Fernandez Rosario, Pena Emma, Navascues Joaquin, Casafont Iñigo, Lafarga Miguel, Berciano Maria T
Department of Physiology and Pharmacology, Faculty of Medicine, University of Cantabria, Santander, Spain.
Glia. 2002 Dec;40(3):378-88. doi: 10.1002/glia.10157.
It is well established that forskolin-induced elevation of cAMP results in activation of DNA synthesis in Schwann cell cultures. This promitotic response is partially mediated by the Cdk2, which is required for the transition from the G1 to the S phase of the cell cycle. In the present study, we analyze the effects of cAMP elevation in cultured Schwann cells on the transcriptional activity and on the organization of two nuclear compartments involved in pre-mRNA processing: Cajal bodies (CBs) and splicing factor compartments. Our immunofluorescence and quantitative studies show that forskolin treatment induces a 5.6-fold increase in the proportion of S phase Schwann cells, detected by a short pulse (20 min) of BrdU incorporation. This increase in DNA synthesis correlates with an activation of global transcription, as is indicated by the higher nuclear incorporation of BrU in nascent RNA. Forskolin treatment significantly increases the percentage of Schwann cells containing typical CBs, which concentrate spliceosomal snRNPs and the survival motor neuron (SMN) protein. This increase in the number of CBs closely correlates with the activation of transcription. Moreover, the occurrence of CBs is significantly higher in BrdU (+) cells than in BrdU (-) cells, indicating that entry in the S phase promotes the formation of CBs. During the S phase, Schwann cell nuclei display higher Cdk2 nuclear staining and concentrate this kinase in CBs. Forskolin also induces a redistribution of the pre-mRNA splicing factors in Schwann cells. Primary cultures of Schwann cells provide an excellent physiological model to demonstrate that the assembly of CBs is a transcription- and replication-dependent cellular event. Moreover, the S phase accumulation of Cdk2 observed in Schwann cells supports a functional link between CBs and DNA replication, which is mediated by the possible participation of CBs in the regulatory control of histone gene expression.
已充分证实，福斯高林诱导的环磷酸腺苷（cAMP）升高会导致雪旺细胞培养物中DNA合成的激活。这种促有丝分裂反应部分由细胞周期蛋白依赖性激酶2（Cdk2）介导，Cdk2是细胞周期从G1期过渡到S期所必需的。在本研究中，我们分析了培养的雪旺细胞中cAMP升高对转录活性以及参与前体mRNA加工的两个核区室组织的影响：卡哈尔体（CBs）和剪接因子区室。我们的免疫荧光和定量研究表明，通过短脉冲（20分钟）掺入溴脱氧尿苷（BrdU）检测到，福斯高林处理可使处于S期的雪旺细胞比例增加5.6倍。DNA合成的这种增加与整体转录的激活相关，新生RNA中更高的溴尿嘧啶（BrU）核掺入量表明了这一点。福斯高林处理显著增加了含有典型CBs的雪旺细胞百分比，CBs集中了剪接体小核核糖核蛋白（snRNPs）和存活运动神经元（SMN）蛋白。CBs数量的这种增加与转录激活密切相关。此外，BrdU（+）细胞中CBs的发生率明显高于BrdU（-）细胞，表明进入S期促进了CBs的形成。在S期，雪旺细胞核显示出更高的Cdk2核染色，并将这种激酶集中在CBs中。福斯高林还诱导雪旺细胞中前体mRNA剪接因子的重新分布。雪旺细胞的原代培养提供了一个极好的生理模型，以证明CBs的组装是一个依赖转录和复制的细胞事件。此外，在雪旺细胞中观察到的Cdk2的S期积累支持了CBs与DNA复制之间的功能联系，这可能是由CBs参与组蛋白基因表达的调控控制介导的。
