Phelps D E, Xiong Y
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599, USA.
Cell Growth Differ. 1998 Aug;9(8):595-610.
Terminal differentiation of many cell lineages involves an exit from the mitotic cycle and entry into, and maintenance of, a permanent state of G1 arrest. We found that during terminal differentiation of mouse 3T3-L1 preadipocytes, the level of cyclin-dependent kinase 4 (CDK4) remained constant, but the subunit composition of the CDK4 complex underwent a dynamic rearrangement. As 3T3-L1 cells differentiated, the levels of cyclin D1 and cyclin D1-CDK4 complexes declined to negligible levels. Meanwhile, cyclins D2 and D3 levels and their associations with CDK4 increased transiently and persistently, respectively, with cyclin D3 becoming the predominant cyclin partner of CDK4 in mature adipocytes. At least five CDK inhibitors are expressed during the differentiation program of 3T3-L1 cells. Both p15INK4b and p16INK4a continuously declined to undetectable levels immediately after differentiation induction. p21 was transiently expressed during the exit of 3T3-L1 cells from mitotic clonal expansion and then decreased to undetectable levels in mature adipocytes. The level of p27KiP1 and p27-CDK4 complexes remain high during differentiation and in mature adipocytes. Distinctly, there is a remarkable induction of p18INK4c mRNA and protein that was not seen in the closely related nondifferentiating 3T3-C2 cell line, suggesting that p18 induction in 3T3-L1 cells is related to cell differentiation, not cell cycle arrest. The pRb kinase activity of cyclin D3 and CDK4 was not detected in quiescent 3T3-L1 cells and was then induced as the cells entered the mitotic clonal expansion phase. Unexpectedly, cyclin D3 and CDK4 pRb kinase activity remained high after 3T3-L1 cells completed their mitotic division and was still readily detectable in mature adipocytes. Our study reveals an active regulation, rather than passive inhibition, of CDK4 activity during adipocyte differentiation. Two central features of this complex regulation are switching of activating cyclin D subunits and concurrent binding by the p18 and p27 CDK inhibitors.
许多细胞谱系的终末分化涉及退出有丝分裂周期，并进入并维持永久性的G1期阻滞状态。我们发现，在小鼠3T3-L1前脂肪细胞的终末分化过程中，细胞周期蛋白依赖性激酶4（CDK4）的水平保持恒定，但其复合物的亚基组成发生了动态重排。随着3T3-L1细胞的分化，细胞周期蛋白D1和细胞周期蛋白D1-CDK4复合物的水平下降到可忽略不计的水平。与此同时，细胞周期蛋白D2和D3的水平及其与CDK4的结合分别短暂且持续增加，细胞周期蛋白D3成为成熟脂肪细胞中CDK4的主要细胞周期蛋白伴侣。在3T3-L1细胞的分化过程中至少表达了五种CDK抑制剂。分化诱导后，p15INK4b和p16INK4a均持续下降至无法检测的水平。p21在3T3-L1细胞从有丝分裂克隆扩增退出过程中短暂表达，然后在成熟脂肪细胞中降至无法检测的水平。在分化过程中和成熟脂肪细胞中，p27KiP1和p27-CDK4复合物的水平仍然很高。明显的是，p18INK4c mRNA和蛋白有显著诱导，而在密切相关的未分化3T3-C2细胞系中未观察到这种现象，这表明3T3-L1细胞中p18的诱导与细胞分化有关，而不是与细胞周期阻滞有关。在静止的3T3-L1细胞中未检测到细胞周期蛋白D3和CDK4的pRb激酶活性，当细胞进入有丝分裂克隆扩增阶段时其活性被诱导。出乎意料的是，3T3-L1细胞完成有丝分裂后，细胞周期蛋白D3和CDK4的pRb激酶活性仍然很高，并且在成熟脂肪细胞中仍然很容易检测到。我们的研究揭示了脂肪细胞分化过程中CDK4活性的主动调节，而不是被动抑制。这种复杂调节的两个核心特征是激活细胞周期蛋白D亚基的转换以及p18和p27 CDK抑制剂的同时结合。
