Ablation of Cdk2 uncovers kinase-independent functions of Cyclin E1 for liver regeneration in mice

The liver has a strong capacity to regenerate in response to injury. In this process, remnant hepatocytes leave their quiescent state, re-enter the cell cycle and proliferate until liver mass and functions are restored. In vitro studies have indicated that cyclin-dependent kinase 2 (Cdk2) is an important cell cycle mediator driving initiation and progression of DNA-synthesis via complex formation with E- and A-type cyclins respectively. The aim of the current study was to evaluate the role of Cdk2 in hepatocyte proliferation. Hepatocyte-specific Cdk2 deleted mice (Cdk2^Δhepa) and wild type (WT) controls were subjected to partial hepatectomy (PH) and analyzed for liver regeneration. Following PH, liver regeneration was unaffected by the loss of Cdk2 and resulted in normal hepatocyte proliferation in Cdk2^Δhepa mice comparable to WT controls. We thus analyzed the mechanisms responsible for compensation of Cdk2-deficiency. Liver regeneration in Cdk2^Δhepa mice was associated with elevated levels of cyclin D1 (CcnD1) and cyclin E1 (CcnE1). Stronger CcnD1 expression resulted in accelerated and enhanced CcnD1-dependent kinase activity whereas over-expression of CcnE1 in Cdk2^Δhepa mice was not associated with kinase activation. A proposed and in part kinase-independent function of CcnE1 is the involvement in pre-replication complex (pre-RC) formation via interaction with minichromosome maintenance (MCM) proteins. We provide evidence that mitogenic activated, primary Cdk2^-/- hepatocytes displayed an earlier onset of pre-RC formation associated with premature expression of the CcnE1, Cdt1 and MCM2 and their physical interaction with chromatin. Futher, we show that combined inhibition of Cdk2 and Cdk1 using roscovitine efficiently blocked hepatocyte proliferation in WT and Cdk2^Dhepa mice demonstrating that hepatocyte proliferation depends on Cdk1, but not on Cdk2. However, over-expression of CcnE1 in vitro or in vivo partially rescued Roscovitine-mediated cell cycle arrest further pointing to kinase-independent functions of CcnE1 in the liver. Thus, we conclude that over-expression of CcnD1 and CcnE1 compensate Cdk2-deficiency via two mechanisms involving increased CcnD1-related kinase activity and a premature, cyclin E-dependent initiation of the pre-replication complex.