Protection Against Liver Damage by Cardiotrophin ... - Gastroenterology

GASTROENTEROLOGY 2003;125:192–201

Protection Against Liver Damage by Cardiotrophin-1: A Hepatocyte Survival Factor Up-Regulated in the Regenerating Liver in Rats MATILDE BUSTOS,* NAIARA BERAZA,* JUAN–JOSE LASARTE,* ELENA BAIXERAS,* PILAR ALZUGUREN,* THIERRY BORDET,‡ and JESUS PRIETO* *Department of Medicine, Division of Hepatology and Gene Therapy, Clinica Universitaria and Medical School, University of Navarra; and the ‡Institut National de la Sante ´ et de la Recherche Me´dicale (INSERM), Institut Cochin de Geńe´tique Molećulaire, Paris, France

Background & Aims: Cardiotrophin-1 (CT-1) is a member of the interleukin 6 (IL-6) family of cytokines, which protects cardiac myocytes against thermal and ischemic insults. In this study, we investigated the expression of CT-1 by liver cells and its possible hepatoprotective properties. Methods: We analyzed the production, signaling, and antiapoptotic properties of CT-1 in hepatocytes and the expression of this cytokine during liver regeneration. We also investigated whether CT-1 might exert protective effects in animal models of liver damage. Results: We found that CT-1 is up-regulated during liver regeneration and exerts potent antiapoptotic effects on hepatocytic cells. Hepatocytes cultured under serum starvation or stimulated with the proapoptotic cytokine transforming growth factor ␤ (TGF-␤) produce CT-1, which behaves as an autocrine/paracrine survival factor. Treatment with an adenovirus encoding CT-1 efficiently protects rats against fulminant liver failure after subtotal hepatectomy, an intervention that causes 91% mortality in control animals whereas 54% of those receiving CT-1 gene therapy were long-term survivors. This protective effect was associated with reduced caspase-3 activity and activation of the antiapoptotic signaling cascades signal transducer and activator of transcription (Stat-3), extracellular regulated kinases (Erk) 1/2, and Akt in the remnant liver. Gene transfer of CT-1 to the liver also abrogated Concanavalin A (Con-A) liver injury and activated antiapoptotic pathways in the hepatic tissue. Similar protection was obtained by treating the animals with 5 ␮g of recombinant CT-1 given intravenously before Con-A administration. Conclusions: We show that CT-1 is a hepatocyte survival factor that efficiently reduces hepatocellular damage in animal models of acute liver injury. Our data point to CT-1 as a new promising hepatoprotective therapy.

cute liver failure may result from severe toxic, immunologic, ischemic, or metabolic insults to the liver or from extensive hepatic resections. In these situations the regenerative potential of the residual hepato-

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cytes may be compromised because of work overload, microcirculatory disturbances, hypercytokinemia, and direct cytotoxicity of the etiologic factor.1,2 Fulminant hepatic failure lacks effective medical therapy and has very poor survival when emergency liver transplantation is not feasible.3 Therefore, therapies aiming at reducing the extent of hepatocellular necrosis/apoptosis and protecting against liver failure are needed urgently. Cardiotrophin-1 (CT-1) is a member of the interleukin-6 (IL-6) family of cytokines. It was first identified by its ability to induce hypertrophy of cardiac myocytes4 and it was later shown to support survival and proliferation of immature cardiomyocytes5,6 and developing motoneurons7,8 and to protect cardiac cells against thermal and ischemic insults by minimizing the degree of apoptosis.9,10 The CT-1 receptor contains the protein chain gp130 (in common with the other members of the IL-6 family), and the leukemia inhibitory factor (LIF) receptor subunit ␤. In addition, CT-1 interacts with a lipid-anchored nonsignaling receptor conferring an increased sensitivity and specificity to this cytokine.11 CT-1 activates several signaling routes including the Janus-activated kinase (Jak)/signal transducer and activator of transcription (Stat)-3 pathway,11 extracellular regulated kinases (Erk) 1/2,9 and phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway.12,13 Available evidence indicates that all these pathways are involved in the cytoprotective properties of CT-1.14 Abbreviations used in this paper: Con-A, Concanavalin A; CT-1, cardiotrphin 1; ELISA, enzyme-linked immunosorbent assay; Erk, extracellular regulated kinases; IL, interleukin; Jak, Janus-activated kinase; LIF, leukemia inhibitory factor; MTT, 3-(4,5-dimethyl thiazoyl-2-yl)2,5-diphenyltretazolium bromide; pfu, plaque-forming units; PI3K, phosphatidylinositol 3-OH kinase; RT-PCR, reverse-transcription polymerase chain reaction; Stat3, signal transducer and activator of transcription; TGF, transforming growth factor; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling. © 2003 by the American Gastroenterological Association 0016-5085/03/$30.00 doi:10.1016/S0016-5085(03)00698-X

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CT-1 induces acute-phase protein gene expression in human and rat hepatoma cells and in primary rat hepatocytes.15 It also increases the size of the liver when administered chronically to mice.16 Here we show that CT-1 exerts antiapoptotic effects on hepatocytic cells. Furthermore, CT-1 is induced and secreted to the medium by cultured hepatocytes when subjected to proapoptotic stimuli and acts as an autocrine hepatocyte surviving factor under stressful conditions. Finally, we found that CT-1 gene transfer to the liver increases the survival of rats after subtotal hepatectomy and that pretreatment with intravenous CT-1 or adenovirus encoding this cytokine markedly reduces liver injury in mice challenged with Con-A.

Materials and Methods Cell Culture Rat hepatoma cell line H35 was maintained in Dulbecco’s modified Eagle medium (Gibco BRL, Gaithersburg, MD) containing 10% fetal calf serum. For cell signaling experiments H35 was plated in 6-well plates with 0.6 ⫻ 106 cells/well and maintained in Dulbecco’s modified Eagle medium with 10% of serum for 2 hours. Cells were cultured in serum-free medium 18 hours and then 50 ng/mL of CT-1 (R&D Systems Inc., Minneapolis, MN) was added to the culture and cells were obtained at different time points. Rat primary hepatocytes were obtained by a collagenase perfusion method, isolated and cultured in minimum essential medium (Gibco BRL) with 5% fetal calf serum for 4 hours, and then changed to serum-free medium. For cell signaling experiments, hepatocytes were plated (0.7 ⫻ 106 cells/well) and after 48 hours of culture under serum-free conditions they were stimulated with CT-1 (50 ng/mL) and cells were obtained at different time points.

Cell Survival Assays and Assessment of Apoptosis H35 cells were trypsinized and apoptosis was analyzed by different methods. Cell cycle was assessed by DNA content after staining with propidium iodide as described.17 Cells were stained with annexin V–fluorescein isothiocyanate conjugate following the manufacturer’s protocol (BD-Pharmingen, San Diego, CA) and analyzed by flow cytometry on a FACScan. Annexin V–fluorescein isothiocyanate–positive cells were considered apoptotic. Primary hepatocytes seeded at 0.7 ⫻ 106 cells/well in serum-free medium were incubated in the presence or absence of CT-1 (50 or 100 ng/mL) for 12 hours and then were treated with transforming growth factor ␤ (TGF-␤) (BD-Pharmingen) at 5 ng/mL for 12 hours. Apoptosis of cultured hepatocytes was assessed by determination of caspase 3 activity as described elsewhere.18 Briefly, the cells were scraped off in phosphatebuffered saline, collected by centrifugation at 2500g for 5 minutes, and lysed at 4°C in 5 mmol/L Tris/HCl, pH 8.0, 20

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mmol ethylenediaminetetraacetic acid, 0.5% Triton X-100. Lysates were clarified by centrifugation at 13,000g for 10 minutes. Reaction mixture contained 30 ␮g cellular lysates, 1000 ␮L assay buffer (20 mmol/L HEPES, pH 7.5, 10% glycerol, 2 mmol/L dithiothreitol), and 20 ␮mol/L caspase-3 substrate (Ac-DEVD-AMC) (BD Pharmingen). After 2 hours’ incubation in the dark, enzymatic activity was measured in a Luminiscence Spectophotometer (LS-50; Perkin Elmer, Norwalk, CT) (␭ excitation, 380 nm; ␭ emission, 440 nm). In confirmatory experiments apoptosis also was assessed by the presence of soluble histone DNA complexes measured by the Cell Death Detection Assay (Boehringer Mannheim, Mannheim, Germany) performed according to the manufacturer’s instructions. Cell survival was measured by 3-(4,5-dimethyl thiazoyl-2-yl)-2,5-diphenyltretazolium bromide (MTT) (Sigma, St. Louis, MO) and viability was determined by colorimetry. Apoptosis in liver sections from mice was estimated using the In situ Cell Death Detection Kit, POD (Boehringer Mannheim) according to the manufacturer’s instructions. Caspase-3 activity in liver homogenates was determined in liver extracts using the CaspACE assay (Promega, Madison, WI) according to the manufacturer’s protocol. Protein concentration was determined by Bradford assay (Bio-Rad, Mu¨ nchen, Germany).

Immunoblotting and Immunoprecipitation Cells and frozen liver tissues were lysed in cold-lysis buffer (20 mmol/L Tris pH 7.5; 150 mmol/L NaCl; 1 mmol/L ethylene glycol-bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid; 1 mmol/L ethylenediaminetetraacetic acid; 1% Triton X-100; 2.5 mmol/L sodium pyrophosphate; 1 mmol/L Na3VO4 , and a cocktail of antiproteases). Protein from lysates was heat-denatured in double-strength sodium dodecyl sulfate sample buffer containing dithiothreitol before resolution in 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Protein immunoblotting and Akt immunoprecipitation were performed as previously described.17 Anti-AKT, anti–phospho-AKT (Ser473P), anti–Stat-3, anti–phospho-Stat-3 (Tyr705P), (Ser727P) anti-Erk1/2, and anti–phospho-Erk1/2 (Thr202/Tyr204P) were from Cell Signaling Technologies (Beverly, MA). Antiphosphotyrosine 4G10 horseradish-peroxidase conjugate was from Upstate Biotech (Lake Placid, NY). Densitometric analysis was performed using Molecular Analyst software (Bio-Rad).

Cardiotrophin-1 Messenger RNA and Enzyme-Linked Immunosorbent Assay for Cardiotrophin-1 in Rat Liver and in Cultured Hepatocytes: Cytokine Messenger RNA Profile in Liver Tissue Total RNA was isolated19 and Northern blotting was performed using 20 ␮g of total RNA and a rat CT-1 complementary DNA probe obtained by reverse-transcription polymerase chain reaction (RT-PCR) using specific oligonucleotides primers (5⬘-AGCATGAGCCAGAGGGAGGGAA-3⬘ and 5⬘-TATGCAGACCAATTG CTGGAGGAA-3⬘) from GenBank (accession number NM_017129). RT-PCR for CT-1

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was performed in cultured hepatocytes with the primers indicated earlier and 18S was used as a control. Enzyme-linked immunosorbent assay (ELISA) for CT-1 was performed in rat liver extracts and supernatant from cultured hepatocytes as described.20 Cytokine mRNA profiles were analyzed using a multiprobe ribonuclease protection assay template set mCK-3b Riboquant (BD-Pharmingen) according to the manufacturer’s instructions.

Recombinant Adenoviruses AdCT-1 and AdLacZ were generated21 and expanded as previously described.22

Animals Wistar rats (Harlan Interfauna Iberica, Barcelona, Spain) (n ⫽ 32) were subjected to two-thirds partial hepatectomy and liver samples were obtained at 0, 1, 3, 6, 10, 24, 48, 72, and 144 hours after surgery. Ninety-percent hepatectomy was performed by removing the median, left lateral, right upper, and right lower lobes by ligation leaving only the caudate lobe. These animals were divided into 3 groups that received, respectively, 108 plaque-forming units (pfu) of AdCT-1 (n ⫽ 13) or AdLacZ (n ⫽ 14) in 1 mL of PBS or only 1 mL of phosphate-buffered saline (n ⫽ 10) 48 hours before 90% partial hepatectomy by the tail vein. Rats were followed-up for survival. Additional rats (n ⫽ 5 per group) were killed 1 hour after 90% partial hepatectomy and liver samples were frozen and stored at ⫺80°C. BALB/c mice (IFFA Credo, Barcelona, Spain) were injected by the tail vein with 107 pfu of AdCT-1, AdLacZ, or saline 24 hours before the administration of Concanavalin A (Con-A) (Sigma) (100 mg/kg in 250 ␮L of saline) and were killed at 1 hour after Con-A challenge (n ⫽ 5 per group) to study signaling and at 6 hours (n ⫽ 5 per group) to asses liver injury by determining serum alanine transaminase (ALT) levels and the rate of cell apoptosis in liver biopsy specimens using terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. Liver sections were placed in OCT (Sakura, Zoeterwoude, the Netherlands) and frozen in cold 2-metylbutane (Merck, Darmstadt, Germany). ALT levels were determined by colorimetry (Sigma). In additional experiments, serum ALT level was measured at later time points (12, 30, and 48 hours) after Con-A administration in animals pretreated with AdCT-1, AdLacZ, or saline (n ⫽ 5 per group). Finally, 2 groups of mice (n ⫽ 5 per group) were given intravenously 5 ␮g of CT-1 in 500 ␮L of saline, or saline alone, 20 minutes before Con-A administration and serum ALT levels and the rate of apoptotic nuclei in liver tissue were assessed at 6 hours after Con-A challenge.

Statistical Analysis Mann–Whitney U tests were used for comparison between 2 groups. A P value of less than 0.05 was considered significant.

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Results Antiapoptotic Properties of Cardiotrophin-1 on Liver Cells To analyze whether CT-1 displayed cytoprotective effects on hepatocytic cells, rat hepatoma H35 cells were cultured under serum deprivation conditions in the absence or in the presence of 50 ng/mL of CT-1 added every 48 hours. After 7 days of culture without CT-1, 28.4% ⫾ 7.5% cells underwent apoptosis as judged by DNA content in the sub-G0/G1 phase, whereas cells treated with CT-1 were arrested in the G0/G1 phase and apoptosis was seen in only 10.6% ⫾ 4.4% of them (Figure 1A ). The reduction of apoptosis by CT-1 also was reflected by a decrease in the percentage of annexin V binding cells, which was 47.3% ⫾ 10.4% in the absence and 14.3% ⫾ 7.1% in the presence of CT-1 (Figure 1A ). Previous studies have reported that CT-1 mediated survival of cardiac myocytes through signaling pathways involving Stat-3, Erk1/2, and Akt factors.14 To investigate whether CT-1 also could activate these molecules in hepatocytic cells, H35 cells were stimulated with CT-1 for different periods of time. As it was reported for cardiac myocytes, we found that CT-1 induced tyrosine and serine phosphorylation of Stat-3, phosphorylation of Erk 1/2, and activation of Akt with maximum intensity between 15 and 30 minutes (Figure 1B). As observed with hepatoma cells, incubation of primary rat hepatocytes with CT-1 resulted in the activation of Stat-3, Erk 1/2, and Akt pathways (Figure 2A ). To analyze the antiapoptotic potential of CT-1 in primary hepatocytes, cells that had been preincubated for 12 hours in the presence or absence of CT-1 (50 or 100 ng/mL) were treated with TGF-␤ (5 ng/mL), and caspase-3 activity, a mediator of TGF-␤– induced apoptosis,23 was measured 12 hours later. We found that TGF-␤ resulted in a marked increase of caspase-3 activity as compared with control hepatocytes cultured with medium alone and that preincubation with CT-1 inhibited caspase-3 activation in a dose-dependent manner (Figure 2B). CT-1 by itself did not modify caspase-3 activity significantly as compared with cells incubated with medium alone (data not shown). These results were confirmed by the presence of soluble histone DNA complex using the Cell Death Detection assay and also by determining cell survival using the MTT assay (data not shown). Our findings suggest the existence of cross-talk between the signaling pathways of TGF-␤ and CT-1.

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Figure 2. Effects of CT-1 on cultured rat hepatocytes. (A) Western blot analysis showing phosphorylation of Stat-3, Erk 1/2, and Akt after stimulation with CT-1 (50 ng/mL) of primary hepatocytes maintained in serum-free culture for 48 hours. The figure is representative of 3 similar experiments. (B) Primary hepatocytes cultured for 12 hours in the absence or in the presence of CT-1 (at 2 different concentrations, 50 and 100 ng/mL) were challenged with TGF-␤1 (5 ng/mL) for 12 additional hours and apoptosis was estimated by determining caspase-3 activation. Control indicates unchallenged cells cultured with medium alone. The value of caspase-3 activity of hepatocytes treated with TGF-␤1 alone (TGF-␤ in the graph) was normalized to 100%. Data from 5 independent experiments are presented as mean ⫾ SEM. *P ⬍ 0.05; **P ⬍ 0.01 vs. TGF-␤.

Expression of Cardiotrophin-1 in Cultured Hepatocytes and in the Regenerating Liver

Figure 1. Effects of CT-1 on H35 cells. (A) Analysis by flow cytometry of cell cycle and annexin V binding of H35 cells cultured without serum in the presence or in the absence of 50 ng/mL of CT-1 for 7 days. Representative experiment run 4 times. Percentages correspond to hypodiploid cells (left panels) or to annexin V–positive cells (right panels). Apoptosis is associated with increased hypodiploid peak (as a result of nuclear fragmentation) and with increased annexin V binding (as a result of phosphatidyl-serine exposure on the outer layer of the cell membrane). The mean values ⫾ SEM of the proportion of hypodiploid cells and annexin V–positive cells of 4 independent experiments are shown in the graphs. *P ⬍ 0.05 vs. medium alone. (B) Western blot showing phosphorylation on Stat-3 in whole-cell lysate, Erk 1/2, and Akt after treatment with CT-1 at the indicated time. Subsequent stripping of blots followed by incubation with antibodies against corresponding specific molecules (lower respective panels) was performed to monitor protein load. The blots are representative of 4 experiments with similar results.

We investigated whether CT-1 expression could be induced in hepatocytes when subjected to stressful conditions. By using RT-PCR, we observed that freshly isolated hepatocytes expressed CT-1 messenger RNA (mRNA) and that the levels of this transcript clearly increased after 24 hours in culture in the absence of serum (Figure 3A). At this time point, CT-1 also could be detected by ELISA in the culture supernatant (Figure 3B). Similarly we observed that proapoptotic stimuli such as TGF-␤ (5 ng/mL) enhanced both transcriptional expression (Figure 3A) and secretion of CT-1 to the medium (Figure 3B). The CT-1 secreted by stressed hepatocytes appeared to reduce hepatocyte apoptosis because addition of anti–CT-1 antibody at 4 and 8 ␮g/mL reduced cell viability of primary hepatocytes cultured for 24 hours in the absence of serum to 77.6% ⫾ 8% and 68.4% ⫾ 10% of control values, respectively (Figure 3C). Taken together, these data indicate that under

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Figure 3. CT-1 is a hepatocyte survival–promoting factor up-regulated in cultured hepatocytes and in regenerating liver. (A) RT-PCR for CT-1 in primary hepatocytes cultured in serum-free conditions. (B) Levels of CT-1 as estimated by ELISA in the supernatant of primary hepatocytes maintained in serum-free culture for 24 hours in the absence (medium) or presence of TGF-␤ (5 ng/mL) added to the culture 6 hours before cell harvesting. Data are the mean values ⫾ SEM of 4 independent experiments with triplicates in each assay. **P ⬍ 0.05. (C) MTT assay of primary hepatocytes cultured in serum-free conditions for 24 hours showing the influence on cell survival of anti–CT-1 antibody (4 and 8 ␮g/mL) added to the medium. Data are means ⫾ SEM of 7 independent experiments performed in triplicate. *P ⬍ 0.01 vs. control. (D) Northern blot analysis of CT-1 gene expression in rat liver (C) and at different time points after partial hepatectomy (representative blot of 4 animals per time point).

stressful conditions hepatocytes can synthesize CT-1, which can act autocrinally to promote survival. Liver regeneration is orchestrated by a diversity of factors that convey pro-apoptotic and antiapoptotic signals to liver cells.24 We investigated whether CT-1 might participate in the events occurring in the regenerating liver. To this purpose we analyzed CT-1 mRNA expression by Northern blot in samples from normal liver and at different time points after partial hepatectomy. We found that CT-1 mRNA was detectable in the liver of adult rats and increased markedly 24 hours after partial hepatectomy, showing a progressive decline between this time point and 76 hours posthepatectomy, reaching basal levels 144 hours after surgery (Figure 3D). CT-1 mRNA expression from sham-operated rats did not show differences with respect to normal livers at any of the time points studied (data not shown). Adenovirus-Mediated Gene Transfer of Cardiotrophin-1 Protects Rats Against Lethal Hepatic Failure After Massive Hepatic Resection The results described earlier led us to investigate whether overexpression of CT-1 in the liver could have a potential therapeutic role in situations of acute severe


liver injury. Adenoviral vectors possess strong hepatotropism and provide an efficient means to achieve high expression of the transgene in hepatic tissue. We used a recombinant adenovirus encoding CT-1 (AdCT-1)21,25 in an attempt to improve survival in rats subjected to 90% hepatectomy. In preliminary experiments we found that the levels of CT-1 protein in the liver of control rats (treated with AdLacZ or saline, n ⫽ 4) were 0.16 ⫾ 0.04 ng/␮g of protein, whereas values of 3.6 ⫾ 0.18 ng/␮g of protein were detected in animals receiving 108 pfu of AdCT-1 48 hours before sampling. Ninety-percent hepatectomy provided a reproducible and well-characterized model of fulminant hepatic failure with high mortality. Forty-eight hours before this intervention different groups of rats were treated with AdCT-1 (108 pfu), or the same dose of AdLacZ or saline. We found that all rats treated with saline (n ⫽ 10) and 80% of those treated with AdLacZ (n ⫽ 14) died during the first 24 hours after hepatectomy. In sharp contrast, 53.8% of animals treated with AdCT-1 (n ⫽ 13) showed long-term survival (Figure 4A). These differences were statistically significant (P ⬍ 0.01). To elucidate the mechanism of the protection afforded by AdCT-1, we analyzed the activation of antiapoptotic signaling pathways in the livers of rats at 1 hour posthepatectomy. A marked increase in phosphorylation of Stat-3, Erk1/2, and Akt was observed in the livers of animals treated with AdCT-1 as compared with the normal liver and with the 2 posthepatectomy control groups (Figure 4B). Caspases 3, 6, and 7 are involved in the execution of apoptosis in response to many stimuli including extensive hepatectomy. Caspase 3 is upstream of caspase 6 and 7 and constitutes one of the key executioners of apoptosis.26 After 90% hepatectomy, rats treated with AdCT-1 showed a significant reduction of caspase 3 activity in the liver as compared with control rats that received saline (P ⬍ 0.05). AdLacZ-treated rats showed intermediate values, suggesting that the adenovirus per se might activate the production of cytokines or other mediators in the liver that might exert some weak protective effect (Figure 4C). Treatment With AdCT-1 Protects Mice From Liver Damage Induced by Concanavalin A Intravenous administration of Con-A to BALB/c mice induced activation of T cells and increased production of cytotoxic cytokines, mainly tumor necrosis factor ␣ and interferon ␥, with apoptosis of hepatocytes and release of transaminases within 6 – 8 hours after the insult.27 This model resembles the hepatocellular damage that occurs in inflammatory liver diseases of viral, auto-

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Figure 4. Effect of AdCT-1 therapy in rats with fulminant hepatic failure after 90% hepatectomy. (A) Survival of rats at 48 hours after 90% hepatectomy. No more deaths occurred beyond this time point. Two days before the surgery, rats received through the tail vein (●) AdCT-1 (n ⫽ 13), (‚) control vector AdLacZ (n ⫽ 14), or (E) saline (n ⫽ 10). P ⬍ 0.01 AdCT-1 group vs. AdLacZ or saline groups. (B) Western blot analysis of homogenates obtained from normal rat liver and from liver tissue sampled 1 hour after 90% hepatectomy in rats receiving AdCT-1, AdLacZ, or saline (n ⫽ 5 per group) 2 days before the surgery. Antibodies against total and phosphorylated Stat-3, Erk 1/2, and Akt were used. The blots are representative of 5 repeated experiments with consistent results. (C) Caspase-3 activity in homogenates of liver tissue sampled 1 hour after 90% hepatectomy in rats that received AdCT-1 or control adenovirus AdLacZ or vehicle 2 days before the surgery (n ⫽ 5 per group) *P ⬍ 0.05 vs. saline.

immune, or toxic origin. To study the cytoprotective effect of CT-1 in animals with Con-A–induced hepatitis, BALB/c mice were treated by intravenous injection of 1 ⫻ 107 pfu of AdCT-1, AdLacZ, or saline 24 hours before Con-A administration. The levels of CT-1 in liver extracts as determined by ELISA were 1.7 ⫾ 0.19 ng/␮g


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of protein in the AdCT-1–treated group (n ⫽ 5) and 0.17 ⫾ 0.022 ng/␮g of protein in controls (n ⫽ 4). Six hours after challenge with Con-A, hepatic injury was assessed by serum ALT levels and by analyzing cell apoptosis in liver biopsy specimens using TUNEL staining. We found that treatment with AdCT-1 was able to prevent the increase of serum transaminase levels and to reduce markedly the number of apoptotic hepatocytes in mice challenged with Con-A (Figure 5A–B). To ascertain that the protection afforded by CT-1 gene transfer was maintained during all the time-course of Con-A hepatitis, serum transaminase levels were evaluated at different moments after Con-A administration in 3 additional groups of mice (n ⫽ 4 each) pretreated with AdCT-1, AdLacZ, or saline. We found that while ALT values were below 150 SFU/mL at all time points in AdCT-1–treated mice, the levels of ALT at 12, 30, and 48 hours were 2000 ⫾ 661, 1535 ⫾ 765, and 549 ⫾ 299 in mice that received AdLacZ and 4732 ⫾ 143, 3789 ⫾ 39, and 1000 ⫾ 191 in those given saline. The marked protection afforded by CT-1 in this model of liver injury was not caused by decreased production of proinflammatory cytokines in AdCT-1–treated mice because hepatic levels of tumor necrosis factor ␣ and interferon ␥ mRNAs, as estimated by the RNAse protection assay, were similar in the 3 experimental groups (data not shown). However, in the liver of mice receiving AdCT-1 there was an intense activation of the antiapoptotic signaling pathways Erk 1/2 and Akt, in clear contrast with the other 2 groups (Figure 5C), indicating that protection against cytokine-induced hepatotoxicity mainly was owing to the direct antiapoptotic effects of CT-1. Because adenoviruses have some limitations for clinical use, we wished to know whether CT-1 given as a recombinant protein could provide protection against Con-A hepatitis similar to that observed with AdCT-1. We found that 5 ␮g of recombinant CT-1 given intravenously to mice (n ⫽ 4) 20 minutes before Con-A injection attenuated the increase in serum ALT levels and the rate of apoptotic nuclei in the liver biopsy specimen at 6 hours after Con-A challenge as compared with mice (n ⫽ 4) that received saline instead of CT-1 (Figure 6). These data indicate that the recombinant protein displays hepatoprotective effects comparable with those of AdCT-1.

Discussion Several studies have confirmed the survival-promoting activities of CT-1 on cardiomyocytes5,6 and motoneurons.21,25 This article shows that this cytokine ex-

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Figure 5. Protective effect of AdCT-1 against acute liver injury induced in mice by administration of Con-A. (A) Serum ALT level 6 hours after Con-A challenge in mice pretreated with AdCT-1, AdLacZ, or saline. **P ⬍ 0.01 vs. saline or AdLacZ. (B) TUNEL staining in liver sections 6 hours after challenge with Con-A in mice pretreated with AdCT-1, AdLacZ, or saline. (C) Representative Western blotting of liver extracts at 1 hour after challenge with Con-A in animals pretreated with AdCT-1, AdLacZ, or saline (n ⫽ 5 per group). Antibodies against total and phosphorylated Erk 1/2 and Akt were used.

erts potent cytoprotective effects in models of acute severe liver damage. We also show that CT-1 is upregulated during liver regeneration and is produced actively by stressed hepatocytes acting autocrinally to activate survival signals and maintain cell viability. Our data have identified CT-1 as a new defense mechanism of hepatocytes against apoptosis. Four lines of evidence support this contention: (1) culture of hepatocytes under serum-free conditions or proapoptotic stimuli such as TGF-␤ activate the expression of CT-1; (2) CT-1 suppresses TGF-␤–induced apoptotic death in a dose-dependent manner; (3) the presence of anti–CT-1 in the medium reduces the survival of hepatocytes under serum-deprivation conditions; and, finally, (4) CT-1 induces activation of the 3 main antiapoptotic signaling pathways Stat-3, Erk 1/2, and Akt in hepatocytic cells. In common with CT-1, IL-6, another gp130 cytokine, has been shown to activate these 3 pathways in hepatoma cells and to promote cell survival, an effect that can be abolished by inhibitors of the PI3K/Akt signaling path-


way.28 Similarly, in cardiac myocytes both CT-1 and LIF (another cytokine signaling through gp130) activate Stat-3, Erk 1/2, and PI3K/Akt pathways and exert cytoprotective effects that can be blocked by inhibitors of Jak or PI3K.12,13,29 The finding that proapoptotic signals stimulate the secretion of CT-1 by hepatocytes and that neutralization of secreted CT-1 increases liver cell death points to the existence of an as yet undescribed biologic response of liver parenchymal cells to counteract injury. It is possible that the antiapoptotic CT-1 reaction of hepatocytes might operate in a diversity of pathologic conditions. In fact, we observed a marked transcriptional up-regulation of CT-1 in rats with CCl4-induced cirrhosis (our unpublished observations), an experimental model characterized by strong oxidant stress and enhanced synthesis of proinflammatory mediators. Interestingly, we found that CT-1 expression is activated during liver regeneration, a process orchestrated by a great diversity of cytokines, hormones, and growth factors that enter into play in a timely coordinated manner.24 CT-1 is a member of the IL-6 family of cytokines, and so far only IL-6 has been shown to participate in liver regeneration. Although IL-6 is up-regulated during the priming phase, the wave of CT-1 expression (which peaks at 24 hours with progressive decline until 72 hours) takes place in later stages, coinciding with the period of maximal cell division (24 –36

Figure 6. Protective effect of recombinant CT-1 against acute liver injury induced in mice by administration of Con-A. (A) Serum ALT level 6 hours after Con-A challenge in mice pretreated with 5 ␮g of intravenous CT-1 or saline 20 minutes before Con-A challenge (n ⫽ 4 per group). *P ⬍ 0.05. (B) TUNEL staining in liver sections 6 hours after challenge with Con-A in mice pretreated with CT-1 or saline.

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hours posthepatectomy).30 It is noteworthy that the upsurge of CT-1 during regeneration occurs in a coordinated manner with the transcriptional wave of TGF-␤, which begins at 12 hours and persists until 48 hours.31 In the regenerating liver, TGF-␤ modulates angiogenesis, stimulates the formation of extracellular matrix, and displays proapoptotic activities.32 The temporal pattern of expression of TGF-␤ and CT-1 after partial hepatectomy, together with our findings showing stimulation of CT-1 production by TGF-␤ and suppression of TGF-␤– induced apoptosis by CT-1, suggest that during liver regeneration this cytokine can counteract the proapoptotic and growth-inhibitory effects of TGF-␤. Thus, CT-1 might promote survival of newly generated hepatocytes, a view that is strengthened by the reported augmented resistance to apoptosis of the regenerating liver after partial hepatectomy33 and by the observed resistance to TGF-␤–induced apoptosis of hepatocytes isolated from actively regenerating liver.34 The antiapoptotic potential of CT-1 on liver cells also was shown in in vivo models of acute severe liver damage. One of the models used in this study was acute liver failure induced by 90% hepatectomy. This intervention caused massive apoptosis of residual hepatocytes owing to hypercytokinemia, endotoxemia, reactive oxygen species, and microcirculatory disturbances, with resulting breakdown of liver function and high mortality.35 The significant improvement in survival observed in animals treated with AdCT-1 before the hepatectomy was associated with reduced apoptotic activity (as reflected by lower caspase 3 activation) and phosphorylation of Stat-3, Akt, and Erk1/2 as soon as 1 hour after the surgery. All 3 signaling pathways have been reported to be involved in suppression of apoptosis and in cell cycle progression.36 –38 These cascades might contribute to protect residual hepatocytes against apoptosis, thus facilitating the regeneration of the remaining liver. AdCT-1 also defended liver cells against Con-A–induced hepatocellular injury. In this model, the protection afforded by AdCT-1 is not associated with changes in the production of tumor necrosis factor ␣ or interferon ␥ in the liver after Con-A challenge, but with an intense activation of antiapoptotic signaling pathways, namely Erk 1/2 and Akt. These data indicate that CT-1 reduces liver damage acting mainly as a potent survival factor. It has been shown recently that apoptotic hepatocytes generate CXC chemokines and promote inflammation in the liver.39,40 Thus, the antiapoptotic properties of CT-1 might also convert this cytokine into a modulator of liver inflammation.


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IL-6, another member of the same cytokine family, also has shown hepatoprotective properties in the Con-A model of hepatitis.41 IL-6 binds to a specific membrane receptor (IL-6R or gp80) triggering homodimerization of gp130 and activation of gp130-dependent pathways. More potent activation of gp130 intracellular signaling using hyper–IL-6, a superagonistic fusion protein consisting of IL-6 linked to a soluble form of IL-6R, was shown to reduce liver injury significantly in the Dgalactosamine model of acute liver failure in rats and mice.42,43 In this model the protection afforded by hyper–IL-6 is not paralleled by IL-6, which was much less efficient than the superagonistic designer cytokine. Future studies are needed to compare the hepatoprotective potential of CT-1 with the effects of IL-6 or hyper–IL-6. The activities of CT-1 on liver cells suggest that this cytokine might find therapeutic applications in different clinical settings. Thus, the ability of AdCT1 to prevent liver failure and death after massive hepatic resection points to a potential use of CT-1 in situations in which an extensive hepatectomy is needed. This may happen in surgery for primary and metastatic liver cancer or in transplantation using living donors or cadaveric split liver. On the other hand, because CT-1 can efficiently reduce acute hepatocellular damage, it might prove useful to prevent or to treat acute liver injury of various etiologies.

References 1. Mochida S, Ogata I, Hirata K, Ohta Y, Yamada S, Fujiwara K. Provocation of massive hepatic necrosis by endotoxin after partial hepatectomy in rats. Gastroenterology 1990;92:1174–1180. 2. Hasegawa S, Kubota T, Fukuyama N, Kurosawa H, Sekido H, Togo S, Nakazawa H, Shimada H. Apoptosis of hepatocytes is a main cause of inducing lethal hepatic failure after excessive hepatectomy in rats. Transplant Proc 1999;31:558 –559. 3. Bernuau J, Benhamou J. Fulminant and subfulminant liver failure. In: Bircher J, Benhamou JP, McIntyre N, Rizzetto M, Rodes J, eds. Textbook of clinical hepatology. Vol. 2. Oxford Medical Publications, 1999:1– 40. 4. Pennica D, King KL, Shaw KJ, Luis E, Rullamas J, Luoh SM, Darbonne WC, Knutzon DS, Yen R, Chien KR, Baker JB, Wood WI. Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy. Proc Natl Acad Sci U S A 1995;92: 1142–1146. 5. Wollert KC, Taga T, Saito M, Narazaki M, Kishimoto T, Glembotski CC, Vernallis AB, Heath JK, Pennica D, Wood WI, Chien KR. Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy. Assembly of sarcomeric units in series VIA gp130/ leukemia inhibitory factor receptor-dependent pathways. J Biol Chem 1996;271:9535–9545. 6. Sheng Z, Pennica D, Wood WI, Chien KR. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development 1996;122:419 – 428. 7. Pennica D, Arce V, Swanson TA, Vejsada R, Pollock RA, Armanini M, Dudley K, Phillips HS, Rosenthal A, Kato AC, Henderson CE. Cardiotrophin-1, a cytokine present in embryonic muscle, sup-

200

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

BUSTOS ET AL.

ports long-term survival of spinal motoneurons. Neuron 1996;17: 63–74. Oppenheim RW, Wiese S, Prevette D, Armanini M, Wang S, Houenou L J, Holtmann B, Gotz R, Pennica D, Sendtner M. Cardiotrophin-1, a muscle-derived cytokine, is required for the survival of subpopulations of developing motoneurons. J Neurosci 2001;21:1283–1291. Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, Chien KR. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway. Divergence from downstream CT-1 signals for myocardial cell hypertrophy. J Biol Chem 1997;272:5783–5791. Stephanou A, Brar B, Heads R, Knight RD, Marber MS, Pennica D, Latchman DS. Cardiotrophin-1 induces heat shock protein accumulation in cultured cardiac cells and protects them from stressful stimuli. J Mol Cell Cardiol 1998;30:849 – 855. Robledo O, Fourcin M, Chevalier S, Guillet C, Auguste P, Pouplard-Barthelaix A, Pennica D, Gascan H. Signaling of the cardiotrophin-1 receptor. Evidence for a third receptor component. J Biol Chem 1997;272:4855– 4863. Oh H, Fujio Y, Kunisada K, Hirota H, Matsui H, Kishimoto T, Yamauchi-Takihara K. Activation of phosphatidylinositol 3-kinase through glycoprotein 130 induces protein kinase B and p70 S6 kinase phosphorylation in cardiac myocytes. J Biol Chem 1998; 273:9703–9710. Kuwahara K, Saito Y, Kishimoto I, Miyamoto Y, Harada M, Ogawa E, Hamanaka I, Kajiyama N, Takahashi N, Izumi T, Kawakami R, Nakao K. Cardiotrophin-1 phosphorylates akt and BAD, and prolongs cell survival via a PI3K-dependent pathway in cardiac myocytes. J Mol Cell Cardiol 2000;32:1385–1394. Brar BK, Stephanou A, Liao Z, O’Leary RM, Pennica D, Yellon DM, Latchman DS. Cardiotrophin-1 can protect cardiac myocytes from injury when added both prior to simulated ischaemia and at reoxygenation. Cardiovasc Res 2001;51:265–274. Peters M, Roeb E, Pennica D, Meyer zum Buschenfelde KH, Rose-John S. A new hepatocyte stimulating factor: cardiotrophin-1 (CT-1). FEBS Lett 1995;372:177–180. Jin H, Yang R, Keller GA, Ryan A, Ko A, Finkle D, Swanson TA, Li W, Pennica D, Wood WI, Paoni NF. In vivo effects of cardiotrophin-1. Cytokine 1996;8:920 –926. Baixeras E, Jeay S, Kelly PA, Postel-Vinay MC. The proliferative and antiapoptotic actions of growth hormone and insulin-like growth factor-1 are mediated through distinct signaling pathways in the Pro-B Ba/F3 cell line. Endocrinology 2001;142:2968 – 2977. Herrera B, Fernandez M, Alvarez AM, Roncero C, Benito M, Gil J, Fabregat I. Activation of caspases occurs downstream from radical oxygen species production, Bcl-xL down-regulation, and early cytochrome C release in apoptosis induced by transforming growth factor beta in rat fetal hepatocytes. Hepatology 2001;34: 548 –556. Chomczynski P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 1993;15:532–534, 536 –537. Lesbordes JC, Bordet T, Haase G, Castelnau-Ptakhine L, Rouhani S, Gilgenkrantz H, Kahn A. In vivo electrotransfer of the cardiotrophin-1 gene into skeletal muscle slows down progression of motor neuron degeneration in pmn mice. Hum Mol Genet 2002; 11:1615–1625. Bordet T, Schmalbruch H, Pettmann B, Hagege A, CastelnauPtakhine L, Kahn A, Haase G. Adenoviral cardiotrophin-1 gene transfer protects pmn mice from progressive motor neuronopathy. J Clin Invest 1999;104:1077–1085. Qian C, Bilbao R, Bruna O, Prieto J. Induction of sensitivity to ganciclovir in human hepatocellular carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase. Hepatology 1995;22:118 –123.


23. Chen RH, Chang TY. Involvement of caspase family proteases in transforming growth factor-beta-induced apoptosis. Cell Growth Differ 1997;8:821– 827. 24. Fausto N. Liver regeneration. In: Arias I, ed. The liver, biology and pathobiology. Philadelphia: Lippincott Williams and Wilkins, 2001:591– 610. 25. Bordet T, Lesbordes JC, Rouhani S, Castelnau-Ptakhine L, Schmalbruch H, Haase G, Kahn A. Protective effects of cardiotrophin-1 adenoviral gene transfer on neuromuscular degeneration in transgenic ALS mice. Hum Mol Genet 2001;10:1925–1933. 26. Hayami S, Yaita M, Ogiri Y, Sun F, Nakata R, Kojo S. Change in caspase-3-like protease in the liver and plasma during rat liver regeneration following partial hepatectomy. Biochem Pharmacol 2000;60:1883–1886. 27. Tiegs G, Hentschel J, Wendel A. A T cell-dependent experimental liver injury in mice inducible by concanavalin A. J Clin Invest 1992;90:196 –203. 28. Chen RH, Chang MC, Su YH, Tsai YT, Kuo ML. Interleukin-6 inhibits transforming growth factor beta-induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways. J Biol Chem 1999;274:23013–23019. 29. Yasukawa H, Hoshijima M, Gu Y, Nakamura T, Pradervand S, Hanada T, Hanakawa Y, Yoshimura A, Ross J, Chien KR. Suppressor of cytokine signaling-3 is a biomechanical stress-inducible gene that suppresses gp-130-mediated cardiac myocyte hypertrophy and survival pathways. J Clin Invest 2001;108:1459 – 1467. 30. Fausto N. Liver regeneration. J Hepatol 2000;32:19 –31. 31. Ujike K, Shinji T, Hirasaki S, Shiraha H, Nakamura M, Tsuji T, Koide N. Kinetics of expression of connective tissue growth factor gene during liver regeneration after partial hepatectomy and D-galactosamine-induced liver injury in rats. Biochem Biophys Res Commun 2000;277:448 – 454. 32. Bissell DM, Roulot D, George J. Transforming growth factor beta and the liver. Hepatology 2001;34:859 – 867. 33. Takehara T, Hayashi N, Mita E, Kanto T, Tatsumi T, Sasaki Y, Kasahara A, Hori M. Delayed Fas-mediated hepatocyte apoptosis during liver regeneration in mice: hepatoprotective role of TNF alpha. Hepatology 1998;27:1643–1651. 34. Houck KA, Michalopoulos GK. Altered responses of regenerating hepatocytes to norepinephrine and transforming growth factor type beta. J Cell Physiol 1989;141:503–509. 35. Kobayashi N, Fujiwara T, Westerman KA, Inoue Y, Sakaguchi M, Noguchi H, Miyazaki M, Cai J, Tanaka N, Fox IJ, Leboulch P. Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes. Science 2000;287:1258 –1262. 36. Fukada T, Ohtani T, Yoshida Y, Shirogane T, Nishida K, Nakajima K, Hibi M, Hirano T. STAT3 orchestrates contradictory signals in cytokine-induced G1 to S cell-cycle transition. EMBO J 1998;17: 6670 – 6677. 37. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N. Cyclin D expression is controlled posttranscriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J Biol Chem 1998;273:29864 –29872. 38. Rescan C, Coutant A, Talarmin H, Theret N, Glaise D, GuguenGuillouzo C, Baffet G. Mechanism in the sequential control of cell morphology and S phase entry by epidermal growth factor involves distinct MEK/ERK activations. Mol Biol Cell 2001;12: 725–738. 39. Faouzi S, Burckhardt BE, Hanson JC, Campe CB, Schrum LW, Rippe RA, Maher JJ. Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway. J Biol Chem 2001;276: 49077– 49082. 40. Jaeschke H. Inflammation in response to hepatocellular apoptosis. Hepatology 2002;35:964 –966.

July 2003

41. Mizuhara H, O’Neil E, Seki N, Ogawa T, Kusunoki C, Otsuka K, Satoh S, Niwa M, Senoh H, Fujiwara H. T cell activationassociated hepatic injury: mediation by tumor necrosis factors and protection by interleukin-6. J Exp Med 1994;179:1529 – 1537. 42. Galun E, Zeira E, Pappo O, Peters M, Rose-John S. Liver regeneration induced by a designer human IL6/sIL-6R fusion protein reverses severe hepatocellular injury. FASEB J 2000, 14:1979 – 1987. 43. Hecht N, Pappo O, Shouval D, Rose-John S, Galun E, Axelrod JH. Hyper-IL-6 gene therapy reverses fulminant hepatic failure. Mol Ther 2001;3:683– 687.


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Received July 30, 2002. Accepted March 27, 2003 Address requests for reprints to: Jesus Prieto, M.D., or Matilde Bustos, M.D., Department of Medicine and Liver Unit, Clinica Universitaria, 31008 Pamplona, Spain. e-mail: [email protected] or mbustos@ unav.es; fax: (34) 948-29-67-85. M.B. and N.B. contributed equally to this work. Supported by a Fondo Investigaciones Sanitarias (FIS) grant from the Ministerio de Sanidad y Consumo 01/723 (to M.B.), grant from the Gobierno de Navarra (to M.B.), and grant SAF 2002-0327 from the Ministerio de Sanidad y Consumo (to J.P.) and Instituto de Salud Carlos III C02/03. N.B. was supported by the FIS.