Hauser P J, Agrawal D, Flanagan M, Pledger W J
Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37240, USA.
Cell Growth Differ. 1997 Feb;8(2):203-11.
We have studied the regulation of cyclins and cyclin-dependent kinase activities during differentiation of primary mouse keratinocytes. Differentiation was induced by placing primary murine keratinocytes into suspension culture, under conditions which prevent cells from attaching to any surface. This treatment induces synthesis of keratin 1, one of the earliest known markers of keratinocyte differentiation, and also results in a profound change in the regulation of G1 and S-phase cyclins and their associated proteins as well as their activities. The placement of cells in suspension culture reduced cyclin A, D1, and E kinase activity within 6 h, accompanied by the cessation of DNA synthesis. K1 mRNA levels were observed to increase after this period, supporting the hypothesis that cell cycle withdrawal precedes the differentiation program. Our data further revealed that the p27kip1 protein level and associated cyclin-dependent kinase inhibitory activity increased when keratinocytes were induced to differentiate. Pretreatment of adherent keratinocytes with p27kip1 antisense oligonucleotides dramatically reduced the accumulation of p27kip1 protein upon subsequent suspension culturing and prevented the onset of differentiation independently of the loss of cyclin-dependent kinase activities. Although antisense oligonucleotide treatment inhibited differentiation, it did not prevent growth arrest. Therefore, the differentiation of primary mouse keratinocytes required a function of Kip other than the inhibition of cyclin-associated activities, and we suggest that this requirement may reflect a novel Rb kinase activity present in Kip immune complexes, which is dependent on the presence of cyclin D3. Thus, the placement of keratinocytes in suspension induces a program that includes loss of cyclin activity, which is linked to terminal growth arrest, and an induction of p27kip1, which is linked to the differentiation program.
我们研究了原代小鼠角质形成细胞分化过程中细胞周期蛋白及细胞周期蛋白依赖性激酶活性的调控。将原代小鼠角质形成细胞置于悬浮培养中诱导分化，培养条件可防止细胞附着于任何表面。这种处理诱导了角蛋白1的合成，角蛋白1是已知最早的角质形成细胞分化标志物之一，同时也导致G1期和S期细胞周期蛋白及其相关蛋白的调控以及它们的活性发生深刻变化。将细胞置于悬浮培养中6小时内，细胞周期蛋白A、D1和E激酶活性降低，同时DNA合成停止。在此之后观察到K1 mRNA水平升高，支持了细胞周期退出先于分化程序的假说。我们的数据进一步显示，当角质形成细胞被诱导分化时，p27kip1蛋白水平及相关的细胞周期蛋白依赖性激酶抑制活性增加。用p27kip1反义寡核苷酸预处理贴壁角质形成细胞，可显著降低随后悬浮培养时p27kip1蛋白的积累，并独立于细胞周期蛋白依赖性激酶活性的丧失而阻止分化的开始。尽管反义寡核苷酸处理抑制了分化，但并未阻止生长停滞。因此，原代小鼠角质形成细胞的分化需要Kip的一种功能，而不是抑制细胞周期蛋白相关活性，我们认为这种需求可能反映了Kip免疫复合物中存在的一种新的Rb激酶活性，它依赖于细胞周期蛋白D3的存在。因此，将角质形成细胞置于悬浮培养中可诱导一个程序，该程序包括细胞周期蛋白活性丧失，这与终末生长停滞有关，以及p27kip1的诱导，这与分化程序有关。
