Four waves of hepatocyte proliferation linked with three waves of hepatic fat accumulation during partial hepatectomy-induced liver regeneration

Partial hepatectomy (PH) triggers hepatocyte proliferation-mediated liver repair and is widely used to study the mechanisms governing liver regeneration in mice. However, the dynamics of the hepatocyte proliferative response to PH remain unclear. We found that PH-induced mouse liver regrowth was driven by four consecutive waves of hepatocyte replication. The first wave exhibited the highest magnitude followed by two moderate waves and one minor wave. Underlying this continuous hepatocyte replication was persistent activation of cell cycle components throughout the period of liver regeneration. Hepatocyte mitotic activity in the first three proliferative cycles showed a circadian rhythm manifested by three corresponding mitosis peaks, which were always observed at Zeitgeber time 0. The Bmal1-Clock/Wee1/Cdc2 pathway has been proposed by others to govern the circadian rhythm of hepatocyte mitosis during liver regeneration. However, we did not observe the correlations in the expression or phosphorylation of these proteins in regenerating livers. Notably, Bmal1 protein displayed frequent changes in hepatic distribution and cellular localization as the liver regrowth progressed. Further, three waves of hepatic fat accumulation occurred during hepatic regeneration. The first started before and lasted through the first round of hepatocyte proliferation, whereas the second and third occurred concomitantly with the second and third mitotic peaks, respectively. CONCLUSION: PH-induced liver regeneration consists of four continuous waves of hepatocyte proliferation coupled with three waves of hepatic fat accumulation. Bmal1, Wee1, and Cdc2 may not form a pathway regulating the circadian rhythm of hepatocyte mitosis during liver regeneration.     

Cdk2 plays a critical role in hepatocyte cell cycle progression and survival in the setting of cyclin D1 expression in vivo

Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2-/- mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication, and survival. Cdk2-/- hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2-/- liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2-/- hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2-/- mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation, and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.     

Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice

Background

Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration.

Methods

We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes.

Results

At 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment.

Conclusion

IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.     

Cdc2-cyclin E complexes regulate the G1/S phase transition

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.     

High levels of Myc expression are required for the robust proliferation of hepatocytes,but not for the sustained weak proliferation

In contrast to the robust proliferation exhibited following acute liver injury, hepatocytes exhibit long-lasting proliferative activity in chronic liver injury. The mechanistic differences between these distinct modes of proliferation are unclear. Hepatocytes exhibited robust proliferation that peaked at 2 days following partial hepatectomy in mice, but this proliferation was completely inhibited by hepatocyte-specific expression of MadMyc, a Myc-suppressing chimeric protein. However, Myc suppression induced weak but continuous hepatocyte proliferation, thereby resulting in full restoration of liver mass despite an initial delay. Late-occurring proliferation was accompanied by prolonged suppression of proline dehydrogenase (PRODH) expression, and forced PRODH overexpression inhibited hepatocyte proliferation. In hepatocytes in chronic liver injury, Myc was not activated but PRODH expression was suppressed in regenerating hepatocytes. In liver tumors, PRODH expression was often suppressed, especially in the highly proliferative tumors with distinct Myc expression. Our results indicate that the robust proliferation of hepatocytes following acute liver injury requires high levels Myc expression and that there is a compensatory Myc-independent mode of hepatocyte proliferation with the regulation of proline metabolism, which might be relevant to liver regeneration in chronic injury.     

Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome

Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.     

IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: regulation by SOCS3

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3+/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3+/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.     

Tubulin Polymerization Promoting Protein 1 (Tppp1) Phosphorylation by Rho-associated Coiled-coil Kinase (Rock) and Cyclin-dependent Kinase 1 (Cdk1) Inhibits Microtubule Dynamics to Increase Cell Proliferation

Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G₁/S-phase and the mitosis to G₁-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G₁-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.     

Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G2/M transition

Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) –dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed. In fission yeast, the Cdc14 homologue Flp1/Clp1 regulates the stability of the mitotic inducer Cdc25 at the end of mitosis to ensure Cdk1 inactivation before cytokinesis. We have recently reported that human Cdc14A, the Cdc14 isoform located at the centrosomes during interphase, down-regulates Cdc25 activity at the G2/M transition to prevent premature activation of Cdk1-Cyclin B1 complexes and untimely entry into mitosis. Here we speculate about new molecular mechanisms for Cdc14A and discuss the current evidence suggesting that Cdc14 phosphatase plays a role in cell cycle control in higher eukaryotes.     

Blocking of G1/S transition and cell death in the regenerating liver of Hepatitis B virus X protein transgenic mice

The Hepatitis B virus X (HBx) protein has been strongly implicated in the carcinogenesis of hepatocellular carcinoma (HCC). However, effects of the HBx protein on cell proliferation and cell death are controversial. This study investigates the effects of the HBx protein on liver regeneration in two independent lines of HBx transgenic mice, which developed HCC at around 14 to 16 months of age. High mortality, lower liver mass restoration, and impaired liver regeneration were found in the HBx transgenic mice post-hepatectomy. The levels of alanine aminotransferase and alpha-fetoprotein detected post-hepatectomy increased significantly in the HBx transgenic livers, indicating that they were more susceptible to damage during the regenerative process. Prolonged activation of the immediate-early genes in the HBx transgenic livers suggested that the HBx protein creates a strong effect by promoting the transition of the quiescent hepatocytes from G0 to G1 phase. However, impaired DNA synthesis and mitosis, as well as inhibited activation of G1, S, and G2/M markers, were detected. These results indicated that HBx protein exerted strong growth arrest on hepatocytes and imbalanced cell-cycle progression resulting in the abnormal cell death; this was accompanied by severe fat accumulation and impaired glycogen storage in the HBx transgenic livers. In conclusion, this study provides the first physiological evidence that HBx protein blocks G1/S transition of the hepatocyte cell-cycle progression and causes both a failure of liver functionality and cell death in the regenerating liver of the HBx transgenic mice.     

Involvement of IQGAP3, a regulator of Ras/ERK‐related cascade,in hepatocyte proliferation in mouse liver regeneration and development

The spatio‐temporal regulation of hepatocyte proliferation is a critical issue in liver regeneration. Here, in normal and regenerating liver as well as in developing liver, we examined its expression/localization of IQGAP3, which was most recently reported as a Ras/Rac/Cdc42‐binding proliferation factor associated with cell–cell contacts in epithelial‐type cells. In parallel, the expression/localization of Rac/Cdc42‐binding IQGAP1/2 was examined. IQGAP3 showed a specific expression in proliferating hepatocytes positive for the proliferating marker Ki‐67, the levels of expressions of mRNAs and proteins were significantly increased in hepatocytes in liver regeneration and development. In immunofluorescence, IQGAP3 was highly enriched at cell–cell contacts of hepatocytes. IQGAP1 and IQGAP2 were exclusively expressed in Kupffer and sinusoidal endothelial cells, respectively, in normal, regenerating, and developing liver. The expression of IQGAP1, but not of IQGAP2, was increased in CCl₄‐induced (but not in partial hepatectomy‐induced) liver regeneration. Exclusive expression/localization of IQGAP3 to hepatocytes in the liver likely reflects the specific involvement of the IQGAP3/Ras/ERK signaling cascade in hepatocyte proliferation in addition to the previously identified signaling pathways, possibly by integrating cell–cell contact‐related proliferating signaling events. On the other hand, the Rac/Cdc42‐binding properties of IQGAP1/2/3 may be related to the distinct modes of remodeling due to the different strategies which induced proliferation of liver cells; partial hepatectomy, CCl₄ injury, or embryonic development. Thus, the functional orchestration of Ras and the Ras homologous (Rho) family proteins Rac/Cdc42 likely plays a critical role in liver regeneration and development. J. Cell. Physiol. 220: 621–631, 2009. © 2009 Wiley‐Liss, Inc.