Depoortere F, Van Keymeulen A, Lukas J, Costagliola S, Bartkova J, Dumont J E, Bartek J, Roger P P, Dremier S
Institute of Interdisciplinary Research, Université Libre de Bruxelles, Campus Erasme, B-1070 Brussels, Belgium.
J Cell Biol. 1998 Mar 23;140(6):1427-39. doi: 10.1083/jcb.140.6.1427.
In different systems, cyclic adenosine monophosphate (cAMP) either blocks or promotes cell cycle progression in mid to late G1 phase. Dog 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 thyrotropin (TSH). The cAMP-dependent mitogenic pathway is unique as it is independent of mitogen-activated protein kinase activation and differs from growth factor-dependent pathways at the level of the expression of several protooncogenes/transcription factors. This study examined the involvement of D-type G1 cyclins and their associated cyclin-dependent kinase (cdk4) in the cAMP-dependent G1 phase progression of dog thyroid cells. Unlike epidermal growth factor (EGF)+serum and other cAMP-independent mitogens, TSH did not induce the accumulation of cyclins D1 and D2 and partially inhibited the basal expression of the most abundant cyclin D3. However, TSH stimulation enhanced the nuclear detection of cyclin D3. This effect correlated with G1 and S phase progression. It was found to reflect both the unmasking of an epitope of cyclin D3 close to its domain of interaction with cdk4, and the nuclear translocation of cyclin D3. TSH and EGF+serum also induced a previously undescribed nuclear translocation of cdk4, the assembly of precipitable cyclin D3-cdk4 complexes, and the Rb kinase activity of these complexes. Previously, cdk4 activity was found to be required in the cAMP-dependent mitogenic pathway of dog thyrocytes, as in growth factor pathways. Here, microinjections of a cyclin D3 antibody showed that cyclin D3 is essential in the TSH/ cAMP-dependent mitogenesis, but not in the pathway of growth factors that induce cyclins D1 and D2. The present study (a) provides the first example in a normal cell of a stimulation of G1 phase progression occurring independently of an enhanced accumulation of cyclins D, (b) identifies the activation of cyclin D3 and cdk4 through their enhanced assembly and/or nuclear translocation, as first convergence steps of the parallel cAMP-dependent and growth factor mitogenic pathways, and (c) strongly suggests that this new mechanism is essential in the cAMP-dependent mitogenesis, which provides the first direct demonstration of the requirement for cyclin D3 in a G1 phase progression.
在不同的系统中，环磷酸腺苷（cAMP）在G1期中期到后期既可以阻断也可以促进细胞周期进程。原代培养的犬甲状腺上皮细胞构成了一个模型，其中cAMP作为促甲状腺激素（TSH）的第二信使，对DNA合成起始和G0-S复制前阶段进程进行正向调控。cAMP依赖性有丝分裂途径是独特的，因为它不依赖于丝裂原活化蛋白激酶的激活，并且在几个原癌基因/转录因子的表达水平上与生长因子依赖性途径不同。本研究检测了D型G1细胞周期蛋白及其相关的细胞周期蛋白依赖性激酶（cdk4）在犬甲状腺细胞cAMP依赖性G1期进程中的作用。与表皮生长因子（EGF）+血清及其他cAMP非依赖性有丝分裂原不同，TSH并未诱导细胞周期蛋白D1和D2的积累，反而部分抑制了最丰富的细胞周期蛋白D3的基础表达。然而，TSH刺激增强了细胞周期蛋白D3的核内检测。这种效应与G1期和S期进程相关。结果发现，这既反映了细胞周期蛋白D3靠近其与cdk4相互作用结构域的一个表位的暴露，也反映了细胞周期蛋白D3的核转位。TSH和EGF+血清还诱导了cdk4先前未被描述的核转位、可沉淀的细胞周期蛋白D3-cdk4复合物的组装以及这些复合物的Rb激酶活性。此前发现，与生长因子途径一样，cdk4活性在犬甲状腺细胞的cAMP依赖性有丝分裂途径中是必需的。在此，显微注射细胞周期蛋白D3抗体表明，细胞周期蛋白D3在TSH/cAMP依赖性有丝分裂中是必需的，但在诱导细胞周期蛋白D1和D2的生长因子途径中并非必需。本研究（a）在正常细胞中首次提供了一个独立于细胞周期蛋白D积累增强而刺激G1期进程的例子，（b）通过细胞周期蛋白D3和cdk4组装增强和/或核转位来确定其激活，作为平行的cAMP依赖性和生长因子有丝分裂途径的首个汇聚步骤，并且（c）强烈表明这种新机制在cAMP依赖性有丝分裂中是必不可少的，这首次直接证明了细胞周期蛋白D3在G1期进程中的必要性。
