pRb. Relative to insulin, carbamylcholine offers the significant experimental advantage that its signaling cascades can be immediately deactivated by the muscarinic antagonist atropine. In the presence of carbamylcholine, the elimination of (Bu) 2 cAMP blocked within 2 h the entry of cells into DNA synthesis phase, but the addition of atropine still permitted the entry of cells in S phase. These data support our view that the progression in G1 phase stimulated by cAMP consists of at least two essential actions that are clearly dissociated: in a first stage, depending on the supportive activity of an agent that stimulates the required cyclin D3 accumulation, cAMP induces the assembly and nuclear translocation of cyclin D3-cdk4 complexes, and then cAMP can exert alone the last crucial control that determines the cell commitment toward DNA replication. (Endocrinology 142: [1251][1252][1253][1254][1255][1256][1257][1258][1259] 2001) D OG THYROID epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for TSH (1). In this system, as well as in human thyrocytes and in the FRTL-5 rat thyroid cell line, the stimulation of DNA synthesis and proliferation by TSH depends on the presence of IGF-1 (insulin-like growth factor 1) or insulin (2-4). As the real physiological stimulator of thyroid cells is TSH, the comitogenic role of insulin or IGF-1 is described as permissive for the proliferative action of TSH in dog and human thyrocytes (5). However, in various in vitro thyroid models the respective roles of TSH and insulin/IGF-1 are a matter of major controversy. A central question is whether TSH and insulin/IGF-1 through distinct signaling cascades exert complementary functions required for cell proliferation, or whether one of these factors exerts priming actions making the cell more competent to respond to the other one.In dog thyrocytes primary culture, the roles of TSH (cAMP) and insulin/IGF-1 on cell cycle progression have been recently found to be distinct and complementary (6). Their signaling pathways are largely independent. TSH, unlike insulin/IGF-1 and/or growth fa...