Proto-oncogene expression in regenerating liver is simulated in cultures of primary adult rat hepatocytes

Proto-oncogene fos mRNA levels are rapidly and transiently elevated 12-fold in regenerating liver 10-60 min following partial hepatectomy. This response, and the induction of fos protein synthesis, has been simulated qualitatively and quantitatively in long term primary cultures of quiescent adult rat hepatocytes where proliferative transitions can be initiated directly in serum-free medium by known hepatocyte mitogens like epidermal growth factor. Expression of a second proto-oncogene, c-rasH, in proliferatively activated hepatocyte cultures between 6 and 24 h also simulates the delayed hepatic response that occurs in vivo following partial hepatectomy. These results suggest that sequential proto-oncogene expression during liver regeneration is caused directly by hepatocellular interactions with specific mitogens. In addition, a role for monovalent cations in the regulation of hepatocyte gene expression is implicated from findings that Na+ deprivation inhibits induction of fos expression in cultured hepatocytes by epidermal growth factor under chemically defined conditions.     

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.     

Modulation of c-myc and c-fos gene expression in regenerating rat liver by 2-mercaptopropionylglycine

Expression of the two proto-oncogenes, c- myc and c- fos, in proliferating liver, and the modulation of their expression by 2-mercaptopropionylglycine (MPG) has been investigated. A significant increase in the expression of both c-myc and c-fos was observed, attaining a peak at 30 min, followed by a gradual decline for 8h after partial hepatectomy. Treatment of partially hepatectomized animals with MPG resulted in a significant decrease in the expression of both genes, the decrease being more marked in c- myc mRNA levels. At later time points, there was a very small rise in the mRNA levels of these genes in MPG-treated animals but they still remained much lower than the peak levels observed in both genes 30 min after surgery. A decline in the recovery of liver weight in MPG-treated animals was also observed.     

CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

Background

Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.

Methods

Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.

Results

We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a “high” dose that replicated serum levels found after I/R injury and a “low” dose that was similar to that found after hepatectomy. Mice receiving the “high” dose had reduced levels of hepatocyte proliferation and regeneration whereas the “low” dose promoted hepatocyte proliferation and regeneration.

Conclusions

Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.     

Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats.

Vascular endothelial growth factor (VEGF) plays a major role in angiogenesis, which is essential for both healing of injured tissue and proliferation of carcinoma cells. In this study we elucidated the expression and role of VEGF in rat liver regeneration after partial hepatectomy. VEGF expression was mainly detected in periportal hepatocytes and reached a maximal level 48-72 hr after partial hepatectomy by both immunohistochemistry and in situ hybridization. Similarly, immunohistochemistry for Ki-67 showed that the proliferative activity of sinusoidal endothelial cells was highest in the periportal area and reached a maximal level 72 hr after partial hepatectomy. Moreover, neutralization of VEGF significantly inhibited proliferative activity of hepatocytes (p<0. 0001), as well as sinusoidal endothelial cells (p<0.001), at 48 and 96 hr after partial hepatectomy. Conversely, injection of VEGF significantly promoted proliferative activity of hepatocytes (p<0. 0001) as well as sinusoidal endothelial cells (p<0.0005) at 48 hr after partial hepatectomy. These results suggest that VEGF promotes proliferation of hepatocytes through reconstruction of liver sinusoids by proliferation of sinusoidal endothelial cells. Furthermore, these data point to a new therapeutic strategy, the use of VEGF and other hepatocyte growth factors in fulminant or severe acute hepatitis.     

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.     

The Cdk inhibitor p21 is required for necrosis,but it inhibits apoptosis following toxin-induced liver injury

Liver injury and repair were examined in wild type, p21Waf1/Cip1, and p27Kip1-deficient mice following carbon tetrachloride (CCl4) administration. In wild type liver, p21 expression is induced in a biphasic manner following injection of CCl4, with an early peak of p21 expression occurring in pericentral hepatocytes at 6 h, prior to evidence of injury, and a second peak succeeding regenerative proliferation. In contrast, p27 is present throughout the quiescent liver, but its expression decreases following CCl4 injection. Surprisingly, p21-deficient animals were resistant to CCl4-induced necrotic injury, indicating that rapid induction of p21 in pericentral hepatocytes following CCl4 injection contributes to subsequent necrosis. Expression of cytochrome P450 2E1, which plays an essential role in CCl4-induced necrotic injury, was not affected in p21-deficient mice. Although they had the least injury, p21-deficient mice had the highest levels of hepatic proliferation that correlated with increases in hyperphosphorylated retinoblastoma protein and Cyclin A gene expression. Increased replication in p21-deficient livers was counteracted by an increase in hepatocyte apoptosis as detected by caspase-3 activation. p21 plays distinct and opposing roles regulating hepatocyte survival during injury and subsequent repair, with early induction of p21 contributing to necrotic injury and later expression to cessation of proliferation and hepatocyte survival.     

c-myc and c-fos expression in differentiating mouse primary keratinocytes.

Expression of the myc and fos genes has been monitored in mouse primary keratinocytes after induction of terminal differentiation by calcium or tetradecanoylphorbol acetate (TPA). myc RNA levels in growing cells are very high and remain elevated even at late times after calcium-induced differentiation. Thus, keratinocytes provide the first example of normal primary cells with persistent c-myc expression irrespective of their proliferative or differentiated state. fos expression is also relatively unaffected by addition of calcium. In contrast to calcium, TPA-induced differentiation is accompanied by dramatic changes in proto-oncogene expression: marked c-fos induction and considerable although transient decrease in c-myc expression. These effects might be important for the keratinocyte response to TPA: TPA treatment of a keratinocyte cell line (RBK) resistant to this substance has no effect on c-myc expression and leads only to minimal c-fos induction. In these cells full fos induction can still be triggered by addition of fresh medium. Thus, the fos gene in normal keratinocytes is inducible through at least two independent mechanisms, only one of which has been lost during derivation of the TPA-resistant cell line.     

Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.

Hepatocyte Growth Factor (HGF) is a potent complete mitogen for primary cultures of hepatocytes in vitro. There is strong evidence that this novel growth factor may mediate hepatocyte regeneration after liver damage. We have shown previously that the amount of immunoreactive HGF markedly increases in the serum of rats soon after partial hepatectomy or CCl4 administration. In the present paper, we demonstrate that the level of HGF mRNA in rat liver also dramatically increases from 3 to 6 hours post hepatectomy, peaks at 12 hr and gradually returns to undetectable levels by 72 to 96 hours post hepatectomy. In separate experiments, DNA synthesis (in vivo) was determined in rat liver remnants after partial hepatectomy. DNA synthesis peaked 24 hr after hepatectomy, 12 hr after the peak of HGF mRNA expression. These results suggest that HGF may be one of the major early signals that triggers hepatocyte proliferation during liver regeneration.     

TNF-driven inflammation during mouse liver regeneration after partial hepatectomy and its role in growth regulation of liver.

To determine the role of TNF-driven inflammation in self-regulation of cell growth and differentiation, mouse liver regeneration after partial hepatectomy was examined for TNF-driven inflammation. Hepatectomy provoked priming state for TNF production in both whole body and liver on day 3 when the peak mitotic response occurred. Histochemical studies of liver also showed an inflammatory symptom; hepatocellular necrotic foci appeared by 6 hours after hepatectomy. TNF itself was secreted spontaneously in liver transiently on day 1 to 2 after hepatectomy just before the proliferation of hepatocytes. Dexamethasone reduced both TNF secretion and hepatocyte proliferation after hepatectomy. Recombinant murine TNF stimulated the in vitro proliferation of hepatocytes. These findings indicate that hepatectomy induces short-term secretion of TNF in liver and TNF-driven inflammation has an important role in liver regeneration, at least in part by the direct stimulation of hepatocyte proliferation.     

TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice

Background & Aims

Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH.

Methods

To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies.

Results

In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12–8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes.

Conclusions

TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.     

TEC酪氨酸激酶基因是一种与肝再生调控相关的早期反应基因

 肝再生过程中立即早期反应基因的表达在成熟肝细胞由G0 期向G1期的转变中起着关键作用 .为探讨肝再生早期基因表达的变化 ,利用表达性差异显示分析 (RDA)技术研究了 2 3肝部分切除后 1h再生肝选择性基因表达 ,发现一株TEC酪氨酸激酶同源序列存在于差减产物中 ,RNA狭缝杂交证实确为差异表达基因 .从大鼠肝cDNA文库中分离其全长cDNA ,序列分析结果表明 ,该基因为小鼠 人TEC酪氨酸激酶的同源体 ,进而以该cDNA为探针 ,用Northern杂交证实 2 3肝部分切除后TEC酪氨酸激酶基因在 1h内呈现瞬间表达增加 ,其表达水平较基础水平增高 2 5倍 ;在原代培养大鼠肝细胞体系中 ,EGF可迅速诱导TEC基因表达 ,且不被蛋白合成抑制剂阻断 .结果表明 ,TEC基因是一种与肝再生调控密切相关的早期反应基因 .     

Annexin A3 expression increases in hepatocytes and is regulated by hepatocyte growth factor in rat liver regeneration

Annexin (Anx) A3 increases and plays important roles in the signalling cascade in hepatocyte growth in cultured hepatocytes. However, no information is available on its expression and role in rat liver regeneration. In the present study, AnxA3 expression was investigated to determine whether it also plays a role in the signalling cascade in rat liver regeneration. AnxA3 protein and mRNA level both increase in liver after administration of carbon tetrachloride (CCl4) or 70% partial hepatectomy. AnxA3 protein level increases in isolated parenchymal hepatocytes, but not in non-parenchymal liver cells, in these rat liver regeneration models. AnxA3 mRNA increases in hepatocytes after CCl4 administration. Anti-hepatocyte growth factor antibody suppresses this increase in AnxA3 mRNA level. These results demonstrate that AnxA3 expression increases in hepatocytes through a hepatocyte growth factor-mediated pathway in rat liver regeneration models, suggesting that AnxA3 plays an important role in the signalling cascade in rat liver regeneration.     

Differential expression of apoptosis-associated genes post-hepatectomy in cirrhotic vs. normal rats

Liver regeneration after partial hepatectomy or liver injury is controlled by a wide variety of growth factors that are proven activators or inhibitors of hepatocyte proliferation. Liver regeneration post-hepatectomy has been proven to be decreased and delayed in cirrhotic vs. normal liver. Apoptosis seems to play an important role in cellular proliferation and in liver regeneration. Therefore, this study has analyzed the expression of apoptosis-associated genes following 2/3 hepatectomy in cirrhotic vs. normal rats. Cirrhosis was induced by a weekly intragastric administration of CCl4 for 16 weeks followed by hepatectomy and histological examination of the resected liver. Rats were sacrificed at 6 h, 12 h, 24 h, or 72 h after liver resection. The expression of proapoptotic (Bad, Bak, Bax) and antiapoptotic (Bcl-2, Bcl-XL) genes was analyzed by quantitative RT-PCR. We have observed an early increase in antiapoptotic mRNA levels and a delayed increase in proapoptotic mRNA levels in normal liver following hepatectomy. Before resection, proapoptotic mRNA levels were significantly higher in cirrhotic vs. normal liver. After hepatectomy, apoptotic mRNA levels were decreased and delayed as compared with that observed following hepatectomy in normal liver. These results indicate that apoptosis takes place in liver during CCl4-induced cirrhosis and could participate in the impaired regenerative response observed in cirrhotic liver.     

Down-regulation of microRNA-26a promotes mouse hepatocyte proliferation during liver regeneration

Background

Inadequate liver regeneration (LR) is still an unsolved problem in major liver resection and small-for-size syndrome post-living donor liver transplantation. A number of microRNAs have been shown to play important roles in cell proliferation. Herein, we investigated the role of miR-26a as a pivotal regulator of hepatocyte proliferation in LR.

Methodology/Principal Findings

Adult male C57BL/6J mice, undergoing 70% partial hepatectomy (PH), were treated with Ad5-anti-miR-26a-LUC or Ad5-miR-26a-LUC or Ad5-LUC vector via portal vein. The animals were subjected to in vivo bioluminescence imaging. Serum and liver samples were collected to test liver function, calculate liver-to-body weight ratio (LBWR), document hepatocyte proliferation (Ki-67 staining), and investigate potential targeted gene expression of miR-26a by quantitative real-time PCR and Western blot. The miR-26a level declined during LR after 70% PH. Down-regulation of miR-26a by anti-miR-26a expression led to enhanced proliferation of hepatocytes, and both LBWR and hepatocyte proliferation (Ki-67⁺ cells %) showed an increased tendency, while liver damage, indicated by aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin (T-Bil), was reduced. Furthermore, CCND2 and CCNE2, as possible targeted genes of miR-26a, were up-regulated. In addition, miR-26a over-expression showed converse results.

Conclusions/Significance

MiR-26a plays crucial role in regulating the proliferative phase of LR, probably by repressing expressions of cell cycle proteins CCND2 and CCNE2. The current study reveals a novel miRNA-mediated regulation pattern during the proliferative phase of LR.