Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture

Abstract

The proliferation of most normal cells depends on the synergistic interaction of several growth factors and hormones, but the cell cycle basis for this combined requirement remains largely uncharacterized. We have addressed the question of the requirement for insulin/ IGF-1 also observed in many cell culture systems in the physiologically relevant system of primary cultures of dog thyroid epithelial cells stimulated by TSH, which exerts its mitogenic activity only via cAMP. The induction of cyclin A and cdc2, the phosphorylation of cdk2, the nuclear translocation of cdk4 and the assembly of cyclin D3-cdk4 complexes required the synergy of TSH and insulin. Cyclin D3 (the most abundant cyclin D) was necessary for the proliferation stimulated by TSH in the presence of insulin as shown by microinjection of a neutralizing antibody. Cyclin D3 accumulation and activity were dierentially regulated by insulin and TSH, which points out this cyclin as an integrator that ranks these comitogenic pathways as supportive and activatory, respectively. Paradoxically TSH alone strongly repressed cyclin D3 accumulation. This inhibition was overridden by insulin, which markedly stimulated cyclin D3 mRNA and protein accumulation, but failed to assemble cyclin D3-cdk4 complexes in the absence of TSH. TSH unmasked the DCS-22 epitope of cyclin D3 and assembled cyclin D3-cdk4 in the presence of insulin. These data demonstrate that cyclin D synthesis and cyclin D-cdk assembly can be dissociated and complementarily regulated by dierent agents and signalling pathways.

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

“…The different mitogenic stimulations (TSH, cAMP, growth factors) require the activity of CDK4 , and converge on the inactivating phosphorylation of pRb and related proteins p107 and p130 (Coulonval et al, 1997;Van Keymeulen et al, 1999), on the phosphorylation and nuclear translocation of CDK2, and on the induction of cyclin A and CDK1 (Baptist et al, 1996). These effects are dependent on insulin (Coulonval et al, 1997;Van Keymeulen et al, 1999).…”

“…In dog thyrocytes, the formation and the nuclear translocation of essential cyclin D3-CDK4 complexes depend on the synergistic interaction of TSH and insulin. The presence of the required cyclin D3 depends on insulin/IGF-I (Van Keymeulen et al, 1999), whereas TSH stimulates the assembly of the cyclin D3-CDK4 complexes and their import into nuclei (Depoortere et al, 1998;Van Keymeulen et al, 1999) where these complexes are anchored by their association with p27 kip1 (Depoortere et al, 2000;. This also sequesters p27 away of CDK2 complexes (Depoortere et al, 2000), thus contributing to CDK2 activation.…”

“…The investigation of cell cycle regulatory proteins has thus clearly established that both CDK4 activation and pRb phosphorylation result from distinct but complementary actions of TSH and insulin, rather than from their interaction at an earlier step of the signalling cascades (Kimura et al, 2001;Van Keymeulen et al, 1999;Van Keymeulen et al, 2001) (Fig. 1A and 3).…”

Distinct Specificities of pRb Phosphorylation by CDK4 Activated by Cyclin D1 or Cyclin D3: Differential Involvement in the Distinct Mitogenic Modes of Thyroid Epithelial Cells

“…23 The expression of cyclin D3 supported by facilitative comitogenic factors (insulin and carbachol) is specifically required in the cAMP-dependent cell cycle, but not in the mitogenic stimulation by growth factors that induce cyclin D1. 23,49 cAMP promotes the assembly of cyclin D3-CDK4 complexes, 23,49 their nuclear import associated with their binding to p27, 50,28 and then stimulates the Thr172 phosphorylation and activity of CDK4 within this complex. 25 In this study, two new essential features of these distinct mitogenic modes are reported, initially deriving from the same experiments.…”

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

“…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.…”