Garnier Delphine, Loyer Pascal, Ribault Catherine, Guguen-Guillouzo Christiane, Corlu Anne
Institut National de la Santé et de la Recherche Médicale, U522, Centre Hospitalier Universitaire Pontchaillou, Rennes, France.
Hepatology. 2009 Dec;50(6):1946-56. doi: 10.1002/hep.23225.
Liver regeneration is a unique process to restore hepatic homeostasis through rapid and synchronous proliferation of differentiated hepatocytes. Previous studies have shown that hepatocyte proliferation is characterized by high expression levels of the "mitotic" cyclin-dependent kinase 1 (Cdk1) during S-phase compared to other mammalian cells. In the light of findings showing that Cdk1 compensates for the loss of Cdk2 and drives S-phase in Cdk2-deficient cells derived from Cdk2 knockout mice, we took advantage of the models of liver regeneration following partial hepatectomy and primary cultures of normal rat hepatocytes to further examine the involvement of Cdk1 during DNA replication in hepatocytes and to dissect specific cell cycle regulation in hepatocytes compared to control human foreskin fibroblasts. In hepatocytes, Cdk1 exhibited a biphasic activation pattern correlating S-phase and G(2)/M transition, bound to cyclin A or B1 and localized to the nucleus during DNA replication. Importantly, small interfering RNA (siRNA)-mediated silencing of Cdk1 led to a strong decrease in DNA synthesis without affecting centrosome duplication. Furthermore, in hepatocytes arrested by the iron chelator O-Trensox in early S-phase prior to DNA replication, Cdk1/cyclin complexes were active, while replication initiation components such as the minichromosome maintenance 7 (Mcm7) protein were loaded onto DNA. Moreover, Mcm7 expression and loading onto DNA were not modified by Cdk1 silencing. Conversely, in fibroblasts, Cdk1 expression and activation were low in S-phase and its silencing did not reduce DNA synthesis.
Cdk1 is essential for DNA replication downstream formation of replication initiation complexes in hepatocytes but not in fibroblasts and, as such, our data exemplify crucial differences in the cell cycle regulation between various mammalian cell types.
肝脏再生是一个独特的过程，通过分化的肝细胞快速同步增殖来恢复肝脏内稳态。先前的研究表明，与其他哺乳动物细胞相比，肝细胞增殖的特征是在S期“有丝分裂”细胞周期蛋白依赖性激酶1（Cdk1）表达水平较高。鉴于有研究发现Cdk1可补偿Cdk2的缺失并驱动来自Cdk2基因敲除小鼠的Cdk2缺陷细胞进入S期，我们利用部分肝切除术后肝脏再生模型和正常大鼠肝细胞原代培养，进一步研究Cdk1在肝细胞DNA复制过程中的作用，并剖析与对照人包皮成纤维细胞相比肝细胞中特定的细胞周期调控机制。在肝细胞中，Cdk1呈现双相激活模式，与S期和G(2)/M期转换相关，在DNA复制期间与细胞周期蛋白A或B1结合并定位于细胞核。重要的是，小干扰RNA（siRNA）介导的Cdk1沉默导致DNA合成显著减少，而不影响中心体复制。此外，在DNA复制前被铁螯合剂O-Trensox阻滞在早S期的肝细胞中，Cdk1/细胞周期蛋白复合物具有活性，而诸如微小染色体维持蛋白7（Mcm7）等复制起始成分则加载到DNA上。而且，Mcm7的表达及其加载到DNA上的过程不受Cdk1沉默的影响。相反，在成纤维细胞中，Cdk1在S期的表达和激活水平较低，其沉默不会降低DNA合成。
Cdk1对于肝细胞中复制起始复合物下游的DNA复制至关重要，而对成纤维细胞则不然，因此，我们的数据例证了不同哺乳动物细胞类型在细胞周期调控方面的关键差异。
