Overexpression of the gene for transmembrane 4 superfamily member 4 accelerates liver damage in rats treated with CCl4

Journal of Hepatology 46 (2007) 266–275 www.elsevier.com/locate/jhep

Overexpression of the gene for transmembrane 4 superfamily member 4 accelerates liver damage in rats treated with CCl4 Jie Qiu1, Zhanwu Liu1, Liang Da1, Ying Li1, Haixing Xuan1, Qishui Lin1, Feng Li2, Yifei Wang2, Zaiping Li1, Mujun Zhao1,* 1

State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People’s Republic of China 2 Department of Mathematics, Shanghai University, 99 Shangda Road, Shanghai 200444, People’s Republic of China

Background/Aims: Transmembrane 4 superfamily member 4 (TM4SF4) is up-regulated in regenerating liver after partial hepatectomy in rats, but the in vivo functions of this protein are still largely unknown. Therefore, we investigated the role of TM4SF4 during liver injury. Methods: Expression of TM4SF4 was analyzed by RT-PCR and Western blotting in normal and CCl4-injured rats. Overexpression or reduced expression of TM4SF4 in the liver was achieved by injection of sense or antisense TM4SF4 expression plasmids. Assessment of liver injury (histology, serum ALT and AST levels), apoptosis by TUNEL assay were performed. Expression of injury-related genes was analyzed by quantitative real-time PCR. Results: Overexpression of TM4SF4 in rats after CCl4 treatment showed extensive liver damage and increased levels of serum ALT and AST. Decreased TM4SF4 gene expression showed minimal liver necrosis and depressed ALT and AST levels. Increased expression of TM4SF4 affected the expression levels of growth factors and receptors, such as TNF-a, TNFR1 and c-met. Furthermore, pro-apoptotic and anti-apoptotic gene expression was altered after TM4SF4 administration. Conclusions: Rat TM4SF4 is overexpressed in acutely injured liver induced by CCl4 and plays a crucial role in accelerating liver injury, which may be mediated by the TNF-a and HGF/c-met signaling pathways.  2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: TM4SF4; Carbon tetrachloride; Acute liver injury

1. Introduction Rat TM4SF4 (transmembrane 4 superfamily member 4) was first identified as a gene product that was overReceived 23 January 2006; received in revised form 20 July 2006; accepted 21 August 2006; available online 2 October 2006 * Corresponding author. Tel.: +86 21 54921115; fax: +86 21 54921011. E-mail address: [email protected] (M. Zhao). Abbreviations: TM4SF4, transmembrane 4 superfamily member 4; CCl4, carbon tetrachloride; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SA liposome, stearylamine liposome; TUNEL, terminal deoxynucleotidyl transferase-mediated uDP nickend labeling assay.

expressed in regenerating liver after partial hepatectomy [1]. Rat TM4SF4 closely resembles human il-TMP, which mediates cell density-associated inhibition of proliferation [2]. TM4SF4 belongs to a large family of ubiquitously expressed membrane proteins that have four putative membrane-spanning domains, short N- and C-terminal cytoplasmic domains, a small intracellular loop and two extracellular domains [3]. Recent studies have shown that transmembrane 4 superfamily proteins associate with each other and with membrane and intracellular signaling molecules such as integrins, pro-growth factors and their receptors, and protein kinase-C [4–9]. These interactions facilitate the formation of membrane

0168-8278/$32.00  2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2006.08.011

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275

signaling microdomains, which regulate cell motility and proliferation, trigger homotypic cell aggregation, and participate in various types of cell fusion and signaling [10–13]. However, the in vivo function conferred by TM4SF4 is still largely unknown. Carbon tetrachloride (CCl4)-mediated liver injury is a well-established model for studying liver regeneration in rodents. It is mainly characterized by acute hepatocellular necrosis caused by alterations in permeability of cellular, mitochondrial, and lysosomal membranes, and it has been shown to induce hepatocyte apoptosis [14–17]. CCl4 injury activates many growth factors, such as tumor necrosis factor alpha (TNFa), transforming growth factor beta (TGF-b) and hepatocyte growth factor (HGF) [18–21]. TNF-a plays an ambiguous role in CCl4-induced liver injury, in which its effect can be either damaging or protective [15,22–24]. TGF-b acts as a potent growth inhibitor and a pro-fibrotic cytokine after CCl4 exposure [23,25,26]. HGF inhibits liver injury by stimulating hepatocyte proliferation and protecting hepatocytes from apoptosis [27]. The TNF-a pathway, TGF-b pathway and HGF pathway might play a critical role in CCl4-mediated liver injury. In the present study we examined the in vivo functions of rat TM4SF4 in CCl4-induced acute liver injury and further analyzed the signaling pathways in which TM4SF4 may be involved.

2. Materials and methods 2.1. Animals Six-week-old male Sprague–Dawley rats with a body weight of 180 ± 20 g were obtained from the Shanghai Experimental Animal Center (Shanghai, China) and had free access to water and food. Animals received humane care in compliance with the institutional guidelines of Shanghai Institutes for Biological Sciences.

2.2. Plasmid construction The rat cDNA for TM4SF4 gene was originally cloned by our laboratory and was deposited in the GenBank database under Accession No. AF205717, as described by Liu et al. [1]. To obtain the sense TM4SF4 expression plasmid, the cDNA fragment containing the entire open-reading frame (ORF) of TM4SF4 gene was inserted into pcDNA3 plasmid using EcoRI and XhoI restriction sites. The antisense TM4SF4 plasmid (pTM4SF4-AS) was constructed by cloning the TM4SF4 ORF in the reverse direction into the pcDNA3 plasmid using the BamHI and EcoRI restriction sites.

2.3. CCl4-induced liver injury Rats were given 500 ll of CCl4 in corn oil (3:7 v/v) per 100 g of body weight using an oral gastric catheter. Control rats received the same volume of corn oil alone. Animals were sacrificed at 12, 24, 48 or 72 h after CCl4 treatment. The tissues from control and CCl4-treated rats were removed and immediately frozen in liquid nitrogen until further needed.

267

2.4. Treatment with sense or antisense TM4SF4 gene injection Seventy-two CCl4-injured rats were randomly divided into four groups, receiving pTM4SF4 plasmid, pTM4SF4-AS plasmid, pcDNA3 vehicle or PBS solution through tail-vein injection, respectively. Each group contains 18 rats and each rat received the therapeutic complex, containing 200 ll stearylamine liposome (SA-liposome, 2 mg/ml) [28] and 500 lg of recombinant plasmid or 600 ll PBS, at 6 h following intragastric administration of CCl4. Animals were sacrificed at 24, 48 and 72 h after CCl4 injury (six rats per group at each time point, respectively), serum and liver tissues from each rat were collected. Part of the liver tissues were cut into pieces and put into fixation solution (4% paraformaldehyde) for histological assay, and the rest were snap frozen in liquid nitrogen for verifying the specific mRNA or protein expression.

2.5. ALT and AST assay Serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with an autoanalyzer (Hitachi 7020 automatic analyzer, Hitachi Co., Tokyo, Japan) and expressed in IU/l.

2.6. Histological examination In all studies of acute injury, formalin-fixed, paraffin-embedded liver sections (5 lm) were stained with hematoxylin and eosin (HE, Amresco Inc., Solon, Ohio). Degrees of centrilobular necrosis and inflammatory infiltration were evaluated on a 4-point scale (Table 1) [29,30] in 20 random fields at 10· magnification per animal (n = 6 per group) by a blinded pathologist.

2.7. Immunoblots Preparation of whole liver extracts and Western blot analysis were carried out as previously described [1]. Primary antibodies used were anti-TM4SF4 [1] (1:2000 dilution), anti-TNF receptor I (1:500 dilution, Abcam, Cambridge, United Kingdom), anti-a-tubulin (H-300) and anti-Met (C-28) (1:1000 dilution, Santa Cruz Biotechnology Inc., CA, USA). Respective secondary antibodies were used (1:5000 dilution, Santa Cruz). a-Tubulin protein expression was used as a loading control. Bands were visualized with an ECL detection system (Perfect Biotech, Shanghai, PR China) and quantified by scanning densitometry using UVP LABWORKS Imaging Analysis software (UVP LABWORKS, Upland, CA).

2.8. RNA isolation, RT-PCR and quantitative real-time PCR Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). Five micrograms of total RNA was reverse-transcribed with SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. To ensure that no DNA contaminations were in the RNA samples, the non-reversetranscribed RNA extracts were checked by PCR, to make sure no target fragment produced. PCRs were performed for 30 cycles with the following cycling conditions: 95 C for 30 s, 55 C for 30 s, 72 C for 30 s and finished with 10 min at 72 C. Primers used to amplify the specific transcripts are shown in Table 2. PCR products were analyzed on 1.2% agarose gel and bands were evaluated with UVP LABWORKS Imaging Analysis Software. Quantitative real-time PCR was carried out to estimate the mRNA levels using the Quantitect SYBR Green kit (Qiagen, Valencia, CA) and the DNA Engine 2 Opticon Continuous Fluorescence Detector (MJ Research Incorporated, Boston). Primers for detection of the mRNA levels of target genes are shown in Table 2 and 10 ng cDNA was used as the template. Rat liver cDNA was fivefold serially diluted

268

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275

Table 1 Histological assessment of livers following CCl4 injury and plasmid administration Score

Centrilobular necrosis

0 1 2

No morphologic evidence of injury Scattered pericentral necrosis seen occasionally, only within a five-hepatocyte circumference around the central vein (zone 3) Pericentral necrosis seen uniformly, but only within a five-hepatocyte circumference around the central vein, with the other areas undamaged Widespread hepatocellular necrosis, extending beyond a five-hepatocyte circumference around the central vein, frequently reaching to the midzone, with confluent and/or bridging necrosis

3

Inflammation in centrilobular areas 0 1 2 3

None Mild: inflammatory infiltrate affecting <50% centrilobular areas Moderate: inflammatory infiltrate affecting >50% and <75% centrilobular areas Severe: dense inflammatory infiltrate affecting >75% centrilobular areas

Blinded pathologist examined 20, 10· fields per animal (n = 6 per group) and scored centrilobular necrosis and inflammation independently on 4-point scale (0–3). Data were analyzed using Mann–Whitney U test and represented graphically as means ± SD in Fig. 3.

to generate a standard curve including five points. Quantitative RTPCR was performed including the standard curve, no-template controls and samples in triplicate. The levels of GAPDH mRNA were quantified as internal controls [31]. The mRNA levels of target genes were normalized according to the GAPDH mRNA levels. PCR products were visualized with gel electrophoresis to confirm a single product of the correct size.

respectively. As a control for method specificity, CCl4/pTM4SF4 treated livers were stained with labeling solution (without terminal transferase) instead of the TUNEL reaction mixture, as negative controls. Five (200· magnification) fields were randomly selected per slide and 100 hepatocytes counted per field. The mean percent of apoptotic hepatocytes was calculated and compared between different study groups.

2.9. TUNEL assay

2.10. Statistical analysis

The terminal deoxynucleotidyl transferase-mediated uDP nick-end labeling (TUNEL) assay was used to assess the degree of apoptosis (Cell Death Detection Kit; Roche) [32,33]. Quiescent and DNAse I pretreated normal livers were used as negative and positive controls,

All data were presented as means ± standard deviation (X ± SD). Significant differences were determined by Mann–Whitney U test. Statistical significance was set at P < 0.05. Statistical analyses were performed using SPSS software.

Table 2 Primer sequences for genes associated with liver injury Gene TM4SF4 GAPDH TNF-a TNFR1 TGF-b1 TGF-b R II HGF c-met Bfl-1 Bcl-XL Bak Bax

Primers 0

Tm (C) 0

F: 5 -GTG GTG AGG AGC CTT TGA T-3 R: 5 0 -TAG GAC CCT TGT TGA TGG A-3 0 F: 5 0 -GGC AAG TTC AAC GGC ACA G-3 0 R: 5 0 -ATG AGC CCT TCC ACG ATG-3 0 F: 5 0 -CAT CTT CTC AAA ACT CGA GTG ACA A-3 0 R: 5 0 -TGG GAG TAG ATA AGG TAC AGC CC-3 0 F: 5 0 -TGC TGT ATG CTG TGG TGG AT-3 0 R: 5 0 -CGA GTG GGC AGG GCT TTC TA-3 0 F: 5 0 -AGG ACC TGG GTT GGA AGT GG-3 0 R: 5 0 -AGT TGG CAT GGT AGC CCT TG-3 0 F: 5 0 -CCT GAG CCC TAC TCT GTC TGT-3 0 R: 5 0 -TCT GGA TGC CCT GGT GGT-3 0 F: 5 0 -CAT CAT TGG TAA AGG AGG CA-3 0 R: 5 0 -TGT CAC AGA CTT CGT AGC GTA-3 0 F: 5 0 -GTA AGT GCC CGA AGT GTA AGC-3 0 R: 5 0 -TTG GGT TAT GAG TCT CGT TTC-3 0 F: 5 0 -CGT CCA GAG TGC TAC AGA GAG TT-3 0 R: 5 0 -GCC AGC CAG ATT TAG GTT CA-3 0 F: 5 0 -GGC TGG CGA TGA GTT TGA-3 0 R: 5 0 -TCC TTG TCT ACG CTT TCC AC-3 0 F: 5 0 -GAG GCT TCT CGG GCT AAA-3 0 R: 5 0 -TTC TGG AAC TCC GTG TCG T-3 0 F: 5 0 -TCA TCC AGG ATC GAG CAG A-3 0 R: 5 0 -GCA AAG TAG AAG AGG GCA ACC-3 0

55 54 60 58 60 55 53 53 55 54 55 56

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275

3. Results 3.1. Hepatic expression of TM4SF4 is up-regulated after administration of CCl4 The tissue distribution of TM4SF4 in normal (N) and CCl4-injured rats at 24 h after CCl4-treatment (C) was examined by RT-PCR (Fig. 1A). In both normal and injured rats, a PCR product corresponding to TM4SF4 mainly existed in liver and testis, low in intestine and colon, but was not detectable in stomach, brain, heart and lung. Notably, after CCl4 treatment, TM4SF4 expression was significantly enhanced in injured liver. The enhanced expression of TM4SF4 in the livers was further confirmed at both mRNA (Fig. 1B) and protein (Fig. 1C) levels at different time points after CCl4 injury. TM4SF4 mRNA was increased at 12 h after CCl4 and further increased with maximum expression at 24 h. TM4SF4 expression was slightly decreased from peak levels but still remained high at 48–72 h compared with normal liver (Fig. 1B). The expression pattern of TM4SF4 protein was similar to that of TM4SF4 mRNA (Fig. 1C). TM4SF4 protein was increased at 12 h after CCl4 injury, peaked at 24 h and remained highly expressed until 72 h. These results indicate that expres-

269

sion of TM4SF4 in liver is up-regulated by CCl4 during the injury course. 3.2. Overexpression of TM4SF4 enhances CCl4-induced liver injury To investigate the in vivo functions of TM4SF4, we established a procedure to up- or down-regulate the expression of TM4SF4 in vivo. We injected stearylamine-liposomes (SA liposomes) that encapsulate pTM4SF4 (sense TM4SF4), pTM4SF4-AS (antisense TM4SF4), pcDNA3 (vehicle control) plasmids or PBS solution into rats via the tail vein at 6 h after CCl4 treatment. Then the liver toxicity was further detected by ALT/AST assay and histopathological studies. After chemical injury and TM4SF4 plasmid administration, liver tissues were collected from animals at 24, 48, 72 h after CCl4. TM4SF4 protein level in the livers was examined by Western blotting. The results showed that TM4SF4 protein was increased in pTM4SF4 transferred rats, while it was decreased in pTM4SF4-AS injected rats, compared with pcDNA3 vehicle or PBS controls (Fig. 2), indicating that the gene transfer procedure had effectively enhanced or inhibited the TM4SF4 expression.

Fig. 1. Expression of TM4SF4 is increased in rat liver after acute intoxication with CCl4. (A) RT-PCR analysis of TM4SF4 gene expression in different tissues removed from normal rats, N and CCl4-administered rats, C (24 h after CCl4 treatment). Hepatic expression of TM4SF4 was analyzed by RTPCR (B) and Western blot (C) at different time points after a single oral dose of CCl4. Livers from normal control rats or rats at 12, 24, 48, 72 h after CCl4 treatment were indicated. GAPDH and a-tubulin were used as internal controls. The quantitations of increases in TM4SF4 expression are plotted in the lower part of the graph. The densitometric analysis was performed with UVP LABWORKS Imaging Analysis software. mRNA or protein levels of TM4SF4 were normalized to those of the housekeeping gene GAPDH or a-tubulin, respectively. Results are expressed as the ratio between relative values of TM4SF4 in treated rats to those of TM4SF4 in normal rats (set arbitrarily at 1.0). Data from three independent experiments are shown as means ± SD, significantly different (P < 0.01) from normal control rats.

270

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275

to PBS and vehicle control rats (Fig. 3G and H vs. Fig. 3C–F; Fig. 3K and L, P < 0.01). The livers from the rats treated with pTM4SF4-AS resulting in TM4SF4 down-regulated were much less damaged, demonstrating a 68% decrease in inflammation and 70% decrease in necrosis than those of PBS and vehicle control group (Fig. 3I and J vs. Fig. 3C–F; Fig. 3K and L, P < 0.01). These results indicate that increased levels of TM4SF4 could accelerate liver necrosis, while inhibition of TM4SF4 expression may result in an amelioration of liver damage induced by CCl4. 3.3. Dose-dependent amelioration of liver injury by pTM4SF4-AS administration To further confirm the amelioration of liver damage by antisense TM4SF4, different amounts of pTM4SF4AS plasmid were introduced into CCl4-injured rats at 6 h post-CCl4 treatment in a dosage gradient (100, 300 and 500 lg of pTM4SF4-AS plasmid, respectively). The liver toxicity was then detected by histopathological examination (Fig. 4A–H) and ALT/AST assay (Fig. 4I) at 48 h after CCl4 injury. The data showed that 100 lg pTM4SF4-AS administration resulted in partially decreased ALT and AST levels by 28% and 34%, respectively (Fig. 4I, P < 0.01), and a slight reduction of liver necrosis, as compared with CCl4/pcDNA3 control groups (Fig. 4C and D vs. Fig. 4A and B). pTM4SF4AS at a dose of 300 or 500 lg had a marked protective effect on CCl4-induced ALT and AST elevation, resulting in a 60% or 70% decrease in ALT level, a 73% or 80% reduction in AST level (Fig. 4I, P < 0.01); and had a significant reduction of liver damage, shown by less necrosis (Fig. 4E–H vs. Fig. 4A and B). It indicates that the reduced TM4SF4 expression could protect liver from CCl4-induced damage.

Fig. 2. Expression of TM4SF4 protein in livers from the different experimental groups. At the time of 24, 48 or 72 h after CCl4 injury, whole liver extracts were harvested from CCl4-injured rats with PBS, pcDNA3, pTM4SF4 or pTM4SF4-AS treatments as indicated. Representative immunoblot for TM4SF4 demonstrated an increased expression of TM4SF4 in pTM4SF4 administered rats, while a decreased expression in rats receiving pTM4SF4-AS injection. The blot probed for a-tubulin was included to confirm equal protein loading.

As shown in Table 3, pTM4SF4 administration resulted in a marked increase of serum ALT levels (by 5.8-fold at 24 h, by 2.2-fold at 48 h, by 2.0-fold at 72 h) and AST levels (by 5.9-fold at 24 h, by 2.1-fold at 48 h, and by 1.8-fold at 72 h), as compared with pcDNA3 control (P < 0.01); while post-treatment with pTM4SF4-AS plasmids significantly decreased the CCl4-induced elevation of serum ALT and AST levels, which approached the normal level (P < 0.02). These results demonstrate that TM4SF4 expression positively correlates with elevation of serum ALT and AST levels in CCl4-injured rats. Liver sections from animals at 48 h after a single dose of CCl4 and with different plasmid administrations were examined histologically. Overexpression of TM4SF4 through pTM4SF4 plasmid administration resulted in an extensive liver damage, demonstrating a 28% increase in inflammation and 40% increase in necrosis compared

3.4. Effects of TM4SF4 on signaling pathways essential to CCl4-induced liver injury To identify possible mechanisms of TM4SF4 in liver injury, the endogenous hepatic TNF-a, TNFR1, TGF-b1, TGF-b RII, HGF and c-met mRNA were examined by quantitative real-time PCR at 48 h after

Table 3 Serum ALT and AST levels of study groups at different time points after CCl4 administration Time after CCl4 injury Normal CCl4+PBS CCl4+pcDNA3 CCl4+pTM4SF4 CCl4+pTM4SF4-AS

ALT levels (IU/l)

AST levels (IU/l)

24 h

48 h

72 h

24 h

48 h

72 h

44.5 ± 5.2 136.4 ± 55.4 162.2 ± 72.8 949.3 ± 311.2 70.6 ± 23.6

44.4 ± 3.0 162.3 ± 34.9 170.3 ± 22.1 378.2 ± 81.0 53.2 ± 11.2

39.5 ± 5.9 75.9 ± 8.5 71.4 ± 13.0 143.4 ± 43.5 50.8 ± 9.3

204.5 ± 32.8 643.4 ± 125.6 626.8 ± 159.2 3688.1 ± 1041.1 268.3 ± 51.7

229.5 ± 38.2 1053.0 ± 224.8 1073.4 ± 213.8 2272.5 ± 851.3 208.6 ± 24.8

256.2 ± 23.5 389.5 ± 63.9 383.2 ± 39.8 681.5 ± 84.7 220.7 ± 39.4

Data represent means ± SD of six animals in each group. Data were analyzed using Mann–Whitney U test (CCl4+pTM4SF4 group vs. CCl4+PBS group, CCl4+pTM4SF4-AS group vs. CCl4+PBS group; P < 0.02).

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275

CCl4-treatment (Fig. 5A). The expression of TNF-a and TNFR1 was significantly increased (by 3.6-fold and 5.5fold, respectively) in pTM4SF4 administered group and was inhibited post-treatment with pTM4SF4-AS, compared with pcDNA3 control group (P < 0.01). The cmet was reduced by pTM4SF4, but enhanced by 5.6-fold in pTM4SF4-AS treated rats (P < 0.01). However, hepatic

271

TGF-b1, HGF and TGF-b RII mRNA were not significantly altered in CCl4/pTM4SF4 and CCl4/pTM4SF4AS treated animals. The changed expression of TNFR1 and c-met protein was further confirmed by Western blotting (Fig. 5B). Taken together, these data clearly demonstrate that TM4SF4 affects the TNF-a, TNFR1 and c-met expression. 3.5. Effects of TM4SF4 on hepatocyte apoptosis and on apoptosis-related genes in injured rats TNF-a is a critical and powerful mediator of cell death/apoptosis in liver [15,34,35]. Since we observed that TM4SF4 could enhance TNF-a expression in CCl4-injured liver (Fig. 5A), we investigated whether TM4SF4 could affect apoptosis and regulate apoptosis-related gene expression. Livers receiving pTM4SF4 demonstrated a 2.0-fold increase in hepatocyte apoptosis as assessed by TUNEL-staining compared to the pcDNA3 control group (Fig. 6C and D vs. Fig. 6A and B; Fig. 6K, P < 0.05), while pTM4SF4-AS administration resulted in a 63% reduction of hepatocyte apoptosis (Fig. 6E and F vs. Fig. 6A and B; Fig. 6K, P < 0.05). We further examined the expression levels of the proapoptotic gene Bak and Bax, and anti-apoptotic gene Bfl-1 and Bcl-XL by quantitative real-time RT-PCR (Fig. 7). We observed that Bax transcripts were increased by 3.3-fold and Bcl-XL transcripts were reduced by 47% after TM4SF4 induction, compared to pcDNA3 control group (Fig. 7, *P < 0.01, **P < 0.05). The Bfl-1 and Bcl-XL transcripts were increased (by 1.8-fold and 4.2-fold, respectively) after pTM4SF4-AS administration (Fig. 7, P < 0.01). No significant change of Bak mRNA level was detected after pTM4SF4 or pTM4SF4-AS treatments. These results suggest that changing TM4SF4 expression can affect hepatocyte apoptosis and regulate the expression of some apoptosis-related genes.

b Fig. 3. pTM4SF4 administration accelerates liver damage in CCl4injured rats. Liver morphologies were examined in the livers from normal (non-injured) rats (A, 40·; B, 100·, original magnification), CCl4-injured rats (C, 40·; D, 100·), CCl4/pcDNA3 control rats (E, 40·; F, 100·), CCl4/pTM4SF4 administered rats (G, 40·; H, 100·) and CCl4/ pTM4SF4-AS administered rats (I, 40·; J, 100·). The injured livers were harvested at 48 h after CCl4 induction. Portions of the liver were fixed, sectioned and stained with hematoxylin and eosin. CCl4/TM4SF4 group showed extensive liver damage, while CCl4/pTM4SF4-AS rats showed minimal liver injury. Arrows indicate inflammation and necrosis. Inflammation and perivenular necrosis were graded on a 4-point scale (Table 1) by a blinded pathologist in 20 random high power fields per animal (n = 6 per group) and represented graphically in (K) and (L). Data represent means ± SD of six animals in each group, significantly different (P < 0.01) from CCl4/PBS control group. (This figure appears in color on the web.)

272

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275

Fig. 4. Reduced liver damage following antisense TM4SF4 (pTM4SF4AS) administration in CCl4-injured rats in a dose-dependent manner. Liver morphologies were examined in livers from CCl4/pcDNA3 control rats (A, 40·; B, 100·, original magnification), CCl4-injured rats with 100 lg pTM4SF4-AS administration (C, 40·; D, 100·), CCl4-injured rats with 300 lg pTM4SF4-AS administration (E, 40·; F, 100·) and CCl4-injured rats with 500 lg pTM4SF4-AS administration (G, 40·; H, 100·). Livers were harvested at 48 h after CCl4 treatments. Portions of the liver were fixed, sectioned and stained with hematoxylin and eosin. Representative photomicrographs from six rats per group are shown; arrows show the hepatocellular necrosis. Serum levels of ALT and AST in CCl4/pcDNA3 and CCl4/pTM4SF4-AS (with 100, 300 or 500 lg pTM4SF4-AS, respectively) administered rats were examined and shown in histogram (I). The reduced TM4SF4 expression could protect liver from CCl4induced damage biochemically as well as histologically. Data represent means ± SD of six animals in each group, significantly different (P < 0.01) from CCl4/pcDNA3 treated rats. (This figure appears in color on the web.)

Fig. 5. (A) Real-time RT-PCR analysis of six mRNA values including TNF-a, TNFR1, TGF-b, TGF-b RII, HGF and c-met in the livers of CCl4-injured rats with various treatments. Livers were harvested from the rats of experimental groups as indicated at 48 h after CCl4 injury. Total RNA was extracted and processed as described in Section 2 methods. Following the amplification, mRNA levels of target genes were normalized to those of the housekeepinggene GAPDH. Results are expressed as the ratio between relative values of target mRNA in treated rats to those of target mRNA in PBS-treated CCl4-injured rats set arbitrarily at 1.0. Data represent means ± SD of six animals in each group, significantly different (*P < 0.01) from CCl4/pcDNA3-treated rats. (B) The expression of cmet, TNFR1 protein in livers of the rats after TM4SF4 administration was analyzed by Western blotting with their specific antibodies as indicated. The expression of a-tubulin protein was examined as internal control.

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275

273

4. Discussion Rat TM4SF4 was the first transmembrane 4 superfamily member found to be associated with liver regeneration [1], however, its in vivo functions are still largely unknown. In this study, we investigated the role of

Fig. 7. Real-time RT-PCR analysis of four mRNA values including Bax, Bak, Bcl-XL and Bfl-1 in the livers from rats with various treatments. Livers were harvested from the rats of experimental groups as indicated at 24 h after CCl4 injury. Total RNA was extracted and processed as described in Section 2. Following the amplification, mRNA levels of target genes were normalized to those of the housekeeping-gene GAPDH. Results are expressed as the ratio between relative values of target mRNA in treated rats to those of target mRNA in PBS-treated CCl4-injured rats set arbitrarily at 1.0. Data represent means ± SD of six animals in each group, significantly different (*P < 0.01, **P < 0.05) from CCl4/pcDNA3-treated rats.

TM4SF4 in liver damage using a rat acute liver injury model. First, we investigated the expression pattern of TM4SF4 in normal or CCl4-injured rats and found that TM4SF4 gene is significantly up-regulated in CCl4-injured liver (Fig. 1). We then examined the function of TM4SF4 in CCl4-induced acute liver injury in vivo, the

b Fig. 6. Effects of TM4SF4 on hepatocyte apoptosis. TUNEL staining was performed on liver sections from normal rats (without any treatment), or CCl4-intoxicated rats with pcDNA3, pTM4SF4 or pTM4SF4-AS treatments. Livers were harvested at 24 h after CCl4 treatments. CCl4-injured rats receiving pTM4SF4 (C) showed more apoptosis than pcDNA3 control group (A), while less apoptosis in CCl4/ pTM4SF4-AS rats (E) (magnification 200·), DAB (3,3-diaminobenzidine) with hematoxylin counterstaining. Higher power magnification (400·) demonstrates more TUNEL-positive nuclei in animals receiving pTM4SF4 (D) and less in animals with pTM4SF4-AS administration (F), comparing with those in animals receiving pcDNA3 (B). Arrows denote TUNEL-positive cells. No TUNEL-positive nuclei were found in normal rat livers (G, 200·; H, 400·. TUNEL staining of the CCl4/ pTM4SF4 administered livers without terminal transferase treatment was also included as a negative control (I, 200·; J, 400·). (K) Five (200· magnification) fields were randomly selected per slide and 100 hepatocytes counted per field. Mean percent of apoptotic hepatocytes was calculated and compared between different study groups. Bars represent means ± SD from six rats in each group, significantly different (P < 0.05) from CCl4/pcDNA3 group. LPF, low-power field. (This figure appears in color on the web.)

274

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275

expression of endogenous TM4SF4 was enhanced or inhibited in rats through the injection of sense (pTM4SF4) or antisense (pTM4SF4-AS) TM4SF4 expression plasmids. We examined morphological and biochemical changes at 24–72 h after CCl4 injury. Our observations clearly demonstrate that overexpression of TM4SF4 could accelerate CCl4-induced liver damage biochemically as well as histologically (Table 3 and Figs. 3 and4). We have also examined the effect of TM4SF4 administration on normal (non-injured) rats. We found that normal rats did not show significant changes in either ALT levels or liver morphology after pTM4SF4 or pTM4SF4-AS treatment, in spite of changing TM4SF4 expression levels (Supplementary material 1). It implies that TM4SF4 itself may be not an initiator for liver injury. After CCl4 induction, CCl4 initiates lipid peroxidation and leads to hepatocellular membrane damage [18,36]. TM4SF4, as a transmembrane protein, might be rapidly changed in its conformation or localization, which may convert it into a damage factor. To explore the possible molecular mechanism for TM4SF4 action, we investigated the expression levels of liver injury-related genes after TM4SF4 treatment. Previous studies have shown that CCl4 activates a series of growth factors and receptors, including TNF-a/TNFR1, TGF-b/TGF-b RII and HGF/c-met [19,21,37]. We found that endogenous expression of TNF-a, TNFR1 and c-met was changed by pTM4SF4 or pTM4SF4-AS injection (Fig. 5). In contrast, the expression of genes involved in TGF-b pathway, such as TGF-b RII and TGF-b1, was not altered. It suggests that TM4SF4 may affect the TNF-a and HGF/c-met signaling pathway. TNF-a/TNFR has been shown to increase the expression of pro-apoptotic genes (Bax, Bak) [15,34], and downregulate the expression of anti-apoptotic genes (Bfl-1, Bcl-XL) [15,35]. HGF/c-met protects cells from apoptosis by inducing the expression of Bcl-XL [38]. Our data demonstrate that overexpression of TM4SF4 enhances apoptosis, up-regulates the expression of proapoptotic gene Bax, and down-regulates anti-apoptotic gene Bfl-1 and Bcl-XL (Figs. 6 and 7), suggesting that TM4SF4 could affect TNF-a-induced apoptosis. Moreover, pTM4SF4-AS administration could protect rats against CCl4 hepatotoxicity, shown by decreased serum ALT/AST level and histological necrosis (Figs. 3 and 4; Table 3). These data suggest that TM4SF4 gene may be a therapeutic target for chemical liver damage. However, this attractive presumption needs to be explored further. In conclusion, our study shows that TM4SF4 gene is overexpressed in acutely injured liver. TM4SF4 overexpression is found to be associated with more elevated ALT/AST, increased necrosis and enhanced apoptosis. Our findings suggest that TM4SF4 plays an important role in accelerating CCl4 liver injury, which may be mediated by the TNF-a and HGF/c-met signaling pathways.

Acknowledgments This work was supported by grants from the National High Technology Research and Development of China (863 Program) No. 2002BA711A02 and special funds for Major State Basic Research of China (973 Program), No. 2001CB510205. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jhep. 2006.08.011.

References [1] Liu ZW, Zhao MJ, Yokoyama KK, Li ZP. Molecular cloning of a cDNA for rat TM4SF4, a homolog of human il-TMP (TM4SF4), and enhanced expression of the corresponding gene in regenerating rat liver. Biochim Biophys Acta 2001;1518:183–189. [2] Wice BM, Gordon JI. A tetraspan membrane glycoprotein produced in the human intestinal epithelium and liver that can regulate cell density-dependent proliferation. J Biol Chem 1995;270:21907–21918. [3] Boucheix C, Rubinstein E. Tetraspanins. Cell Mol Life Sci 2001;58:1189–1205. [4] Claas C, Wahl J, Orlicky DJ, Karaduman H, Schnolzer M, Kempf T, et al. The tetraspanin D6.1A and its molecular partners on rat carcinoma cells. Biochem J 2005;389:99–110. [5] Furuya M, Kato H, Nishimura N, Ishiwata I, Ikeda H, Ito R, et al. Down-regulation of CD9 in human ovarian carcinoma cell might contribute to peritoneal dissemination: morphologic alteration and reduced expression of beta1 integrin subsets. Cancer Res 2005;65:2617–2625. [6] Gesierich S, Paret C, Hildebrand D, Weitz J, Zgraggen K, Schmitz-Winnenthal FH, et al. Colocalization of the tetraspanins, CO-029 and CD151, with integrins in human pancreatic adenocarcinoma: impact on cell motility. Clin Cancer Res 2005;11: 2840–2852. [7] Nishiuchi R, Sanzen N, Nada S, Sumida Y, Wada Y, Okada M, et al. Potentiation of the ligand-binding activity of integrin alpha3beta1 via association with tetraspanin CD151. Proc Natl Acad Sci USA 2005;102:1939–1944. [8] Shigeta M, Sanzen N, Ozawa M, Gu J, Hasegawa H, Sekiguchi K. CD151 regulates epithelial cell–cell adhesion through PKC- and Cdc42-dependent actin cytoskeletal reorganization. J Cell Biol 2003;163:165–176. [9] Sridhar SC, Miranti CK. Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases. Oncogene 2006;25:2367–2378. [10] Hemler ME. Specific tetraspanin functions. J Cell Biol 2001;155: 1103–1107. [11] Hemler ME. Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol 2003;19:397–422. [12] Levy S, Shoham T. The tetraspanin web modulates immunesignalling complexes. Nat Rev Immunol 2005;5:136–148. [13] Yunta M, Lazo PA. Tetraspanin proteins as organisers of membrane microdomains and signalling complexes. Cell Signal 2003;15:559–564.

J. Qiu et al. / Journal of Hepatology 46 (2007) 266–275 [14] Boll M, Weber LW, Becker E, Stampfl A. Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites. Z Naturforsch (C) 2001;56:649–659. [15] Horn TL, O’Brien TD, Schook LB, Rutherford MS. Acute hepatotoxicant exposure induces TNFR-mediated hepatic injury and cytokine/apoptotic gene expression. Toxicol Sci 2000;54: 262–273. [16] Luckey SW, Petersen DR. Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats. Exp Mol Pathol 2001;71:226–240. [17] Sun F, Hamagawa E, Tsutsui C, Ono Y, Ogiri Y, Kojo S. Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver. Biochim Biophys Acta 2001;1535:186–191. [18] Weber LW, Boll M, Sampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 2003;33:105–136. [19] Grasl-Kraupp B, Rossmanith W, Ruttkay-Nedecky B, Mullauer L, Kammerer B, Bursch W, et al. Levels of transforming growth factor beta and transforming growth factor beta receptors in rat liver during growth, regression by apoptosis and neoplasia. Hepatology 1998;28:717–726. [20] Okano J, Shiota G, Kawasaki H. Protective action of hepatocyte growth factor for acute liver injury caused by D-galactosamine in transgenic mice. Hepatology 1997;26:1241–1249. [21] Simeonova PP, Gallucci RM, Hulderman T, Wilson R, Kommineni C, Rao M, et al. The role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl Pharmacol 2001;177:112–120. [22] Morio LA, Chiu H, Sprowles KA, Zhou P, Heck DE, Gordon MK, et al. Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice. Toxicol Appl Pharmacol 2001;172:44–51. [23] Saile B, Matthes N, Knittel T, Ramadori G. Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells. Hepatology 1999;30:196–202. [24] Sudo K, Yamada Y, Moriwaki H, Saito K, Seishima M. Lack of tumor necrosis factor receptor type 1 inhibits liver fibrosis induced by carbon tetrachloride in mice. Cytokine 2005;29: 236–244. [25] Date M, Matsuzaki K, Matsushiat M, Tahashi Y, Furukawa F, Inoue K. Modulation of transforming growth factor beta function in hepatocytes and hepatic stellate cells in rat liver injury. Gut 2000;46:719–724.

275

[26] Tahashi Y, Matsuzaki K, Date M, Yoshida K, Furukawa F, Sugano Y, et al. Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury. Hepatology 2002;35:49–61. [27] Huh CG, Factor VM, Sanchez A, Uchida K, Conner EA, Thorgeirsson SS. Anti-hepatocyte growth factor antibody inhibits hepatocyte proliferation during liver regeneration. J Pathol 1998;185:298–302. [28] Wang D, Jing NH, Lin QS. Stearylamine liposome as a new efficient reagent for DNA transfection of eukaryotic cells. Biochem Biophys Res Commun 1996;226:450–455. [29] Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Guadat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22:696–699. [30] Yamakawa Y, Takano M, Patel M, Tien N, Takada T, Bulkley GB. Interaction of platelet activating factor, reactive oxygen species generated by xanthine oxidase, and leukocytes in the generation of hepatic injury after shock/resuscitation. Ann Surg 2000;231:387–398. [31] Pohjanvirta R, Niittynen M, Linden J, Boutros PC, Moffat ID, Okey AB. Evaluation of various housekeeping genes for their applicability for normalization of mRNA expression in dioxintreated rats. Chem Biol Interact 2006;160:134–149. [32] Dubska L, Matalova E, Misek I. Detection of apoptosis in paraffin embedded tissues: the influence of tissue type and fixation. Acta Veterinaria Brno 2002;71:529–533. [33] Negoescu A, Guillermet Ch, Lorimier Ph, Barnbilla E, LabatMoleur F. TUNEL apoptotic cell detection in archived paraffinembedded tissues. Biochemica 1998;3:36–41. [34] Suyama E, Kawasaki H, Taira K. Identification of a caspase 3independent role of pro-apoptotic factor Bak in TNF-alphainduced apoptosis. FEBS Lett 2002;528:63–69. [35] Fox SA, Kusmiaty, Loh SS, Dharmarajan AM, Garlepp MJ. Cisplatin and TNF-alpha downregulate transcription of Bcl-XL in murine malignant mesothelioma cells. Biochem Biophys Res Commun 2005;337:983–991. [36] Haouzi D, Lekehal M, Moreau A, Moulis C, Feldmann G, Robin MA, et al. Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes. Hepatology 2000;32:303–311. [37] Taniguchi M, Takeuchi T, Nakatsuka R, Watanabe T, Sato K. Molecular process in acute liver injury and regeneration induced by carbon tetrachloride. Life Sci 2004;75:1539–1549. [38] Liu Y. Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms. Am J Physiol 1999;277:F624–F633.