Gene 537 (2014) 230–237

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

Interleukin 18 accelerates the hepatic cell proliferation in rat liver regeneration after partial hepatectomy Jihong Zhang a,b, Chengkai Ma a,b, Yunqing Liu a,b, Gang Yang a,b, Yun Jiang b, Cunshuan Xu a,b,⁎ a b

College of Life Science, Henan Normal University, Xinxiang 453007, Henan Province, China Key Laboratory for Cell Differentiation Regulation, Xinxiang 453007, Henan Province, China

a r t i c l e

i n f o

Article history: Accepted 30 December 2013 Available online 9 January 2014 Keywords: Liver regeneration Gene expression profile Interleukin-18 Hepatocyte proliferation

a b s t r a c t Interleukin 18 (IL-18) is a proinflammatory cytokine with an ability to accelerate cell proliferation through activating other factors. However, little is yet understood of the role of IL-18 in the regulation of liver regeneration (LR). To study the effect of IL-18 on LR, the gene expression profiles of hepatocytes isolated from rat regenerative liver were determined by Rat Genome 230 2.0 microarray. Next, the synergistic effects of genes associated to IL18 pathway were analyzed by expression profile function Et. Then, the expression level of IL-18 was examined by RT-PCR and ELISA. Finally, the effect of IL-18 on hepatocyte proliferation was detected by injecting recombinant rat IL-18 (rrIL-18) into rats immediately after partial hepatectomy (PH) and the rate of hepatocyte proliferation was detected by BrdU labeling. The microarray result showed that the expressions of 13 genes of IL-18 pathway and 49 cell proliferation genes regulated by the pathway were significantly altered at transcriptional level. The Et values of three branches of IL-18 pathway, NF-κB, p38 and JNK, were markedly enhanced during the priming and progressing phases of rat LR. The mRNA level of IL-18 was significantly elevated at 2 and 36 h, and its level in plasma was also significantly increased at 2 h, and reached the peaks at 12 h and 48 h after PH (p b 0.05). The number of BrdU positive cells was dramatically increased in rats treated with IL-18 compared to PBS control group (p b 0.01). In conclusion, IL-18 promotes rat hepatocyte proliferation in the LR priming and progressing phases after PH. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Hepatocyte is the major cells in the liver, accounting for 70–80% of hepatic mass and 65% of total hepatic cells (Sell, 2003). They have many physiological functions including substance metabolism, amino acid utilization, biotransformation, bile secretion, oxidation protection, and detoxification (Fausto et al., 1995). In normal liver, the majority of adult liver cells are in the quiescent state and only 0.0012–0.01% hepatocytes undergo mitosis (Chen et al., 2013; Khan and Mudan, 2007). However, the hepatocytes are rapidly activated and enter cell cycle to compensate for the lost liver mass to its original level after partial hepatectomy (PH), which is called liver regeneration (LR) (Taub, 2004). LR is a tightly orchestrated process of compensatory growth and rodent

Abbreviation: BrdU, bromodeoxyuridine; DAPI, 4′6′-diamidino-2-phenylindole dihydrochloride; EDTA, ethylene diamine tetraacetic acid; ELISA, enzyme-linked immuno-sorbent assay; IGIF, interferon-γ-inducing factor; IL-18, interleukin 18; IL-18R, interleukin 18 receptor; IPA, Ingenuity Pathway Analysis; LR, liver regeneration; PH, partial hepatectomy; rrIL-18, recombinant rat interleukin 18; RT-PCR, real-time polymerase chain reaction; SO, sham operation. ⁎ Corresponding author at: College of Life Science, Henan Normal University, No. 46, East of Construction Road, Xinxiang 453007, Henan Province, China. Tel.: + 86 373 3326001; fax: +86 373 3326524. E-mail address: [email protected] (C. Xu). 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.12.062

liver 2/3 PH is a well-established liver regeneration model. Generally, LR process can be defined as priming phase (0.5–6 h after PH), progressing phase (6–72 h after PH) and terminal phase (72–168 h after PH) (Ma et al., 2009). It has been shown that hepatocytes are activated to proliferate mainly during priming and progressing phases (Fausto et al., 2006; Michalopoulos, 2007). Interleukin-18 (IL-18), formerly called interferon-γ-inducing factor (IGIF), was initially found as a potent proinflammatory cytokine and played a critical role in the host defense against infections by intracellular microbes (Sugihara, 2000). Prior studies also showed that IL-18 serum level is closely connected with the severity of several liver diseases (Tsutsui et al., 2003) and increased significantly after PH (Shiraki et al., 2007). Using IL-18 transgenic mouse, IL-18 was found with anti-apoptotic effect on fas-mediated liver injury. IL-18 can increase the activities of NF-κB and X-linked inhibitors of apoptosis (XIAP) but inhibit caspase-3 activity (Yamamoto et al., 2008). The pleiotropic activities of IL-18 are mediated by IL-18 receptor (IL-18R). Upon binding to IL-18R, IL-18 initiates a signaling cascade that results in the activation of NF-κB (Kojima et al., 1998). It has also been shown that IL-18 has a role in the regulation of cell proliferation. When primary cultured rat chondrocytes and dog osteoblasts were treated with exogenous IL-18, the number of cells labeled with H3 thymidine was increased at 24 h in a dose-dependent manner (Cornish et al., 2003).

J. Zhang et al. / Gene 537 (2014) 230–237

To study the effect of IL-18 on hepatocyte proliferation, hepatocytes were isolated from rat regenerative liver, mRNA was prepared for Rat Genome 230 2.0 microarray. The results revealed 13 genes related-IL-18 signaling pathway and 49 genes related to hepatocyte proliferation were significantly changed. The synergistic effects of these genes on hepatocyte proliferation were analyzed by expression profile function Et, a method established by Xu et al. (2010). The Et values of three signaling branches of IL-18 pathway, NF-κB, p38 and JNK, were significantly enhanced during the priming phase (2–6 h after PH) and progressing phase (12–72 h after PH) (p b 0.05) of rat LR. In support, the expression level of IL-18 mRNA was significantly raised at 2 and 36 h after PH and its plasma level was significantly increased at 2 h, and reached the peaks at 12 h and 48 h after PH, (p b 0.05). To further study the direct effect of IL-18 on hepatocyte proliferation, rats were injected with an activated form of recombinant IL-18 immediately after PH, the number of dividing cells was significantly increased in rats treated with IL18 compared to PBS control group (p b 0.01). Collectively, IL-18 promotes rat hepatocyte proliferation via the branches NF-κB, p38 and JNK branches in the LR priming and progressing phases after PH. 2. Materials and methods 2.1. Animals and partial hepatectomy Sprague–Dawley (SD) male rats (6-week old), weighted 200 ± 10 g, were obtained from animal center of Henan Normal University. Rats were kept at 21 ± 2 °C, relative humidity 60 ± 10%, illuminated 12 h/d (8:00–20:00) with free access to standard rat chow and water. They were subjected to two-third hepatectomy (PH) under ether anesthesia condition (Higgins and Anderson, 1931). Rats in the sham operation (SO) received the same operation procedures but no liver lobes were removed. The rats and all experimental procedures used here were handled in compliance with the Animal Protection Law of China. 2.2. Isolation of hepatocytes Rats were sacrificed at 0, 2, 6, 12, 24, 30, 36, 72, 120 and 168 h after hepatectomy, and their hepatocytes were isolated by conventional two-step perfusion and Percoll density gradient centrifugation (Vondran et al., 2008). Briefly, rats were anesthetized by ether and sterilized with 75% alcohol. Abdominal cavity was opened. Vena cava below and above the liver were ligated following the catheterization of the liver portal vein. The liver was perfused with calcium-free perfusate preheated at 37 °C via the portal vein till the liver surface turned gray–white, and then with 15 ml of 0.05% collagenase IV solution. The dispersed liver cells were collected and washed three times with PBS at 37 °C. Cells were resuspended in PBS at 1 × 108 cells/ml. 6 ml of mixed cell suspension was loaded onto 4 ml 60% Percoll in a 10 ml tube (Pharmacia, Biotech AB, Uppsala, Sweden) and centrifuged at 200 g, 4 °C for 15 min. The sediment was enriched with hepatocytes. Hepatocytes were identified by immunocytochemistry by their marker proteins ALB and G6P (Wang et al., 2008), and their viability was measured by trypan blue staining. Purity and viability of the hepatocytes used in this study were over 95%.

231

the manufacturer. cRNA fragments of 35–200 bp were prepared by fragmentation reagent treatment. The Rat Genome 230 2.0 array was hybridized with the cRNA fragments, which were pretreated. Then, they were stained, washed automatically using GeneChip fluidics station 450 (Affymetrix Inc., Santa Clara, CA, USA), scanned using GeneChip scanner 3000 (Affymetrix Inc., Santa Clara, CA, USA), and the spots were converted into signal values using Affymetrix GCOS 2.0 software (Guo et al., 2008). The signal values were normalized according to manufacturer's instruction. The p-values were determined based on the probe signal. When the p-value of a gene is b0.05, this gene is defined as present (P), b0.065 is marginal (M), and N0.065 is absent (A). To minimize the experimental operation and microarray test differences, each sample was repeated three times, and the average value was used for statistical analysis. 2.4. Real-time polymerase chain reaction (RT-PCR) mRNA was prepared from the purified hepatocytes as described above. Primers for Il-18, mapk10, ccnd1, agt, hmox1 and β-actin, were designed using Primer Express 5.0 software. Their first chain of cDNA was synthesized by SuperScript II RT reverse transcription system (Promega, USA). The PCR was performed by the conditions with SYBR Green I: 2 min at 95 °C, followed with 40 cycles for 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. Each sample was performed in triplicates. Then PCR products were analyzed by melting curve to confirm the specificity of amplification. β-actin was used as an internal control. 2.5. Confirmation of the significant-expressed genes and liver regeneration-related genes in rat liver regeneration The genes with ratio values of PH/SO ≥ 2 or ≤ 0.33 and a p-value ≤ 0.01 were considered as significant expressed genes (Fukuhara et al., 2003; Nault et al., 2013). This ratio value cut-off was chosen in order to include genes with a low change in expression and, consequently, a low p-value was used in order to reduce the likelihood of false positives. In addition, the genes, at least at one of PH time points in LR, with F-test difference 0.01 ≤ P b 0.05 or P ≤ 0.01 between PH and SO (de Menezes et al., 2004) were considered as LR-related genes. 2.6. Construction of the interaction network of IL-18 signaling pathways The terms of “IL-18 signaling pathway” were entered to the websites NCBI (www.ncbi.nlm.nih.gov) and RGD (www.rgd.mcw.edu) to find out IL-18 signaling pathway-associated genes. Then, the genes were collated according to physiological pathway maps embodied by Pathway Maps (http://pathwaymaps.com/maps/) and Ingenuity Pathway Analysis 9.0 (IPA) software (Kong et al., 2011; Xu et al., 2012). Subsequently, the transcription factors of IL-18 signaling pathway were input into TRED (http://rulai.cshl.edu/cgi-bin/ TRED/tred.cgi?process = searchTF GeneForm) and Lymph TF DB (http://www.iupui.edu/~tfinterx/ activity.php) to search for their downstream target genes (Childress et al., 2007; Jiang et al., 2007), which were then matched against proliferation-related genes by Gene Ontology (http://www.ncbi.nlm. nih.gov/gene) to select the target genes only related to cell proliferation. Literature review was performed to confirm the function of these genes identified by NCBI database.

2.3. Rat Genome 230 2.0 microarray and data analysis

2.7. Gene synergy analysis of IL-18 signaling pathway

Total RNA was extracted and purified by the protocol previously described (Twigger et al., 2006). The cDNA first chain was synthesized by SuperScript II RT reverse transcription system, and the second chain was synthesized according to the guideline of Affymetrix cDNA kit. Biotinlabeled cRNA was prepared using GeneChip IVT kit as instructed by

According to the gene expression abundance of IL-18 signaling pathway in hepatocytes of regenerating liver detected by Rat Genome 230 2.0 Array, expression profile function (Et), a mathematical model, was used to measure gene synergy between the related genes with in time series analysis (Tong, 1978), correlation analysis (rik) (Eisen et al.,

232

J. Zhang et al. / Gene 537 (2014) 230–237

1998) based on the multivariate statistical method (McGuigan, 2006). This mathematical model describes how physiological activities are governed by gene synergy (Wang et al., 2009).

n X n h  i X ðt Þ ðt Þ Xi þ Xk  rik

Et ¼

i¼l k¼l

nðn þ 1Þ

In the formula, the gene correlation coefficient (rik) was defined as:

m

m X

! ðt Þ

Xi Xk

ðt Þ

−

m X

! Xi

ðt Þ

m X

! Xk

ðtÞ

t¼1 t¼1 t¼1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi rik ¼ v" !#" !# u m m m m X X X 2 2 u X 2 ðt Þ ðtÞ ðtÞ ðt Þ 2 t m m Xi − Xi Xk − Xk t¼1

t¼1

t¼1

t¼1

“E” means gene synergy value which refers to physiological activity. (t) “t” represents order time series. “X(t) i ” and “Xk ” are abundances of gene at t. “n” is the total number of genes. Physiological activities in LR are more strengthened than in control when 0 b Et of PH N |Et| of SO; the activities are suppressed or lowered when Et of PH b 0 or Et of PH b |Et| of SO, and the activities are similar in two groups when Et of PH b 0 or 0 b Et of PH b |Et| of SO. 2.8. Preparation of plasma Rats were sacrificed at 0, 2, 6, 12, 24, 48, 72 h after PH. The abdominal cavity was opened and 2 ml of blood was collected from inferior vena cava using EDTA as an anticoagulant. Plasma was prepared according to the manufacturer's instruction (Mouse/Rat HGF Quantikine ELISA, R&D Systems). Briefly, the blood samples were centrifuged for 20 min at 2000 g at room temperature. Supernatant was then transferred to a fresh tube and again centrifuged for 10 min at 10,000 g, 2–8 °C for completely removing platelets.

at 8-μm thickness. After de-waxed and rehydrated according to standard procedure, sections were treated in 0.1% tyrisin at 37 °C for 1 h and denatured in 1 M HCl at 56 °C for 15 min, incubated with antiBrdU monoclonal antibody (Sigma, USA) at 37 °C for 40 min and FITCsecondary antibody (Sigma, USA) for 35 min at room temperature. Cell nuclei were stained with 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, USA). At each step described above, the sections were rinsed in PBS 3 times. Under a fluorescence microscope, 10 random non-overlapping fields were selected and the number of BrdU-positive and nuclei in the same fields was counted using Image-Pro Plus 6.0 software. The ratio of proliferating cells was expressed as the proportion of BrdU-positive cells to total number of cells. Data are expressed as mean ± SD. Statistical significance was determined by One-Way ANOVA using SPSS 13.0 software. P value b 0.05 was considered as statistically significant. 3. Results 3.1. Reliability of the microarray results Rat Genome 230 2.0 microarray, contained 24,618 rat genes, was used to detect hepatocyte genes expression abundance at each time point in rat liver regeneration. To validate reliability of these results, four genes, CCND1, AGT, MAPK10 and HMOX1, were randomly selected for RT-PCR analysis. It was found that the results of RT-PCR and Rat Genome 230 2.0 Array were generally consistent (Fig. 1). 3.2. Expression changes of genes related to IL-18 signaling pathway during rat LR Based on gene information in NCBI, RGD and IL-18 biological pathway map in Pathway Maps and IPA, there are three branches with 33 genes involved in IL-18 signaling pathway. The microarray results showed that 13 of these genes were changed significantly during rat LR (Fig. 2). Of them, 11 genes were up-regulated, MAP2K6 was downregulated and CCNA2 was up/down-regulated during the course of LR. 9 of them were identified as LR-related genes by comparing the gene differential expression in PH and SO group (Table 1). The results above showed that the expression of majority of genes involved in IL-18 signaling pathway was increased in rat LR after PH and may be involved in regulating hepatocyte proliferation.

2.9. Enzyme-linked immuno-sorbent assay (ELISA) IL-18 concentration in plasma was assayed by ELISA kit (Invitrogen, USA). Standard dilution curve was prepared in parallel with the samples. The assay was performed following the manufacturer's specifications. The absolute amount of IL-18 was calculated based on known quantities of recombinant cytokine standards provided by ELISA kit. 2.10. Treatment with rat recombinant IL-18 Rats were randomly divided into five groups with six rats per group: SO + PBS, PH + PBS, PH + IL-18 (0.5 μg), PH + IL-18 (1 μg) and PH + IL-18 (4 μg). Those in the SO + PBS and PH + PBS groups were treated with 2 ml sterile PBS by tail vein, whereas those in the PH + IL-18 (0.5 μg), PH + IL-18 (1 μg) and PH + IL-18 (4 μg) groups were treated with 2 ml sterile PBS containing 0.5 μg, 1 μg or 4 μg of recombinant rat IL-18 (rrIL-18, R&D Systems, USA).

3.3. Expression changes of cell proliferation genes regulated by IL-18 signaling pathway during rat LR The results showed that the transcription factors, NF-κB and c-JUN, were activated in hepatocytes by IL-18 signaling pathway during rat LR. NF-κB was up-regulated rapidly at 2 h and then rose again at 120 h after PH. c-JUN was up-regulated at 2-6 h, 24 h, 72 h and 168 h during rat LR (Table 1). Their expression level was significantly different between PH and SO group detected by F test (p b 0.05). Based on the information from TRED and Lymph TF DB, a total of 395 downstream genes can be regulated by transcription factors NF-κB, ATF2 and c-JUN. Rat Genome 230 2.0 microarray contained 322 of these genes, 103 of which were related to hepatocyte proliferation. Of the 103 genes, 49 genes showed significant expression change in hepatocytes of rat regenerating liver: 43 genes were up-regulated, 3 genes were down-regulated and 3 genes were up/down regulated (Supplemental table).

2.11. BrdU labeling and immunohistochemistry To assess hepatocyte proliferation in vivo, DNA synthesis activity was monitored by bromodeoxyuridine (BrdU, Sigma, USA) assay. After treatment with rrIL-18, BrdU at 50 mg/kg body weight was injected into rat peritoneum 1 h prior to sacrifice. Liver was then harvested and fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned

3.4. The correlation of IL-18 signaling activity with hepatocyte proliferation activity during rat LR In this paper, expression profile function Et was used to analyze the synergistic effects of genes related with IL-18 signaling pathway and genes related with hepatocyte proliferation in regenerative liver. The

J. Zhang et al. / Gene 537 (2014) 230–237

233

Fig. 1. Relative mRNA levels. mRNA levels of gene-related IL-18 signaling pathway were detected by Affymetrix Rat Genome 230 2.0 Array (solid lines) and real-time quantitative PCR (dotted lines) in rat regenerating liver during LR. Every sample of RT-PCR was tested in triplicate from three independent experiments. Three results are similar, and one set of data was presented here.

results indicated that three branches of IL-18 signaling pathway were consistent trend with hepatocyte proliferation during rat LR. Of them, the Et values of NF-κB signaling branch and its downstream genes related to cell proliferation were higher than that of SO groups during 2–36 h and PH 6–72 h after PH, respectively. Similarly, the Et values of p38 branch were higher than that of in SO groups at 2 h, 24–120 h, and the downstream cell proliferation target genes regulated by translation factor ATF2 were enhanced at 2–72 h after PH. The Et values of JNK branch were higher than that of in SO groups at 2 h, 12–72 h, and

downstream cell proliferation target genes regulated by translation factor c-JUN were also enhanced at 6–24 h, 72 h (Fig. 3). Together, these results indicated that IL-18 may promote proliferation of hepatocytes at the priming phase and progressing phase via NF-κB, p38 and JNK branches. 3.5. The expression changes of IL-18 during rat LR The mRNA level of IL-18 increased at 2 h after PH, and then decreased at 24 h. Subsequently, the level went on elevated at 36 h and returned to normal level at 72 h (Fig. 4A). The concentration of IL-18 in plasma began to increase at 2 h after PH, climbed to 33.9 pg/ml at 6 h and reached 38.5 pg/ml at 12 h after PH, both of which were significantly higher than the normal control group 16.5 pg/ml (p b 0.05 or p b 0.01, respectively). The IL-18 concentration in plasma was then declined to normal level at 24 h after PH. Interestingly, it went up again at 48 h to 29.9 pg/ml and returned to normal level at 72 h (Fig. 4B). The results indicated that the expression profiles of IL-18 mRNA and protein have a similar pattern in general after PH. 3.6. The effect of rrIL-18 on hepatocyte proliferation after PH and the metabolism in vivo

Fig. 2. Three branches of IL-18 signaling pathway. The network of IL-18 signaling pathway was obtained by “Pathway Designer” of IPA software according to the information from Pathway Maps and IPA software. Symbols in red, green and blue represent upregulated, down-regulated and up/down-regulated genes in hepatocytes during LR.

The pharmacokinetics of rrIL-18 was determined using rat IL-18 ELISA kit (Invitrogen corporation, Camarillo US). The results showed that IL-18 plasma level was significantly increased at 1 h after the rrIL18 treatment and returned to baseline at 24 h (Fig. 5 B). The concentration of total IL-18 was increased in a dosage-dependent manner to the amount of injected rrIL-18. The effect of rrIL-18 on rat hepatocyte proliferation was examined after PH. BrdU was as a marker of S-phase activity. The results revealed that the mitotic indexes of regenerating liver were significantly increased at 24 h after the rats were treated with 4 μg of IL-18 protein (about 20 μg/kg) (p b 0.01 PH vs PBS control). However, no significant differences were observed in rats treated with low dosages of rrIl-18, either with 0.5 μg or 1 μg (p b 0.5, PH + rrIL-18 verse SO + PBS). A negligible number of labeled cells was observed at 24 h in the liver of

234

J. Zhang et al. / Gene 537 (2014) 230–237

Table 1 Expression changes of IL-18 pathway related genes during rat LR.

1, 2 and 3 represent NF-κB, p38 and JNK branch of IL-18 signaling pathway, respectively; the values in red and green represent the expression abundance of up-regulated, down-regulated genes and other values are insignificantly changed. ▲ represents the gene related with LR.

SO + PBS (b0.01%) (Figs. 5 A and C). The results suggested that IL-18 at a high concentration can accelerate hepatocyte proliferation in rat LR after PH. 4. Discussion Liver regeneration is regulated by a sequence of orchestrated events including activation/expression of a series of cytokines and growth factors, accompanied by cell proliferation, cell differentiation and dedifferentiation, and tissue structure reconstruction, etc. To obtain the overall information of genes involved in rat LR induced by PH, Rat Genome 230 2.0 with 24,618 genes was used to determine the gene expression profiles in hepatocytes. In this paper, the gene synergistic effects were analyzed by a mathematical model (Et) to measure gene synergy of every branch in the IL-18 signaling pathway (Xu et al., 2010). The results showed that the gene synergy value of the three branches, NF-κB, JNK, and p38, were significantly higher in PH group than that of in the SO

group during the early stages of LR covering from 2 to 72 h after PH. The gene synergy value of hepatocyte proliferation genes regulated by the three branches was also increased during the same period of LR, meaning that they possibly take part in proliferation of hepatocytes during 2–72 h of LR. In NF-κB branch, IL18R1 was increased at 2 h, and MYD88 began to rise at 2 h and peaked at 6 h after PH. Meanwhile, NFKB2 was also quickly up-regulated at 2 h during LR after PH. In addition, activation of NF-κB is initiated when I-κB is phosphorylated by I-κB kinase (IKK). The microarray results showed that IKK was significantly up-regulated during rat LR whereas the expression of I-kB was only moderately increased. Kojima et al. reported that IL18R1, specifically binding of IL-18, may activate or increase the transfer of transcription factor NFκB from cytoplasm to nucleus (Kojima et al., 1998) so as to regulate downstream target gene transcription. It has also been proved that IL18 binding to its receptor can result in a rapid activation of NF-κB at 1 h after PH and promote cell cycle progression of the hepatocytes

Fig. 3. The proliferation of hepatocytes regulated by IL-18 signaling pathway during rat LR. The bars in red, blue, grey and yellow show signaling activities of IL-18 pathway, NF-κB, JNK and JNK branch, respectively. The white bar shows their corresponding physiological activities at the recovery time after PH.

J. Zhang et al. / Gene 537 (2014) 230–237

235

Fig. 4. Expression levels of IL-18 during rat LR. (A) Dotted lines represent the results obtained with Affymetrix rat Genome 230 2.0 microarrays. Solid black line represents the results detected by RT-PCR. (B) Histogram represents IL-18 protein level in plasma during LR after rat PH. Bar graphs show mean ± SD for three individual animals from three independent experiments. The differences among groups after PH were assessed using one-way ANOVA test with SPSS 13.0 software. * P b 0.05, ** P b 0.01.

(Beg et al., 1995; Iimuro et al., 1998; Marquez et al., 2010) which is generally consistent with our results. MYD88, a universal adapter molecular, is a critical component in the signaling cascade that is mediated by IL-18 receptor and then signals through IRAK to activate the transcription factor NF-κB (Adachi et al., 1998). Thus, the overall outcome may favor the activation of NF-κB during LR. In JNK branch, our microarray results showed that GADD45G (encoding GADD45γ), MAPK8, MAPK10 (encoding JNK) and JUN

(encoding c-JUN) were all promptly increased at 2 h after PH and stayed going up almost throughout the whole LR process. GADD45γ encoded by GADD45G mediates activation of the p38/JNK pathway responding to environmental stresses, and JNK (c-JUN N-terminal kinase) is involved in a wide variety of cellular processes such as proliferation, differentiation etc. Researchers have proved that overexpression of c-JUN exhibits accelerated cell proliferation by decreased level of p53 and p21 (Schreiber et al., 1999). c-JUN can also protect hepatocytes from apoptosis, as

Fig. 5. Effect of IL-18 treatment on rat hepatocyte proliferation assessed by BrdU assay. (A) Rats were prepared for PH, treated with rrIL-18, and sacrificed at 24 h after the treatment. Rat liver sections were prepared and stained for BrdU-positive cells (A–E). The total numbers of cells were determined by DAPI staining (a–e). (B) IL-18 levels in plasma of rat liver were detected using rat IL-18 ELISA kit (R&D systems). Gray and white showed the results at 1 h and 24 h after rrIL-18 injection in rats by tail vein, respectively. (C) BrdU-positive cells and nucleus were counted as described in “Materials and methods”. Grey color was for total cell number and white was for BrdU-positive cells in regenerating liver at 24 h after treatment with IL-18 protein. The experiments were repeated twice with three rats per group. Statistical analysis was performed by one-way ANOVA. * indicates P b 0.05, ** indicates P b 0.01.

236

J. Zhang et al. / Gene 537 (2014) 230–237

hepatocytes lacking c-Jun showed increased sensitivity to TNFα-induced apoptosis (Eferl et al., 2003). Alcorn et al. showed that c-JUN was upregulated at 0.5 h after 30% liver resection in mouse (Alcorn et al., 1990; Westwick et al., 1995). Moreover, the role of JNK pathway in proliferation of hepatocytes has been reported by Schwabe et al., who proved that JNK pathway was strongly activated after PH and its downstream target gene, CCND1, was up-regulated to drive G0 to G1 transition in hepatocytes during LR (Schwabe et al., 2003), which is in accordance with our results. When JNK was blocked, cell proliferation and cell-cycle transition were inhibited (Ma et al., 2012). Both our microarray results and previous studies confirmed that JNK branch may be involved in regulating hepatocyte proliferation in rat LR. In p38 branch, MAPK14 (encoding p38α), CCNA2 (encoding Cyclin A2), MAP2K6 (encoding MKK6) were down-regulated during rat LR after PH. p38α kinase can be activated by proinflammatory cytokines (such as interleukin-1) and play a dual role, acting as either a mediator of cell survival or cell death depending on the cell type and the stimuli. While MAP2K6 is an essential component of p38 MAP kinase mediated signal transduction pathway by dual phosphorylation p38 on Thr180 and Try182 (Raingeaud et al., 1995; Raingeaud et al., 1996). Our microarray results showed that MAP2K6 was down-regulated from 2 h to 24 h, and MAPK14 was weakly increased at 6 h after PH. Additionally, activation of p38α kinase was implicated in activation of transcription factor ATF2, mediating proliferation signals (Lewis et al., 2005; Recio and Merlino, 2002). However, in our microarray data ATF2 did not present significant change during LR. Nonetheless, CCNA2, a downstream target gene of ATF2 (Shimizu et al., 1998), started to up-regulate at 2 h, and had a significantly higher level from 24 to 120 h after PH than the control in our microarray data. CCNA2 can promote both cell cycle G1/S and G2/M transitions. Thus, the results above may favor the activation of p38 branch during rat LR, which may participate in regulating hepatocyte proliferation. In addition, we confirmed that IL-18 level was changed during the course of rat LR, at both mRNA level and protein level. These changes occurred mainly during the early stages of LR, which was in parallel with the peak of hepatocyte proliferation activity. However, the expression level of IL-18 in mRNA and protein during rat LR is not entirely consistent that may be due to a time difference of translation from mRNA to protein. Shiraki et al. reported that serum level of IL-18 in the liver donors was increased significantly in the second day after liver resection, and was 4-fold higher than preoperative level in the fourth day and then, returned to the preoperative level in the seventh day (Shiraki et al., 2007). Subsequently, they reported that IL-18 could inhibit apoptosis of hepatic cells after liver injury (Yamamoto et al., 2008). The results of ours and Shiraki showed that the change of IL-18 level has a positive correlation with hepatic cell proliferation. More importantly, we further discovered that the injection of rrIL-18 can directly accelerate hepatocyte proliferation. Other studies showed that the addition of exogenous IL-18 to primary cultured rat chondrocytes and dog osteoblasts increased the incorporation of H3-thymidine with a dose-dependent manner, suggesting that IL-18 acts as a mitogen and promotes the proliferation of chondrocytes and osteoblasts (Cornish et al., 2003). IL-18 is expressed in rat testis and can also promote germ cell growth (Strand et al., 2005). In conclusion, IL-18 has a positive role in the regulation of rat hepatocyte proliferation after PH, which is supported by the increased expression of IL-18, its signaling activities and downstream genes after PH, as well as the accelerated hepatocyte proliferation by exogenous rrIL-18. These results provide strong evidence that IL-18 can promote cell proliferation. Our findings suggested that IL-18 may serve as a positive regulator of hepatocyte proliferation, and we will further confirm the results of the study using gene addition, knock-out, RNAi. Future, regulation of IL-18 production or manipulation of IL-18 signaling pathway may have therapeutic potential for acute liver failure and liver regeneration. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.12.062.

Conflict of interest No conflict of interest to disclose. Acknowledgment Funding: This work was supported by the National Basic Research 973 Pre-research Program of China (No. 2012CB722304). References Adachi, O., et al., 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9 (1), 143–150. Alcorn, J.A., Feitelberg, S.P., Brenner, D.A., 1990. Transient induction of c-jun during hepatic regeneration. Hepatology 11 (6), 909–915. Beg, A.A., Sha, W.C., Bronson, R.T., Ghosh, S., Baltimore, D., 1995. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 376 (6536), 167–170. Chen, X.G., Xu, C.S., Liu, Y.M., 2013. Involvement of ERK1/2 signaling in proliferation of eight liver cell types during hepatic regeneration in rats. Genet. Mol. Res. 12 (1), 665–677. Childress, P.J., Fletcher, R.L., Perumal, N.B., 2007. LymphTF-DB: a database of transcription factors involved in lymphocyte development. Genes Immun. 8, 360–365. Cornish, J., Gillespie, M.T., Callon, K.E., Horwood, N.J., Moseley, J.M., Reid, I.R., 2003. Interleukin-18 is a novel mitogen of osteogenic and chondrogenic cells. Endocrinology 144 (4), 1194–1201. de Menezes, R.X., Boer, J.M., van Houwelingen, H.C., 2004. Microarray data analysis: a hierarchical T-test to handle heteroscedasticity. Appl. Bioinformatics 3 (4), 229–235. Eferl, R., et al., 2003. Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell 112 (2), 181–192. Eisen, M.B, Spellman, P.T., Brown, P.O., Botstein, D., 1998. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. U. S. A. 95, 14863–14868. Fausto, N., Laird, A.D., Webber, E.M., 1995. Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration. FASEB J. 9 (15), 1527–1536. Fausto, N., Campbell, J.S., Riehle, K.J., 2006. Liver regeneration. Hepatology 43 (2 Suppl 1), S45–S53. Fukuhara, Y., et al., 2003. Gene expression profile in the regenerating rat liver after partial hepatectomy. J. Hepatol. 38 (6), 784–792. Guo, W., Cai, C., Wang, C., Zhao, L., Wang, L., Zhang, T., 2008. A preliminary analysis of genome structure and composition in Gossypium hirsutum. BMC Genomics 9, 314. Higgins, G.M., Anderson, R.M., 1931. Experimental pathology of the liver: restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12, 186–202. Iimuro, Y., et al., 1998. NFkappaB prevents apoptosis and liver dysfunction during liver degeneration. J. Clin. Invest. 101 (4), 802–811. Jiang, C., Xuan, Z., Zhao, F., Zhang, M.Q., 2007. TRED: a transcriptional regulatory element database, new entries and other development. Nucleic Acids Res. 35, D137–D140. Khan, A.Z., Mudan, S.S., 2007. Liver regeneration: mechanisms, mysteries and more. ANZ J. Surg. 77 (1–2), 9–14. Kojima, H., et al., 1998. Interleukin-18 activates the IRAK-TRAF6 pathway in mouse EL-4 cells. Biochem. Biophys. Res. Commun. 244 (1), 183–186. Kong, B.W., Lee, J.Y., Bottje, W.G., Lassiter, K., Lee, J., Foster, D.N., 2011. Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line. BMC Genomics 12, 571. Lewis, J.S., et al., 2005. Activation of cyclin D1 by estradiol and spermine in MCF-7 breast cancer cells: a mechanism involving the p38 MAP kinase and phosphorylation of ATF-2. Oncol. Res. 15 (3), 113–128. Ma, X.M., Dong, X.S., Zhao, H.L., 2009. Research progress of liver regeneration-related molecules. Int. J. Surg. 36, 760–763. Ma, J., et al., 2012. Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis induced by EET in pulmonary artery endothelial cells. J. Lipid Res. 53 (6), 1093–1105. Marquez, R.T., Wendlandt, E., Galle, C.S., Keck, K., McCaffrey, A.P., 2010. MicroRNA-21 is upregulated during the proliferative phase of liver regeneration, targets Pellino-1, and inhibits NF-kappaB signaling. Am. J. Physiol. Gastrointest. Liver Physiol. 298 (4), G535–G541. McGuigan, K., 2006. Studying phenotypic evolution using multivariate quantitative genetics. Mol. Ecol. 15 (4), 883–896. Michalopoulos, G.K., 2007. Liver regeneration. J. Cell. Physiol. 213 (2), 286–300. Nault, R., Kim, S., Zacharewski, T.R., 2013. Comparison of TCDD-elicited genome-wide hepatic gene expression in Sprague–Dawley rats and C57BL/6 mice. Toxicol. Appl. Pharmacol. 267 (2), 184–191. Raingeaud, J., et al., 1995. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J. Biol. Chem. 270 (13), 7420–7426. Raingeaud, J., Whitmarsh, A.J., Barrett, T., Dérijard, B., Davis, R.J., 1996. MKK3- and MKK6regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol. Cell Biol. 16 (3), 1247–1255. Recio, J.A., Merlino, G., 2002. Hepatocyte growth factor/scatter factor activates proliferation in melanoma cells through p38 MAPK, ATF-2 and cyclin D1. Oncogene 21 (7), 1000–1008.

J. Zhang et al. / Gene 537 (2014) 230–237 Schreiber, M., et al., 1999. Control of cell cycle progression by c-jun is p53 dependent. Genes Dev. 13 (5), 607–619. Schwabe, R.F., et al., 2003. c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration. Hepatology 37 (4), 824–832. Sell, S., 2003. The hepatocyte: heterogeneity and plasticity of liver cells. Int. J. Biochem. Cell Biol. 35 (3), 267–271. Shimizu, M., et al., 1998. Activation of the rat cyclin A promoter by ATF2 and Jun family members and its suppression by ATF4. Exp. Cell Res. 239 (1), 93–103. Shiraki, K., Yamaguchi, Y., Uemoto, S., 2007. Serum interleukin-18 during liver regeneration. Hepatogastroenterology 54 (76), 1. Strand, M.L., Wahlgren, A., Svechnikov, K., Zetterström, C., Setchell, B.P., Söder, O., 2005. Interleukin-18 is expressed in rat testis and may promote germ cell growth. Mol. Cell. Endocrinol. 240 (1–2), 64–73. Sugihara, I., 2000. Interleukin-18 (IL-18) and infectious disease, with special emphasis on disease induced by intracellular pathogen. Microbes Infect. 2 (10), 1257–1263. Taub, R., 2004. Liver regeneration: from myth to mechanism. Nat. Rev. Mol. Cell Biol. 5 (10), 836–847. Tong, H., 1978. On a threshold model, pattern recognition and signal processing. In: Chen, C.H. (Ed.), NATO ASI series E: Applied Sc No. 29. Sijthoff & Noordhoff, Alphen aan den Rijn, The Netherland. Tsutsui, H., Adachi, K., Seki, E., Nakanishi, K., 2003. Cytokine-induced inflammatory liver disease. Curr. Mol. Med. 3 (6), 545–559.

237

Twigger, S.N., S Smith, J., Zuniga-Meyer, A., Bromberg, S.K., 2006. Exploring phenotypic data at the rat genome database. Curr. Protoc. Bioinforma. (Chapter 1, Unit 1.14). Vondran, F.W., et al., 2008. Isolation of primary human hepatocytes after partial hepatectomy: criteria for identification of the most promising liver specimen. Artif. Organs 32 (3), 205–213. Wang, W.B., Xie, L.F., Wang, W., Wang, L., Xu, C.S., 2008. Isolation, purity and identification of hepatocytes in rat normal liver and regenerating liver. Henan Sci. 26, 1492–1498. Wang, W.B., Fan, J.M., Zhang, X.L., Xu, J., Yao, W., 2009. Serial expressing analysis of liver regeneration-related genes in rat regenerating liver. Mol. Biotechnol. 43, 221–231. Westwick, J.K., Weitzel, C., Leffert, H.L., Brenner, D.A., 1995. Activation of Jun kinase is an early event in hepatic regeneration. J. Clin. Invest. 95 (2), 803–810. Xu, C., et al., 2010. Transcriptome analysis of hepatocytes after partial hepatectomy in rats. Dev. Genes Evol. 220 (9–10), 263–274. Xu, C.S., Wang, G.P., Zhang, L.X., Chang, C.F., Zhi, J., Hao, Y.P., 2011. Correlation between liver cancer occurrence and gene expression profiles in rat liver tissue. Genet. Mol. Res. 10 (4), 3480–3513. Xu, C.S., Yang, Y.J., Yang, J.Y., Chen, X.G., Wang, G.P., 2012. Analysis of the role of the integrin signaling pathway in hepatocytes during rat liver regeneration. Cell. Mol. Biol. Lett. 17, 274–288. Yamamoto, N., et al., 2008. Protective role of interleukin-18 against Fas-mediated liver injury. Int. J. Mol. Med. 22 (1), 43–48.