The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G0/G1-S cell-cycle transition of primed hepatocytes is regulated by complexes of cyclin-dependent kinase 2 (Cdk2) with E-type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E-cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for liver regeneration after partial hepatectomy (PH) by generating corresponding double- and triple-knockout (KO) mouse mutants. We demonstrate that conditional deletion of Cdk2 alone in hepatocytes resulted in accelerated induction of CcnE1, but otherwise normal initiation of S phase in vivo and in vitro. Excessive CcnE1 did not contribute to a noncanonical kinase activity, but was located at chromatin together with components of the pre-replication complex (pre-RC), such as the minichromosome maintenance (MCM) helicase. Concomitant ablation of Cdk2 and CcnE1 in hepatocytes caused a defect in pre-RC formation and further led to dramatically impaired S-phase progression by down-regulation of cyclin A2 and cell death in vitro and substantially reduced hepatocyte proliferation and liver regeneration after PH in vivo. Similarly, combined loss of CcnE1 and CcnE2, but also the Cdk2/CcnE1/CcnE2 triple KO in liver, significantly inhibited S-phase initiation and liver mass reconstitution after PH, whereas concomitant ablation of CcnE2 and Cdk2 had no effect. Conclusion: In the absence of Cdk2, CcnE1 performs crucial kinase-independent functions in hepatocytes, which are capable of driving MCM loading on chromatin, cyclin A2 expression, and S-phase progression. Thus, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S-phase machinery in vivo. (Hepatology 2014;59:651–660)
The mammalian cell-cycle machinery is controlled by cyclin-dependent kinases (Cdks) and cyclins (Ccn), which act as Cdk-regulatory subunits. In the presence of extracellular mitogenic signals, D-type cyclins (CcnD1, CcnD2, and CcnD3) drive G1-phase progression through activation of Cdk4 and Cdk6, leading to phosphorylation and thus inactivation of the retinoblastoma protein (Rb). Rb phosphorylation is completed by CcnE/Cdk2 kinase complexes shortly before entry into S phase, eventually leading to activation of E2F transcription factors and induction of cell-cycle–related genes. After initiation of DNA synthesis, Cdk2 interacts with CcnA and triggers S-phase progression. Accurate DNA replication depends on assembly of pre-replicative complexes (pre-RCs), which are formed immediately after exit from mitosis in continuously dividing cells, or in the G1 phase of previously quiescent cells. Pre-RC formation involves recruitment of the origin recognition complex (ORC), chromatin licensing and DNA replication factor 1 (Cdt1), and Cdc6 to replication origins and subsequent loading of the minichromosome maintenance complex (MCM2-7), which acts as the replicative helicase.[5-8]
Surprisingly, genetic knockout (KO) experiments in mice revealed that none of the interphase Cdks (Cdk2, Cdk4, and Cdk6) are essential for cell proliferation or development in vivo.[9, 10] Moreover, Cdk2 and Cdk4 are largely dispensable for liver regeneration.[11-13] Similarly, single genetic inactivation of D-type cyclins (CcnD1-3) or E-type cyclins (CcnE1-2) does not affect viability or development in mice.[14, 15] However, E-cyclins were shown to be essential for the transition from the quiescent state into the active cell cycle, because CcnE1/E2 double-deficient fibroblasts are unable to reenter the cell cycle from starvation-induced quiescence.
Liver regeneration after partial (70%) hepatectomy (PH) in mice is one of the best experimental models for analyzing cell-cycle regulation in vivo. Because of the high regenerative capacity of the liver, resting remnant hepatocytes leave their quiescent state and undergo one to two synchronized rounds of cell cycle with a peak of DNA synthesis after approximately 40-48 hours, resulting in restoration of original liver mass within 7-10 days. We have recently demonstrated that CcnE1 and CcnE2 have unique functions after PH and even play antagonistic roles. CcnE2−/− livers showed accelerated and sustained DNA synthesis and hepatomegaly, whereas ablation of CcnE1 provoked only a minor delay of hepatocyte proliferation.
Here, we aimed to define the relevance of all Cdk2/CcnE complex components (Cdk2, CcnE1, and CcnE2) for DNA synthesis and cell-cycle reentry in vivo. We generated viable mouse mutants harboring all possible combinations of CcnE1/CcnE2 and Cdk2 KO alleles and studied the consequences for hepatocyte proliferation in vitro and in vivo. We demonstrate that CcnE1—but not its homolog, CcnE2—is essential for driving G0/S-phase transition in hepatocytes lacking Cdk2 and identify minimal genetic requirements concerning Cdk2 and E-cyclins to drive the hepatic cell cycle.
The classical paradigm of mammalian cell-cycle control suggested that Cdk2 and its associated cyclins should be absolutely essential for G0/G1/S transition. However, extensive work on gene KO mouse models in vivo revealed that Cdk2, CcnE1, and CcnE2 are largely dispensable for mitotic cell-cycle regulation, presumably because of high redundancy and overlapping functions of residual interphase Cdks and cyclins.
Here, we used the model of PH in mice to study S-phase reentry and cell-cycle progression in vivo. For our study, we used combinations of conditional and constitutive mouse mutants that allowed the stepwise deletion of the complete CcnE/Cdk2 complex, including CcnE1, CcnE2, and Cdk2, in hepatocytes in vitro and in vivo. Our ultimate aim was to identify the minimal requirements essential for blocking S-phase initiation in liver.
As a starting point, we generated mice with conditional Cdk2 deletion specifically in hepatocytes. In agreement with earlier studies using constitutive Cdk2 KO mice, we confirmed that hepatocytes do not require Cdk2 for S-phase transition and liver regeneration after PH. However, our experiments revealed several novel findings. In WT cells, Cdk2 forms kinase complexes with CcnE (CcnE1 or CcnE2) at the late G1 phase and completes Rb phosphorylation. Surprisingly, Rb phosphorylation (Ser807/811) was performed in Cdk2Δhepa mice to the same extent as observed in WT mice after PH. Our systematic analysis of residual Cdk activities suggests that early Cdk2 function (36 hours post-PH) is substituted by CcnD1/Cdk4 and CcnD1/Cdk6 kinases, whereas at the peak of G0/S-phase transition (48 hours post-PH), only Cdk1 kinase activity was detectable in Cdk2Δhepa liver. Therefore, we conclude that Cdk1 substitutes loss of Cdk2 in this setting, presumably by interacting with residual CcnA2. It has been suggested that in the absence of Cdk2, more CcnA2 molecules are available for binding to Cdk1 and thus enhance Cdk1 activity. Consistent with this idea, a recent study using hepatocyte-specific Cdk1 KO mice demonstrated that livers lacking Cdk1 have elevated Cdk2/CcnA2 activity resulting from enhanced binding of CcnA2 to Cdk2.
One of our key findings was the accelerated induction and enhanced expression of CcnE1 in regenerating livers and primary hepatocytes of Cdk2Δhepa mice. Previous work demonstrated that in the absence of Cdk2, G1/S-phase transition is regulated by a noncanonical interaction of CcnE1 with Cdk1 at least in spleen and thymus. However, we did not detect any CcnE1-related noncanonical kinase activity in Cdk2-deficient liver after PH. Instead, this excessive CcnE1 physically interacts with Cdt1 and is predominantly localized at pre-RCs on chromatin. This kinase-independent, enhanced association of CcnE1 with chromatin is absolutely essential for S-phase reentry in Cdk2-deficient hepatocytes, because combined ablation of Cdk2 and CcnE1 abolished MCM loading and induction of the S phase. In addition, isolated hepatocytes lacking Cdk2 and CcnE1 revealed poor survival after onset of the S phase, which was not observed in vivo, as discussed below. Therefore, we conclude that CcnE1 contributes to liver regeneration in a kinase-independent manner in Cdk2Δhepa mice through enhanced MCM loading.
Our data further indicate that CcnE1 is required for inducing CcnA2 and CcnE2 gene expression in a Cdk2-independent manner, although the underlying mechanisms remain, in part, unclear. Individual loss of Cdk2 allowed normal CcnA2/CcnE2 gene induction in liver after PH and in mitogen-stimulated hepatocytes, whereas combined ablation of both factors resulted in a substantial inhibition of CcnA2 and CcnE2. Our data suggest that CcnE1 physically interacts with the CcnA2 promoter, whereas concomitant lack of CcnE1 and Cdk2 completely abrogated the formation of a yet unknown protein complex at a well-characterized E2F-binding element described earlier. Therefore, we speculate that in the absence of Cdk2, CcnE1 might be important for the activation of the CcnA2 promoter and may also regulate CcnE2 expression through a similar mechanism, as indicated by our earlier studies. However, detailed analysis of the CcnA2/CcnE2 promoters was beyond the scope of this study and will be addressed in future work. We conclude that S-phase inhibition in cells lacking Cdk2 and CcnE1 is the consequence of at least two independent mechanisms involving poor MCM loading and blocking of transcriptional CcnA2 and CcnE2 activation.
In agreement with our results from isolated hepatocytes, Cdk2ΔhepaCcnE1−/− livers reveal strongly impaired CcnA2 expression, resulting in substantially reduced S phase and poor liver mass reconstitution. However, we did not observe excessive cell death or enhanced liver injury after PH, as anticipated from our results in primary hepatocytes. Because Cdk2 is only deleted in albumin-expressing parenchymal cells, we hypothesize that, in vivo, Cdk2-expressing progenitor cells could contribute to residual hepatic cell proliferation, improved survival, and liver function until these cells are fully differentiated into mature (albumin-expressing), but Cdk2-deficient hepatocytes. This hypothesis is supported by transient Cdk2 reexpression and significantly higher expression of AFP and Sox9 in Cdk2ΔhepaCcnE1−/− mice. Involvement of Sox9-positive precursors in liver regeneration after injury was recently demonstrated. Thus, we speculate that Sox9- or AFP-positive, Cdk2-expressing progenitor cells could contribute to residual cell proliferation Cdk2ΔhepaCcnE1−/− liver. Besides that, our data suggest that partial liver mass reconstitution in Cdk2ΔhepaCcnE1−/− mice is mediated through hypertrophic cell growth of hepatocytes.
The contribution of CcnE2 for liver regeneration appears complex. Cdk2ΔhepaCcnE2−/− mice displayed normal DNA synthesis and liver regeneration, pointing to a negligible role of CcnE2 for this process. CcnE2 induction was already largely abolished in Cdk2ΔhepaCcnE1−/− hepatocytes after mitogen stimulation. This could explain why additional genetic ablation of CcnE2 in Cdk2ΔhepaCcnE1ΔhepaCcnE2−/− mice did not result in further impairment of S phase and liver regeneration after PH, but rather reflected the phenotype observed in Cdk2ΔhepaCcnE1−/− mice. However, the severely impaired DNA replication and liver regeneration observed in CcnE1ΔhepaCcnE2−/− mice—which was not observed in CcnE1−/− or CcnE2−/− mice17—clearly demonstrated that CcnE2 shares at least some overlapping functions with CcnE1. This result represents the first in vivo confirmation of recent in vitro studies, which demonstrated that combined genetic ablation of both CcnE1 and CcnE2 blocks cell-cycle reentry of mouse embryonic fibroblasts from quiescence.
Our main conclusions are illustrated in Supporting Fig 6. In WT hepatocytes, Cdk2 interacts with CcnE1, CcnE2, and CcnA2 and contributes to G1/S transition and S-phase progression, respectively. In addition, CcnE1 performs a second, Cdk2-independent function in MCM loading. In the absence of CcnE1, CcnA2/Cdk2—and, to a much lesser extent, CcnE2/Cdk2—substitute kinase activity. Proper S-phase transition without Cdk2 requires at least the kinase-independent function of CcnE1 for pre-RC assembly and CcnA2/Cdk1 kinase, as postulated earlier. Simultaneous genetic inactivation of CcnE1 and Cdk2 in hepatocytes results in secondary inhibition of CcnA2 and CcnE2. As a consequence, MCM proteins cannot be sufficiently loaded on replication origins and DNA replication is further inhibited as a result of the lack of CcnA and related kinase activity, eventually leading to reduced DNA synthesis of hepatocytes and inefficient liver regeneration.
In summary, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S-phase machinery. It remains to be elucidated whether this mechanism is restricted to hepatocytes and the liver or is also effective in other cell types and organs.