* Department of Toxicology, College of Pharmacy, The University of Louisiana at Monroe, 700 University Avenue, Sugar Hall #306B, Monroe, Louisiana 71209-0495; and
Pathology Associates International, National Center For Toxicological Research, Jefferson, Arkansas 72079
Received April 29, 2002; accepted June 10, 2002
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ABSTRACT |
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Key Words: Epidermal growth factor receptor; EGFR; hepatocyte growth factor; HGF; IL-6; TGF-; thioacetamide; tissue repair; TNF-
.
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INTRODUCTION |
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The primary objective of this study was to investigate the critical role played by proinflammatory cytokine- and growth factor-mediated signaling in stimulation of liver tissue repair in DR rats upon TA challenge. The molecular mechanisms of liver regeneration have been studied extensively both in partial hepatectomy models and to some extent after chemical hepatectomy (Dalu et al., 1995; Diehl, 2000
; Fausto et al., 1995
). Extensive research has shown that this process is highly regulated by cytokines and growth factors expressed by the remaining liver cells in temporally and spatially controlled fashion. Various studies have highlighted the role of proinflammatory cytokines such as tumor necrosis factor-
(TNF-
), interleukin-6 (IL-6), and growth factors such as transforming growth factor-
(TGF-
) and hepatocyte growth factor (HGF) (Diehl, 2000
; Fausto et al. 1995
; Michalopoulos and DeFrances, 1997
). It was hypothesized that increased expression of cytokines and growth factors in DR rats stimulates the enhanced tissue repair response in these rats after TA administration. We studied five signaling pathways known to be involved in liver regeneration. TNF-
pathway and Janus-activated kinase signal transducer (JAK-STAT) pathway are the proinflammatory cytokine pathways. Mitogen-activated protein kinases (MAPK) and HGF/c-met are the growth factor pathways, and the nitric oxide (NO) pathway involves free radical signaling. Plasma TNF-
and IL-6 and hepatic expression of TGF-
and HGF were assessed in livers of AL and DR rats after TA administration over a time course. Epidermal growth factor receptor (EGFR) protein levels were assessed in livers of DR and AL rats after TA administration. Inducible NO synthase (iNOS), the primary enzyme responsible for production of NO, plays a significant role in liver regeneration (Rai et al., 1998
). The role of iNOS and NO was evaluated by estimation of iNOS activity in livers from TA-intoxicated AL and DR rats over a time course. We report here that upon toxic challenge, all pathways studied except TNF-
are inhibited in AL rats, whereas DR rats exhibit upregulation of JAK-STAT (IL-6), MAPK, HGF/c-met, and iNOS pathways. This upregulated promitogenic signaling leads to timely and robust liver cell division, tissue repair, and survival.
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MATERIALS AND METHODS |
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Animals and treatment.
Male Sprague-Dawley rats (250290 g) were subjected to moderate DR as described previously (Ramaiah et al. 1998b). Briefly, DR rats were allowed to eat 65% of the AL food consumption (Harlen Teklad Rat chow No. 7001, Madison, WI: protein 25%, fat 4.25%, fiber 4.67%, vitamins and minerals supplemented, calories 3.94 Kcal/g) and were allowed free access to water for 21 days. AL rats had free access to food and water at all times. On day 22, rats in both groups received a single normally lethal dose of TA (600 mg/kg ip in 6 ml saline/kg). For lethality studies, rats were observed for 14 days twice daily, and observations were made regarding signs of toxicity and deaths. For the time course studies, separate groups of AL and DR rats were treated with the lethal dose and sacrificed at various time points. Blood was collected from the dorsal aorta in heparinized tubes and plasma was obtained by centrifugation (1000 x g for 20 min) and stored at 70°C till further analysis. Part of the left large lobe of the liver was fixed in 10% neutral buffered formalin. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals published by National Institutes of Health.
Estimation of liver injury and cell division.
Plasma was separated by centrifugation and used for the estimation of alanine aminotransferase activity (ALT; EC 2.6.1.2.) as marker of liver injury using Sigma kit No. 59 UV (ALT) (Sigma Chemical Co., St. Louis, MO). Liver cell division was estimated by incorporation of 3H-thymidine (3H-T) in hepatonuclear DNA, as previously described (Chang and Looney, 1965). Rats were treated with 50 µCi 3H-T/rat (ip) 2 h prior to sacrifice at each time point. Total DNA content was measured by diphenylamine reaction.
Assessment of apoptotic cells.
Apoptotic cells were visualized and counted by TUNEL assay using ApoTag kit (Oncor, Gaithersburg, MD) according to the manufacturers protocol. Paraffinized liver sections of samples collected from AL and DR rats over the 072 h time course after TA administration were used. Three slides per group per time point were stained and apoptotic cells identified by dark brown staining were counted under a microscope.
TNF- and IL-6 ELISA.
TNF- levels were estimated in the plasma of AL and DR rats treated with TA using a rat-specific TNF-
sandwich ELISA (Amersham Life Sciences, Piscataway, NJ) according to manufacturers protocol. Briefly, heparinized plasma samples (n = 4 per time point) were incubated with antiTNF-
capture antibody for 1 h followed by biotinylated detection antibody. Visualization was carried out by peroxidase reaction, and color intensity was measured at 450 nm in a BioRad Model 550 microplate reader (BioRad, Hercules, CA). IL-6 was measured in plasma samples collected at different time points using a rat-specific IL-6 ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturers protocol similar to TNF-
ELISA.
Immunohistochemistry.
TGF-, HGF, and IL-6 protein were evaluated by immunohistochemical method using paraffinized sections of liver samples collected over 096 h time course. Briefly, antigen retrieval was carried out by flooding the liver sections with 0.05% saponin for 30 min followed by treatment with TGF-
specific antibody (Ab-1, Oncogene, Cambridge, MA) at a concentration of 1:500 for 18 h at 4°C. Antigen retrieval for HGF was performed by citrate buffer treatment; no antigen retrieval for IL-6 was necessary. The concentration of primary antibody was 1:50 for both HGF and IL-6 immunohistochemistry. After treatment with secondary antibody (concentration 1:10,000) and streptavidine conjugate, visualization was achieved by peroxidase reaction in all cases. To get a quantitative estimate of the growth factor production from the staining intensity, a scoring scheme was devised using a scale of 04 staining intensity. This type of scoring scheme for staining intensity has been used by other investigators to get a quantitative estimate of immunohistochemical analysis (Roberts et al., 1991
; Williams et al, 1996
). Four slides were stained and coded per time point. Slides were observed under a light microscope, 1015 central veins were observed per slide, and staining intensity was graded in single-blinded manner at a scale of 04 (0, no staining; 1, low staining intensity; 2, medium staining intensity; 3, high staining intensity; and 4, very high staining intensity).
iNOS assay.
iNOS activity was determined according to the method of Bredt and Snyder (1994). Briefly, the reaction mixture composed of the iNOS assay medium (400 µl) containing 100 mM HEPES, pH 7.2; 2 mM NADPH; [3H]-arginine (0.2 µCi/ml), and 400 µg of liver cytosolic protein was incubated for 30 min at 37°C. The reaction was stopped by addition of stop buffer containing 20 mM HEPES and 10 mM EGTA at pH 5.5. The entire reaction mixture was passed through Dowex AG 50 Wx-8 resin (Na+ form) to elute the fraction containing [3H]-citrulline, which was counted in a Beckman 6000 liquid scintillation counter.
Preparation of cell lysate and Western blot analysis.
Liver cell lysate was prepared in lysis buffer (1% Triton-X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 2 mM Na vanadate, 0.2 mM phenylmethylsulfonylfluoride, 1 mM HEPES, 1 µg/ml leupeptin, 1 µg/ml aprotinin), and protein concentration was estimated using a BioRad protein assay kit (BioRad, Hercules, CA). Western blot analysis of EGFR was conducted according to Dalton et al.(2000). Briefly, 50 µg of cell lysates was resolved by electrophoresis on a 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gel (100 V, 1.5 h) in a running gel buffer containing 25 mM Tris, pH 8.3, 162 mM glycine, and 0.1% SDS. The samples were transferred to nitrocellulose membrane for 3 h at 500 mA using 25 mM Tris, 192 mM glycine buffer with methanol reduced to 10% and 0.02% SDS included to facilitate transfer of large molecular weight proteins. The membranes were blocked with 5% nonfat dry milk in TTBS (1 h, 20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween-20) and probed with an anti-EGFR antibody (Calbiochem, La Jolla, CA) diluted 1:800 in TTBS with 5% nonfat dry milk. Membranes were subsequently probed with HRP-conjugated secondary antibody (2 h in TTBS with 1.5% nonfat dry milk) and visualized by an ECL kit (Pierce, Rockford, IL). Densitometric evaluation was done using Quantity One software from BioRad.
Statistical analysis.
Group comparisons were performed using independent t-test. A one-way analysis of variance was used to determine statistical significance that might exist between more than two distributions or sample groups. Statistical analyses were made using SPSS 10.0 software (SPSS Inc., Chicago, IL). Statistical significance was set at p 0.05.
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RESULTS |
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DISCUSSION |
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TNF- has been implicated as a priming factor during liver regeneration, stimulating progression of quiescent hepatocytes from G0 gap phase to G1 (Webber et al., 1998
; Yamada and Fausto, 1998
). TNF-
levels in plasma increased after TA administration over the time course and remained higher until 96 h in both AL and DR groups but there was no significant difference between the two groups. Hepatic expression of TNF-
mRNA measured by semiquantitative RT-PCR indicated that DR rats have significantly higher mRNA at 48 and 72 h after TA administration (data not shown). These data indicate that TNF-
mediated signaling may play a role in the stimulation of tissue repair induced by TA injury, but it does not explain the enhanced compensatory liver tissue repair in DR rats over and above the AL rats. TNF-
is also known to stimulate production of TGF-
, which further stimulates hepatocytes to move from G1 to M phases (Gallucci et al., 2000
; Webber et al., 1998
). Our data support this notion. Increase in TNF-
levels precedes an increase in TGF-
after TA administration in both the groups.
Gene knockout experiments have revealed the critical role played by IL-6 during liver regeneration. IL-6-/- mice have delayed regeneration, and other experiments including human studies with post-liver transplantation regeneration have highlighted crucial involvement of IL-6 during liver tissue repair (Galun et al., 2000; Rai et al., 1998
; Streetz et al., 2000
). IL-6 expression was suppressed in the AL rats after treatment with a high dose of TA. DR rats had significantly higher IL-6 than AL rats as early as 12 h after TA administration that further increased, reaching a peak at 48 h where the IL-6 levels were 6-fold higher in DR rats. The increase in plasma levels of IL-6 was further corroborated by immunohistochemical analysis of IL-6 in livers of AL and DR rats after TA administration. Sinusoidal endothelial cells were identified as the primary source of IL-6 in the liver. The increase in IL-6 coincides with increase in S-phase stimulation in the DR rats, indicating potential involvement of IL-6 in liver tissue repair in DR rats after toxic insult.
The role of TGF- and HGF has been particularly well studied during regeneration after toxic insult (Jiang and Hiscox, 1997
; Lindroos et al., 1991
; Michalopoulos and DeFrances, 1997
; Scotte et al., 1997
). TGF-
expression has been extensively studied in hepatocarcinomas in human subjects and in partial and chemical hepatectomy in animal models (Dalu et al., 1995
; Harada et al., 1999
; Tomiya et al., 1998
). TGF-
and c-myc double knockout mice lose their ability to regenerate liver after partial hepatectomy (Factor et al., 1997
). TGF-
signals via EGFR with downstream activation of MAPK (Boylan and Gruppuso, 1994
; Stromblad et al., 1993). Immunohistochemical localization of TGF-
indicated hepatocytes as the primary source of TGF-
, with the cells immediately surrounding the necrotic areas staining positive. Both AL and DR rats had similar expression of TGF-
during the initial time points (036 h), but in AL rats, TGF-
expression decreased and diminished during the later time points. In DR rats, it remained consistently higher during later time points (4896 h). These data support the observations of other investigators that TGF-
expression in hepatocytes increases during tissue repair after toxicant administration (Burr et al., 1993
; Dalu et al., 1995
; Kobayashi et al., 2000
). Western blot analysis of EGFR indicated AL rats had lower initial expression, which increased comparable to that in DR rats at 24 h, but diminished to undetectable levels at 48 h after TA treatment. Higher EGFR protein was observed initially in DR rats, which further increased slightly at 24 h and remained higher 48 h after TA administration. Both the ligand (TGF-
) and its receptor (EGFR) are upregulated in DR rats, and their temporal correlation with increased cell division points toward an important role of these factors in stimulation of liver tissue repair after toxic insult in DR rats.
HGF is a potent mitogen for a number of cell types including hepatocytes and was thought to be produced in extrahepatic tissues such as salivary glands and Brunners gland in the intestine and transported to liver (Wolf et al., 1991). However, recent investigations suggest that under certain conditions such as embryonic and fetal development, endotoxin challenge, and cirrhosis, the liver parenchymal cells can also express HGF. It is known that cytokines such as TNF-
and IL-6 are capable of stimulating expression of HGF (Masson et al., 2001
). In our study, hepatocytes, Kupffer cells, and infiltrated macrophages stained positive for HGF. HGF is produced in an inactive monomer form and is processed to a dimer in hepatocytes and stimulates the cells in autocrine and paracrine fashion using the transcription factor AP-1 (Gao et al., 1999
; Pediaditakis et al., 2001
). The antibody used in the present study stains the unprocessed form of HGF. Immunohistochemical localization indicated that in AL rats HGF expression increased after TA challenge and attained similar levels as in DR rats at 24 h, but decreased by 48 h and was diminished at 72 h after TA administration. DR rats had slightly higher HGF initially, which quickly increased further after TA treatment and remained higher until 72 h. Western blot analysis of c-met, the receptor for HGF, indicates higher c-met protein at 48 h in DR rats after TA administration (data not shown). The early increase during the first 12 h and persistent expression of growth factors and their receptors during late time points (3672 h) coinciding with cell division indicate that HGF-mediated signaling may have an important role in enhanced cell division in DR rats.
NO is a versatile molecule playing an important role in a variety of physiological processes including liver regeneration. In liver, the primary source of NO is iNOS, which is present at minimal levels in normal liver. During liver regeneration, induction of iNOS via cytokines leads to higher release of NO, further stimulating a variety of genes involved in regeneration and priming of hepatocytes for division. NO plays a dual role during liver regeneration; it stimulates cell division along with inhibition of apoptosis induced by factors such as TNF- (Diez-Fernandez et al., 1997
; Rai et al., 1998
). Estimation of iNOS activity in AL and DR rats revealed an overall inhibition of iNOS induction in the AL rats treated with a high dose of TA. Diet restriction induced iNOS activity 2-fold, which plays a significant role in early priming of cells to divide. iNOS activity in DR rats decreased after TA administration, with a small surge again at 48 h. This may be important for stimulation of additional cells during later time points. The DR-mediated induction of iNOS activity might be related to inhibition of apoptosis in DR rats. During diet restriction, liver cells undergo apoptosis to cope with changes in the energy budget (Frame et al., 1998
; Klaunig and Kamendulis, 1999
). iNOS induction may be controlling the extent of apoptosis and thus play a significant role in establishing homeostasis after caloric restriction. This is consistent with our observation that apoptotic cells increased in DR rats after TA administration from 12 to 48 h, during which a decrease in iNOS activity was observed. At 48 h, iNOS activity increased and the number of apoptotic cells decreased at 60 h. In AL rats, iNOS was suppressed and no increase in apoptotic cells was observed, indicating a deficiency in signaling. TUNEL analysis of AL and DR liver sections after TA administration revealed no significant increase in apoptotic cell in AL rats, which is consistent with our previous observation. DR rats have significantly higher apoptotic cell death after TA administration. Apoptosis has been recognized as a controlled, programmed cell death leading to minimal damage to the surrounding tissue (Barros et al., 2001
). Increased apoptosis in DR rats after TA treatment increased the efficiency of tissue repair by removing the weak and damaged cells that would otherwise undergo oncosis and contribute to progression of injury. Therefore, regulated increase in apoptosis in tandem with stimulated robust cell division in DR rats after toxic challenge with TA appears to facilitate efficient tissue repair, leading to recovery from markedly higher liver injury.
These data support our observation that the high dose of TA inhibits tissue repair in the AL rats because of inhibited signaling (Fig. 11). This is clear from the decreased expression of cytokines, growth factors, and EGFR after TA administration, leading to a delayed repair response. The mechanisms by which the high dose inhibits signal transduction in AL rats are not entirely clear. On the other hand, elimination of this delay and disrepair in DR rats upon TA challenge is due to a combination of several molecular mechanisms. These involve upregulation of cytokines, especially IL-6, and growth factors, both TGF-
and HGF, along with EGFR and iNOS. This sequential orchestration, evident with increase in TNF-
, IL-6 and iNOS during the early periods after TA treatment, was followed by increases of TGF-
, HGF and IL-6 during later time points. The interaction of these molecules orchestrates the complex process of liver cell division and tissue repair in DR rats. The increase in apoptosis after TA treatment in DR rats further enhances the regeneration process. Our studies provide substantial evidence that upregulated promitogenic signaling via cytokines and growth factors is a potential mechanism of stimulated repair in DR rats that disallows progression of injury on one hand and allows for restoration of liver structure and function on the other. Other possible mechanisms, such as involvement of fat mobilization and increased fatty acid metabolism and its relation to cell division (Chanda and Mehendale, 1996
; Yamashita et al., 2000
), that may modulate cell division in DR rats remain to be investigated.
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ACKNOWLEDGMENTS |
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NOTES |
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1 Present address: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas Veterinary Medical Center, Texas A&M University, TX 77843-4467.
2 To whom correspondence should be addressed. Fax: (318) 342-1686. E-mail: mehendale{at}ulm.edu.
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