Serine peptidase inhibitor Kazal type I (SPINK1) promotes BRL‐3A cell proliferation via p38, ERK,and JNK pathways

Serine peptidase inhibitor Kazal type I (SPINK1) has the similar spatial structure as epidermal growth factor (EGF); EGF can interact with epidermal growth factor receptor (EGFR) to promote proliferation in different cell types. However, whether SPINK1 can interact with EGFR and further regulate the proliferation of hepatocytes in liver regeneration remains largely unknown. In this study, we investigated the role of SPINK1 in a rat liver hepatocyte line of BRL‐3A in vitro. The results showed the upregulation of endogenous Spink1 (gene addition) significantly increased not only the cell viability, cell numbers in S and G₂/M phase, but also upregulated the genes/proteins expression related to cell proliferation and anti‐apoptosis in BRL‐3A. In contrast, the cell number in G₁ phase and the expression of pro‐apoptosis‐related genes/proteins were significantly decreased. The similar results were observed when the cells were treated with exogenous rat recombinant SPINK1. Immunoblotting suggested SPINK1 can interact with EGFR. By Ingenuity Pathway Analysis software, the SPINK1 signalling pathway was built; the predicted read outs were validated by qRT‐PCR and western blot; and the results showed that p38, ERK, and JNK pathways‐related genes/proteins were involved in the cell proliferation upon the treatment of endogenous Spink1 and exogenous SPINK1. Collectively, SPINK1 can associate with EGFR to promote the expression of cell proliferation‐related and anti‐apoptosis‐related genes/proteins; inhibit the expression of pro‐apoptosis‐related genes/proteins via p38, ERK, and JNK pathways; and consequently promote the proliferation of BRL‐3A cells. For the first time, we demonstrated that SPINK1 can associate with EGFR to promote the proliferation of BRL‐3A cells via p38, ERK, and JNK pathways. This work has direct implications on the underlying mechanism of SPINK1 in regulating hepatocytes proliferation in vivo and liver regeneration after partial hepatectomy.     

The role of hepatocytes and oval cells in liver regeneration and repopulation

The liver has the unique capacity to regulate its growth and mass. In rodents and humans, it grows rapidly after resection of more than 50% of its mass. This growth process, as well as that following acute chemical injury is known as liver regeneration, although growth takes place by compensatory hyperplasia rather than true regeneration. In addition to hepatocytes and non-parenchymal cells, the liver contains intra-hepatic "stem" cells which can generate a transit compartment of precursors named oval cells. Liver regeneration after partial hepatectomy does not involve intra or extra-hepatic (hemopoietic) stem cells but depends on the proliferation of hepatocytes. Transplantation and repopulation experiments have demonstrated that hepatocytes, which are highly differentiated and long-lived cells, have a remarkable capacity for multiple rounds of replication. In this article, we review some aspects of the regulation of hepatocyte proliferation as well as the interrelationships between hepatocytes and oval cells in different liver growth processes. We conclude that in the liver, normally quiescent differentiated cells replicate rapidly after tissue resection, while intra-hepatic precursor cells (oval cells) proliferate and generate lineage only in situations in which hepatocyte proliferation is blocked or delayed. Although bone marrow stem cells can generate oval cells and hepatocytes, transdifferentiation is very rare and inefficient.     

Timing of protooncogene expression varies in toxin-induced liver regeneration

Hepatic expression of the protooncogenes c-fos and c-myc occurs within 2 h after partial hepatectomy, and these immediate early genes are thought to prime the hepatocytes for subsequent proliferation. To examine whether such gene activation occured in the setting of hepatocyte proliferation after toxic liver injury, protooncogene expression was examined during the regenerative response following liver injury from carbon tetrachloride (CCI₄) or galactosamine (GaIN). The pattern of protooncogene expression after CCI₄ mirrored that seen after partial hepatectomy, with rises in c-fos and c-myc mRNA content within 2 h, and then a rapid return to baseline levels. In contrast, early c-fos and c-myc expression did not occur after GaIN injury. Instead GaIN-induced regeneration led to a delayed and prolonged c-fos an c-myc activation which peaked 24–48 h after injury. Increase in c-jun, jun-B, and jun-D mRNA levels also occured in both models at times similar to the rises of c-fos and c-myc expression. Although the timing of DNA synthesis was identical after GaIN or CCI₄ treatment the proliferative response after GaIN injury was significantly less than that of CCI₄, and marked by the histologic appearance of oval cells. The coadministration of 2-acetylaminofluorene, an inhibitor of differentiated hepatocyte proliferation, together with CCI₄ altered the usual pattern of post-CCI₄ protooncogene expression to one resembling that seen after GaIN injury. Thus, the timing of protooncogene expression during liver regeneration may vary considerably. These variations may influence the nature of the proliferative response in terms of which cell types(s) proliferates, and the amount of regeneration that ensures. © 1993 Wiley-Liss, Inc.     

Peculiarities of liver regeneration in Chinese hamster <Emphasis Type="Italic">Cricetulus griseus</Emphasis>

With the aid of cytofluorimetry and interference microscopy, the ploidy level and the hepatocyte ploidy class distribution were studied and the dry mass of hepatocytes was measured in hepatocytes in liver of Chinese hamsters Cricetulus griseus and of Balb/c mice before and one month after partial hepatectomy. The mean ploidy level in hepatocytes of the Chinese hamster normal liver amounted to 2.35 ± 0.03 c. The modal class was mononuclear hepatocytes with diploid nuclei (82.4 ± 1.3%). The mean dry mass of hepatocytes amounted to 605.2 ± 4.8 pg. In the process of liver regeneration in the Chinese hamsters, the ratio of ploidy classes and the hepatocyte dry mass did not change. After a similar liver resection in the mice, a significant polyploidization of liver parenchyma occurred. The mean ploidy level in hepatocytes rose by 32%. Instead of 4cx2-hepatocytes, the modal class became mononuclear octaploid cells the relative portion of which increased, on average, by five times. The portion of binuclear hepatocytes with octaploid nuclei in mouse liver rose by more than five times. Thus, in the Chinese hamsters Cricetulus griseus, unlike mice, regeneration of liver occurred exclusively at the expense of proliferation of hepatocytes.     

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.     

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.     

Vasoactive intestinal peptide induces proliferation of human hepatocytes

Objectives

Proliferation of hepatocytes in vitro can be stimulated by growth factors such as epidermal growth factor (EGF), but the role of vasoactive intestinal peptide (VIP) remains unclear. We have investigated the effect of VIP on maintenance and proliferation of human hepatocytes.

Materials and methods

Human hepatocytes were isolated from liver specimens obtained from patients undergoing liver surgery. Treatment with VIP or EGF was started 24 h after plating and continued for 3 or 5 d. DNA replication was investigated by Bromodeoxyuridine (BrdU) incorporation and cell viability detected by MTT assay. Cell lysate was analysed by western blotting and RT‐PCR. Urea and albumin secretion into the culture supernatants were measured.

Results

VIP increased DNA replication in hepatocytes in a dose‐dependant manner, with a peak response at day 3 of treatment. VIP treatment was associated with an increase in mRNA expression of antigen identified by monoclonal antibody Ki‐67 (MKI‐67) and Histone Cluster 3 (H3) genes. Western blotting analysis showed that VIP can induce a PKA/B‐Raf dependant phosphorylation of extracellular signal‐regulated kinases (ERK). Although EGF can maintain hepatocyte functions up to day 5, no marked efffect was found with VIP.

Conclusions

VIP induces proliferation of human hepatocytes with little or no effect on hepatocyte differentiation. Further investigation of the role of VIP is required to determine if it may ultimately support therapeutic approaches of liver disease.

     

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.     

PLCγ2 promotes apoptosis while inhibits proliferation in rat hepatocytes through PKCD/JNK MAPK and PKCD/p38 MAPK signalling

Objectives

The PLCG2 (PLCγ2) gene is a member of PLC gene family encoding transmembrane signalling enzymes involved in various biological processes including cell proliferation and apoptosis. Our earlier study indicated that PLCγ2 may be involved in the termination of regeneration of the liver which is mainly composed of hepatocytes, but its exact biological function and molecular mechanism in liver regeneration termination remains unclear. This study aims to examine the role of PLCγ2 in the growth of hepatocytes.

Materials and methods

A recombinant adenovirus expressing PLCγ2 was used to infect primary rat hepatocytes. PLCγ2 mRNA and protein levels were detected by qRT‐PCR and Western blot. The subcellular location of PLCγ2 protein was tested by an immunofluorescence assay. The proliferation of hepatocytes was measured by MTT assay. The cell cycle and apoptosis were analysed by flow cytometry. Caspase‐3, ‐8 and ‐9 activities were measured by a spectrophotometry method. Phosphorylation levels of PKCD, JNK and p38 in the infected cells were detected by Western blot. The possible mechanism underlying the role of PLCγ2 in hepatocyte growth was also explored by adding a signalling pathway inhibitor.

Results

Hepatocyte proliferation was dramatically reduced, while cell apoptosis was remarkably increased. The results demonstrated that PLCγ2 increased the phosphorylation of PKCD, p38 and JNK in rat hepatocytes. After PKCD activity was inhibited by the inhibitor Go 6983, the levels of both p‐p38 and p‐JNK MAPKs significantly decreased, and PLCγ2‐induced cell proliferation inhibition and cell apoptosis were obviously reversed.

Conclusions

This study showed that PLCγ2 regulates hepatocyte growth through PKCD‐dependently activating p38 MAPK and JNK MAPK pathways; this result was experimentally based on the further exploration of the effect of PLCγ2 on hepatocyte growth in vivo.

     

Nrf2 Is Involved in Maintaining Hepatocyte Identity during Liver Regeneration

Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH). To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3), depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration.     

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.     

The Protective Roles of IL‐6 Trans‐Signaling Regulated by ADAM9 on the Liver in Carbon Tetrachloride‐Induced Liver Injury in Mice

Our study was undertaken to evaluate the important role that a disintegrin and metalloproteinase 9 (ADAM9) regulates IL‐6 trans‐signaling in carbon tetrachloride (CCl₄)‐induced liver injury in mice. Mice were divided into four groups. Each group respectively received mineral oil injection, CCl₄ injection, anti‐ADAM9 monoclonal antibody (mAb) pretreatment and CCl₄ injection, anti‐ADAM9 mAb and recombinant mouse ADAM9 molecules pretreatment with CCl₄ injection. Our results showed that anti‐ADAM9 mAb pretreatment significantly aggravated liver injury, inhibited IL‐6 trans‐signaling, which led to downregulation of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), upregulation of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocytes apoptosis at 24 h after CCl₄ injection. Recombinant ADAM9 molecules pretreatment reversed the impact of anti‐ADAM9 mAb pretreatment in mice. In conclusion, our study suggested that ADAM9 could regulate the hepatocytes proliferation, apoptosis, angiogenesis, and CYP2E1 expression by activating IL‐6 trans‐signaling and play important protective roles during CCl₄‐induced liver injury in mice.     

Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation

Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)^–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.     

How Does a Single Cell Know When the Liver Has Reached Its Correct Size?

The liver is a multi-functional organ that regulates major physiological processes and that possesses a remarkable regeneration capacity. After loss of functional liver mass the liver grows back to its original, individual size through hepatocyte proliferation and apoptosis. How does a single hepatocyte ‘know’ when the organ has grown to its final size? This work considers the initial growth phase of liver regeneration after partial hepatectomy in which the mass is restored. There are strong and valid arguments that the trigger of proliferation after partial hepatectomy is mediated through the portal blood flow. It remains unclear, if either or both the concentration of metabolites in the blood or the shear stress are crucial to hepatocyte proliferation and liver size control. A cell-based mathematical model is developed that helps discriminate the effects of these two potential triggers. Analysis of the mathematical model shows that a metabolic load and a hemodynamical hypothesis imply different feedback mechanisms at the cellular scale. The predictions of the developed mathematical model are compared to experimental data in rats. The assumption that hepatocytes are able to buffer the metabolic load leads to a robustness against short-term fluctuations of the trigger which can not be achieved with a purely hemodynamical trigger.     

Alrp,a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats

Abstract

Augmenter of Liver Regeneration (Alrp) enhances, through unknown mechanism/s, hepatocyte proliferation only when administered to partially hepatectomized (PH) rats. Liver resection, besides stimulating hepatocyte proliferation, induces reactive oxygen species (ROS), triggering apoptosis. To clarify the role of Alrp in the process of liver regeneration, hepatocyte proliferation, apoptosis, ROS-induced parameters and morphological findings of regenerating liver were studied from PH rats Alrp-treated for 72 h after the surgery. The same parameters, evaluated on regenerating liver from albumin-treated PH rats, were used as control. The results demonstrated that Alrp administration induces the anti-apoptotic gene expression, inhibits hepatocyte apoptosis and reduces ROS-induced cell damage. These and similar data from in vitro studies and the presence of ‘Alrp homologous proteins’ in viruses as well as in mammals (i) allow to hypothesize that Alrp activity/ies may not be exclusive for regenerating liver and (ii) suggest the use of Alrp in the treatment of oxidative stress-related diseases.     

Modulation of mitogen-independent hepatocyte proliferation during the perinatal period in the rat

Summary Late gestation fetal rat hepatocytes can proliferate under defined in vitro conditions in the absence of added mitogens. However, this capacity declines with advancing gestational age of the fetus from which the hepatocytes are derived. The present studies were undertaken to investigate this change in fetal hepatocyte growth regulation. Examination of E19 fetal hepatocyte primary cultures using immunocytochemistry for 5-bromo-2′-deoxyuridine (BrdU) incorporation showed that approximately 80% of these cells traverse S-phase of the cell cycle over the first 48 h in culture. Similarly, 65% of E19 hepatocytes maintained in culture under defined mitogen-free conditions for 24 h showed nuclear expression of proliferating cell nuclear antigen (PCNA). These in vitro findings correlated with a high level of immunoreactive PCNA in immunofluorescent analyses of E19 liver. In contrast, E21 (term) liver showed little immunoreactive PCNA. The in vivo finding was recapitulated by in vitro studies showing that E21 hepatocytes had low levels of BrdU incorporation during the first day in culture and were PCNA negative shortly after isolation. However, within 12 h of plating, E21 hepatocytes showed cytoplasmic staining for PCNA. Although maintained under mitogen-free conditions, PCNA expression progressed synchronously to a nucleolar staining pattern at 24 to 48 h in culture followed by intense, diffuse nuclear staining at 60 h which disappeared by 72 h. This apparently synchronous cell cycle progression was confirmed by studies showing peak BrdU incorporation on the third day in culture. Whereas DNA synthesis by both E19 and E21 hepatocytes was potentiated by transforming growth factor α (TGFα), considerable mitogen-independent DNA synthesis was seen in hepatocytes from both gestational ages. These results may indicate that fetal hepatocytes come under the influence of an exogenous, in vivo growth inhibitory factor as term approaches and that this effect is relieved when term fetal hepatocytes are cultured.