Jirawatnotai Siwanon, Moons David S, Stocco Carlos O, Franks Roberta, Hales Dale B, Gibori Geula, Kiyokawa Hiroaki
Department of Molecular Genetics, University of Illinois College of Medicine, Chicago, Illinois 60607, USA.
J Biol Chem. 2003 May 9;278(19):17021-7. doi: 10.1074/jbc.M301206200. Epub 2003 Feb 27.
The timing of cellular exit from the cell cycle during differentiation is specific for each cell type or lineage. Granulosa cells in the ovary establish quiescence within several hours after the ovulation-inducing luteinizing hormone surge, whereas they undergo differentiation into corpora lutea. The expression of Cdk inhibitors p21(Cip1/Waf1) and p27(Kip1) is up-regulated during this process, suggesting that these cell cycle inhibitors are involved in restricting proliferative capacity of differentiating granulosa cells. Here we demonstrate that the lack of p27(Kip1) and p21(Cip1) synergistically renders granulosa cells extended an proliferative life span. Immunohistochemical analyses demonstrated that corpora lutea of p27(Kip1), p21(Cip1) double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27(Kip1) single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21(Cip1)-deficient mice ceased proliferation similarly to those in wild-type mice. Interestingly, granulosa cells isolated from p27(Kip1), p21(Cip1) double-null mice exhibited markedly prolonged proliferative life span in culture, unlike cells with other genotypes. Cultured p27(Kip1), p21(Cip1) double-null granulosa cells maintained expression of steroidogenic enzymes and gonadotropin receptors through 8-10 passages and could undergo further differentiation in responses to cAMP accumulation. Thus, the cooperation of p27(Kip1) and p21(Cip1) is critical for withdrawal of granulosa cells from the cell cycle, in concert with luteal differentiation and possibly culture-induced senescence.
细胞在分化过程中退出细胞周期的时间对于每种细胞类型或谱系而言都是特定的。卵巢中的颗粒细胞在诱导排卵的促黄体生成素激增后的数小时内进入静止状态，随后它们会分化为黄体。在此过程中，细胞周期蛋白依赖性激酶（Cdk）抑制剂p21（Cip1 / Waf1）和p27（Kip1）的表达上调，这表明这些细胞周期抑制剂参与限制分化中的颗粒细胞的增殖能力。在此，我们证明p27（Kip1）和p21（Cip1）的缺失协同作用使颗粒细胞的增殖寿命延长。免疫组织化学分析表明，p27（Kip1）、p21（Cip1）双敲除小鼠的黄体中有大量细胞掺入溴脱氧尿苷且增殖细胞核抗原表达较高，这比p27（Kip1）单敲除小鼠中显示出适度增殖增强的情况更为显著。相比之下，p21（Cip1）缺陷小鼠中正在分化的颗粒细胞与野生型小鼠中的细胞一样停止增殖。有趣的是，与其他基因型的细胞不同，从p27（Kip1）、p21（Cip1）双敲除小鼠中分离出的颗粒细胞在培养中表现出明显延长的增殖寿命。培养的p27（Kip1）、p21（Cip1）双敲除颗粒细胞在8 - 10代中维持类固醇生成酶和促性腺激素受体的表达，并能在cAMP积累的反应中进一步分化。因此，p27（Kip1）和p21（Cip1）的协同作用对于颗粒细胞退出细胞周期至关重要，这与黄体分化以及可能的培养诱导衰老相一致。
