Carey Rebecca G, Li Baogang, DiCicco-Bloom Emanuel
Department of Neuroscience and Cell Biology, Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
J Neurosci. 2002 Mar 1;22(5):1583-91. doi: 10.1523/JNEUROSCI.22-05-01583.2002.
Generation of distinct cell types and numbers in developing cerebral cortex is subject to regulation by extracellular factors that positively or negatively control precursor proliferation. Although signals stimulating proliferation are well described, factors halting cell cycle progression are less well defined. At the molecular level, production and association of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) regulate cycle progression. We now report that the endogenous peptide, pituitary adenylate cyclase activating polypeptide (PACAP), negatively regulates the cell cycle by inhibiting p57Kip2-dependent CDK2 activity in embryonic cortex. Protein levels of CDK2 and members of the CIP/KIP family of CKIs (p27Kip1, p57Kip2) were detected in developing rat cortex from embryonic day 13.5 through postnatal day 2. With advancing development, CDK2 protein levels decreased, whereas CKI expression increased, suggesting that stimulatory and inhibitory cycle proteins control cell cycle exit. Using a well defined, nonsynchronized, 8 hr precursor culture, PACAP decreased the fraction of cells crossing the G1/S boundary, inhibiting DNA synthesis by 35%. CDK2 kinase activity was inhibited 75% by PACAP, whereas kinase protein and its regulatory cyclin E subunit were unaffected. Moreover, decreased kinase activity was accompanied by a twofold increase in levels of p57Kip2 protein, but not p21Cip1 or p27Kip1, suggesting that p57Kip2 mediates PACAP anti-mitogenic effects. Indeed, immunoprecipitation of CDK2 complex revealed increased p57Kip2 association with the kinase and concomitant reduction in free inhibitor after PACAP exposure, suggesting that p57Kip2 interactions directly regulate CDK2 activity. These observations establish a mechanism whereby anti-mitogenic signals actively induce cell cycle withdrawal in developing cortex.
在发育中的大脑皮层中，不同细胞类型的产生和数量受到细胞外因子的调控，这些因子对前体细胞增殖具有正向或负向控制作用。尽管刺激增殖的信号已得到充分描述，但使细胞周期进程停止的因子却不太明确。在分子水平上，细胞周期蛋白、细胞周期蛋白依赖性激酶（CDK）和CDK抑制剂（CKI）的产生及相互作用调节着细胞周期进程。我们现在报告，内源性肽垂体腺苷酸环化酶激活多肽（PACAP）通过抑制胚胎皮层中p57Kip2依赖的CDK2活性来负向调节细胞周期。在胚胎第13.5天至出生后第2天的发育中的大鼠皮层中检测到了CDK2以及CKI的CIP/KIP家族成员（p27Kip1、p57Kip2）的蛋白水平。随着发育进程的推进，CDK2蛋白水平下降，而CKI的表达增加，这表明刺激性和抑制性细胞周期蛋白控制着细胞周期退出。使用定义明确的、非同步化的8小时前体细胞培养体系，PACAP降低了跨越G1/S边界的细胞比例，使DNA合成减少了35%。PACAP使CDK2激酶活性抑制了75%，而激酶蛋白及其调节性细胞周期蛋白E亚基未受影响。此外，激酶活性降低伴随着p57Kip2蛋白水平增加两倍，但p21Cip1或p27Kip1未增加，这表明p57Kip2介导了PACAP的抗有丝分裂作用。事实上，CDK2复合物的免疫沉淀显示，PACAP作用后，p57Kip2与激酶的结合增加，游离抑制剂随之减少，这表明p57Kip2的相互作用直接调节CDK2活性。这些观察结果建立了一种机制，即抗有丝分裂信号在发育中的皮层中积极诱导细胞周期退出。
