- Email: [email protected]
Peroxynitrite-mediated matrix metalloproteinase-2 activation in human hepatic stellate cells
FEBS 29609
FEBS Letters 579 (2005) 3119–3125
Peroxynitrite-mediated matrix metalloproteinase-2 activation in human hepatic stellate cells Kiyoshi Migitaa,*, Yumi Maedaa, Seigo Abirua, Atsumasa Komoria, Terufumi Yokoyamaa, Yasushi Takiia, Minoru Nakamuraa, Hiroshi Yatsuhashia, Katsumi Eguchib, Hiromi Ishibashia a
Clinical Research Center, NHO Nagasaki Medical Center, Kubara 2-1001-1, Omura 856-8562, Japan b First Department of Internal Medicine, Nagasaki University School of Medicine, Japan Received 24 December 2004; revised 29 March 2005; accepted 1 April 2005 Available online 12 May 2005 Edited by Veli-Pekka Lehto
Abstract To investigate whether hepatic stellate cells (HSCs) alter their expression of MMPs after exposure to nitrogen oxide intermediate (NOI), a human hepatic stellate cell line, LI90 cells, was stimulated with an NO donor, SNAP, or a peroxynitrite donor, SIN-1, and the culture supernatants were analyzed by gelatin zymography or anti-MMPs immunoblot. Although SIN-1 did not enhance the secretions of MMP-1 and 13, SIN1 induced the NF-jB activation, MT1-MMP expression and the secretion of activated MMP-2 in LI90 cells. These results suggest that peroxynitrite may contribute to the remodeling of the extracellular matrix in liver by activating pro-MMP-2. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Hepatic stellate cell; Peroxynitrite; Matrix metalloproteinase; Nitric oxide
species (ROS) occurs in the process of liver fibrosis [9]. ROS may represent direct or indirect pro-fibrogenic stimuli for HSCs [10]. Moreover, signs of oxidative stress and lipid peroxidation are concomitant or precede HSCs activation and collagen deposition [11]. It was demonstrated that HSCs express non-phagocytic NADPH oxidase and inducible NO synthase (iNOS) [12], which generate superoxide and NO, once exposed to effective stimuli. Furthermore, the simultaneous production of NO and superoxide by HSCs or Kupffer cells in liver may lead to the generation of peroxynitrite (ONOO ) [13]. The present study was designed to more clearly elucidate the biological effects elicited by oxidative stress on HSCs. In particular, we investigated whether nitrogen oxide intermediate (NOI) may affect the synthesis of MMPs and their related inhibitors (TIMP-1 and TIMP2) in HSCs.
2. Materials and methods 1. Introduction Liver fibrosis occurs following most chronic liver injuries and results from changes in the balance between the synthesis and degradation of extracellular matrix (ECM) components [1,2]. Hepatic stellate cells (HSCs) play a pivotal role in the cellular and molecular events that lead to fibrosis [3]. Following liver injury, these cells undergo changes toward a myofibroblastic-like phenotype and secrete extracellular matrix components [4]. In addition, activated HSCs are involved in ECM degradation by providing MMPs [5]. During liver fibrogenesis, a number of MMPs, such as MMP-1, MMP-2, and MMP-13, are expressed in the liver by hepatic stellate cells [6]. Therefore, it has been suggested that the activation mechanism of HSCs and the secretion of MMPs may contribute to the process of liver fibrosis [7]. Evidence of oxidative stress has also been detected in clinical condition of liver fibrosis with different etiology [8]. It has become obvious that an increase in the levels of reactive oxygen * Corresponding author. Fax: +81 957 54 0292. E-mail address: [email protected] (K. Migita).
Abbreviations: ECM, extracellular matrix; HSCs, hepatic stellate cells; IjB-a, IkappaB-a; MAPK, mitogen activated protein kinase; MMP, matrix metalloproteinases; SEAP, secretory alkaline phosphatase; SNAP, S-nitroso-N-acetyl-DL -penicillamine; SIN-1, N-morpholino sydnonimine hydrochloride
2.1. Reagents An NO donor, SNAP (S-nitroso-N-acetyl-DL -penicillamine), and a peroxynitrite donor, SIN-1 (N-morpholino sydnonimine hydrochloride), were purchased from Dojindo Laboratories (Kumamoto, Japan). Mouse anti-human membrane-type 1 matrix metalloproteinase (MT1-MMP) monoclonal antibody and mouse anti-human tissue inhibitor metalloproteinase–1 (TIMP-1) and TIMP-2 antibodies were obtained from Fuji Chemical Co. (Takaoka, Japan). Mouse monoclonal anti-a-smooth muscle actin antibody was purchased from Novacastra Laboratories (Newcastle, UK). Mouse monoclonal anti-nitrotyrosine antibody (1A6) was purchased from UBI (Lake Placid, USA). Purified pro-MMP-2 was purchased from Calbiochem (San Diego, USA). Activation with p-aminophenylmercuric acetate (APMA, Sigma Chemical) was done by incubation of purified pro-MMP-2 with 1 mM APMA at 37 C for 15 min [14]. All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). 2.2. Cells LI90 cells, human HSCs established by Dr. Murakami, were purchased by JCRB (Japanese Collection of Research Bioresources, Osaka, Japan). Cells were propagated in DulbeccoÕs minimal essential medium (DMEM) supplemented with 10% fetal calf serum (Gibco BRL, Gaithersburg, MD). LI90 cells were stimulated with SIN-1 at different concentrations (0–500 lM) for 24 h in serum-free DMEM. Culture supernatants were subjected to the analysis for gelatin zymography or immunoblot. 2.3. Gelatin zymography LI90 cells-conditioned culture media (serum-free) were incubated at 37 C for 20 min in sodium dodecyl sulfate (SDS) sample buffer free of reducing agents and then electrophoresed on 8% polyacrylamide gels containing 0.5 mg/ml gelatin at 4 C. After electrophoresis, gels were
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.04.071
3120 washed in 2.5% Triton-X 100 to remove SDS, and incubated with 50 mM Tris–HCl buffer (pH 7.5) containing 0.15 M NaCl, 10 mM CaCl2, and 0.02% NaN3 for 16 h at 37 C, and stained with 0.1% Coomassie Blue R250.
2.4. Immunoblot analysis The expression of MT1-MMP on LI90 cells and the secretions of MMP-1, MMP-13 and TIMPs from synovial cells were analyzed by immunoblot. For this purpose, cells were washed with cold PBS and lysed by the addition of a lysis buffer containing 1% Nonidet P-40, 50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM EDTA, 10 lg/ml aprotinin, and 10 lg/ml leupeptin for 20 min at 4 C. Insoluble materials were removed by centrifugation at 1500 · g for 15 min at 4 C. The supernatant was saved and the protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). An identical amount of protein (30 lg) from each lysate or the culture supernatant was subjected to 8% SDS–polyacrylamide gel electrophoresis (PAGE). The fractionated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL), and the filters were blocked for 1.5 h using non-fat dried milk in Trisbuffered saline (TBS: 50 mM Tris, 0.15 M NaCl, pH 7.5) containing 0.1% Tween 20, washed with TBS, and incubated at room temperature for 2 h at a 1:150 dilution of mouse anti-MT1-MMP or MMPs monoclonal antibodies (mAb). The membranes were further incubated with a 1:2000 dilution of horseradish peroxidaseconjugated donkey anti-mouse immunoglobulin G (IgG) antibody (Promega, Madison, WI) for 20 min. An enhanced chemiluminescence (ECL) system (Amersham) was used for detection. The blots were analyzed by densitometer (Fuji Film, Tokyo, Japan). Images from densitometer were transferred to a personal computer and analyzed using Science Lab software (Fuji Film). The density of the target protein band of untreated cells was assigned the value of 1.0 and each result was calculated as relative units.
2.5. RNA preparation and quantitative PCR assay Total cellular RNA was extracted from LI90 cells using guanidium thiocyanate and phenol (RNAzol B, Cinna/Biotek Labs Int. Inc., Friendswood, TX). First-strand cDNA was synthesized by reverse transcription at 45 C for 45 min in a 50 ll reaction mixture containing 1 lg of total RNA and MuLV reverse transcriptase (Invitrogen). After denaturing at 99 C for 5 min followed by cooling at 5 C, the cDNA was amplified using PCR. The quantification of MT1-MMP transcripts in LI90 cells was carried out with the LightCycler PCR system (Roche Diagnostics, Meylan, France) using the DNA binding SYBR Green I dye for the detection of PCR products. A serial dilution was used to generate each standard curve. For real time quantitative PCR, each reaction contained: 1· of LightCycler-DNA master SYBR Green I, specific primer (Roche), and 2 ll of cDNA matrix, in a final volume of 20 ll. After 2 min of denaturation at 95 C, the reactions were cycled 40 times for 5 s at 95 C, 10 s at the annealing temperature, and 15 s or 7 s at 72 C for MT1-MMP and GAPDH, respectively. Product specificity was determined by melting curve analysis as described in the LightCycler handbook. The amount of transcripts in each sample is given as copy number. The results are expressed as ratios of MT1-MMP transcripts to GAPDH transcripts.
2.6. Plasmids and transfection pNFjB-SEAP (CLONTECH Laboratories, Inc., Palo Alto, CA) is designed to monitor the activation of NF-jB and NF-jB-mediated signal transduction pathways. pNFjB-SEAP contains four tandem copies of the jB4 enhancer fused to the HSV-TK promoter. pTAL-SEAP (CLONTECH Laboratories) was used as a negative control to determine the background signals associated with the culture medium. Cells were grown to approximately 50% confluence on 30-mm plates. Transfections were done using the calcium phosphate reagents and following the manufacturerÕs instructions (CLONTECH Laboratories), and cells were treated as described above. Chemiluminescence detection of SEAP activity was performed according to the manufacturerÕs instructions (CLONTECH Laboratories) using a plate fluorometer (Berthold, Bad Wildbach, Germany).
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125 2.7. Statistical analysis Differences in the data were analyzed by 1-way ANOVA combined with the BonferroniÕs test, and all values were expressed as means ± S.D. The differences between groups were considered to be significant at P < 0.05.
3. Results The effects of NO to human hepatic stellate cells were investigated by incubating LI90 cells with an NO donor, SNAP, which generates NO in vitro. As shown in Fig. 1, SNAP treatment did not modulate the gelatinase secretions from LI90 cells. Next, we incubated the cells with SIN-1, which generates ONOO by releasing NO and O2 simultaneously for 24 h and the culture supernatants were subjected to gelatin zymography. SIN-1 was not toxic to LI90 cells at the concentrations (500 lM) used in this experiment (data not shown). Gelatin zymography demonstrated that in addition to the secretion of a late form of MMP-2 (Mr = 72 kd), the activated form of MMP-2 (Mr = 66 kd) was detected when LI90 cells were treated with SIN-1 (Fig. 2A and B). This gelatinolytic band with lower molecular weight (Mr = 66 kd) was similar to APMAactivated MMP-2 in that it migrated at 66 kd (Fig. 2C). Also, these gelatinolytic activities were completely inhibited by EDTA demonstrating that these are metalloproteinases (Fig. 2D). In contrast, the SIN-1 treatment did not significantly affect the secretion of MMP-1 (Fig. 3A and B) and MMP-13 (Fig. 3C and D). It was demonstrated that peroxynitrite is capable of degrading TIMP-1, a process which could potentiate MMP-2 activation [15]. Therefore, we examined the effects of SIN-1 on TIMP-1 secretion from LI90 cells. The molecular forms of TIMP-1 and its secretion levels were not affected by SIN-1 treatment (Fig. 3E and F). TIMP-2 was not detected in LI90-conditioned media with or without SIN-1 treatment by anti-TIMP-2 immunoblot analysis (data not shown). Because peroxynitrite has the functional property of inducing nitration, we examined whether SIN-1-treated cells contain proteins with nitrated tyrosine residues [16]. The level of tyrosine nitration was clearly increased after 4 h of treatment with SIN-1. This effect was smaller in SNAP-treated LI90 cells (Fig. 4A). Expression of a-smooth muscle actin (a-SMA) is considered as an indicator of HSCs activation [17]. The expression of a-SMA was also examined using LI90 cells treated with SIN-1
Fig. 1. Effects of SNAP on gelatinase secretions from LI90 cells. LI90 cells were cultured with SANP in serum-free culture media for 24 h. The conditioned media were analyzed by gelatin zymography. The data shown are representatives of at least three independent experiments.
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125
Fig. 2. Effects of SIN-1 on gelatinase secretion from LI90 cells. (A) LI90 cells were cultured with various concentrations of SIN-1 in serum-free culture media for 24 h. The conditioned media were analyzed by gelatin zymography. Note the activated MMP-2 with lower molecular weights in addition to 72 Kda MMP-2 in the SIN-1treated LI90 cells-conditioned media. (B) Densitometric analysis of A is presented as the relative ratio of active form/pro-form of MMP-2. These data represent means ± S.D. of three independent experiments. \ P < 0.01 versus untreated LI90 cells. (C) Gelatinolytic activity of LI90 cells-conditioned media detected by zymography. Lane 1: untreated LI90 cells-conditioned media, Lane 2: SIN-1 (500 lM)-treated LI90 cells-conditioned media, Lane 3: purified pro-MMP-2 standards (20 ng), Lane 4 pro-MMP-2 standards (20 ng) were activated with 1 mM APMA at 37 C for 15 min. The data shown are representatives of at least three independent experiments. (D) LI90 cells were cultured with SIN-1 in serum-free culture media for 24 h. Zymogram of LI90 cells-conditioned media was incubated in the presence or absence of EDTA (10 mM) for 16 h at 37 C. Gelatinolytic activities were completely inhibited by EDTA.
for 24 h. SIN-1 treatment resulted in an increased a-SMA expression on LI90 cells (Fig. 4B and C). As shown in Fig. 5, the incubation of LI90 cells-conditioned media containing pro-MMP-2 with SIN-1 for 24 h did not result in the activation of pro-MMP-2. This result indicates that SIN-1-induced pro-MMP-2 activation requires the presence of pro-MMP-2 producing IL90 cells. It has been suggested that the conversion of pro-MMP-2 to active MMP-2 requires cell membrane-associated processing by membrane-type matrix
3121
Fig. 3. Effects of SIN-1 on MMPs or TIMP-1 secretions from LI90 cells. LI90 cells were cultured with various concentrations of SIN-1 for 24 h. The conditioned media were analyzed by anti-MMP-1 (A), MMP-13 (C), and TIMP-1 (E) immunoblot. MMP-1 (B), MMP-13 (D) and TIMP-1 (F) bands were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
metalloproteinase (MT-MMP) [18]. Therefore, we examined whether MT-MMP is involved in the SIN-1-induced proMMP-2 activation. LI90 cells were cultured with SIN-1 for
3122
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125
Fig. 6. MT1-MMP expression in SIN-1-treated LI90 cells. (A) LI90 cells were cultured with various concentrations of SIN-1 for 24 h. The cells were lysed and equal amounts of cellular lysates were analyzed by immunoblot using antibodies to human MT1-MMP. The band of MT1-MMP of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. (B) MT1-MMP bands were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
Fig. 4. (A) Tyrosine nitration in LI90 cells. Immunoblot analysis of LI90 cells extracts with anti-nitrotyrosine antibody showed SIN-1 induced tyrosine nitration on several proteins. Immunoblot analysis of a-smooth muscle actin expression in LI90 cells. (B) LI90 cells were treated with SIN-1 or SNAP with the indicated concentrations for 24 h. Cellular lysates were analyzed by immunoblot using anti-asmooth muscle actin antibodies. (C) Bands of a-smooth muscle actin were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
activated form of MMP-2 in SIN-1-treated LI90 cells-conditioned media (Fig. 2A and B). We further investigated the peroxynitrite-mediated signaling pathway in LI90 cells. We examined the effects of SIN-1 on the state of ERK1/2, p38 and JNK1/2. The state of phosphorylation of these MAPKs was examined by immunoblot using anti-phospho-specific MAPKs antibodies. The phosphorylation of ERK1/2, p38 and JNK1/2 was not increased but rather decreased in SIN-1-treated LI90 cells (Fig. 8A–F). Immunoblot of b-actin is shown as a control for equal loading of samples (Fig. 8G). We also evaluated IjB-a proteolysis. The protein levels of IjB-a in SIN-1-treated LI90 were measured by immunoblot analysis (Fig. 9A and B). The SIN-1
24 h. The cells were lysed and the lysates were subjected to immunoblot using anti-MT1-MMP antibodies. As shown in Fig. 6, MT1-MMP was barely detected in unstimulated LI90 cells and the intensity of MT1-MMP band was increased when the LI90 cells were treated with SIN-1. We also analyzed MT1MMP mRNA expression by real-time quantitative RT-PCR analysis. As shown in Fig. 7, MT1-MMP mRNA expression in LI90 cells was increased by SIN-1 treatment. This SIN-1-induced MT1-MMP expression was parallel to the induction of
Fig. 5. In vitro effects of SIN-1 on gelatinase. LI90 cells-conditioned media were incubated with SIN-1 for 24 h at 37 C. Incubated media were analyzed by gelatin zymography. The data shown are representatives of at least three independent experiments.
Fig. 7. MT1-MMP mRNA expression in SIN-1-treated LI90 cells. LI90 cells were stimulated by SIN-1 for 6 h. Total cellular RNA was analyzed by real-time RT-PCR using specific primers for MT1-MMP. Results are means of three separate experiments. \P < 0.01 versus untreated LI90 cells.
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125
3123
Fig. 8. Effects of SIN-1 on the MAP kinase phosphorylation of LI90 cells. LI90 cells were stimulated by SIN-1 (500 lM) for indicated times. Equal amounts of cellular lysates were analyzed by immunoblot using anti-phospho-specific ERK1/2 (A), p38 (C), JNK1/2 (E) and b-actin (G) antibodies. Bands of ERK1/2 (B), p38 (D), and JNK1/2 (F) were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
stimulation induced substantial IjB-a degradation in a timedependent manner, suggesting the activation of NF-jB in LI90 cells. However, the SNAP treatment did not affect the protein levels of IjB-a. To determine whether SIN-1 treatment modulates the transcriptional activity of NF-jB, LI90 cells were transiently transfected with a reporter gene construct, pNFjB-SEAP. Cells were subsequently treated with SIN-1, and SEAP in the culture supernatants was assayed. As shown in Fig. 10A, SIN-1 treatment increased the SEAP levels compared to those of control cells. SNAP treatment did not activate the NF-jB transcriptional activity (Fig. 10B).
4. Discussion Hepatic stellate cells (HSCs) play a central role in the development of liver fibrosis [3]. After chronic liver injury, HSCs proliferate and transform into myofibroblastic cells (activated HSCs) that are the major source of collagens that accumulate
in the fibrotic liver [4]. This process is promoted by a variety of cytokines and soluble factors released by Kupffer cells and inflammatory cells as well as HSCs [19]. Mayoral et al. [20] demonstrated an increased hepatic expression of iNOS in bile duct ligated rats, model animals for hepatic fibrosis. Furthermore, stimulation of NO synthesis with L -arginine treatment augmented hepatic fibrosis. These results suggest that NO may be involved in the development of fibrosis. We found that peroxynitrite induces the secretion of activated MMP-2 in cultured human HSCs. A number of studies have emphasized the profibrogenic functions of MMP-2 [21,22]. However, the exact mechanisms leading to MMP-2 activation have not been clearly delineated. The MMPs activity is closely regulated at 3 levels, namely gene transcription, proteolytic activation, and inhibition by endogenous inhibitors [23]. As well, proteolytic activation of MMP-2 is induced by membrane-type MMPs (MT-MMP) [24]. Previous reports suggest that MT1-MMP-activated MMP-2 exerted a profibrogenic role during hepatic fibrosis [25]. It is possible
3124
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125
Fig. 9. SIN-1 stimulated IjB-a degradation in LI90 cells. (A) LI90 cells were stimulated with SIN-1 (500 lM) and SNAP (500 lM) for the indicated times. Cells were lysed and the cellular lysates were assessed by anti-IjB-a immunoblot analysis. (B) Bands of IjB-a were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
that peroxynitrite generated by SIN-1 directly nitrates MMP-2 and induces its enzymatic activation. However, SIN-1 did not activate pro-MMP-2 in the absence of HSCs in vitro, suggesting that the interaction between peroxynitrite and HSCs is required for this MMP-2 activation. The hepatic expression of MMP-2 and MT1-MMP is increased in human fibrotic livers, and activated HSCs have been identified as a source of both proteins [26,27]. Therefore, we investigated whether the SIN-1 treatment increases MT1MMP expression in HSCs. Our data showed that MMP-2 activation in SIN-1-treated HSCs is associated with the increased expression of MT1-MMP by these cells. In response to peroxynitrite, HSCs may express MT1-MMP on their surface thereby stimulating pro-MMP-2 activation. The mechanism leading to MT1-MMP expression in SIN-1-treated HSCs is not known. The finding that SIN-1 leads to the rapid degradation of IjB-a suggests that the peroxynitrite may activate NF-jB in HSCs. It can be speculated that the peroxynitrite-mediated NF-jB dependent pathway might be involved in the MT1MMP induction as reported previously [28,29]. Oxidative stress has been detected in fibrotic liver and acts as a mediator of the ECM remodeling responsible for liver fibrosis [30]. In liver injury, iNOS is upregulated in liver cell populations including HSCs, and the NO generation is increased [31]. The pathogenic action of NO seems to occur as the interaction of NO with O2 to generate peroxynitrite. The effects of peroxynitrite can be deleterious, since it induces the oxidation and nitration of biological macromolecules [32]. Our data suggest that peroxynitrite may activate MMP-2 secreted from HSCs by inducing MT1-MMP. ECM remodeling in fibrotic liver could be accelerated by MMP-2 activation, because this enzyme degrades the normal subendothelial matrix, hastening its replacement by collagens [33]. The result of this investigation may help to elucidate the mechanism by which oxidative stress activates MMP-2 in the development of liver fibrosis.
Fig. 10. SEAP expression of LI90 cells transfected with pNFjBSEAP. LI90 cells were transfected with pNFjB-SEAP and a control plasmid, pTAL-SEAP, and stimulated with SIN-1 (A) or SNAP (B) for indicated time. SIN-1 treatment significantly increased SEAP expression in pNFjB-SEAP-transfected LI90 cells. Columns represent means ± S.D. of data obtained from three independent and duplicate experiments. \P < 0.01 versus untreated LI90 cells.
In conclusion, our study shows that peroxynitrite promotes the activation of MMP-2 secreted by human hepatic stellate cell (HSC) line and that this activation is at least in part a result of MT1-MMP induction. These findings suggest that oxidative stress represents pro-fibrogenic stimuli for HSCs leading to MT1-MMP expression, thereby stimulating MMP-2 activation and ECM remodeling in hepatic fibrosis development. References [1] Arthur, M.J. (1995) Collagenases and liver fibrosis. J. Hepatol. 22 (Suppl. 2), 43–48. [2] Friedman, S.L. (2000) Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem. 275, 2247–2250. [3] Brenner, D.A., Waterboer, T., Choi, S.K., Lindquist, J.N., Stefanovic, B., Burchardt, E., Yamauchi, M., Gillan, A. and Rippe, R.A. (2000) New aspects of hepatic fibrosis. J. Hepatol. 32 (Suppl. 1), 32–38. [4] Bataller, R. and Brenner, D.A. (2001) Hepatic stellate cells as a target for the treatment of liver fibrosis. Semin Liver Dis. 21, 437– 451. [5] Arthur, M.J., Mann, D.A. and Iredale, J.P. (1998) Tissue inhibitors of metalloproteinases, hepatic stellate cells and liver fibrosis. J. Gastroenterol. Hepatol. 13 (Suppl.), S33–S38. [6] Benyon, R.C. and Arthur, M.J. (2001) Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis. 21, 373–384.
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125 [7] Wu, J. and Zern, M.A. (2000) Hepatic stellate cells: a target for the treatment of liver fibrosis. J. Gastroenterol. 35, 665– 672. [8] Pietrangelo, A. (1998) Iron, oxidative stress and liver fibrogenesis. J. Hepatol. 28 (Suppl. 1), 8–13. [9] Parola, M. and Robino, G. (2001) Oxidative stress-related molecules and liver fibrosis. J. Hepatol. 35, 297–306. [10] Svegliati-Baroni, G., Saccomanno, S., van Goor, H., Jansen, P., Benedetti, A. and Moshage, H. (2001) Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells. Liver 21, 1–12. [11] Svegliati Baroni, G., DÕAmbrosio, L., Ferretti, G., Casini, A., Di Sario, A., Salzano, R., Ridolfi, F., Saccomanno, S., Jezequel, A.M. and Benedetti, A. (1998) Fibrogenic effect of oxidative stress on rat hepatic stellate cells. Hepatology 27, 720–726. [12] Bataller, R., Schwabe, R.F., Choi, Y.H., Yang, L., Paik, Y.H., Lindquist, J., Qian, T., Schoonhoven, R., Hagedorn, C.H., Lemasters, J.J. and Brenner, D.A. (2003) NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J. Clin. Invest. 112, 1383–1394. [13] Akaike, T., Suga, M. and Maeda, H. (1998) Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO. Proc. Soc. Exp. Biol. Med. 217, 64–73. [14] Okada, Y., Morodomi, T., Enghild, J.J., Suzuki, K., Yasui, A., Nakanishi, I., Salvesen, G. and Nagase, H. (1990) Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. Eur. J. Biochem. 194, 721–730. [15] Frears, E.R., Zhang, Z., Blake, D.R., OÕConnell, J.P. and Winyard, P.G. (1996) Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite. FEBS Lett. 381, 21–24. [16] Beckman, J.S. and Koppenol, W.H. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271 (5 Pt 1), C1424–C1437. [17] Rockey, D.C., Boyles, J.K., Gabbiani, G. and Friedman, S.L. (1992) Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J. Submicrosc. Cytol. Pathol. 24, 193–203. [18] Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E. and Seiki, M. (1994) A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370, 61– 65. [19] Maher, J.J. (2001) Interactions between hepatic stellate cells and the immune system. Semin. Liver Dis. 21, 417–426. [20] Mayoral, P., Criado, M., Hidalgo, F., Flores, O., Arevalo, M.A., Eleno, N., Sanchez-Rodriguez, A., Lopez-Novoa, J.M. and Esteller, A. (1999) Effects of chronic nitric oxide activation or inhibition on early hepatic fibrosis in rats with bile duct ligation. Clin. Sci. 96, 297–305.
3125 [21] Takahara, T., Furui, K., Funaki, J., Nakayama, Y., Itoh, H., Miyabayashi, C., Sato, H., Seiki, M., Ooshima, A. and Watanabe, A. (1995) Increased expression of matrix metalloproteinase-II in experimental liver fibrosis in rats. Hepatology 21, 787–795. [22] Arthur, M.J. (2000) Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am. J. Physiol. Gastrointest. Liver Physiol. 279, G245–G249. [23] Corcoran, M.L., Hewitt, R.E., Kleiner, D.E. and Stetler-Stevenson, W.G. (1996) MMP-2: expression, activation and inhibition. Enzyme Protein 49, 7–19. [24] Murphy, G., Stanton, H., Cowell, S., Butler, G., Knauper, V., Atkinson, S. and Gavrilovic, J. (1990) Mechanisms for pro matrix metalloproteinase activation. APMIS 107, 38–44. [25] Preaux, A.M., Mallat, A., Nhieu, J.T., DÕOrtho, M.P., Hembry, R.M. and Mavier, P. (1999) Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions. Hepatology 30, 944–950. [26] Takahara, T., Furui, K., Yata, Y., Jin, B., Zhang, L.P., Nambu, S., Sato, H., Seiki, M. and Watanabe, A. (1997) Dual expression of matrix metalloproteinase-2 and membrane-type 1-matrix metalloproteinase in fibrotic human livers. Hepatology 26, 1521–1529. [27] Watanabe, T., Niioka, M., Ishikawa, A., Hozawa, S., Arai, M., Maruyama, K., Okada, A. and Okazaki, I. (2001) Dynamic change of cells expressing MMP-2 mRNA and MT1-MMP mRNA in the recovery from liver fibrosis in the rat. J. Hepatol. 35, 465–473. [28] Kim, H. and Koh, G. (2000) Lipopolysaccharide activates matrix metalloproteinase-2 in endothelial cells through an NF-jBdependent pathway. Biochem. Biophys. Res. Commun. 269, 401–405. [29] Yoon, S.O., Park, S.J., Yoon, S.Y., Yun, C.H. and Chung, A.S. (2002) Sustained production of H(2)O(2) activates pro-matrix metalloproteinase-2 through receptor tyrosine kinases/phosphatidylinositol 3-kinase/NF-kappa B pathway. J. Biol. Chem. 277, 30271–30282. [30] Poli, G. (2000) Pathogenesis of liver fibrosis: role of oxidative stress. Mol. Aspects Med. 21, 49–98. [31] Mihm, S., Fayyazi, A. and Ramadori, G. (1997) Hepatic expression of inducible nitric oxide synthase transcripts in chronic hepatitis C virus infection: relation to hepatic viral load and liver injury. Hepatology 26, 451–458. [32] Virag, L., Szabo, E., Gergely, P. and Szabo, C. (2003) Peroxynitrite-induced cytotoxicity: mechanism and opportunities for intervention. Toxicol Lett. 140–141, 113–124. [33] Theret, N., Lehti, K., Musso, O. and Clement, B. (1999) MMP2 activation by collagen I and concanavalin A in cultured human hepatic stellate cells. Hepatology 30, 462–468.
FEBS Letters 579 (2005) 3119–3125
Peroxynitrite-mediated matrix metalloproteinase-2 activation in human hepatic stellate cells Kiyoshi Migitaa,*, Yumi Maedaa, Seigo Abirua, Atsumasa Komoria, Terufumi Yokoyamaa, Yasushi Takiia, Minoru Nakamuraa, Hiroshi Yatsuhashia, Katsumi Eguchib, Hiromi Ishibashia a
Clinical Research Center, NHO Nagasaki Medical Center, Kubara 2-1001-1, Omura 856-8562, Japan b First Department of Internal Medicine, Nagasaki University School of Medicine, Japan Received 24 December 2004; revised 29 March 2005; accepted 1 April 2005 Available online 12 May 2005 Edited by Veli-Pekka Lehto
Abstract To investigate whether hepatic stellate cells (HSCs) alter their expression of MMPs after exposure to nitrogen oxide intermediate (NOI), a human hepatic stellate cell line, LI90 cells, was stimulated with an NO donor, SNAP, or a peroxynitrite donor, SIN-1, and the culture supernatants were analyzed by gelatin zymography or anti-MMPs immunoblot. Although SIN-1 did not enhance the secretions of MMP-1 and 13, SIN1 induced the NF-jB activation, MT1-MMP expression and the secretion of activated MMP-2 in LI90 cells. These results suggest that peroxynitrite may contribute to the remodeling of the extracellular matrix in liver by activating pro-MMP-2. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Hepatic stellate cell; Peroxynitrite; Matrix metalloproteinase; Nitric oxide
species (ROS) occurs in the process of liver fibrosis [9]. ROS may represent direct or indirect pro-fibrogenic stimuli for HSCs [10]. Moreover, signs of oxidative stress and lipid peroxidation are concomitant or precede HSCs activation and collagen deposition [11]. It was demonstrated that HSCs express non-phagocytic NADPH oxidase and inducible NO synthase (iNOS) [12], which generate superoxide and NO, once exposed to effective stimuli. Furthermore, the simultaneous production of NO and superoxide by HSCs or Kupffer cells in liver may lead to the generation of peroxynitrite (ONOO ) [13]. The present study was designed to more clearly elucidate the biological effects elicited by oxidative stress on HSCs. In particular, we investigated whether nitrogen oxide intermediate (NOI) may affect the synthesis of MMPs and their related inhibitors (TIMP-1 and TIMP2) in HSCs.
2. Materials and methods 1. Introduction Liver fibrosis occurs following most chronic liver injuries and results from changes in the balance between the synthesis and degradation of extracellular matrix (ECM) components [1,2]. Hepatic stellate cells (HSCs) play a pivotal role in the cellular and molecular events that lead to fibrosis [3]. Following liver injury, these cells undergo changes toward a myofibroblastic-like phenotype and secrete extracellular matrix components [4]. In addition, activated HSCs are involved in ECM degradation by providing MMPs [5]. During liver fibrogenesis, a number of MMPs, such as MMP-1, MMP-2, and MMP-13, are expressed in the liver by hepatic stellate cells [6]. Therefore, it has been suggested that the activation mechanism of HSCs and the secretion of MMPs may contribute to the process of liver fibrosis [7]. Evidence of oxidative stress has also been detected in clinical condition of liver fibrosis with different etiology [8]. It has become obvious that an increase in the levels of reactive oxygen * Corresponding author. Fax: +81 957 54 0292. E-mail address: [email protected] (K. Migita).
Abbreviations: ECM, extracellular matrix; HSCs, hepatic stellate cells; IjB-a, IkappaB-a; MAPK, mitogen activated protein kinase; MMP, matrix metalloproteinases; SEAP, secretory alkaline phosphatase; SNAP, S-nitroso-N-acetyl-DL -penicillamine; SIN-1, N-morpholino sydnonimine hydrochloride
2.1. Reagents An NO donor, SNAP (S-nitroso-N-acetyl-DL -penicillamine), and a peroxynitrite donor, SIN-1 (N-morpholino sydnonimine hydrochloride), were purchased from Dojindo Laboratories (Kumamoto, Japan). Mouse anti-human membrane-type 1 matrix metalloproteinase (MT1-MMP) monoclonal antibody and mouse anti-human tissue inhibitor metalloproteinase–1 (TIMP-1) and TIMP-2 antibodies were obtained from Fuji Chemical Co. (Takaoka, Japan). Mouse monoclonal anti-a-smooth muscle actin antibody was purchased from Novacastra Laboratories (Newcastle, UK). Mouse monoclonal anti-nitrotyrosine antibody (1A6) was purchased from UBI (Lake Placid, USA). Purified pro-MMP-2 was purchased from Calbiochem (San Diego, USA). Activation with p-aminophenylmercuric acetate (APMA, Sigma Chemical) was done by incubation of purified pro-MMP-2 with 1 mM APMA at 37 C for 15 min [14]. All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). 2.2. Cells LI90 cells, human HSCs established by Dr. Murakami, were purchased by JCRB (Japanese Collection of Research Bioresources, Osaka, Japan). Cells were propagated in DulbeccoÕs minimal essential medium (DMEM) supplemented with 10% fetal calf serum (Gibco BRL, Gaithersburg, MD). LI90 cells were stimulated with SIN-1 at different concentrations (0–500 lM) for 24 h in serum-free DMEM. Culture supernatants were subjected to the analysis for gelatin zymography or immunoblot. 2.3. Gelatin zymography LI90 cells-conditioned culture media (serum-free) were incubated at 37 C for 20 min in sodium dodecyl sulfate (SDS) sample buffer free of reducing agents and then electrophoresed on 8% polyacrylamide gels containing 0.5 mg/ml gelatin at 4 C. After electrophoresis, gels were
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.04.071
3120 washed in 2.5% Triton-X 100 to remove SDS, and incubated with 50 mM Tris–HCl buffer (pH 7.5) containing 0.15 M NaCl, 10 mM CaCl2, and 0.02% NaN3 for 16 h at 37 C, and stained with 0.1% Coomassie Blue R250.
2.4. Immunoblot analysis The expression of MT1-MMP on LI90 cells and the secretions of MMP-1, MMP-13 and TIMPs from synovial cells were analyzed by immunoblot. For this purpose, cells were washed with cold PBS and lysed by the addition of a lysis buffer containing 1% Nonidet P-40, 50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM EDTA, 10 lg/ml aprotinin, and 10 lg/ml leupeptin for 20 min at 4 C. Insoluble materials were removed by centrifugation at 1500 · g for 15 min at 4 C. The supernatant was saved and the protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). An identical amount of protein (30 lg) from each lysate or the culture supernatant was subjected to 8% SDS–polyacrylamide gel electrophoresis (PAGE). The fractionated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL), and the filters were blocked for 1.5 h using non-fat dried milk in Trisbuffered saline (TBS: 50 mM Tris, 0.15 M NaCl, pH 7.5) containing 0.1% Tween 20, washed with TBS, and incubated at room temperature for 2 h at a 1:150 dilution of mouse anti-MT1-MMP or MMPs monoclonal antibodies (mAb). The membranes were further incubated with a 1:2000 dilution of horseradish peroxidaseconjugated donkey anti-mouse immunoglobulin G (IgG) antibody (Promega, Madison, WI) for 20 min. An enhanced chemiluminescence (ECL) system (Amersham) was used for detection. The blots were analyzed by densitometer (Fuji Film, Tokyo, Japan). Images from densitometer were transferred to a personal computer and analyzed using Science Lab software (Fuji Film). The density of the target protein band of untreated cells was assigned the value of 1.0 and each result was calculated as relative units.
2.5. RNA preparation and quantitative PCR assay Total cellular RNA was extracted from LI90 cells using guanidium thiocyanate and phenol (RNAzol B, Cinna/Biotek Labs Int. Inc., Friendswood, TX). First-strand cDNA was synthesized by reverse transcription at 45 C for 45 min in a 50 ll reaction mixture containing 1 lg of total RNA and MuLV reverse transcriptase (Invitrogen). After denaturing at 99 C for 5 min followed by cooling at 5 C, the cDNA was amplified using PCR. The quantification of MT1-MMP transcripts in LI90 cells was carried out with the LightCycler PCR system (Roche Diagnostics, Meylan, France) using the DNA binding SYBR Green I dye for the detection of PCR products. A serial dilution was used to generate each standard curve. For real time quantitative PCR, each reaction contained: 1· of LightCycler-DNA master SYBR Green I, specific primer (Roche), and 2 ll of cDNA matrix, in a final volume of 20 ll. After 2 min of denaturation at 95 C, the reactions were cycled 40 times for 5 s at 95 C, 10 s at the annealing temperature, and 15 s or 7 s at 72 C for MT1-MMP and GAPDH, respectively. Product specificity was determined by melting curve analysis as described in the LightCycler handbook. The amount of transcripts in each sample is given as copy number. The results are expressed as ratios of MT1-MMP transcripts to GAPDH transcripts.
2.6. Plasmids and transfection pNFjB-SEAP (CLONTECH Laboratories, Inc., Palo Alto, CA) is designed to monitor the activation of NF-jB and NF-jB-mediated signal transduction pathways. pNFjB-SEAP contains four tandem copies of the jB4 enhancer fused to the HSV-TK promoter. pTAL-SEAP (CLONTECH Laboratories) was used as a negative control to determine the background signals associated with the culture medium. Cells were grown to approximately 50% confluence on 30-mm plates. Transfections were done using the calcium phosphate reagents and following the manufacturerÕs instructions (CLONTECH Laboratories), and cells were treated as described above. Chemiluminescence detection of SEAP activity was performed according to the manufacturerÕs instructions (CLONTECH Laboratories) using a plate fluorometer (Berthold, Bad Wildbach, Germany).
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125 2.7. Statistical analysis Differences in the data were analyzed by 1-way ANOVA combined with the BonferroniÕs test, and all values were expressed as means ± S.D. The differences between groups were considered to be significant at P < 0.05.
3. Results The effects of NO to human hepatic stellate cells were investigated by incubating LI90 cells with an NO donor, SNAP, which generates NO in vitro. As shown in Fig. 1, SNAP treatment did not modulate the gelatinase secretions from LI90 cells. Next, we incubated the cells with SIN-1, which generates ONOO by releasing NO and O2 simultaneously for 24 h and the culture supernatants were subjected to gelatin zymography. SIN-1 was not toxic to LI90 cells at the concentrations (500 lM) used in this experiment (data not shown). Gelatin zymography demonstrated that in addition to the secretion of a late form of MMP-2 (Mr = 72 kd), the activated form of MMP-2 (Mr = 66 kd) was detected when LI90 cells were treated with SIN-1 (Fig. 2A and B). This gelatinolytic band with lower molecular weight (Mr = 66 kd) was similar to APMAactivated MMP-2 in that it migrated at 66 kd (Fig. 2C). Also, these gelatinolytic activities were completely inhibited by EDTA demonstrating that these are metalloproteinases (Fig. 2D). In contrast, the SIN-1 treatment did not significantly affect the secretion of MMP-1 (Fig. 3A and B) and MMP-13 (Fig. 3C and D). It was demonstrated that peroxynitrite is capable of degrading TIMP-1, a process which could potentiate MMP-2 activation [15]. Therefore, we examined the effects of SIN-1 on TIMP-1 secretion from LI90 cells. The molecular forms of TIMP-1 and its secretion levels were not affected by SIN-1 treatment (Fig. 3E and F). TIMP-2 was not detected in LI90-conditioned media with or without SIN-1 treatment by anti-TIMP-2 immunoblot analysis (data not shown). Because peroxynitrite has the functional property of inducing nitration, we examined whether SIN-1-treated cells contain proteins with nitrated tyrosine residues [16]. The level of tyrosine nitration was clearly increased after 4 h of treatment with SIN-1. This effect was smaller in SNAP-treated LI90 cells (Fig. 4A). Expression of a-smooth muscle actin (a-SMA) is considered as an indicator of HSCs activation [17]. The expression of a-SMA was also examined using LI90 cells treated with SIN-1
Fig. 1. Effects of SNAP on gelatinase secretions from LI90 cells. LI90 cells were cultured with SANP in serum-free culture media for 24 h. The conditioned media were analyzed by gelatin zymography. The data shown are representatives of at least three independent experiments.
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125
Fig. 2. Effects of SIN-1 on gelatinase secretion from LI90 cells. (A) LI90 cells were cultured with various concentrations of SIN-1 in serum-free culture media for 24 h. The conditioned media were analyzed by gelatin zymography. Note the activated MMP-2 with lower molecular weights in addition to 72 Kda MMP-2 in the SIN-1treated LI90 cells-conditioned media. (B) Densitometric analysis of A is presented as the relative ratio of active form/pro-form of MMP-2. These data represent means ± S.D. of three independent experiments. \ P < 0.01 versus untreated LI90 cells. (C) Gelatinolytic activity of LI90 cells-conditioned media detected by zymography. Lane 1: untreated LI90 cells-conditioned media, Lane 2: SIN-1 (500 lM)-treated LI90 cells-conditioned media, Lane 3: purified pro-MMP-2 standards (20 ng), Lane 4 pro-MMP-2 standards (20 ng) were activated with 1 mM APMA at 37 C for 15 min. The data shown are representatives of at least three independent experiments. (D) LI90 cells were cultured with SIN-1 in serum-free culture media for 24 h. Zymogram of LI90 cells-conditioned media was incubated in the presence or absence of EDTA (10 mM) for 16 h at 37 C. Gelatinolytic activities were completely inhibited by EDTA.
for 24 h. SIN-1 treatment resulted in an increased a-SMA expression on LI90 cells (Fig. 4B and C). As shown in Fig. 5, the incubation of LI90 cells-conditioned media containing pro-MMP-2 with SIN-1 for 24 h did not result in the activation of pro-MMP-2. This result indicates that SIN-1-induced pro-MMP-2 activation requires the presence of pro-MMP-2 producing IL90 cells. It has been suggested that the conversion of pro-MMP-2 to active MMP-2 requires cell membrane-associated processing by membrane-type matrix
3121
Fig. 3. Effects of SIN-1 on MMPs or TIMP-1 secretions from LI90 cells. LI90 cells were cultured with various concentrations of SIN-1 for 24 h. The conditioned media were analyzed by anti-MMP-1 (A), MMP-13 (C), and TIMP-1 (E) immunoblot. MMP-1 (B), MMP-13 (D) and TIMP-1 (F) bands were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
metalloproteinase (MT-MMP) [18]. Therefore, we examined whether MT-MMP is involved in the SIN-1-induced proMMP-2 activation. LI90 cells were cultured with SIN-1 for
3122
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125
Fig. 6. MT1-MMP expression in SIN-1-treated LI90 cells. (A) LI90 cells were cultured with various concentrations of SIN-1 for 24 h. The cells were lysed and equal amounts of cellular lysates were analyzed by immunoblot using antibodies to human MT1-MMP. The band of MT1-MMP of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. (B) MT1-MMP bands were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
Fig. 4. (A) Tyrosine nitration in LI90 cells. Immunoblot analysis of LI90 cells extracts with anti-nitrotyrosine antibody showed SIN-1 induced tyrosine nitration on several proteins. Immunoblot analysis of a-smooth muscle actin expression in LI90 cells. (B) LI90 cells were treated with SIN-1 or SNAP with the indicated concentrations for 24 h. Cellular lysates were analyzed by immunoblot using anti-asmooth muscle actin antibodies. (C) Bands of a-smooth muscle actin were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
activated form of MMP-2 in SIN-1-treated LI90 cells-conditioned media (Fig. 2A and B). We further investigated the peroxynitrite-mediated signaling pathway in LI90 cells. We examined the effects of SIN-1 on the state of ERK1/2, p38 and JNK1/2. The state of phosphorylation of these MAPKs was examined by immunoblot using anti-phospho-specific MAPKs antibodies. The phosphorylation of ERK1/2, p38 and JNK1/2 was not increased but rather decreased in SIN-1-treated LI90 cells (Fig. 8A–F). Immunoblot of b-actin is shown as a control for equal loading of samples (Fig. 8G). We also evaluated IjB-a proteolysis. The protein levels of IjB-a in SIN-1-treated LI90 were measured by immunoblot analysis (Fig. 9A and B). The SIN-1
24 h. The cells were lysed and the lysates were subjected to immunoblot using anti-MT1-MMP antibodies. As shown in Fig. 6, MT1-MMP was barely detected in unstimulated LI90 cells and the intensity of MT1-MMP band was increased when the LI90 cells were treated with SIN-1. We also analyzed MT1MMP mRNA expression by real-time quantitative RT-PCR analysis. As shown in Fig. 7, MT1-MMP mRNA expression in LI90 cells was increased by SIN-1 treatment. This SIN-1-induced MT1-MMP expression was parallel to the induction of
Fig. 5. In vitro effects of SIN-1 on gelatinase. LI90 cells-conditioned media were incubated with SIN-1 for 24 h at 37 C. Incubated media were analyzed by gelatin zymography. The data shown are representatives of at least three independent experiments.
Fig. 7. MT1-MMP mRNA expression in SIN-1-treated LI90 cells. LI90 cells were stimulated by SIN-1 for 6 h. Total cellular RNA was analyzed by real-time RT-PCR using specific primers for MT1-MMP. Results are means of three separate experiments. \P < 0.01 versus untreated LI90 cells.
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125
3123
Fig. 8. Effects of SIN-1 on the MAP kinase phosphorylation of LI90 cells. LI90 cells were stimulated by SIN-1 (500 lM) for indicated times. Equal amounts of cellular lysates were analyzed by immunoblot using anti-phospho-specific ERK1/2 (A), p38 (C), JNK1/2 (E) and b-actin (G) antibodies. Bands of ERK1/2 (B), p38 (D), and JNK1/2 (F) were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
stimulation induced substantial IjB-a degradation in a timedependent manner, suggesting the activation of NF-jB in LI90 cells. However, the SNAP treatment did not affect the protein levels of IjB-a. To determine whether SIN-1 treatment modulates the transcriptional activity of NF-jB, LI90 cells were transiently transfected with a reporter gene construct, pNFjB-SEAP. Cells were subsequently treated with SIN-1, and SEAP in the culture supernatants was assayed. As shown in Fig. 10A, SIN-1 treatment increased the SEAP levels compared to those of control cells. SNAP treatment did not activate the NF-jB transcriptional activity (Fig. 10B).
4. Discussion Hepatic stellate cells (HSCs) play a central role in the development of liver fibrosis [3]. After chronic liver injury, HSCs proliferate and transform into myofibroblastic cells (activated HSCs) that are the major source of collagens that accumulate
in the fibrotic liver [4]. This process is promoted by a variety of cytokines and soluble factors released by Kupffer cells and inflammatory cells as well as HSCs [19]. Mayoral et al. [20] demonstrated an increased hepatic expression of iNOS in bile duct ligated rats, model animals for hepatic fibrosis. Furthermore, stimulation of NO synthesis with L -arginine treatment augmented hepatic fibrosis. These results suggest that NO may be involved in the development of fibrosis. We found that peroxynitrite induces the secretion of activated MMP-2 in cultured human HSCs. A number of studies have emphasized the profibrogenic functions of MMP-2 [21,22]. However, the exact mechanisms leading to MMP-2 activation have not been clearly delineated. The MMPs activity is closely regulated at 3 levels, namely gene transcription, proteolytic activation, and inhibition by endogenous inhibitors [23]. As well, proteolytic activation of MMP-2 is induced by membrane-type MMPs (MT-MMP) [24]. Previous reports suggest that MT1-MMP-activated MMP-2 exerted a profibrogenic role during hepatic fibrosis [25]. It is possible
3124
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125
Fig. 9. SIN-1 stimulated IjB-a degradation in LI90 cells. (A) LI90 cells were stimulated with SIN-1 (500 lM) and SNAP (500 lM) for the indicated times. Cells were lysed and the cellular lysates were assessed by anti-IjB-a immunoblot analysis. (B) Bands of IjB-a were calculated as a relative unit by densitometer. The band of untreated cells was assigned the value of 1.0 and data were expressed as the relative units. These data represent means ± S.D. of three independent experiments. \P < 0.01 versus untreated LI90 cells.
that peroxynitrite generated by SIN-1 directly nitrates MMP-2 and induces its enzymatic activation. However, SIN-1 did not activate pro-MMP-2 in the absence of HSCs in vitro, suggesting that the interaction between peroxynitrite and HSCs is required for this MMP-2 activation. The hepatic expression of MMP-2 and MT1-MMP is increased in human fibrotic livers, and activated HSCs have been identified as a source of both proteins [26,27]. Therefore, we investigated whether the SIN-1 treatment increases MT1MMP expression in HSCs. Our data showed that MMP-2 activation in SIN-1-treated HSCs is associated with the increased expression of MT1-MMP by these cells. In response to peroxynitrite, HSCs may express MT1-MMP on their surface thereby stimulating pro-MMP-2 activation. The mechanism leading to MT1-MMP expression in SIN-1-treated HSCs is not known. The finding that SIN-1 leads to the rapid degradation of IjB-a suggests that the peroxynitrite may activate NF-jB in HSCs. It can be speculated that the peroxynitrite-mediated NF-jB dependent pathway might be involved in the MT1MMP induction as reported previously [28,29]. Oxidative stress has been detected in fibrotic liver and acts as a mediator of the ECM remodeling responsible for liver fibrosis [30]. In liver injury, iNOS is upregulated in liver cell populations including HSCs, and the NO generation is increased [31]. The pathogenic action of NO seems to occur as the interaction of NO with O2 to generate peroxynitrite. The effects of peroxynitrite can be deleterious, since it induces the oxidation and nitration of biological macromolecules [32]. Our data suggest that peroxynitrite may activate MMP-2 secreted from HSCs by inducing MT1-MMP. ECM remodeling in fibrotic liver could be accelerated by MMP-2 activation, because this enzyme degrades the normal subendothelial matrix, hastening its replacement by collagens [33]. The result of this investigation may help to elucidate the mechanism by which oxidative stress activates MMP-2 in the development of liver fibrosis.
Fig. 10. SEAP expression of LI90 cells transfected with pNFjBSEAP. LI90 cells were transfected with pNFjB-SEAP and a control plasmid, pTAL-SEAP, and stimulated with SIN-1 (A) or SNAP (B) for indicated time. SIN-1 treatment significantly increased SEAP expression in pNFjB-SEAP-transfected LI90 cells. Columns represent means ± S.D. of data obtained from three independent and duplicate experiments. \P < 0.01 versus untreated LI90 cells.
In conclusion, our study shows that peroxynitrite promotes the activation of MMP-2 secreted by human hepatic stellate cell (HSC) line and that this activation is at least in part a result of MT1-MMP induction. These findings suggest that oxidative stress represents pro-fibrogenic stimuli for HSCs leading to MT1-MMP expression, thereby stimulating MMP-2 activation and ECM remodeling in hepatic fibrosis development. References [1] Arthur, M.J. (1995) Collagenases and liver fibrosis. J. Hepatol. 22 (Suppl. 2), 43–48. [2] Friedman, S.L. (2000) Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem. 275, 2247–2250. [3] Brenner, D.A., Waterboer, T., Choi, S.K., Lindquist, J.N., Stefanovic, B., Burchardt, E., Yamauchi, M., Gillan, A. and Rippe, R.A. (2000) New aspects of hepatic fibrosis. J. Hepatol. 32 (Suppl. 1), 32–38. [4] Bataller, R. and Brenner, D.A. (2001) Hepatic stellate cells as a target for the treatment of liver fibrosis. Semin Liver Dis. 21, 437– 451. [5] Arthur, M.J., Mann, D.A. and Iredale, J.P. (1998) Tissue inhibitors of metalloproteinases, hepatic stellate cells and liver fibrosis. J. Gastroenterol. Hepatol. 13 (Suppl.), S33–S38. [6] Benyon, R.C. and Arthur, M.J. (2001) Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis. 21, 373–384.
K. Migita et al. / FEBS Letters 579 (2005) 3119–3125 [7] Wu, J. and Zern, M.A. (2000) Hepatic stellate cells: a target for the treatment of liver fibrosis. J. Gastroenterol. 35, 665– 672. [8] Pietrangelo, A. (1998) Iron, oxidative stress and liver fibrogenesis. J. Hepatol. 28 (Suppl. 1), 8–13. [9] Parola, M. and Robino, G. (2001) Oxidative stress-related molecules and liver fibrosis. J. Hepatol. 35, 297–306. [10] Svegliati-Baroni, G., Saccomanno, S., van Goor, H., Jansen, P., Benedetti, A. and Moshage, H. (2001) Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells. Liver 21, 1–12. [11] Svegliati Baroni, G., DÕAmbrosio, L., Ferretti, G., Casini, A., Di Sario, A., Salzano, R., Ridolfi, F., Saccomanno, S., Jezequel, A.M. and Benedetti, A. (1998) Fibrogenic effect of oxidative stress on rat hepatic stellate cells. Hepatology 27, 720–726. [12] Bataller, R., Schwabe, R.F., Choi, Y.H., Yang, L., Paik, Y.H., Lindquist, J., Qian, T., Schoonhoven, R., Hagedorn, C.H., Lemasters, J.J. and Brenner, D.A. (2003) NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J. Clin. Invest. 112, 1383–1394. [13] Akaike, T., Suga, M. and Maeda, H. (1998) Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO. Proc. Soc. Exp. Biol. Med. 217, 64–73. [14] Okada, Y., Morodomi, T., Enghild, J.J., Suzuki, K., Yasui, A., Nakanishi, I., Salvesen, G. and Nagase, H. (1990) Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. Eur. J. Biochem. 194, 721–730. [15] Frears, E.R., Zhang, Z., Blake, D.R., OÕConnell, J.P. and Winyard, P.G. (1996) Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite. FEBS Lett. 381, 21–24. [16] Beckman, J.S. and Koppenol, W.H. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271 (5 Pt 1), C1424–C1437. [17] Rockey, D.C., Boyles, J.K., Gabbiani, G. and Friedman, S.L. (1992) Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J. Submicrosc. Cytol. Pathol. 24, 193–203. [18] Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E. and Seiki, M. (1994) A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370, 61– 65. [19] Maher, J.J. (2001) Interactions between hepatic stellate cells and the immune system. Semin. Liver Dis. 21, 417–426. [20] Mayoral, P., Criado, M., Hidalgo, F., Flores, O., Arevalo, M.A., Eleno, N., Sanchez-Rodriguez, A., Lopez-Novoa, J.M. and Esteller, A. (1999) Effects of chronic nitric oxide activation or inhibition on early hepatic fibrosis in rats with bile duct ligation. Clin. Sci. 96, 297–305.
3125 [21] Takahara, T., Furui, K., Funaki, J., Nakayama, Y., Itoh, H., Miyabayashi, C., Sato, H., Seiki, M., Ooshima, A. and Watanabe, A. (1995) Increased expression of matrix metalloproteinase-II in experimental liver fibrosis in rats. Hepatology 21, 787–795. [22] Arthur, M.J. (2000) Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am. J. Physiol. Gastrointest. Liver Physiol. 279, G245–G249. [23] Corcoran, M.L., Hewitt, R.E., Kleiner, D.E. and Stetler-Stevenson, W.G. (1996) MMP-2: expression, activation and inhibition. Enzyme Protein 49, 7–19. [24] Murphy, G., Stanton, H., Cowell, S., Butler, G., Knauper, V., Atkinson, S. and Gavrilovic, J. (1990) Mechanisms for pro matrix metalloproteinase activation. APMIS 107, 38–44. [25] Preaux, A.M., Mallat, A., Nhieu, J.T., DÕOrtho, M.P., Hembry, R.M. and Mavier, P. (1999) Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions. Hepatology 30, 944–950. [26] Takahara, T., Furui, K., Yata, Y., Jin, B., Zhang, L.P., Nambu, S., Sato, H., Seiki, M. and Watanabe, A. (1997) Dual expression of matrix metalloproteinase-2 and membrane-type 1-matrix metalloproteinase in fibrotic human livers. Hepatology 26, 1521–1529. [27] Watanabe, T., Niioka, M., Ishikawa, A., Hozawa, S., Arai, M., Maruyama, K., Okada, A. and Okazaki, I. (2001) Dynamic change of cells expressing MMP-2 mRNA and MT1-MMP mRNA in the recovery from liver fibrosis in the rat. J. Hepatol. 35, 465–473. [28] Kim, H. and Koh, G. (2000) Lipopolysaccharide activates matrix metalloproteinase-2 in endothelial cells through an NF-jBdependent pathway. Biochem. Biophys. Res. Commun. 269, 401–405. [29] Yoon, S.O., Park, S.J., Yoon, S.Y., Yun, C.H. and Chung, A.S. (2002) Sustained production of H(2)O(2) activates pro-matrix metalloproteinase-2 through receptor tyrosine kinases/phosphatidylinositol 3-kinase/NF-kappa B pathway. J. Biol. Chem. 277, 30271–30282. [30] Poli, G. (2000) Pathogenesis of liver fibrosis: role of oxidative stress. Mol. Aspects Med. 21, 49–98. [31] Mihm, S., Fayyazi, A. and Ramadori, G. (1997) Hepatic expression of inducible nitric oxide synthase transcripts in chronic hepatitis C virus infection: relation to hepatic viral load and liver injury. Hepatology 26, 451–458. [32] Virag, L., Szabo, E., Gergely, P. and Szabo, C. (2003) Peroxynitrite-induced cytotoxicity: mechanism and opportunities for intervention. Toxicol Lett. 140–141, 113–124. [33] Theret, N., Lehti, K., Musso, O. and Clement, B. (1999) MMP2 activation by collagen I and concanavalin A in cultured human hepatic stellate cells. Hepatology 30, 462–468.