FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2066410366> ?p ?o ?g. }

• W2066410366 endingPage "28280" @default.
• W2066410366 startingPage "28274" @default.
• W2066410366 abstract "Redox-regulated processes are important elements in various cellular functions. Reducing agents, such asN-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-β, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-β receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that “redox therapy” is promising for fibrosis-related disease. Redox-regulated processes are important elements in various cellular functions. Reducing agents, such asN-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-β, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-β receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that “redox therapy” is promising for fibrosis-related disease. vascular smooth muscle cell(s) N-acetyl-l-cysteine platelet-derived growth factor platelet-derived growth factor receptor hepatic stellate cell l-buthionine-(S,R)-sulfoximine l-3-carboxy-2,3-trans-epoxypropionyl-(4-guanidine)butane N-[N-(l-3-trans-carboxirane-2-carbonyl)-l-leucyl]-3-methylbutylamine ethyl-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)-butylcarbamoyl]-2-oxiranecarboxylate N-(l-3-trans-prorylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline 2-mercaptoethanol transforming growth factor transforming growth factor-β receptor smooth muscle α-actin thioacetamide immunoprecipitation Western blot mitogen-activated protein carbobenzoxy-l-isoleucyl-γ-t-butyl-l-glutamyl-l-alamyl-l-leucinal carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal Reactive oxygen species are known to activate intracellular signal molecules (1Lander H. FASEB J. 1997; 11: 118-124Crossref PubMed Scopus (823) Google Scholar). Extracellular signal-regulated kinase is one of the reactive oxygen species-responsive serine/threonine kinases (2Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. Hiroi Y. Yazaki Y. J. Clin. Invest. 1997; 100: 1813-1821Crossref PubMed Scopus (630) Google Scholar). A recent study showed that hydrogen peroxide (H2O2) directly attacks Gαo and Gαi to activate extracellular signal-regulated kinase in rat neonatal cardiomyocytes (3Nishida M. Maruyama Y. Tanaka R. Kontani K. Nagao T. Kurose H. Nature. 2000; 408: 492-495Crossref PubMed Scopus (229) Google Scholar). In addition to the direct activation of signal molecules, H2O2 was also reported to contribute to the phosphorylation of mitogen-activated protein kinase in vascular smooth muscle cells (VSMC)1 under the stimulation of the platelet-derived growth factor (PDGF) (4Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2313) Google Scholar). However, recent studies have forced reconsideration of this hypothesis. In HepG2 cells, PDGF receptor (PDGFR) was autophosphorylated independently of H2O2 production (5Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (570) Google Scholar). In epithelial cells, interleukin-1β activated NF-κB without H2O2production (6Bonizzi G. Dejardin E. Piret B. Piette J. Merville M.P. Bours V. Eur. J. Biochem. 1996; 242: 544-549Crossref PubMed Scopus (68) Google Scholar). These evidences confirmed that reactive oxygen species are not always involved in ligand-induced signaling pathways. The hepatic stellate cell (HSC), a liver-specific pericyte, plays a central role in liver fibrogenesis (7Friedman S.L. N. Engl. J. Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar). When undergoing activation, the HSC proliferates and generates a large amount of extracellular matrix materials including fibril-forming collagens, fibronectin, and proteoglycans, resulting in septum formation in chronically damaged liver. Because recent studies have confirmed that PDGF is the most potent mitogen for HSC (8Pinzani M. Gesualdo L. Sabbah G.M. Abboud H.E. J. Clin. Invest. 1989; 84: 1786-1793Crossref PubMed Scopus (427) Google Scholar), regulation of PDGF-stimulated HSC proliferation would serve as a therapeutic target for liver fibrosis. Here we show that reducing agents, includingN-acetyl-l-cysteine (NAC), have the potential to disturb PDGF-dependent signal transductions and DNA synthesis in HSC. Molecular analysis revealed that NAC triggered extracellular proteolysis of PDGFR-β through the activation of a thiol-protease, cathepsin B, independently of its H2O2 scavenging action. Collagenase and thioacetamide (TAA) were purchased from Wako Pure Chemical Co. (Osaka, Japan). Pronase E was purchased from Merck. PDGF-BB was purchased from R & D systems (Minneapolis, MN). The polyclonal antibodies against PDGFR-α and PDGFR-β, type I transforming growth factor-β receptor (TGF-βRI), and TGF-βRII were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal antibodies against cathepsin B and phosphotyrosine were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The polyclonal antibodies against phosphoextracellular signal-regulated kinase and phospho-Akt were purchased from New England Biolabs (Beverly, MA). The monoclonal antibody against smooth muscle α-actin (α-SMA), desmin, vimentin, and glial fibrillary acidic protein were purchased from DAKO A/S (Glostrup, Denmark). [3H]Thymidine, [35S]methionine, [α-32P]dCTP, and 125I-PDGF-BB were purchased from Amersham Pharmacia Biotech. E64, E64-c, E64-d, CA074, and pepstatin A were purchased from the Peptide Institute (Osaka, Japan). These drugs were dissolved in Me2SO before use, and, throughout the experiments, the final concentration of Me2SO in cell culture medium was kept at 0.5% including control cultures. Unless specifically indicated, all other reagents were purchased from Sigma. Pathogen-free male Wistar rats were obtained from SLC (Shizuoka, Japan). Animals were housed at a constant temperature and supplied with laboratory chow and water ad libitum. The protocol of experiments was approved by the Animal Research Committee of Osaka City University (Guide for Animal Experiments, Osaka City University). We isolated HSC from Wistar rat livers using Pronase E and the collagenase digestion method as previously described (9Kawada N. Tran-Thi T-A. Klein H. Decker K. Eur. J. Biochem. 1993; 213: 815-823Crossref PubMed Scopus (375) Google Scholar). Cell purity was around 95% as assessed by a typical starlike configuration and by detecting vitamin A autofluorescence. Cells were cultured on plastic dishes in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies). We changed the culture medium every 2 days. We isolated VSMC from Wistar rat thoracic aorta by subcultured explants method as previously described (10Ross R. J. Cell. Biol. 1971; 50: 172-186Crossref PubMed Scopus (1268) Google Scholar). Cells were cultured on plastic dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Passaged cells were used in an in vitroassay. In all experiments, we incubated HSC or VSMC in serum-free Dulbecco's modified Eagle's medium for 24 h before the addition of test agents. We treated confluent HSC with NAC for 24 h, followed by treatment with PDGF-BB (20 ng/ml) for another 24 h. Cells were pulse-labeled with 1.0 µCi/ml [3H]thymidine during the last 6 h. Incorporated radioactivity was counted as previously described (11Kawada N. Ikeda K. Seki S. Kuroki T. J. Hepatol. 1999; 30: 1057-1064Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We lysed HSC cultured in a 60-mm dish (FALCON 3002; Becton Dickinson, Flanklin Lakes, NJ) by 500 µl of radioimmune precipitation buffer (10 mm Tris-HCl, pH 7.4, 1% IGEPAL CA-630, 0.1% SDS, 0.1% sodium deoxycholate). The lysates (500 µg) were incubated with 2 µg of antibody against PDGFR-β at 4 °C for 24 h, followed by incubation with 20 µl of protein G plus agarose (Santa Cruz Biotechnology) at 4 °C for 1 h. We homogenized HSC treated with EDTA (Wako), 1,10-phenanthroline, phenylmethylsulfonyl fluoride, leupeptin, chymostatin, E64, E64-c, E64-d, CA074, and pepstatin A in 1× SDS sample buffer (100 µl/35-mm dish) (62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-β-mercaptoethanol, 1 mm Na3VO4). After heat denaturation, the samples (10 µg of protein) were subjected to SDS-polyacrylamide gel electrophoresis (6–12%) and then transferred onto an Immobilon P membrane (Millipore Corp., Bedford, MA). The membranes were subsequently treated with monoclonal antibody against either α-SMA, PDGFR-β, TGF-βR, phosphotyrosine (4G10), phopsho-Akt, or phospho-MAP kinase. Immunoreactive bands were visualized on Eastman Kodak Co. XAR5 films, using ECL detection reagent (Amersham Pharmacia Biotech). After treatment with Dulbecco's modified Eagle's medium lacking methionine (Life Technologies), we incubated HSC with 0.1 mCi of [35S]methionine for 24 h at 37 °C. After removal of the radioactive medium, they were incubated with NAC for the following 24 h at 37 °C. After incubation, HSC were washed twice with cold phosphate-buffered saline. HSC were lysed by scraping into radioimmune precipitation buffer and prepared for immunoprecipitation. Immunoprecipitations were performed as described above using anti-PDGFR-β antibody followed by adsorption to protein G plus agarose. SDS-polyacrylamide gel electrophoresis was carried out in 6% gels. Gels were dried and exposed to Kodak XAR5 x-ray film at −80 °C for 1 week. We measured the binding capacity of 125I-PDGF-BB to HSC cultured in 35-mm dishes as previously described (12Mori S. Heldin C-H. Claesson-Welsh L. J. Biol. Chem. 1993; 268: 577-583Abstract Full Text PDF PubMed Google Scholar). Briefly, HSC were incubated on ice for 2 h with 1 ng/ml 125I-PDGF-BB in the presence or absence of 400 ng/ml of unlabeled PDGF-BB. After incubation, radioactivity in the lysate was determined in a γ-counter (Aloka Auto well γ system ARC-2000, Tokyo, Japan). The radioactivity in the presence of unlabeled PDGF-BB was subtracted as nonspecific binding. We extracted total RNA from the cells using Isogen (Nippon Gene, Tokyo, Japan) as described previously (17Kristensen D.B. Kawada N. Imamura K. Miyamoto Y. Tateno C. Seki S. Kuroki T. Yoshizato K. Hepatology. 2000; 32: 268-277Crossref PubMed Scopus (205) Google Scholar). Total RNA (20 µg) was separated on a 1% agarose gel (Nippon Gene) and transferred onto a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech). After prehybridization, the membrane was incubated in the buffer supplemented with polymerase chain reaction-amplified double-stranded cDNAs, which were labeled with [α-32P]dCTP using a Rediprime DNA Labeling System (Amersham Pharmacia Biotech), followed by autoradiography on a Kodak XAR5 x-ray film. The following primers were used: PDGFR-β, 5′-GATGTCACTGAGACGACGAT-3′ (forward) and 3′-CCTCAAACACCACCTGCAAC-5′ (reverse); glyceraldehyde-3-phosphate dehydrogenase, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 3′-TCCACCACCCTGTTGCTGTA-5′ (reverse). Identification of amplified DNAs was confirmed by sequencing. We incubated the immunoprecipitated PDGFR-β protein for 1 h at 37 °C with NAC (20 mm) in the presence or absence of either 0.05 unit of cathepsin B, 0.05 unit of cathepsin D, or 1 mm E64. Then the samples were subjected to 6% SDS-polyacrylamide gel electrophoresis, followed by Western blot. We confirmed the immunoprecipitated band at 180 kDa as rat PDGFR-β by amino acid sequencing of the proteins using a Q-TOF mass spectrometer (Micromass, Manchester, United Kingdom). We injected TAA (50 mg/body) twice a week intraperitoneally into rats (n = 15). We administered NAC (100 mg/body) simultaneously either intraperitoneally (n = 5) or orally (n= 5) every day for 6 weeks. In another model, we injected TAA for 9 weeks (n = 15), and NAC (100 mg/body) was administered either intraperitoneally (n = 5) or orally (n = 5) during the last 3 weeks. One day after the final injection, rats were anesthetized by diethylether, and the peritoneal cavity was opened. The liver was perfused with phosphate-buffered saline via the portal vein at a flow rate of 10 ml/min until the blood was completely removed from the whole liver lobules. Subsequently, the liver was removed. A part of the liver was fixed in 10% formaldehyde and used for histologic examination. The remaining part was quickly frozen in liquid nitrogen and stored at −80 °C until use. We laparotomized rats (n = 10) and ligated the common bile ducts at the two different sites. We administered NAC (100 mg/body) intraperitoneally every day for 2 weeks (n = 5). We stained the paraffin-embedded sections (4-µm thickness) with Mallory azan, followed by morphometric analysis of fibrotic area using Mac SCOPE version 2.5 (MITANI Corp.) and Photgrab-2500 for Macintosh FUJIX SH-25/M (FUJIFILM, Tokyo, Japan). Five nonoverlapping areas were evaluated in each group. In immunohistochemistry, tissue sections were treated with 1% hydrogen peroxide, 0.1% proteinase K, and 1% Triton X-100. They were reacted with 1:50-diluted mouse monoclonal antibody against α-SMA overnight at 4 °C, followed by reaction with 1:200-diluted biotin-conjugated rabbit anti-mouse IgG F(ab′)2 for 1 h at room temperature. Data presented as bar graphs are the means ± S.D. of three independent experiments except for in vivo analysis of five independent experiments. Luminograms and autoradiograms are representative of at least three experiments. Statistical analysis was performed by Student's t test (p < 0.05 was considered significant). Treatment of HSC with NAC inhibited PDGF-BB-dependent DNA synthesis in a dose-dependent manner (Fig.1A). NAC impeded the phosphorylation of tyrosine residue of PDGFR-β and the activation of both MAP kinase and Akt dose-dependently under PDGF-BB stimulation (Fig. 1, B–D). Notably, NAC down-regulated the protein level of PDGFR-β dose-dependently (Fig.1 E). 125I-PDGF-BB binding experiments showed that pretreatment with NAC for 24 h decreased the specific binding of PDGF-BB to HSC (Fig. 1 F). These results indicate that NAC blocked PDGF-dependent signaling pathways and DNA synthesis in HSC through the down-regulation of PDGFR-β protein. NAC triggered down-regulation of PDGFR-β protein in a dose- and time-dependent manner (Fig.2, A and B). Since removal of NAC restored PDGFR-β, this effect of NAC was not derived from its cytotoxic action (Fig. 2 C). BSO, an inhibitor for GSH production, did not restore the down-regulation of PDGFR-β (Fig. 2 D). This result indicated that GSH endogenously produced is not involved in this effect of NAC. Northern blot analysis showed that NAC had no effect on the expression of PDGFR-β mRNA (Fig. 2 E). Metabolic labeling experiments revealed that NAC reduced de novo synthesized PDGFR-β (Fig.2 F). These results indicated that this effect of NAC was presumably due to accelerating degradation of PDGFR-β that had already been synthesized. To clarify the mechanism by which NAC down-regulates PDGFR-β protein in HSC, we tested whether protease inhibitors affected NAC-induced degradation of PDGFR-β protein. EDTA and 1,10-phenanthroline are inhibitors of matrix metalloprotease; phenylmethylsulfonyl fluoride inhibits a serine protease; chymostatin inhibits chymotrypsin, papain, and cathepsin A, B, and D; leupeptin inhibits trypsin, plasmin, papain, and cathepsin B; E64 inhibits thiol proteases including cathepsin B, H, and L and calpain; CA074 inhibits cathepsin B; and pepstatin A inhibits cathepsin D. Among these protease inhibitors, chymostatin, leupeptin, E64, CA074, and pepstatin A hampered NAC-induced down-regulation of PDGFR-β (Fig.3A). Proteasome inhibitors such as MG115 and PSI failed to affect the degradation of PDGFR-β by NAC. These results indicate that down-regulation of PDGFR-β protein expression is mediated through its degradation by cathepsin B and/or cathepsin D. Next, we determined whether cathepsin B or cathepsin D could degrade PDGFR-β protein using in vitro degradation assays. Neither NAC alone nor cathepsin B alone had any effect on PDGFR-β protein (Fig. 3 B). However, cathepsin B in combination with NAC induced degradation of PDGFR-β protein in an E64-inhibitable manner. In contrast, cathepsin D had no effect on PDGFR-β protein level with or without NAC. These results indicate that cathepsin B is responsible for the degradation of PDGFR-β and that NAC is required for the activation of cathepsin B. Western blot analysis showed that reducing agents, such asl-cysteine, GSH, 2-mercaptoethanol (2-ME), and dithiothreitol, down-regulated PDGFR-β, while l-cystine and GSSG failed to affect the PDGFR-β level (Fig. 3 C). Trolox (a water-soluble derivative of vitamin E) had no effect either. These results indicate that reducing agents play an essential role in the enhancement of the catalytic activity of cathepsin B, which is consistent with the general character of thiol proteases (13Kirschke H. Barrett A.J. Rawlings N.D. Lysosomal Cysteine Proteinases. 2nd Ed. Oxford University Press, Oxford1998Google Scholar). Next, we tested the site where cathepsin B degraded PDGFR-β. E64-c is a membrane-impermeable derivative of E64 (14Kasai Y.S. Senshu M. Iwashita S. Imahori K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 146-150Crossref PubMed Scopus (57) Google Scholar). E64-d is a membrane-permeable derivative of E64 (15Hill P.A. Buttle D.J. Jones S.J. Boyde A. Murata M. Reynolds J.J. Meikle M.C. J. Cell. Biochem. 1994; 56: 118-130Crossref PubMed Scopus (107) Google Scholar). CA074 is a membrane-impermeable derivative (16Buttle D.J. Murata M. Knight C.G. Barrett A.J. Arch. Biochem. Biophys. 1992; 299: 377-380Crossref PubMed Scopus (172) Google Scholar). Both E64-c and CA074 were able to restore the PDGFR-β protein similarly to E64-d (Fig. 3 D). These results indicate that PDGFR-β might be degraded extracellularly by cathepsin B in the presence of NAC. In fact, mature cathepsin B as well as procathepsin B was present in the culture medium of activated HSC (Fig. 3 E). This is consistent with the report by Kristensen et al. (17Kristensen D.B. Kawada N. Imamura K. Miyamoto Y. Tateno C. Seki S. Kuroki T. Yoshizato K. Hepatology. 2000; 32: 268-277Crossref PubMed Scopus (205) Google Scholar) showing that proteome analysis detected cathepsin B in the culture supernatant of HSC. In sharp contrast to HSC, VSMC secreted procathepsin B, but not mature cathepsin B, into the culture medium (Fig. 3 E). Here, we assumed that NAC might have no effect on the expression of PDGFR-β in VSMC. As expected, NAC failed to affect both the protein level of PDGFR-β and tyrosine phosphorylation of PDGFR-β under PDGF-BB stimulation in VSMC (Fig. 4, Aand B). Accordingly, NAC had no effect on the phosphorylation of MAP kinase and Akt under PDGF-BB stimulation in VSMC (Fig. 4, C and D). However, NAC blocked the DNA synthesis of VSMC under PDGF-BB stimulation in a dose-dependent manner, as previously reported (Fig.4 E) (4Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2313) Google Scholar). These results indicate that NAC may block some signal molecules except for PDGFR-β, MAP kinase, and Akt in VSMC. Next, we examined whether the above mentioned effect of NAC was specific for PDGFR-β. NAC was also found to down-regulate the expression of TGF-βRII (Fig. 5). In contrast, NAC had no effect on the expression of other receptors, such as PDGFR-α and TGF-βRI. NAC also failed to affect the level of cytoskeletous proteins, such as α-SMA, desmin, vimentin, and glial fibrillary acidic protein (Fig. 5). The above mentioned results indicated that NAC may suppress liver fibrosis by inhibiting HSC proliferation through inducing unresponsiveness to growth factors, such as PDGF-BB and TGF-β. To test this hypothesis, we prepared two different liver fibrosis models. First, we examined whether NAC could suppress thioacetamide (TAA)-induced liver fibrosis. Mallory azan staining revealed that fibrosis was well developed in the liver of rats treated with TAA for 6 weeks (fibrotic area 8.16 ± 1.24%) (Fig.6, A and K). NAC treatment either intraperitoneally or orally suppressed fibrosis of the liver (1.79 ± 0.51 or 2.51 ± 0.90%, p < 0.01) (Fig. 6, B, C, and K). Immunostaining of α-SMA, a marker of myofibroblast and activated HSC, showed that, while α-SMA-positive cells were numerous in and around fibrotic septum of TAA-treated livers (Fig. 6 G), administration of NAC suppressed the increase of α-SMA-positive cells (Fig. 6 H). This result indicated that NAC inhibited the proliferation of activated HSC also in in vivo fibrosis models. To further test whether NAC had the potential to restore the liver fibrosis that had already been developed, we started NAC treatment 6 weeks after TAA administration and excised liver tissues for histologic evaluation 3 weeks later. Liver fibrosis progressed with thick bundles of elastic fibers after 9-week administration of TAA (12.44 ± 1.74%) (Fig. 6, D and K). However, NAC treatment during the last 3 weeks either intraperitoneally or orally restored the fibrotic liver tissue to almost physiological architecture (1.79 ± 0.51 or 2.51 ± 0.90%,p < 0.01) (Fig. 6, E, F, andK). Next, we examined the effect of NAC on the bile duct ligation model. Bile duct ligation for 2 weeks induced cholestasis, periductular hepatocyte necrosis with leukocyte accumulation, and fibrosis around the portal area (3.50 ± 2.06%) (Fig. 6, I andK). When we administered NAC on a daily basis, the deposition of extracellular matrix was much less than that in nontreated rats (1.45 ± 1.49%, p < 0.05) (Fig.6, J and K). Finally, we examined whether NAC could down-regulate growth factor receptors in rat livers. Western blot analysis revealed that neither PDGFR-β protein nor TGF-βRII protein was detectable in liver tissues of rats treated with TAA and NAC (Fig. 6 L). In contrast, the protein level of PDGFR-α, TGF-βRI, and α-SMA showed limited suppression by NAC administration. These results indicate that NAC has a potential to induce degradation of both PDGFR-β and TGF-βRII in vivo. Redox regulation by intrinsic reducing suppliers, including GSH and thioredoxin, is essential to maintain intact cellular functions against oxidative stress in many mammalian cells (18Sasada T. Yodoi J. Packer L. Yodoi J. Redox Regulation of Cell Signaling and Its Clinical Application. Marcel Dekker, Inc., New York1999: 1-9Google Scholar). Both GSH and thioredoxin regulate cell growth and death by modulating superoxide-sensitive transcription factors, such as AP-1 and NF-κB (19Okamoto T. Yoshida S. Tetsuka T. Packer L. Yodoi J. Redox Regulation of Cell Signaling and Its Clinical Application. Marcel Dekker, Inc., New York1999: 261-277Google Scholar). Likewise, extrinsic reducing suppliers, such as NAC, are known to regulate cell growth by increasing intracellular GSH production. NAC has been clinically utilized for acute respiratory distress syndrome, cancer, chronic bronchitis, heart disease, heavy metal poisoning, human immunodeficiency virus infection, and acetaminophen-induced liver injury (20Kelly G.S. Alt. Med. Rev. 1997; 3: 114-127Google Scholar). Our current study provides a new insight into the pathophysiological role of redox processes. We found that the extracellular reduced condition may be an important factor for the regulation of growth factor-stimulated cellular response through the degradation of membrane receptors assisted with secreted cathepsin B. However, such a phenomenon was observed after the long term exposure of HSC to NAC. Inin vitro culture, NAC at 20 mm required 24 h for inducing complete degradation of PDGFR-β. In contrast, we found that the exposure of HSC to NAC for 30 min could also inhibit PDGF-dependent signaling without any change of PDGFR-β protein level (data not shown). Moreover, although E64 completely restored the expression of PDGFR-β at the protein level, E64-induced recovery of PDGF-dependent DNA synthesis and signaling was limited (data not shown). These results suggest that NAC may additionally modify the PDGF-induced signaling cascade by some unknown mechanism other than proteolytic action. Sundaresan et al. (4Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2313) Google Scholar) reported that, in rat VSMC, NAC blocked PDGF-induced signaling pathways, such as MAP kinase and DNA synthesis, by scavenging H2O2. Indeed, NAC removed cytosolic H2O2 in rat HSC (data not shown). However, when measured by dichlorodihydrofluolescein diacetate (DCFH-DA), HSC at 4 days after primary culture generated detectable H2O2 without any stimulation, and PDGF-BB failed to increase intracellular H2O2level, while VSMC was reported to produce H2O2only under the stimulation of PDGF (4Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2313) Google Scholar). 2H. Okuyama, Y. Shimahara, N. Kawada, S. Seki, D. B. Kristensen, K. Yoshizato, N. Uyama, and Y. Yamaoka, unpublished data. These results indicate that H2O2 may play a limited role in PDGF-dependent signaling in HSC. Cathepsin B belongs to a family of thiol proteases and localizes in lysosome. Cathepsin B has been reported to digest intracellularly antigens, immunoglobulin heavy chain, insulin A and B chain, glucagon, actin, myosin heavy chain, albumin, collagen, fibronectin, proteoglycans, fibrinogen, proplasminogen activator, and trypsinogen (13Kirschke H. Barrett A.J. Rawlings N.D. Lysosomal Cysteine Proteinases. 2nd Ed. Oxford University Press, Oxford1998Google Scholar). However, some studies reported that malignant cells, fibroblasts, and osteoblasts secrete cathepsin B into the extracellular space (21Mort J.S. Buttle D.J. Int. J. Biochem. Cell. Biol. 1997; 29: 715-720Crossref PubMed Scopus (271) Google Scholar). These cells are reported to secrete procathepsin B but not mature cathepsin B. In contrast, we detected the presence of mature-type cathepsin B in the culture medium of HSC. Some studies showed that cathepsin D is necessary for the processing of procathepsin B to mature cathepsin B (22Nishimura Y. Kawabata T. Yano S. Kato K. Acta Histochem. Cytochem. 1990; 23: 53-64Crossref Scopus (41) Google Scholar). Since pepstatin A impeded PDGFR-β degradation (Fig.3 A), it seems that cathepsin D is also involved in the activation of cathepsin B, leading to the degradation of PDGFR-β by NAC in HSC. Although the protective and antioxidative effect of NAC on hepatocytes should be taken into account, we showed that NAC down-regulated the expression of both PDGFR-β and TGF-βRII also in in vivoliver fibrosis models (23Lauterburg B.H. Corcoran G.B. Mitchell J.R. J. Clin. Invest. 1983; 71: 980-991Crossref PubMed Scopus (298) Google Scholar). TGF-β is the most potent fibrogenic factor for HSC (24Li D. Friedman S.L. J. Gastroenterol. Hepatol. 1999; 14: 618-633Crossref PubMed Scopus (333) Google Scholar). Some studies reported that inhibition of TGF-β action resulted in the suppression of liver fibrosis (25George J. Roulot D. Koteliansky V.E. Bissell D.M. Proc. Natl Acad. Sci. U. S. A. 1999; 96: 12719-12724Crossref PubMed Scopus (322) Google Scholar). Hence, we assumed that cathepsin B-mediated proteolysis of TGF-βRII might contribute to the suppression of liver fibrosis in the in vivo model. More surprisingly, NAC has the potential to reverse established liver fibrosis (Fig. 6, D–F). In this context, we assume that NAC may induce cathepsin B-mediated direct degradation of extracellular matrix. In fact, some studies showed that cathepsin B degraded extracellular matrix (26Buck M.R. Karustis D.G. Day N.A. Honn K.V. Sloane B.F. Biochem. J. 1992; 282: 273-278Crossref PubMed Scopus (367) Google Scholar). Leto et al. (27Leto G. Tumminello F.M. Pizzolanti G. Montalto G. Soresi M. Gebbia N. Oncology. 1997; 54: 79-83Crossref PubMed Scopus (59) Google Scholar) reported that in human serum the concentration of cathepsin B is significantly higher in patients with liver cirrhosis than in normal volunteers, indicating a clinical benefit of NAC for patients with liver fibrosis. We thank Yoshiko Takeda (Hiroshima Proteome Laboratory, Regional Science Promoter Program, JST) for valuable technical assistance in the analysis using a Q-TOF mass spectrometer." @default.
• W2066410366 created "2016-06-24" @default.
• W2066410366 creator A5003263577 @default.
• W2066410366 creator A5015020453 @default.
• W2066410366 creator A5024647870 @default.
• W2066410366 creator A5025363291 @default.
• W2066410366 creator A5029029456 @default.
• W2066410366 creator A5060407711 @default.
• W2066410366 creator A5085790083 @default.
• W2066410366 creator A5087434980 @default.
• W2066410366 date "2001-07-01" @default.
• W2066410366 modified "2023-09-27" @default.
• W2066410366 title "Regulation of Cell Growth by Redox-mediated Extracellular Proteolysis of Platelet-derived Growth Factor Receptor β" @default.
• W2066410366 cites W1537279861 @default.
• W2066410366 cites W1612998887 @default.
• W2066410366 cites W1791153000 @default.
• W2066410366 cites W1805256772 @default.
• W2066410366 cites W1965568495 @default.
• W2066410366 cites W1965957397 @default.
• W2066410366 cites W1987790381 @default.
• W2066410366 cites W1989318637 @default.
• W2066410366 cites W1991863368 @default.
• W2066410366 cites W1993562714 @default.
• W2066410366 cites W2000287530 @default.
• W2066410366 cites W2038348468 @default.
• W2066410366 cites W2038990415 @default.
• W2066410366 cites W2048569323 @default.
• W2066410366 cites W2055426473 @default.
• W2066410366 cites W2084026660 @default.
• W2066410366 cites W2099922198 @default.
• W2066410366 cites W2122530058 @default.
• W2066410366 cites W2150228169 @default.
• W2066410366 cites W2158979977 @default.
• W2066410366 cites W2783716685 @default.
• W2066410366 cites W4235424265 @default.
• W2066410366 cites W4300379263 @default.
• W2066410366 doi "https://doi.org/10.1074/jbc.m102995200" @default.
• W2066410366 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11346654" @default.
• W2066410366 hasPublicationYear "2001" @default.
• W2066410366 type Work @default.
• W2066410366 sameAs 2066410366 @default.
• W2066410366 citedByCount "41" @default.
• W2066410366 countsByYear W20664103662012 @default.
• W2066410366 countsByYear W20664103662013 @default.
• W2066410366 countsByYear W20664103662014 @default.
• W2066410366 countsByYear W20664103662021 @default.
• W2066410366 countsByYear W20664103662023 @default.
• W2066410366 crossrefType "journal-article" @default.
• W2066410366 hasAuthorship W2066410366A5003263577 @default.
• W2066410366 hasAuthorship W2066410366A5015020453 @default.
• W2066410366 hasAuthorship W2066410366A5024647870 @default.
• W2066410366 hasAuthorship W2066410366A5025363291 @default.
• W2066410366 hasAuthorship W2066410366A5029029456 @default.
• W2066410366 hasAuthorship W2066410366A5060407711 @default.
• W2066410366 hasAuthorship W2066410366A5085790083 @default.
• W2066410366 hasAuthorship W2066410366A5087434980 @default.
• W2066410366 hasBestOaLocation W20664103661 @default.
• W2066410366 hasConcept C170493617 @default.
• W2066410366 hasConcept C178790620 @default.
• W2066410366 hasConcept C181199279 @default.
• W2066410366 hasConcept C185592680 @default.
• W2066410366 hasConcept C2775960820 @default.
• W2066410366 hasConcept C2781307694 @default.
• W2066410366 hasConcept C28406088 @default.
• W2066410366 hasConcept C55493867 @default.
• W2066410366 hasConcept C55904794 @default.
• W2066410366 hasConcept C62112901 @default.
• W2066410366 hasConcept C86803240 @default.
• W2066410366 hasConcept C95444343 @default.
• W2066410366 hasConceptScore W2066410366C170493617 @default.
• W2066410366 hasConceptScore W2066410366C178790620 @default.
• W2066410366 hasConceptScore W2066410366C181199279 @default.
• W2066410366 hasConceptScore W2066410366C185592680 @default.
• W2066410366 hasConceptScore W2066410366C2775960820 @default.
• W2066410366 hasConceptScore W2066410366C2781307694 @default.
• W2066410366 hasConceptScore W2066410366C28406088 @default.
• W2066410366 hasConceptScore W2066410366C55493867 @default.
• W2066410366 hasConceptScore W2066410366C55904794 @default.
• W2066410366 hasConceptScore W2066410366C62112901 @default.
• W2066410366 hasConceptScore W2066410366C86803240 @default.
• W2066410366 hasConceptScore W2066410366C95444343 @default.
• W2066410366 hasIssue "30" @default.
• W2066410366 hasLocation W20664103661 @default.
• W2066410366 hasLocation W20664103662 @default.
• W2066410366 hasOpenAccess W2066410366 @default.
• W2066410366 hasPrimaryLocation W20664103661 @default.
• W2066410366 hasRelatedWork W2021099451 @default.
• W2066410366 hasRelatedWork W2068769994 @default.
• W2066410366 hasRelatedWork W2087714342 @default.
• W2066410366 hasRelatedWork W2312579622 @default.
• W2066410366 hasRelatedWork W2322840078 @default.
• W2066410366 hasRelatedWork W2396978324 @default.
• W2066410366 hasRelatedWork W2408218962 @default.
• W2066410366 hasRelatedWork W2886855338 @default.
• W2066410366 hasRelatedWork W2952478468 @default.
• W2066410366 hasRelatedWork W4232552561 @default.
• W2066410366 hasVolume "276" @default.
• W2066410366 isParatext "false" @default.

• next