I. CELL PROLIFERATION IN NORMAL THYROID TISSUE cles and of their supporting mesenchymal tissue and IN VIVO cells, the endothelial cells of the capillaries (20%) and the fibroblasts (10%) (78). Scarce calcitonin-secreting A. Physiological Situation parafollicular cells are located at the periphery of the follicles. After its differentiation in the fetus, the tissue The thyroid tissue is mainly composed of thyroid grows roughly in parallel with body weight and remains follicular cells, the thyrocytes (70954, arranged in folli-at the same size throughout adult life.

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Cyclin-dependent kinase 4 (CDK4) is a master integrator of mitogenic and antimitogenic extracellular signals. It is also crucial for many oncogenic transformation processes. Various molecular features of CDK4 activation remain poorly known or debated, including the regulation of its association with D-type cyclins, its activating Thr172 phosphorylation, and the roles of Cip/Kip CDK "inhibitors" in these processes. Thr172 phosphorylation of CDK4 was reinvestigated using two-dimensional gel electrophoresis in various experimental systems, including human fibroblasts, canine thyroid epithelial cells stimulated by thyrotropin, and transfected mammalian and insect cells. Thr172 phosphorylation of CDK4 depended on prior D-type cyclin binding, but Thr172 phosphorylation was also found in p16-bound CDK4. Opposite effects of p27 on cyclin D3-CDK4 activity observed in different systems depended on its stoichiometry in this complex. Thr172-phosphorylated CDK4 was enriched in complexes containing p21 or p27, even at inhibitory levels of p27 that precluded CDK4 activity. Deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4 in the cytoplasm but did not affect CDK4 phosphorylation. Within cyclin D3 complexes, T-loop phosphorylation of CDK4, but not of CDK6, was directly regulated, identifying it as a determining target for cell cycle control by extracellular factors. Collectively, these unexpected observations indicate that CDK4-activating kinase(s) should be reconsidered.Cyclin-dependent kinase 4 (CDK4) and CDK6 act in G 1 phase as a master integrator of various mitogenic and antimitogenic signals (76, 80). They phosphorylate and inactivate the cell cycle/tumor suppressor proteins of the pRb family (p105 Rb , p107, and p130 Rb2 ) (6,21,22,39,49,92) and Smad3 (55). CDK4 activity is deregulated in many human tumors (61, 77) and was recently found to be crucial for various oncogenic transformation processes (43,56,84,88). Understanding CDK4 regulation is thus of fundamental importance.As initially considered, mitogens activate CDK4/6 by inducing at least one D-type cyclin (D1, D2, and D3) to concentrations allowing an inhibitory threshold imposed by INK4 CDK4/6 inhibitory proteins to be overcome (76). These proteins (p15, p16, p18, and p19) bind to the catalytic domain of the isolated CDK4/6, preventing cyclin association and thus its activation (25,65,78). The functions of CDK inhibitors of the CIP/KIP family (p21 Cip1 , p27 Kip1 , and p57 Kip2 ) in the activation of D-type cyclin-CDK complexes are more complex and debated. Their down-regulation by mitogenic factors and/or their titration by D-type cyclin-CDK complexes participates in cyclin E/A-CDK2 activation (70,78,79). Mostly in in vitro experiments, p21 and p27 were initially observed to similarly inhibit CDK4 activity (26,40,67). Nevertheless, p21 is transiently induced in G 1 by mitogenic factors in different cell systems (42,51,93). Moreover, p21 and p27 were found to be associated with a pRb kinase activity (7,11,44,83), to stabilize cycl...

TSH via cAMP, and various growth factors, in cooperation with insulin or IGF-I stimulate cell cycle progression and proliferation in various thyrocyte culture systems, including rat thyroid cell lines (FRTL-5, WRT, PC Cl3) and primary cultures of rat, dog, sheep and human thyroid. The available data on cell signaling cascades, cell cycle kinetics, and cell cycle-regulatory proteins are thoroughly and critically reviewed in these experimental systems. In most FRTL-5 cells, TSH (cAMP) merely acts as a priming/competence factor amplifying PI3K and MAPK pathway activation and DNA synthesis elicited by insulin/IGF-I. In WRT cells, TSH and insulin/IGF-I can independently activate Ras and PI3K pathways and DNA synthesis. In dog thyroid primary cultures, TSH (cAMP) does not activate Ras and PI3K, and cAMP must be continuously elevated by TSH to directly control the progression through G(1) phase. This effect is exerted, at least in part, via the cAMP-dependent activation of the required cyclin D3, itself synthesized in response to insulin/IGF-I. This and other discrepancies show that the mechanistic logics of cell cycle stimulation by cAMP profoundly diverge in these different in vitro models of the same cell. Therefore, although these different thyrocyte systems constitute interesting models of the wide diversity of possible mechanisms of cAMP-dependent proliferation in various cell types, extrapolation of in vitro mechanistic data to TSH-dependent goitrogenesis in man can only be accepted in the cases where independent validation is provided.