Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.     

Expression of the insulinoma gene rig during liver regeneration and in primary cultured hepatocytes

We have isolated a novel gene, rig (rat insulinoma gene) from rat insulinomas. In the present study, rig was found to be expressed in rat regenerating liver and in primary cultured rat hepatocytes. The level of rig mRNA was increased at the proliferative phase of liver regeneration. In synchronously cultured hepatocytes, the rig mRNA level was elevated at the G1 phase of the cell cycle and the rig-protein was accumulated in the nuclei during the S phase. These results indicated that rig could be involved in a more general way in growth or cell replication.     

Liver cell polyploidization: a pivotal role for binuclear hepatocytes

Polyploidy is a general physiological process indicative of terminal differentiation. During liver growth, this process generates the appearance of tetraploid (4n) and octoploid (8n) hepatocytes with one or two nuclei. The onset of polyploidy in the liver has been recognized for quite some time; however, the cellular mechanisms that govern it remain unknown. In this report, we observed the sequential appearance during liver growth of binuclear diploid (2 x 2n) and mononuclear 4n hepatocytes from a diploid hepatocyte population. To identify the cell cycle modifications involved in hepatocyte polyploidization, mitosis was then monitored in primary cultures of rat hepatocytes. Twenty percent of mononuclear 2n hepatocytes failed to undergo cytokinesis with no observable contractile movement of the ring. This process led to the formation of binuclear 2 x 2n hepatocytes. This tetraploid condition following cleavage failure did not activate the p53-dependent checkpoint in G1. In fact, binuclear hepatocytes were able to proceed through S phase, and the formation of a bipolar spindle during mitosis constituted the key step leading to the genesis of two mononuclear 4n hepatocytes. Finally, we studied the duplication and clustering of centrosomes in the binuclear hepatocyte. These cells exhibited two centrosomes in G1 that were duplicated during S phase and then clustered by pairs at opposite poles of the cell during metaphase. This event led only to mononuclear 4n progeny and maintained the tetraploidy status of hepatocytes.     

Study on the activity of the signaling pathways regulating hepatocytes from G0 phase into G1 phase during rat liver regeneration

Under normal physiological conditions, the majority of hepatocytes are in the functional state (G0 phase). After injury or liver partial hepatectomy (PH), hepatocytes are rapidly activated to divide. To understand the mechanism underlying hepatocyte G0/G1 transition during rat liver regeneration, we used the Rat Genome 230 2.0 Array to determine the expression changes of genes, then searched the GO and NCBI databases for genes associated with the G0/G1 transition, and QIAGEN and KEGG databases for the G0/G1 transition signaling pathways. We used expression profile function (E _t ) to calculate the activity level of the known G0/G1 transition signal pathways, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the interactions among these signaling pathways. The results of our study show that the activity of the signaling pathways of HGF, IL-10 mediated by p38MAPK, IL-6 mediated by STAT3, and JAK/STAT mediated by Ras/ERK and STAT3 are significantly increased during the priming phase (2–6 h after PH) of rat liver regeneration. This leads us to conclude that during rat liver regeneration, the HGF, IL-10, IL-6 and JAK/STAT signaling pathways play a major role in promoting hepatocyte G0/G1 transition in the regenerating liver.     

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 .     

双斑蟋若虫后足的再生观察

研究蟋蟀若虫是否具有再生的能力,在实验室内对直翅目蟋蟀科的双斑蟋Gryllus bimaculata de Geer若虫后足的再生情况进行初步观察。结果表明:双斑蟋若虫的后足确实具有再生的能力,且再生足的伸长生长与正常足一样,均出现于虫体蜕皮时。再生的过程主要分为3个阶段:突起期、“肢芽”期、短小足期。实验结果还表明:再生能力与断肢发生的时间及断肢部位有关;断肢部位离肢体越远,再生能力越强;断肢发生的时间越早,再生能力越强。     

Growth potential of adult hepatocytes in mammals: highly replicative small hepatocytes with liver progenitor-like traits

The liver is one of the few organs that is capable of completely regenerating itself without using a stem cell population. When damaged, growth factors and cytokines are released, stimulating terminally differentiated adult hepatocytes and making them re-enter the cell cycle. We have been developing a series of studies on the growth potential of rat and human hepatocytes to identify a population of hepatocytes that is responsible for the regeneration of the injured liver. For this purpose, we established an appropriate culture method for hepatocytes by which growth and differentiation capacities are practically examined under various experimental conditions. This in vitro assay system allows us to identify small hepatocytes that are prominently replicative compared to large hepatocytes. Non-parenchymal cells play critical roles in the proliferation of small hepatocytes. These hepatocytes are present in both rat and human liver and are located in portal regions there. Phenotypic features were examined at morphological and gene/protein levels in detail, which showed the phenotypic plasticity in vitro. Mammalian liver includes a population of small hepatocytes in normal adults with a minute occupancy rate. We speculate that small hepatocytes play a role in regenerating the injured liver and in compensating for apoptotic hepatocytes in the physiological turnover of hepatocytes.     

Different modes of state transitions determine pattern in the Phosphatidylinositide-Actin system

Background

In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.

Results

In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved.

Conclusions

Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.     

Effect of "vilon" on cirrhotically changed rat liver. Liver regeneration, and status of glycogen-forming function of hepatocytes

Effects of a dipeptide preparation "Vilon" on rehabilitation of functional activity of hepatocytes and regeneration of the cirrhotically altered rat liver were studied. The liver cirrhosis was produced by poisoning of rats for 4 months with carbon tetrachloride (CCl4). On the end of the poisoning with CCl4, one group of animals was not submitted to any further actions, whereas animals of the other group were injected "Vilon" (1.7 micrograms/kg) daily for 5 days. On smears of isolated hepatocytes, contents of total glycogen (TG), and its labile and stable fractions (LF and SF) were determined in addition to cell ploidy levels and the total protein content. In liver homogenates, activities of glucose-6-phosphatase (G6P), glycogen synthase (GS), and glycogen phosphorylase (GP) were measured. In 2 weeks after the drug application, G6P activity being reduced in cirrhosis 1.2 times, elevated under effect of "Vilon". In non-treated rats the contents of TG and its fractions and of G6P activity remained at the level characteristic of the cirrhotic liver prior to "Vilon" administration. In both groups of rats, GP and GS activities in the cirrhotically altered liver did not differ from their control values throughout the entire experiment. "Vilon" has been shown to exert a weak stimulating effect on regeneration of the cirrotically altered rat liver: in hepatocytes of the second group of rats the total protein content and ploidy levels were higher than those in the first group by 4.7 and 11.5%, respectively.     

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.     

Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.     

Role of cell-surface modulator of DNA synthesis in liver regeneration

A cell-surface modulator of DNA synthesis by cultured rat hepatocytes was studied in relation to the liver regeneration process. When rat hepatocytes isolated 24 h after partial hepatectomy were cultured, the first burst of DNA synthesis peaked at 5-8 h and declined until 24 h, followed by the second burst. Rat liver plasma membranes added 2 h after plating inhibited only the second burst, while in the case of the normal hepatocytes where the DNA synthesis began to increase after 5 h, this inhibition was observed at 16 h but not at 8 h. The inhibition did not differ when the membranes obtained from regenerating livers 12 h after partial hepatectomy were used. Epidermal growth factor binding to the cultured hepatocytes was not hindered by the membranes. These results suggest that the modulator inhibits hepatocyte proliferation at the early G1-phase of the cell cycle and that its action might be controlled by other factors in the process of liver regeneration.     

Altered expression of diacylglycerol kinase isozymes in regenerating liver

The liver possesses the capacity to restore its function and mass after injury. Liver regeneration is controlled through complicated mechanisms, in which the phosphoinositide (PI) cycle is shown to be activated in hepatocytes. Using a rat partial hepatectomy (PH) model, the authors investigated the expression of the diacylglycerol kinase (DGK) family, a key enzyme in the PI cycle, which metabolizes a lipid second-messenger diacylglycerol (DG). RT-PCR analysis shows that DGKζ and DGKα are the major isozymes in the liver. Results showed that in the process of regeneration, the DGKζ protein, which is detected in the nucleus of a small population of hepatocytes in normal liver, is significantly increased in almost all hepatocytes. However, the mRNA levels remain largely unchanged. Double labeling with bromodeoxyuridine (BrdU), an S phase marker, reveals that DGKζ is expressed independently of DNA synthesis or cell proliferation. However, DGKα protein localizes to the cytoplasm in normal and regenerating livers, but immunoblot analysis reveals that the expected (80 kDa) and the lower (70 kDa) bands are detected in normal liver, whereas at day 10 after PH, the expected band is solely recognized, showing a different processing pattern of DGKα in liver regeneration. These results suggest that DGKζ and DGKα are involved, respectively, in the nucleus and the cytoplasm of hepatocytes in regenerating liver.