Non-canonical functions of Cyclin E1 for control of hepatic DNA replication and liver regeneration in mice

Canonical cell cycle activation in quiescent hepatocytes prior to liver regeneration involves complex formation of cyclin E1 (CcnE1) with its associated cyclin-dependent kinase 2 (Cdk2), thereby initiating DNA-replication (S-phase). Surprisingly, we previously found that unique loss of either Cdk2 or CcnE1 does not affect hepatocyte proliferation after partial hepatectomy (PH). Thus, the aim of the current study was to evaluate the compensatory mechanisms driving liver regeneration in absence of Cdk2. Formation of the pre-replication complex (pre-RC) and loading of minichromosome maintenance (MCM) proteins on chromatin is a requirement for S-phase initiation. Unique ablation of Cdk2 in mitogenic-stimulated hepatocytes was associated with a premature, CcnE1-dependent initiation of the pre-RC. We assumed that a yet unknown kinase-independent function of CcnE1 may compensate Cdk2-deficiency. To test this hypothesis, we generated CcnE1/Cdk2 double knockout mice (DKO). These mice were subjected to PH and analyzed for hepatocyte proliferation and liver regeneration in comparison to wildtype (WT) controls. Following PH, S-phase in DKO liver and subsequent liver mass reconstitution was dramatically reduced compared to WT controls or single knockout mice. However, all DKO mice survived liver resection for at least 7 days. Combined loss of Cdk2 and CcnE1 inhibited activation of the S-phase mediator cyclin A (CcnA) in the liver following PH or in primary hepatocytes after mitotic stimulation with EGF. In line with these findings, CcnA/Cdk1 kinase activity was dramatically reduced in DKO after PH. In vitro experiments using mitogenic activated primary hepatocytes revealed that loading of MCM proteins on pre-RC was also substantially impaired in Cdk2/CcnE1 DKO hepatocytes. We conclude that CcnE1 performs kinase-independent functions in hepatocytes which are capable of driving MCM loading, CcnA expression and thus S-phase progression in the absence of Cdk2.