ToxSci Advance Access originally published online on January 23, 2006
Toxicological Sciences 2006 90(2):470-477; doi:10.1093/toxsci/kfj096
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Src Tyrosine Kinases Mediate Crystalline Silica-Induced NF-
B Activation through Tyrosine Phosphorylation of IB-
and p65 NF-B in RAW 264.7 Macrophages
* Department of Physiology, Pharmacology, and Biochemistry, Division of Cell Biology, Ewha Medical Research Center, Ewha Womans University College of Medicine, Seoul, Korea
1 To whom correspondence should be addressed at Department of Physiology, College of Medicine, Ewha Womans University, 911–1 Mok-6-dong, Yangcheon-ku, Seoul 158–056, Korea. Fax: 82–2–2650–5717. E-mail: jihee{at}ewha.ac.kr.
Received October 6, 2005; accepted January 3, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein tyrosine kinases (PTKs) and mitogen-activated protein kinases (MAPKs) have been demonstrated to play a crucial role in the signaling pathways induced by silica. In the present study, we investigated whether Src family TKs play a role in crystalline silica-induced NF-
B activation and whether NF-B activation requires Src TK-dependent MAPK activity in RAW 264.7 cells, a mouse peritoneal macrophage cell line. Selective Src TK inhibitors, damnacanthal or PP1, inhibited silica-induced NF-B activation in a dose-dependent manner. Furthermore, these kinase inhibitors suppressed silica-induced tyrosine phosphorylation of IB-
and p65 NF-B. Within a similar time frame, c-Src and Lck were physically associated with IB- and with p65 NF-B. Silica stimulated the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), but not p38 MAPK and c-Jun NH2-terminal kinase 1 and 2 (JNK1/2). Damnacanthal or PP1 substantially blocked the silica-induced activation of ERK1/2. Moreover, PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 MAPK, failed to inhibit silica-induced NF-B activation. These results suggest that c-Src and Lck act for silica-induced NF-B activation by mediating the tyrosine phosphorylations of IB- and p65 NF-B. However, the Src TK-dependent activation of ERK1/2 may not be involved in the silica signaling pathway leading to NF-B activation.
Key Words: crystalline silica; Src tyrosine kinases; NF-B; mitogen activated protein kinases; RAW 264.7 macrophages.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The pulmonary deposition of crystalline silica can result in a cycle of lung damage, i.e., fibroblast proliferation and excess lung collagen production, causing lung fibrosis or silicosis (Craighead et al., 1998
). Evidence presented in recent years from epidemiologic and animal studies has also implicated crystalline silica as a potential carcinogen (Kuempel et al., 2001). The silicon-based free radicals Si·, SiO·, and SiOO· and reactive oxygen species (ROS) generated by silica-mediated reactions play a key role in silica-induced pathogenesis (Vallyathan et al., 1995). These radicals are also associated with the silica-induced activation of the nuclear transcription factor NF-B (Kang et al., 2000a).
NF-B controls a variety of genes involved in immune, inflammatory, and proliferative responses; these include various cytokines, cell-adhesion molecules, and growth factors (Barnes and Karin, 1997; Chen et al., 1999a). Moreover, recent strong evidence of NF-B involvement in lymphoma carcinogenesis has been collected (Ding et al., 2000).
Phosphorylation is an important event in NF-B activation at different levels. The exposure of T cells to hypoxia, reoxygenation, or pervanadate results in the phosphorylation of IB- at tyrosine 42 (Koong et al., 1994; Livolsi et al., 2001). This mechanism of NF-B activation involving tyrosine phosphorylation is quite distinct from the events involved in the serine phosphorylation of IB-, which leads to IB- degradation through the proteasome pathway. Our previous study also supports this mechanism of NF-B activation by silica in RAW 264.7 macrophages via the tyrosine phosphorylation of IB- in the absence of serine phosphorylation (Kang et al., 2000b). In addition to IB-, we found that silica also induced the tyrosine phosphorylation of the p65 subunit of NF-B (Kang et al., 2003). Moreover, tyrosine phosphorylation of p65 NF-B efficiently modulates its transcription activity (Ghosh et al., 1998, Pellegatta et al., 2003).
The Src family tyrosine kinases (TKs) have been shown to play key roles in oxidants-mediated signal transduction in lymphocytes and macrophages (Khadaroo et al., 2003; Nishida et al., 2000; Schieven et al., 1993). The activations of c-Src (Abu-Amer et al., 1998) and Lck (Livolsi et al., 2001) are required for the tyrosine phosphorylations of IB- and NF-B activation in bone marrow macrophages and T cells stimulated with TNF or pervanadate, respectively. The roles of these TKs in silica-induced NF-B activation through the tyrosine phosphorylation of IB- and p65 NF-B are as yet unknown.
Another group of oxidant-dependent signaling molecules potentially important in silica-induced inflammation and proliferative responses are mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), c-Jun-N-terminal kinase 1 and 2 (JNK1/2), and p38 MAPK (Ding et al., 1999, Shukla et al., 2001). Silica has been shown to induce transcription factor activator protein-1 (AP-1) through p38 MAPK and ERK1/2 (Ding et al., 1999). Furthermore, the activation of Src TKs has been reported to be an essential link to Ras signaling, the stimulation of the Ras-Raf-ERK pathway, and the transcriptional transactivation mediated by (AP)-1 (Klein and Schneider, 1997). In addition to AP-1, the MAPK signaling pathway has been indicated to play role in NF-B activity through serine phosphorylation of IB-, leading to IB- degradation via the mediation of IB kinase (IKK) 1 and/or 2 (Chen et al., 1999b; Dhawan and Richmond, 2002; Mukundan et al., 2004; Rangaswami et al., 2004). However, it has not been elucidated whether these MAPKs are involved in NF-B activation in association with condition such as the silica-induced tyrosine phosphorylation of IB- and p65 NF-B. We address this question in the present study.
Thus, in the present study, we investigated (1) whether the Src-related kinases, namely, c-Src and Lck, are required for silica-induced activation of NF-B by mediating the tyrosine phosphorylation of IB- and/or p65 NF-B in RAW 264.7 macrophages, and (2) whether NF-B activation requires the activities of MAPK family members regulated by Src TKs signaling in response to silica.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents.
Crystalline silica (Min-U-Sil, particle size < 5 µm) was obtained from the U.S. Silica Corporation (Berkeley Springs, WV). Prior to use, silica samples were sterilized by heating at 160°C for 90 min in a dry oven. Silica particles were then dispersed in DMEM (Life Technologies, Inc, Madison, WI) containing supplements just before being added to culture plates. The antibodies used in this study were: anti-I
B- rabbit polyclonal, anti-phospho-ERK1/2/ERK1/2, phospho-p38 MAPK/p38 MAPK, phospho-JNK1/2/JNK1 (New England Biolabs, Inc., Beverly, MA), anti-phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-phospho-c-Src (Tyr416), anti-c-Src, anti-Lck, and anti-p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Damnacanthal and PP1(4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) were purchased from Biomol Company (Plymouth Meeting, PA). LPS (Escherichia coli lipopolysaccharide, 055:B5), SB203580, and PD98059 were purchased from the Sigma Chemical Company (St. Louis, MO), and DNA polymerase and dNTP from Life Technologies (Gaithersburg, MD).
Cell line and cell culture.
RAW 264.7 cells, a mouse peritoneal macrophage cell line, were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in DMEM supplemented with 5% fetal bovine serum (FBS) (HyClone, Logan, UT), 2 mM glutamine, and 1,000 units/ml penicillin-streptomycin.
Nuclear extract.
RAW 264.7 cells were cultured in six-well plates at 5 x 106 cells/ml for 2 days. The medium was then replaced with fresh medium, and cells were pretreated with the inhibitors of protein kinases, such as damnacanthal (1.7–170 nM), PP1 (1–100 nM), PD98059 (10–50 µM), or SB203580 (10–50 µM) for 1 or 2 h. These kinase inhibitors have been used previously (Kang et al., 2005; Livolsi et al., 2001; Øvrevik et al., 2004), and significant toxicity was not noted. Cells were then cultured for 4 h with silica (50 µg/cm2) in the absence or presence of an inhibitor. The concentrations of silica and the duration of exposure used in this investigation were determined by previous concentration-response and time-course studies on NF-B activation (Kang et al., 2000a). After 4 h of exposure, the cells were harvested and resuspended in hypotonic buffer A (100 mM HEPES, pH 7.9; 10 mM KCl; 0.1 M EDTA; 0.5 mM dithiothreitol; 1% nonidet P-40; and 0.5 mM phenylmethylsulfonyl fluoride) for 10 min on ice and then vortexed for 10 s. Nuclei were pelleted by centrifugation at 12,000 x g for 30 s and then resuspended in buffer C (20 mM HEPES, pH 7.9; 20% glycerol; 0.42 M NaCl; 1 mM EDTA; and 0.5 mM PMSF) for 30 min on ice. Supernatants containing nuclear proteins were collected by centrifugation at 10,000 x g for 2 min and stored at –70°C.
Electrophoretic mobility shift assay (EMSA).
Binding reaction mixtures (10 µl), containing 5 µg (4 µl) of nuclear extract protein, 2 µg of poly (dI-dC)·poly (dI-dC) (Sigma Co., St. Louis. MO), and 40,000 cpm of 32P-labeled probe in binding buffer (4 mM HEPES pH 7.9; 1 mM MgCl2; 0.5 mM DTT; 2% glycerol; and 20 mM NaCl), were incubated for 30 min at room temperature. Protein-DNA complexes were separated on 5% nondenaturing polyacrylamide gels in 1x Tris-borate/EDTA electrophoresis buffer and autoradiographed overnight.
The oligonucleotide used as a probe for EMSA was a double-stranded DNA, containing the NF-B consensus sequence (5'-CTGTGCTCCGGGAATTTCCCTG-GCC-3') labeled with -32P-dATP (Amersham, Buckinghamshire, UK) using a DNA polymerase Klenow fragment.
Immunoprecipitation.
Confluent cells grown on 100-mm plastic dishes were incubated in DMEM supplemented with 5% FBS, 2 mM glutamine, and 1,000 units/ml penicillin–streptomycin for 3 days. Cells were then treated with silica in the presence or absence of damnacanthal or PP1 and washed with ice-cold phosphate buffered saline (pH 7.4). The washed cells were lysed with 1 ml of ice-cold lysis buffer, containing 50 mM Tris–HCl (pH 8), 150 mM NaCl, 1% nonidet P-40 (NP-40), 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, 1 mM Na3VO4, 5 mM EDTA, and 1 mM benzamidine.
Cell lysates were centrifuged for 5 min at 13,000 x g, and the resulting supernatant was incubated with anti-IB-, anti-p65, or anti-Lck polyclonal at 4°C for 1 h and then at 4°C for 30 min with protein A or G conjugated sepharose (5 µg/ml). The antigen/antibody complexes formed were pelleted by centrifugation at 13,000 x g for 30 s. Pellets were washed three times with ice-cold lysis buffer by centrifugation at 13,000 x g for 30 s, dissolved in 20 µg of Laemmli's sample buffer, and separated on 10% SDS–polyacrylamide gels.
Western blotting.
Immunoprecipitated proteins or total cell lysates were resolved on 10% SDS–polyacrylamide gels and electrophoretically transferred onto a nitrocellulose paper. Antibodies used for immunoblotting these samples were anti-phospho-c-Src (Tyr416), anti-c-Src, anti-Lck, anti-phospho-ERK1/2/ERK1/2, anti-phospho-p38 MAPK/MAPK, anti-phospho-JNK1/2/JNK1, and anti-phosphotyrosine antibody. The antibody labeling of protein bands was detected using enhanced chemiluminescence (ECL) reagents according to the supplier's protocol.
Statistics.
Values are expressed as means ± standard errors. Data were analyzed using one-way analysis of variance (ANOVA) and followed by a Tukey's post hoc test. Significance was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Selective Src Kinase Inhibitors, Damnacanthal and PP1, Block Silica-Induced NF-
B Activation and the Tyrosine Phosphorylations of IB- and p65 NF-BWe investigated whether Src TKs are required for tyrosine phosphorylation–dependent NF-
B activation at the level of IB- or p65 NF-B in silica-stimulated RAW 264.7 macrophages. To address this question, we used the selective Src TK inhibitors, damnacanthal and PP1. RAW 264.7 macrophages were pretreated for 2 h with different concentrations of these inhibitors and then examined for NF-B activation in response to silica (50 µg/cm2) for 4 h at 37°. Damnacanthal (1.7–170 nM) or PP1 (1–100 nM) inhibited the silica-induced binding activity of NF-B to DNA in a dose-dependent manner (Figs. 1A and 1B). The inhibition of 68% was shown by 170 nM damnacanthal and of 95% by 100 nM of PP1.
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An analysis of the tyrosine phosphorylation of I
B- indicated that damnacanthal (170 nM) or PP1 (100 nM) significantly inhibited the silica-induced tyrosine phosphorylation of IB- by approximately 75 and 65%, respectively (Fig. 2A, p < 0.05). In addition, Figure 2B shows significant inhibition of the tyrosine phosphorylation of p65 in cells exposed to silica for 20 min following preincubation with damnacanthal (170 nM) or PP1 (100 nM) (60 and 63% inhibition, respectively, p < 0.05). These findings suggest that Src TK activation results in the increased tyrosine phosphorylations of IB- and p65 NF-B and, thus, contributes to NF-B activation in silica-treated cells.
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c-Src and Lck Directly Interact with I
B- and p65 NF-BPP1 is selectively capable of inhibiting c-Src and Lck at nanomolar concentrations. Damnacanthal is a potent and selective inhibitor of Lck (Faltynek et al., 1995
). Therefore, we first examined whether silica can induce the activation of these Src-related kinases in RAW 264.7 macrophages. Cell lysates were analyzed by Western blotting using a phospho-specific c-Src (Tyr416) antibody, or were used for immunoprecipitation with anti-Lck followed by Western blot analysis with anti-phosphotyrosine mAb at various times after stimulating RAW 264.7 cells with silica. As shown Figure 3A, the phosphorylation of c-Src at Tyr416, an index of c-Src activation, was observed 5 min after silica stimulation, and this further increased after 20 min of silica exposure. Significant increases in the tyrosine phosphorylation of Lck were also observed after 5–20 min of silica stimulation (Fig. 3B, p < 0.05). The ability of silica to cause Lck tyrosine phosphorylation is consistent with its potential contribution to the activation of downstream signaling (Budagian et al., 2003). Basal levels of c-Src in cell lysates and Lck in protein immunoprecipitated with anti-Lck were determined by immunoblotting with c-Src- or Lck-specific antibodies after stimulating RAW 264.7 cells with silica various times.
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We further tested the direct connection of these Src kinases to I
B- or p65 NF-B by examining their interaction in silica-stimulated RAW 264.7 cells. Cell lysates from silica-treated or untreated cells were then immunoprecipitated with IB-- or p65 NF-B-specific antibody followed by Western blot analysis with phospho-c-Src (Tyr416) or specific Lck antibody. The interaction between phospho-c-Src or Lck and IB- reached a peak approximately 20 min after silica stimulation and then declined up to 60 min (Figs. 4A and 4B). Phospho-c-Src and Lck were also found to be physically associated with p65 NF-B. The strong association between phospho-c-Src and p65 NF-B occurred after 10 min of silica stimulation (Fig. 5A), and for Lck this association reached a peak after 30 min of silica stimulation (Fig. 5B).
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ERK1/2 Activity Regulated by Src TK Signaling May Not Be Involved in Silica-Induced NF-
B ActivationAlthough the activation of Src TKs is indispensable for the MAPK pathway (Kitagawa et al., 2002
; Liu et al., 2001; Mukundan et al., 2004; Scapoli et al., 2004; van Vliet et al., 2005), it is likely that MAPKs are not involved in silica-induced NF-B activation, since the NF-B activation pathway in response to silica is dependent on tyrosine phosphorylation on IB- and p65 NF-B. To examine this hypothesis, we first sought to identify the MAP kinases among ERK1/2, p38 MAPK, and JNK1/2 that are activated in RAW 264.7 macrophages in response to silica, and then to determine whether the activation of NF-B activation induced by silica is mediated through a MAPK-dependent signal transduction pathway. Cell lysates were analyzed by Western blot analysis using specific Abs for the above MAPK family members and phospho-specific Abs for phosphorylated MAPK family members after stimulating RAW 264.7 cells with silica. Phosphorylated ERK1/2 levels peaked after 20 min of silica exposure (p < 0.05) and then decreased up to 90 min (Fig. 6A). In contrast, silica did not affect the phosphorylation levels of p38 MAPK or JNK1/2 after exposure for 20 to 90 min, whereas LPS, a known MAPK stimulant, induced significant increases in the phosphorylations of these kinases (Figs. 6B and 6C). Pretreatment with damnacanthal or PP1 resulted in significant inhibitions in the phosphorylation of ERK1/2 in cells exposed to silica for 20 min (Fig. 6D, p < 0.05).
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RAW 264.7 cells were preincubated with different concentrations of a selective MAP kinase (MEK) inhibitor upstream of ERK1/2 (PD98059, 10–50 µM), or with a selective inhibitor of p38 MAPK (SB203580, 10–50 µM), and then examined for NF-
B activation induced by exposure to silica for 4 h. Both PD98059 and SB203580 failed to inhibit silica-induced NF-B activation (Figs. 7A and 7B). These data suggest that the activation of ERK1/2 mediated by Src TKs is not involved in silica-induced NF-B activation in RAW 264.7 macrophages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Src TKs and MAPKs are redox sensitive (Khadaroo et al., 2003
; Nishida et al., 2000) and activated in response to silica (Ding et al., 1999; Øvrevik et al., 2004; Shukla et al., 2001). Their signaling pathways link to activation of nuclear transcription factors, leading to the induction of early response genes that are critical in inflammation and carcinogenesis. However, whether these kinases are required for silica-induced NF-B activation in RAW 264.7 macrophages has not been elucidated. Therefore, we examined first whether Src TKs play a role in the silica induction of the pathway leading to NF-B activation. In the present study, selective Src kinase inhibitors such as damnacanthal and PP1 were used, since damnacanthal inhibits Lck autophosphorylation (IC50 = 17 nM) as well as phosphorylation of an exogenous peptide by Lck (IC50 = 620 nM) (Faltynek et al., 1995), and PP1 inhibits protein tyrosine phosphorylation induced by Src tyrosine kinases at nanomolar concentrations (Hanke et al., 1996). These inhibitors significantly inhibited silica-induced NF-B activation and the tyrosine phosphorylation of the NF-B proteins. These data suggest that Src TKs are required for the tyrosine phosphorylation–dependent activation of NF-B in RAW 264.7 macrophages in response to silica.
It has been reported that Src TKs play an important downstream role in a variety of stimulant-induced NF-B-dependent responses (Huang et al., 2003; Liu et al., 2001; Orlicek et al., 1999). Consistent with these findings, Øvrevik et al. (2004) reported that PP2, a selective Src TK inhibitor, efficiently inhibited silica-induced IL-8 production in the human epithelial lung cell line A549. These data suggest the functional involvement of Src TKs in the silica signaling pathway cascade, leading to the activation of NF-B-dependent pathways and to the resultant production of inflammatory mediators.
c-Src and Lck have been shown to be required for NF-B activation through the tyrosine phosphorylation of IB- in TNF- and pervanadate-stimulated cells (Abu-Amer et al., 1998; Livolsi et al., 2001). Therefore, c-Src and Lck are likely candidates in the silica-induced tyrosine phosphorylation of IB- in RAW 264.7 macrophages. Thus, in the present study we further investigated the ability of silica to induce the activations of these Src TKs in RAW 264.7 cells. c-Src phospho-Tyr416 levels and the tyrosine phosphorylation of Lck by RAW 264.7 cells were increased after silica stimulation.
Data from the present study indicate that phospho-c-Src and Lck interact with IB- and p65 NF-B. In addition, the time frames of the phosphorylations of c-Src or Lck in response to silica were similar to the time frames of interaction between these kinases and IB- or p65 NF-B. Accordingly, Mahabeleshwar and Kundu (2003) and Fan et al. (2003) reported that pervanadate, H2O2, and hypoxia/reoxygenation induce an interaction between c-Src or Lck and tyrosine-phosphorylated IB-. Taken together, we suggest a physiological relationship between the Src TK-mediated tyrosine phosphorylation of these NF-B proteins and the activation of NF-B in response to silica.
Src TKs have been shown to regulate MAPK activation in response to ROS (Nishida et al., 2000) and other extracellular stimuli (Kitagawa et al., 2002; Scapoli et al., 2004). In addition, Src-dependent ERK1/2 activation has been demonstrated in pulmonary epithelial cells in response to silica (Øvrevik et al., 2004). In the present study, therefore, we further evaluated the role of these MAPKs as the downstream effector molecules of Src TK signaling pathway leading to NF-B activation in RAW 264.7 macrophages. Consistently, we found that Src TK signaling regulates silica-induced ERK1/2 activation. However, ERK1/2 were not involved in Src TK-mediated NF-B activation in response to silica. In contrast, ERK1/2 were involved in Src TK-mediated IB- kinase (IKK)/NF-B pathway, including serine phosphorylation of IB- in response to IFN
(Mukundan et al., 2004). These results support our hypothesis that ERK1/2 may not be involved in NF-B activation through the tyrosine phosphorylation of IB-. Indeed, silica does not induce the serine phosphorylation of IB- (Kang et al., 2000b). Taken together, the difference in the regulation of NF-B phosphorylation at different sites in IB- could cause NF-B transcriptional specificity and thereby account for the varied role of NF-B in different diseases.
In conclusion, our findings suggest that Src TKs, such as c-Src and Lck, are key components in silica signaling pathways leading to NF-B activation by mediating the tyrosine phosphorylations of IB- and p65 NF-B. In addition, Src TK-dependent ERK1/2 activation in RAW 264.7 cells in response to silica may not be involved in NF-B activation. Understanding the silica-induced Src TK-dependent signaling pathway leading to NF-B activation is of basic importance and presents a new potential target for the prevention and therapy of both inflammatory and proliferative diseases.
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ACKNOWLEDGMENTS |
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We thank H. S. Lee for expert assistance of the data analysis. This work was supported by grant No. R04-2002–000–00023–0 from the Basic Research Program of the Korea Science & Engineering Foundation and a Korea Research Foundation Grant (KRF-2001–015-FP0055).
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