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

The proliferation of most normal cells depends on the synergistic interacti on of several growth factors and hormones, but the cell cycle basis for thi s combined requirement remains largely uncharacterized, We have addressed t he 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 mitoge nic activity only via cAMP, The induction of cyclin A and cdc2, the phospho rylation of cdk2, the nuclear translocation of cdk4 and the assembly of cyc lin D3-cdk4 complexes required the synergy of TSH and insulin. Cyclin D3 (t he most abundant cyclin D) was necessary for the proliferation stimulated b y TSH in the presence of insulin as shown by microinjection of a neutralizi ng antibody, Cyclin D3 accumulation and activity mere differentially regula ted 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 i nhibition was overridden by insulin, which markedly stimulated cyclin D3 mR NA and protein accumulation, but failed to assemble cyclin D3-cdk4 complexe s in the absence of TSH. TSH unmasked the DCS-22 epitope of cyclin D3 and a ssembled cyclin D3-cdk4 in the presence of insulin. These data demonstrate that cyclin D synthesis and cyclin D-cdk assembly can be dissociated and co mplementarily regulated by different agents and signalling pathways.