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–Mediated Plasminogen Activator Inhibitor-1 Expression via Inhibition of Smad Pathway in Human Mesangial Cells
Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan.
Correspondence to Dr. Masakazu Haneda, Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, 520-2192, Japan. Phone: 81-77-548-2222; Fax: 81-77-543-3858;
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Abstract |
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(TGF-), one of the key cytokines in the progression of renal disease, in human mesangial cells. Ac-SDKP inhibited TGF-1–induced plasminogen activator inhibitor-1 (PAI-1) and alpha2 (I) collagen mRNA. Ac-SDKP suppressed not only TGF-1–induced Smad2 phosphorylation at Ser-465/467 in a dose-dependent manner, but also the nuclear accumulation of receptor-regulated Smads (R-Smad), Smad2 and Smad3. As expected, Ac-SDKP inhibited TGF-–responsive Smad-dependent luciferase reporters, 3TP-luc and 4xSBE-luc. Immunofluorescence analysis revealed that the inhibitory Smad, Smad7, was exported to the cytoplasm from the nucleus by the treatment with Ac-SDKP. These findings provide novel evidence that Ac-SDKP inhibits TGF- signal transduction through the suppression of R-Smad activation via nuclear export of Smad7, highlighting an alternative mechanism involved in the reno-protective efficacy of ACE-I. E-mail: haneda@belle.shiga-med.ac.jp |
Introduction |
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N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of the proliferation of hematopoietic stem cells, which prevents the cells from entering the S phase of the cell cycle and thus maintains the cells in the G0/G1 phase (12). Ac-SDKP is normally present in human plasma and is exclusively hydrolyzed by ACE. The plasma levels of Ac-SDKP were shown to be increased by fivefold after the administration of ACE-I in humans (13). Ac-SDKP was shown to suppress the proliferation of renal fibroblasts (14) and to inhibit DNA synthesis as well as collagen deposition in cardiac fibroblasts (15). In a hypertensive rat model, long-term administration of Ac-SDKP can ameliorate renal fibrosis and ventricular hypertrophy (16,17 ). These observations suggest that the reno-protective effect of ACE-I could be partly mediated by the accumulation of Ac-SDKP in the plasma. However, the underlying mechanism by which Ac-SDKP mediates anti-fibrotic effects remains unclear.
Transforming growth factor- (TGF-) is a cytokine that regulates development, cell proliferation, and matrix protein synthesis (18,19 ). During the glomerular scarring process, TGF- plays a major role in matrix protein accumulation and collagen deposition (20). One of the main target cells for TGF- in the kidney is glomerular mesangial cells (20,21 ). TGF- stimulates the expression of extracellular matrix (ECM) proteins such as collagens, laminin, and fibronectin, while it suppresses the expression of ECM-degrading proteases and increases the synthesis of ECM protease inhibitors, including plasminogen activator inhibitor-1 (PAI-1) (22). Therefore, downregulation of TGF- signaling provides a new therapeutic strategy for inhibiting the progressive renal disease. Indeed, the administration of anti–TGF- antibody was shown to ameliorate the diabetic glomerular lesions in db/db mice (23).
TGF- signaling from the cell membrane to the nucleus is mediated by the intracellular effector molecules, termed Smads (19,24,25 ). After TGF- binding to its receptors, the receptor-regulated Smads (R-Smads), Smad2 and Smad3, are phosphorylated by the TGF- type I receptor and associate with a common partner, Smad4. The heteromultimer translocates to the nucleus, resulting in the expression of TGF- target genes (26). The inhibitory Smad, Smad7, may participate in a negative feedback loop that controls TGF- responses by competitive interaction with the type I receptor (27).
We thus hypothesized that the anti-fibrotic effects of Ac-SDKP are mediated by the modulation of TGF- signaling. To test this hypothesis and to establish a mechanistic basis for the interaction between TGF- and Ac-SDKP, we examined the effect of Ac-SDKP on TGF-–mediated PAI-1 mRNA expression and TGF- signal transduction in human mesangial cells.
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Materials and Methods |
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1 from R&D Systems (Minneapolis, MN); goat polyclonal anti-Smad2/3IgG (N-19), goat polyclonal anti-Smad7IgG (N-19), anti-goat-IgG-horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-phospho-Smad2 IgG from Upstate Biotechnology Inc. (Lake Placid, NY), luciferase assay systems from Promega (Madison, WI), and anti-phospho p42/p44 ERK from New England Biolabs (Beverly, MA). Anti-Myc-monoclonal-antibody was bought from Clontech (Palo Alto, CA), anti-Nucleoporin p62-monoclonal-antibody from Transduction Laboratories (San Diego, CA), anti-GAPDH monoclonal antibody was purchased from Biogenesis Ltd. (Poole, England, UK), anti-mouse and anti-rabbit horseradish peroxidase–conjugated IgG from Amersham Pharmacia Biotech (Piscataway, NJ), and [
-32P] dCTP from New England Nuclear (Boston, MA).
DNA Constructs
The 3TP-luc reporter was kindly provided by Dr. J. Massague; pCMV-Myc-Smad7 was made by subcloning the PCR products into the vector pCMV-Myc (Clontech, Palo Alto, CA), which was confirmed by sequence analysis using dye terminator methods (ABI PRISM, Foster City, CA). pcDNA-Myc-Smad3 was kindly provided by Dr. Yan Chen (28). Dr. C.H. Heldin kindly donated 4xSBE-luc reporter plasmid. pAP-1-luc was bought from Stratagene (La Jolla, CA). Human PAI-1 cDNA was kindly provided by Dr. Y. Eguchi (29). Alpha2 (I) collagen (COL1A2) cDNA was obtained by subcloning the PCR products using primers specific for rat COL1A2 (5'-ATACGCGGACTCTGTTGCTG-3' and 5'-CTTGACCTGGGGTTCCATTC-3') into the vector pCR-TOPO (Invitrogen, Carlsbad, CA), which was also confirmed by sequence analysis.
Cell Culture
Primary culture human mesangial cell (Cryo NHMC) and its corresponding growth medium (CC-3146 MsGM) supplemented with 5% fetal calf serum (FCS) were purchased from Clonetics (San Diego, CA). Cells were cultured on 10-cm culture dish (Corning Incorporation, Corning, NY) at 37°C in a humidified 95% air/5% CO2 atmosphere and were used at passages 5 to 10 for the experiments. Cells were made quiescent by incubation in medium containing 0.2% bovine serum albumin (BSA) for 48 h before the experiments.
Protein Extraction and Western Blot Analyses
The quiescent cells were exposed to 2.5 ng/ml TGF-1 for the indicated intervals after several pretreatment intervals with or without Ac-SDKP. Cells were lysed with sodium dodecyl sulfate (SDS) sample buffer, sonicated for 7 s, and boiled at 100°C for 5 min. After centrifugation at 15,000 rpm for 10 min at 4°C, the supernatant was separated on 12% SDS-polyacrylamide gels, blotted onto nitrocellulose membranes (Immobilon, Bedford, MA) by semi-dry method. After blocking with TBS-T (Tris-buffered saline containing 0.1% Tween 20) containing 5% nonfat milk, the membranes were incubated with 1:400 diluted anti-phospho-Smad2 polyclonal antibody or 1:1000 diluted anti-phospho-ERK antibody at 4°C overnight. The membranes were washed three times and incubated with 1:2000 diluted horseradish peroxide (HRP) conjugated-secondary antibody (Amersham, Buckinghamshire, UK) at room temperature for 1 h. The immunoreactive bands were detected with an enhanced chemiluminescence (ECL) detection system (Perkin Elmer Life Science, Boston, MA). The level of each protein was quantified by scanning densitometry and corrected by reference to the value for Smad2 or ERK2, respectively.
RNA Isolation and Northern Blot Analyses
Human mesangial cells pretreated with or without Ac-SDKP for 2 h were stimulated with TGF-1 for the indicated intervals. Total RNA (12 µg) was isolated by guanidium and phenol extraction (TRIzol Reagent; Life Technologies BRL, Grand Island, NY), electrophoresed on 1% formaldehyde-agarose gels, and transferred onto a nylon membrane (Nytran; Schleider & Schuell, Dassel, Germany). The membranes were hybridized with human plasminogen inhibitor-1 cDNA (29), human Smad7 cDNA, or rat alpha2 (I) collagen cDNA and radiolabeled with [-32P] dCTP by a random primer labeling method (Bca BEST; TAKARA, Shiga, Japan) in hybridization buffer (0.5 M NaPO4, pH 7.0, 1% BSA, 7% SDS, and 1 mM EDTA) at 65°C for 16 h. The membranes were autoradiographed and rehybridized with a radioactive probe of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as an internal standard.
Immunofluorescence
The subconfluent cells on non-coated sterile cover slides in 12-well plates (FALCON, Franklin Lakes, NJ) were transiently transfected with pcDNA-Myc-Smad3 or with pCMV-Myc-Smad7 using a LipofectAMINE reagent (Invitrogen, Carlsbad, CA). Two days after serum starvation, cells were treated with Ac-SDKP or TGF-1 for the indicated intervals. Cells on cover slides were washed once with phosphate-buffered saline (PBS), fixed for 3 min with methanol (-20°C), and air-dried. After blocking with PBS-BSA (2%) for 20 min, these samples were incubated with primary antibody (1:200) in PBS-BSA for 30 min. Samples were exposed to 2% goat serum in PBS-BSA for 20 min and then incubated with fluorescein-conjugated secondary antibody (ICN Pharmaceuticals Inc., Aurora, Ohio) for 30 min in the dark. Before these samples were mounted with mounting medium, cover slides were washed five times with PBS and the nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was detected using a fluorescence microscope with appropriate filters (BX61, OLYMPUS, Tokyo, Japan).
Transfection and Luciferase Assay
The subconfluent cells in six-well plates (FALCON, Franklin Lakes, NJ) were transiently transfected with an indicated reporter plasmid and a CMV--galactosidase-containing plasmid as a control for transfection efficiency, using a LipofectAMINE reagent. Two days after serum starvation, 2.5 ng/ml TGF-1 or vehicle was added to the cells. In some experiments, the transfected cells were pretreated for 2 h with Ac-SDKP before addition of TGF-1. Twenty hours later, the cells were harvested in 300 µl of reporter lysis buffer (Promega, Madison, WI). Luciferase and -galactosidase activities were measured as described previously (30). Luciferase assay results were normalized for -galactosidase activity. Experimental values were obtained in triplicate in at least three independent transfections.
Nuclear Extraction
Human mesangial cells were washed twice with ice-cold PBS and lysed by addition of a hypotonic buffer (10 mM HEPES-KCl, pH 7.9, 1 mM EDTA, 15 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol [DTT], and protease inhibitor cocktail [Boehringer Mannheim, Lewes, UK]) with 0.8% Nonidet P-40, and the lysates were centrifuged at 6000 rpm for 10 min. The pellets were resuspended in high-salt buffer (hypotonic buffer with 420 mM NaCl and 25% glycerol), rotated for 30 min at 4°C, and centrifuged at 15,000 rpm for 30 min. The supernatants were used as nuclear extracts, and the samples were frozen immediately in liquid nitrogen and stored at -80°C until use.
Smad7 Localization by Western Blot Analyses
Human mesangial cells on 10-cm culture dish were transfected with pCMV-Myc-Smad7 expression plasmid using a LipofectAMINE reagent. Two days after serum starvation, cells were treated with Ac-SDKP for 2 h. After collecting with PBS-EDTA (2 mM), cells were homogenized, centrifuged at 2500 rpm for 5 min, and the pellet was used as a nuclear extraction and the supernatant was centrifuged at 15,000 rpm for 30 min. Resultant supernatant was centrifuged at 550,000 rpm for 60 min to sort the membrane fractions from cytoplasm, and the supernatants were used as cytoplasm extraction. The concentrations of the protein in the supernatants were measured by the Bradford method (Bio-Rad, Hercules, CA), and the cell lysates containing 20 µg of protein were boiled in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 50 mM DTT, 0.1% bromophenol blue) for 5 min. Samples were immunodetected by Western blot analysis with anti-Myc antibody. The expression of each protein was quantified by scanning densitometry and corrected by the level of GAPDH and Nucleoporin p62 as loading controls for cytoplasm and nucleus, respectively.
Statistical Analyses
The data are expressed as means ± SEM. ANOVA followed by Scheffe test was used to determine significant difference in multiple comparisons. The nuclear and cytoplasmic distribution of Myc-Smad3 or Myc-Smad7 was compared by 
2 tests. Statistical significance was defined as P < 0.05.
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Results |
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1-Mediated PAI-1, Alpha2 (I) Collagen mRNA Expression1-stimulated expression of PAI-1 mRNA in human mesangial cells. PAI-1 mRNA expression was increased at 1 h after the start of stimulation with TGF-1 and reached its maximum at 6 h (Figure 1A). Thus, to determine the effect of Ac-SDKP on TGF-1-induced PAI-1 mRNA expression, we analyzed PAI-1 mRNA expression at 6 h after the stimulation with or without Ac-SDKP. Ac-SDKP significantly inhibited TGF-1-induced PAI-1 mRNA expression in human mesangial cells (Figure 1B), whereas Ac-SDKP did not inhibit basal PAI-1 mRNA expression. Similarly to the effect on PAI-1 mRNA expression, Ac-SDKP also inhibited TGF-1-stimulated COL1A2 mRNA expression (Figure 1C).
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11-induced PAI-1 mRNA expression is regulated by R-Smad activation (31), and the TGF-/Smad pathway is functional in human mesangial cells (32). To elucidate the molecular mechanism by which Ac-SDKP inhibits TGF-1-induced PAI-1 mRNA expression, we examined the effects of Ac-SDKP on Smad2 phosphorylation induced by TGF-1 in human mesangial cells. TGF-1 stimulated the phosphorylation of Smad2 at Ser-465/467 at 15 min after TGF-1 stimulation (Figure 2A). After 15 min of stimulation, Ac-SDKP suppressed TGF-1-induced phosphorylation of Smad2 in a dose-dependent manner (Figure 2, B and C). Next, we confirmed the influence of incubation interval on the inhibitory effect of Ac-SDKP on TGF-1-stimulated Smad2 phosphorylation. TGF-1-stimulated Smad2 phosphorylation was suppressed by 2-h and 1-h pretreatment with Ac-SDKP, whereas it was not inhibited by 30-min pretreatment (Figure 2D).
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11-mediated PAI-1 mRNA expression (33); we therefore tested the effect of Ac-SDKP on the activation of ERK. Western blot analyses revealed that p42/p44 ERK was phosphorylated at 15 min by TGF-1 (Figure 3A). Ac-SDKP failed to inhibit TGF-1-induced phosphorylation of ERK, and the basal level of the phosphorylated form of ERK was further increased by Ac-SDKP alone (Figure 3A).
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. Incubation with U0126, a MEK inhibitor, completely suppressed the ERK phosphorylation but failed to inhibit the antagonistic effect of Ac-SDKP on TGF-1-induced Smad2 phosphorylation (Figure 3B). These data suggest that the Ras-MEK-ERK pathway does not contribute to the inhibitory effect of Ac-SDKP on TGF- signal transduction.
Effect of Ac-SDKP on Nuclear Accumulation of R-Smad
The phosphorylated R-Smads, Smad2 and Smad3, associate with a common partner, Smad4, and translocate into the nucleus (26,36,37 ). We examined the effects of Ac-SDKP on nuclear translocation of these Smad proteins. Western blot analyses showed that Ac-SDKP inhibited nuclear translocation of Smad2 and Smad3 (Figure 4A). To confirm this result, we next examined the effect of Ac-SDKP on overexpressed-Myc-Smad3 in human mesangial cells. Immunofluorescence analyses also demonstrated that Myc-Smad3 in the cytoplasm was translocated to the nucleus by TGF-1 stimulation, whereas Ac-SDKP suppressed the Smad3 nuclear translocation induced by TGF-1 (Figure 4B). Quantitative analysis demonstrated that the inhibition of TGF-1-induced nuclear accumulation of Myc-Smad3 by Ac-SDKP was statistically significant (TGF-1 alone 66% versus TGF-1 with Ac-SDKP 24%, P < 0.01 by 2 tests; Figure 4C). No fluorescence was observed in the absence of pcDNA-Myc-Smad3 or anti-Myc antibody (Figure 4D).
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1-Induced Transcriptional Activation-Smad signaling, we examined the effect of this peptide on TGF--mediated transcriptional activation with luciferase assay. TGF-1 significantly increased the luciferase activity of the TGF--responsive reporter 3TP-luc by 3.0-fold, whereas Ac-SDKP significantly suppressed the induction of 3TP-luc activity by TGF-1 (Figure 5A). Similarly, Ac-SDKP markedly suppressed the TGF--responsive transcriptional reporter 4xSBE-luc (Figure 5B) in a dose-dependent manner. However, it failed to inhibit the pAP-1-luc (data not shown).
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responses by competitive interaction with the type I receptor (27), via nuclear to cytoplasmic localization (38). To elucidate the inhibitory effect of Ac-SDKP on TGF--induced signal transduction, we first tested the influence of Ac-SDKP on mRNA expression of Smad7. We found that Ac-SDKP did not induce the mRNA expression of Smad7 (Figure 6A). Thus, we next tested the effect of Ac-SDKP on overexpressed-Myc-Smad7 localization by immunofluorescence analysis. Myc-Smad7 was primarily localized in the nucleus and was translocated to the cytoplasm by TGF-1 stimulation (Figure 6B). Interestingly, we found that Myc-Smad7 was localized in the cytoplasm after 2-h incubation with Ac-SDKP (Figure 6B). Figure 6C demonstrates that nuclear Myc-Smad7 began to translocate at 1 h after the start of incubation with Ac-SDKP and was mainly translocated to the cytoplasm at 2 h. Myc-Smad7 remained in the cytoplasm at 4 h after Ac-SDKP treatment (Figure 6C). No fluorescence was observed in the absence of pCMV-Myc-Smad7 or anti-Myc antibody (Figure 6D). As shown in Figure 6E, translocation of Myc-Smad7 to the cytoplasm from nucleus by Ac-SDKP or by TGF-1 was confirmed by counting the number of Myc-positive cells. Figure 6E shows that the cytoplasmic distribution of Myc-Smad7 induced in the presence of Ac-SDKP was compared with the cells in the absence of Ac-SDKP significantly (1 h Ac-SDKP 58%, 2 h Ac-SDKP 72% versus control 24%, P < 0.01 by 2 tests). To further confirm Ac-SDKP-mediated Smad7 translocation, we examined the Western blot analyses of nuclear and cytoplasmic proteins from Myc-Smad7-transfected cells treated with Ac-SDKP. Ac-SDKP significantly (P < 0.01 by 2 tests) increased the cytoplasmic distribution and decreased the nuclear localization of Myc-Smad7 (Figure 6, F and G).
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Discussion |
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action of Ac-SDKP in human mesangial cells and investigated the mechanisms of the action of Ac-SDKP. We demonstrated that Ac-SDKP can inhibit the TGF-1-induced PAI-1 and COL1A2 mRNA expression in human glomerular mesangial cells. Regarding the molecular mechanisms of this inhibitory effect of Ac-SDKP, we found that (1) Ac-SDKP inhibited Smad2 phosphorylation stimulated by TGF-1, (2) the RAS-MEK-ERK pathway did not contribute to the antagonistic effects of Ac-SDKP on TGF-1-induced phosphorylation of Smad2, (3) Ac-SDKP suppressed the transcriptional activity of the 3TP-luc and 4xSBE-luc reporter, and (4) Ac-SDKP induced the cytoplasmic translocation of Smad7 from the nucleus. Taken together, these observations highlight a new mechanism for the beneficial role of ACE-I, whereby Ac-SDKP serves as an anti-fibrotic factor via suppression of the TGF-/Smad signaling pathway.
TGF- is a key cytokine that regulates development, cell proliferation, and matrix protein synthesis (18,19 ). During the glomerular scarring process, TGF- plays a major role in matrix protein accumulation and collagen deposition (20). TGF- signaling from the cell membrane to the nucleus is mediated by intracellular effector molecules, termed Smads. It is well documented that TGF- binds TRII, resulting in the activation of TRI, which causes the phosphorylation of specific Smad proteins, which are called R-Smads, including Smad2 and Smad3 (19,24,25 ). We found that Ac-SDKP inhibited TGF-1-induced PAI-1 mRNA expression, Smad2 phosphorylation, and nuclear localization of R-Smad proteins. We also found that Ac-SDKP inhibited the transcriptional activity of the Smad-dependent reporters, 3TP-luc and 4xSBE, but failed to inhibit the Smad-independent reporter, pAP-1, similar to the findings in cardiac fibroblasts (39). However, other reports indicate that PAI-1 mRNA expression is also regulated by ERK activation (33). This was not found to be the case in the present study, because Ac-SDKP did not inhibit TGF-1-induced activation of p42/p44 ERK. These observations suggest that the effect of Ac-SDKP on the TGF- signaling pathway could be specific for the Smad pathway.
Phosphorylation of R-Smads in the linker region by the Ras-MEK-ERK pathway at multiple -PXSP- consensus sites can inhibit both the nuclear accumulation and transcriptional activity of Smad proteins (34,40 ). Because incubation with Ac-SDKP alone increased the amount of phosphorylated ERK, we examined whether Ac-SDKP-stimulated ERK has a role in the inhibitory effect of Ac-SDKP on TGF-1-induced Smad2 phosphorylation. We observed that incubation with U0126 failed to inhibit the antagonistic effect of Ac-SDKP on TGF-1-mediated Smad2 phosphorylation. These data suggest that the Ras-MEK-ERK pathway does not contribute to the function of Ac-SDKP in TGF- signal transduction.
The activation of TGF- signaling is tightly controlled by several negative feedback mechanisms (27,41–44 ). Among these mechanisms regulating TGF-, we focused here on the inhibitory Smad, Smad7. It is reported that Smad7 is induced by TGF- and participates in a negative feedback loop to control TGF- responses by competitive interaction with the type I receptor (27). We thus examined the effect of Ac-SDKP on the protein and mRNA expression of Smad7. We found that Ac-SDKP could not induce Smad7 mRNA expression. However, we failed to detect endogenous Smad7 protein expression by the antibody we used. These results also indicate that the de novo induction of Smad7 is not a mechanism involved in the inhibitory action of Ac-SDKP. Previous reports indicate that Smad7 is localized in the nucleus and that TGF--mediated nuclear export of Smad7 is associated with its inhibitory effects (38,45 ); we therefore clarified the influence of Ac-SDKP on Smad7 localization. Although we could not clearly detect endogenous Smad7 in human mesangial cells by the antibody we used (data not shown), we confirmed the localization of Smad7 by the transfection study. Surprisingly, Myc-Smad7 was translocated to the cytoplasm by 2 h after incubation with Ac-SDKP. This translocation began at 1 h after treatment, consistent with the result that more than 1 h of preincubation with Ac-SDKP was necessary for its inhibitory effect on Smad2 phosphorylation stimulated by TGF-1. In renal tublar epithelial cells, overexpression of Smad7 inhibited TGF--mediated phosphorylation of Smad2, but the late expression of Smad7 induced by TGF- could not overcome the TGF--induced R-Smad activation (46). These data and our present results suggest that the cytoplasmic translocation of Smad7 by Ac-SDKP before TGF- stimulation might be an important in the inhibitory effect of Ac-SDKP on TGF-/Smad signal transduction. Although the precise mechanisms of Ac-SDKP-mediated translocation of Smad7 need to be clarified further, these observations suggest that Ac-SDKP might inhibit TGF-/Smad signaling via translocation of Smad7 to the cytoplasm from the nucleus.
In glomerular scarring, TGF- plays a major role in matrix protein accumulation and collagen deposition. In fact, several groups (including ours) reported that the Smad pathway is important in the pathogenesis of renal disease (47–49 ). Our present findings that Ac-SDKP inhibits the TGF-1-mediated PAI-1 and COL1A2 mRNA expression via suppression of the Smad pathway and that this inhibitory effect of Ac-SDKP is mediated by the nuclear exportation of Smad7 highlight the involvement of RAS-independent mechanisms in the therapeutic value of ACE-I and also provide a new approach to the design of drugs for progressive renal diseases.
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1 on mesangial cells by overexpression of Smad7. J Am Soc Nephrol 13: 887–893, 2002This article has been cited by other articles:
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