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INFLAMMATION AND IMMUNOPHARMACOLOGY
B Activation and Phosphorylation of p38 Mitogen-Activated Protein Kinase, Extracellular Signal-Regulated Kinase, and c-Jun N-Terminal Kinase in Human Mast Cell Line Cells
College of Oriental Medicine, Kyung Hee University, Dongdaemun-Gu, Seoul, Republic of Korea (S.-J.K., H.-J.J., I.-Y.C., H.-M.K.); Division of Biological Sciences, College of Natural Science, Chonbuk National University, Jeonju, Jeonbuk, Republic of Korea (S.-J.K., K.-M.L.); and Department of Microbiology and Immunology (R.-K.P.) and College of Pharmacy (H.-J.J., I.-Y.C., S.-H.H.), VestibuloCochlear Research Center of Wonkwang University, Iksan, Jeonbuk, Republic of Korea
Received December 23, 2004; accepted March 18, 2005.
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, interleukin (IL)-6, IL-8, vascular endothelial growth factor, COX-2, inducible nitric-oxide synthase, and hypoxia-inducible factor-1 in phorbol 12-myristate 13-acetate plus calcium ionophore A23187
[GenBank]
(PMACI)-stimulated HMC-1. SC-236 suppressed nuclear factor (NF)-
B activation induced by PMACI, leading to suppression of IB- phosphorylation and degradation. SC-236 also suppressed strong induction of NF-B promoter-mediated luciferase activity. In addition, SC-236 suppressed PMACI-induced phosphorylation of the mitogen-activated protein kinase p38, the extracellular-regulated kinase p44, and the c-Jun N-terminal kinase and induced expression of mitogen-activated protein kinase phosphatase-1. These results provide new insight into the pharmacological actions of SC-236 as a potential molecule for therapy of mast cell-mediated inflammatory diseases.
, interleukin (IL)-6, and IL-8 (Gordon et al., 1990
; Murakami et al., 1995). Chronic synthesis and release of TNF- from mast cells may maintain leukocyte migration and promote chronicity in inflammatory lesions (Walsh et al., 1995). IL-8 from mast cells acts on surrounding cells such as neutrophils, T-lymphocytes, and eosinophils and plays a role in activation of inflammatory effector cells (Mukaida, 2000). Although these cytokines are beneficial to the host defense, they can also trigger pathological conditions when expressed in excess. Mast cells can also contribute to various aspects of angiogenesis through the production of vascular endothelial growth factor (VEGF) (Abdel-Majid and Marshall, 2004). Angiogenesis is accompanied by inflammation (Risau, 1997).
In recent years, it has been demonstrated that both cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) play important roles in various tumors and inflammatory diseases (Kong et al., 2002). COX-2, one of the major mediators of the inflammatory reaction, is also strongly induced in activated monocytes/macrophages. Several recent studies demonstrated that PGD2, which is the COX-2 metabolite released from activated mast cells, is also essential for the pathogenesis of eosinophilic airway inflammation (Bochenek et al., 2004). Previously, it was reported that COX-2 inhibitors abolished PGD2 synthesis and attenuated eosinophils accumulation in the airways inflammation (Oguma et al., 2002). Hypoxia-inducible factor (HIF)-1, a transcription factor, is produced or activated in response to hypoxia. There is growing evidence that HIF-1 is involved in the inflammatory process by regulating angiogenesis, and it has been identified as a pivotal transcription factor linking the inflammatory and oncogenic pathways (Jung et al., 2003).
Nuclear factor (NF)-B is a key transcription factor required for the expression of many inflammatory involved genes including COX-2, iNOS, HIF-1, and inflammatory cytokines (Jung et al., 2003). In an inactive state, NF-B is normally sequestered in the cytoplasm of cells, where it is bound by a family of inhibitory proteins known as IB- (Barnes and Karin, 1997). A variety of stimuli modulate signal transduction pathways to activate IB kinases. Stimulation of the activity of these kinases results in phosphorylation, ubiquitination, and degradation of IB-, leading to the nuclear translocation of NF-B (Schaecher et al., 2004). Most agents that activate NF-B mediate their effects through suppression of IB- phosphorylation and degradation (Yamamoto et al., 1999).
In mammalian cells, three major mitogen-activated protein kinases (MAPKs) have been defined: the extracellular signal-regulated kinase (ERK) pathway, the stress-activated pathways of the c-Jun N-terminal kinase (JNK), and the p38 MAPK (Cobb and Goldsmith, 2000). These pathways are central components of the intracellular signaling networks that control many aspects of mammalian cellular physiology including cell proliferation, differentiation, and apoptosis (Lewis et al., 1998). Activation of MAPK results ultimately in the direct or indirect phosphorylation and/or activation of various transcription factors such as Ets-like protein-1, activated protein (AP)-1, activating transcription factor, and alterations in gene expression.
MAPK phosphatase (MKP)-1 is a critical negative regulator in response to inflammatory stimuli and is responsible for switching off the production of proinflammatory cytokines. The three MAP kinases are normally dephosphorylated by MKP-1 (Sakaue et al., 2004). It was reported that inhibition of MKP-1 expression prolonged cytokine production-induced p38 and JNK kinase phosphorylation (Wadgaonkar et al., 2004). Several studies demonstrated that MKP-1 is induced by certain anti-inflammatory drugs/agents and proposed that MKP-1 could be a target for developing novel anti-inflammatory drugs (Chen et al., 2002; Jeong et al., 2003).
A COX-2 inhibitor has recently been approved for the treatment of colon carcinogenesis, gastric cancer, rheumatoid arthritis, and other inflammatory diseases (Kawamori et al., 1998; Everts et al., 2000). SC-236, a COX-2 inhibitor, is a 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1-pyrazol-1-l]benzenesulfonamide compound that is a highly selective and potent COX-2 inhibitor (with an IC50 = 10 nM for COX-2, compared with IC50 = 17.8 µM for COX-1) with antitumor properties (Castano et al., 2000). Recently, there were reports that SC-236 protects against cartilage damage in addition to reducing inflammation and pain in osteoarthritis (Hardy et al., 2002). However, the mechanism involved in the anti-inflammatory effect of SC-236 has not been examined.
To elucidate the mechanism of SC-236 that accounts for its anti-inflammatory effect, we examined the effect of SC-236 on inflammatory gene expression, the NF-B pathway in 12-myristate 13-acetate (PMA) plus calcium ionophore A23187
[GenBank]
(PMACI)-stimulated human mast cell line HMC-1. In addition, we investigated the effects of SC-236 on MAPK activation and MKP-1 expression.
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and VEGF antibody (Ab), biotinylated anti-human TNF- Ab, VEGF, recombinant human TNF-, and VEGF were purchased from R&D Systems (Minneapolis, MN). Anti-human IL-6, IL-8 Ab, biotinylated anti-human IL-6, IL-8 Ab, recombinant human IL-6, and IL-8 were obtained from BD Biosciences PharMingen (San Diego, CA). Abs for anti-human NF-B, IB-, pp38, JNK, ERK, and actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cell Culture. HMC-1 was grown in Iscove's modified Dulbecco's medium supplemented with 100 unit/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum at 37°C under 5% CO2 in air.
MTT Assay. To test the viability of cells, an MTT colorimetric assay was performed as described previously (Kim et al., 2001). Briefly, HMC-1 cells (3 x 105 cells/ml) were incubated for 8 h after stimulation in the absence or presence of SC-236. After addition of MTT solution, the cells were incubated at 37°C for 4 h. The crystallized MTT was dissolved in dimethyl sulfoxide, and the absorbance was measured at 540 nm.
Cytokine Assay. TNF-, IL-6, IL-8, and VEGF secretion were measured by a modified ELISA, as described previously (Kim et al., 2001). Ninety-six-well plates were coated with 100-µl aliquots of anti-human TNF-, IL-6, IL-8, and VEGF monoclonal Abs, respectively, at 1.0 µg/ml in phosphate-buffered saline (PBS) at pH 7.4 and were incubated overnight at 4°C. After additional washes, 100 µl of cell medium or TNF-, IL-6, IL-8, and VEGF standards were added and incubated at 37°C for 2 h. After 2 h of incubation at 37°C, the wells were washed; then, 0.2 µg/ml biotinylated anti-human TNF-, IL-6, IL-8, and VEGF, respectively, was added and again incubated at 37°C for 2 h. After washing the wells, avidin-peroxidase was added, and the plates were incubated for 30 min at 37°C. Wells were again washed, and 2,2-azio-bis (3-ethylbenzthiazoline-6-sulfonic acid) substrate was added. Color development was measured at 405 nm using an automated microplate ELISA reader. A standard curve was run on each assay plate using recombinant TNF-, IL-6, IL-8, and VEGF in serial dilutions. The inhibition percentage of cytokines release was calculated using % inhibition = (A - B) x 100/A, where A is cytokine release without SC-236 and B is cytokine release with SC-236.
RNA Isolation and RT-PCR. Total RNA was isolated from HMC-1 according to the manufacturer's specifications using an easy-BLUE RNA extraction kit (iNtRON Biotech, Seoul, Korea). The concentration of total RNA in the final elutes was determined by spectrophotometry. Total RNA (2.0 µg) was heated at 65°C for 10 min and then chilled on ice. Each sample was reverse-transcribed to cDNA for 90 min at 37°C using a cDNA synthesis kit (Amersham Biosciences Inc., Piscataway, NJ). RT-PCR was carried out with 1 µl of a cDNA mixture, in 20-µl final volume with 2.5 mM MgCl2, 200 mM dNTPs, 25 pM cytokine primers, and 2.5 U of TaqDNA polymerase in the reaction buffer (50 mM KCl, 10 mM Tris-HCl, pH 9, and 0.1% Triton X-100). PCR was performed with the following primers for human TNF- (5'-CAC CAG CTG GTT ATC TCT CA-3' and 5'-CGG GAC GTG GAG CTG GCC GAG GAG-3'), IL-8 (5'-CGA TGT CAG TGC ATA AAG ACA-3' and 5' TGA ATT CTC AGC CCT CTT CAA AAA-3'), IL-6 (5' GAT GGA TGC TTC CAA TCT GGA T-3' and 5'-AGT TCT CCA TAG AGA ACA ACA TA -3'), VEGF (5'-CGG GAT CCC GAT GAA CCT TCT GCT GTC TTG GGT-3' and 5'-CGG AAG CCC GTC ACC GCC TCG GCT TGT-3'), and GAPDH (5'-CAA AAG GGT CAT CAT CTC TG -3' and 5'-CCT GCT TCA CCA CCT TCT TG-3'), which were used to verify if equal amounts of RNA were used for reverse transcription and PCR amplification from different experimental conditions. The annealing temperature was 60°C for TNF-, 60°C for IL-8, 50°C for IL-6, 57°C for VEGF, and 62°C for GAPDH, respectively. Products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide. The relative mRNA amounts were estimated by an FC-26WL image analyzer (Vilber Lourmat, Marne La Vallée, France).
Preparation of Cytoplasmic and Nuclear Extract. Nuclear and cytoplasmic extracts were prepared as described previously (Schoonbroodt et al., 1997). Briefly, after cell activation for the times indicated, cells were washed with ice-cold PBS and resuspended in 60 µl of buffer A (10 mM HEPES/KOH, 2 mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride, pH 7.9). The cells were allowed to swell on ice for 15 min, lysed gently with 2.5 µl of 10% Nonidet P-40, and centrifuged at 2000g for 10 min at 4°C. The supernatant was collected and used as the cytoplasmic extracts. The nuclei pellet was resuspended in 40 µl of buffer B (50 mM HEPES/KOH, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride, pH 7.9), left on ice for 20 min, and inverted. The nuclear debris was then spun down at 15,000g for 15 min. The supernatant (nuclear extract) was collected, frozen in liquid nitrogen, and stored at -70°C until conducting the analysis.
Western Blot Analysis. For analysis of the levels of iNOS, COX-2, HIF-1, NF-B, p-IB-, IB-, p38, p-p38, ERK, p-ERK, JNK, and p-JNK, stimulated cells were rinsed twice with ice-cold PBS and were then lysed in ice-cold lysis buffer (1% Triton, 1% Nonidet P-40, 0.1% SDS, and 1% deoxycholate in PBS). Cell lysates were centrifuged at 15,000g for 5 min at 4°C; the supernatant was then mixed with an equal volume of 2x SDS sample buffer, boiled for 5 min, and then separated through 10% SDS-polyacrylamide gel electrophoresis gels. After electrophoresis, the protein was transferred to nylon membranes by electrophoretic transfer. The membranes were blocked in 5% skim milk for 2 h, rinsed, and incubated overnight at 4°C with primary antibodies in PBS/0.5% Tween 20. Excess primary antibody was then removed by washing the membranes four times in PBS/0.5% Tween 20, and the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (against mouse, goat, or rabbit). After three washes in PBS/0.5% Tween 20, the protein bands were visualized by an enhanced chemiluminesence assay (Amersham Biosciences Inc.) following the manufacturer's instructions.
Transient Transfection and Luciferase Assay. NF-B luciferase reporter gene constructs (pNF-kB-LUC, plasmid containing NF-B binding site; STANTAGEN, Grand Island, NY) were transiently transfected into HMC-1 cells by using Profection Mammalian Transfection System-Calcium phosphate reagent (Promega, Madison, WI). After 48 h, transfected HMC-1 cells (3 x 105) were plated and stimulated with PMACI. SC-236 was added 2 h before stimulation. Cells were harvested after 24-h stimulation and washed in cold PBS before lysis in 100 µl of lysis buffer (Luciferase assay kit; Promega). Luciferase activity was measured with a MicroLumat Plus luminometer, according to the manufacturer's protocol. All transfection experiments were performed in triplicate for at least four separate experiments, with similar results. To control for differences in the uptake of transfected DNA, cells were plated after transfection. Protein content was determined by using bicinchoninic acid solution reagent. This amount will vary depending on the strength of the promoter being studied, and to a lesser extent, on the efficiency of transfection in individual experiments. Luciferase activity was divided by total protein and expressed as relative light units per milligram of protein in the cell lysate.
Immunocytochemistry and Confocal Microscopy. HMC-1 cells were fixed with 3% paraformaldehyde and incubated with 5% bovine serum albumin (BSA) in PBS for 60 min. The preparation was then incubated for 1 h at room temperature with the MKP-1 Ab diluted in 0.1% BSA (1:500). The preparation was subsequently washed three times with PBS and then exposed to the secondary Ab (fluorescein isothiocyanate-conjugated anti-rabbit IgG at 1:200 and 0.1% BSA/PBS) for 60 min. The fluorescent image was viewed with an Olympus confocal microscope (Olympus, Tokyo, Japan).
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, IL-6, IL-8, and VEGF production was considerably increased after stimulation with PMACI in HMC-1. Pretreatment of SC-236 (25–50 µM) significantly inhibited these increases in a concentration-dependent manner (P < 0.05). The maximal inhibition of TNF-, IL-6, IL-8, and VEGF production by SC-236 (50 µM) was approximately 47% (P < 0.05), 42% (P < 0.05), 38% (P < 0.05), and 66% (P < 0.05), respectively.
The enhanced TNF-, IL-6, IL-8, and VEGF mRNA expression induced by PMACI was also inhibited by pretreatment of SC-236 (Fig. 1B). Cell cytotoxicity by SC-236 was not observed (data not shown).
Effect of SC-236 on Inflammatory Gene Expression in PMACI-Stimulated HMC-1. To determine the effect of SC-236 on inflammatory gene expression induced by PMACI, Western blot analyses for COX-2, iNOS, and HIF-1 were performed. The cells were pretreated with SC-236 for 2 h and then treated with PMACI for 24 h. As shown in Fig. 2A, we observed an induction of the COX-2 and iNOS expression by PMACI treatment. SC-236 inhibited COX-2 and iNOS expression levels induced by PMACI.
We investigated whether SC-236 can modulate HIF-1 expression. First, the time course of the PMACI-induced HIF-1 expression in HMC-1 cells is shown in Fig. 2B. HIF-1 expression was transient and peaked at 1 to 4 h after addition of PMACI. SC-236 (25–50 µM) decreased HIF-1 expression induced PMACI at 4 h (Fig. 2C).
Effect of SC-236 on NF-B Activation, IB- Phosphorylation, and Degradation in PMACI-Stimulated HMC-1. To elucidate the effect of SC-236 on NF-B activation, we examined the effect of SC-236 on the cytosolic and nuclear pool of RelA/p65 protein by Western blot analysis in HMC-1. PMACI treatment considerably increased the nuclear RelA/p65 protein level and decreased the cytosolic RelA/p65, which is an indication of the nuclear translocation of RelA/p65. SC-236 (25–50 µM) inhibited the increased nuclear RelA/p65 levels and increased the cytosolic RelA/p65 levels, respectively (Fig. 3A). To determine whether SC-236 is an inhibitor of NF-B-mediated gene transcription, HMC-1 was transfected with the NF-B-luciferase reporter plasmid for 48 h. Transfected cells were incubated with 50 µM SC-236 for 2 h, and then stimulated by PMACI for an additional 24 h. SC-236 significantly inhibited PMACI-stimulated NF-B-mediated transcription activity in HMC-1 (Fig. 3B).
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Effect of SC-236 on MKP-1 Induction. To determine whether SC-236 can modulate MKP-1 expression, a Western blot analysis was performed. Data in Fig. 5A show that treatment of SC-236 (25–50 µM) for 2 h induced MKP-1 expression. Expression of MKP-1 in HMC-1 was visualized using confocal microscopy. Figure 5B shows that SC-236 (50 µM) induced an increase of MKP-1 expression.
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, IL-8, VEGF, COX-2, iNOS, and HIF-1 expression in PMACI-simulated HMC-1 cells. Furthermore, SC-236 suppressed activation of NF-B and MAPK and SC-236-induced MKP-1 expression.
At local inflammatory sites, infiltrated mast cells and their mediators may contribute to the initiation and progression of the distributive inflammatory process (Marone, 1998). VEGF expression in mast cells may be of particular importance at sites of chronic inflammation in which both mast cells numbers and PGE2 levels are elevated (Abdel-Majid and Marshall, 2004). In inflammation, infiltrating inflammatory cells and some resident cells produce VEGF (Iijima et al., 1996). COX-2 is an inducible enzyme found at low concentration in healthy tissues, but it is up-regulated in response to tissue damage during inflammation. It has been reported that COX-2, one of the major mediators of the inflammatory reaction, is also strongly induced in activated monocytes/macrophages. Several studies demonstrated that PGD2, which is the major COX-2 metabolite released from activated mast cells, is also essential for the pathogenesis of eosinophilic airway inflammation (Bochenek et al., 2004). Recent evidence has identified a link between inflammation and HIF-1 expression (Hollander et al., 2001). For example, a knockout of HIF-1 in macrophages and other myeloid lineage cells leads to decreased myeloid cell infiltration and activation, impaired chronic inflammation, and decreased joint inflammation in rheumatoid arthritis (Cramer et al., 2003). In this study, we showed that PMACI-induced mast cell inflammatory gene expression was inhibited by pretreatment of SC-236 in HMC-1 cells. The results indicate that SC-236 has an anti-inflammatory effect, which might explain its beneficial effect in the treatment of mast cell-mediated inflammatory diseases.
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B activation has been linked with anti-inflammation, we postulated that SC-236 mediates its effects at least partly through suppression of NF-B activation. Activation of NF-B is dependent on the phosphorylation and degradation of IB-, an endogenous inhibitor that binds to NF-B in the cytoplasm. Glucocorticoids that have frequently been used for the treatment of inflammatory bowel disease and rheumatoid arthritis were suggested to suppress NF-B activation. Sulfasalazine, a potent and specific inhibitor of NF-B, inhibits NF-B activation. Previously, we reported that aucubin inhibited NF-B activation via phosphorylation and degradation of IB-. Stark et al. (2001) reported that SC-236-mediated regulation of the NF-B pathway might be cell-type specific. In a gastric cancer cell line, SC-236 affected neither the IB- phosphorylation nor degradation, whereas SC-236 significantly inhibited IB- phosphorylation and degradation in colon cancer cell lines (Wong et al., 2003). These results suggest that SC-236 regulates the NF-B pathway through different mechanisms in different cell systems. SC-236 suppresses NF-B transcriptional activation and translocation to the nucleus induced by PMACI in HMC-1. This suppression is also mediated through inhibition of IB- phosphorylation and degradation. Therefore, our results suggest that the anti-inflammatory effect of SC-236 is similar to the mechanism of sulfasalazine or aucubin. Jung et al. (2003) reported that activation of NF-B increased the expression of COX-2. They also showed that up-regulation of HIF-1 was attenuated by COX-2 inhibitor. These results demonstrate that COX-2 is a potent effector on HIF-1 up-regulation. From this, we can presuppose that SC-236 might inhibit COX-2 and HIF-1 expression through suppression of NF-B activation in HMC-1. Hence, it is hypothesized that SC-236 might act as a potent NF-B inhibitor on the mast cell activation induced by PMACI.
MAPK signaling cascade plays an essential role in the initiation of inflammatory responses (Adwanikar et al., 2004). Several recent studies have reported that suppression of MAPKs in mast cells may be a useful tool to reduce mast cell number in the inflammatory response (Koranteng et al., 2004). It was reported that p38 MAPK inhibitor suppressed IL-6 and TNF- production in monocytic or mast cells (Guo et al., 2003; Jeong et al., 2003), and inhibition of p38 MAPK and ERK also attenuated COX activity (Borsch-Haubold et al., 1998). In addition, P38, ERK, and JNK are known to participate in the pathway of iNOS expression (Wang et al., 2004). MAPK activation induces c-Fos and Jun expression by activating different transcription factors such as Ets-like protein-1, AP-1, and activating transcription factor. Previously, it was reported that the MAPK/AP-1 pathway could play an important role in mediating mast cell-derived tryptase effects in allergic inflammation (Temkin et al., 2002). Taken together, we supposed that the MAPK pathway could be an effective target for anti-inflammatory therapy. Therefore, we investigated whether SC-236 affects the MAPK pathway in stimulated HMC-1. We showed that SC-236 abrogated the activation of all the three pathways by PMACI. These results demonstrate that the anti-inflammatory effect of SC-236, at least in part, might be derived through regulation of the MAPK pathway. Although SC-236 attenuated MAPK activation, the effect of SC-236 on other pathway-involved MAPK upstream/downstream is not elucidated in the present study. Thus, further investigation is necessary to clarify the role of SC-236 on the MAPK pathway in HMC-1.
MKP-1 is a critical negative regulator in response to inflammatory stimuli and is responsible for switching off the production of proinflammatory cytokines. Previously, it was found that MKP-1 is induced by certain anti-inflammatory drugs/agents, and it was proposed that MKP-1 could be a target for developing novel anti-inflammatory drugs (Lasa et al., 2002; Jeong et al., 2003). MKP-1 was induced concurrently with the inactivation of p38, ERK, and JNK, whereas blocking MKP-1 induction prevented this inactivation. Overexpression of MKP-1 accelerated JNK and p38 inactivation and substantially inhibited the production of TNF- and IL-6 (Chen et al., 2002). Bokemeyer et al. (1998) reported that anisomycin induced MKP-1 expression and was inhibited by a p38 MAPK inhibitor. In the present study, we showed that SC-236 induced MKP-1 expression. From this, we speculate that PMACI would not activate MAPK because MKP-1 was induced by pretreatment of SC-236 in advance. Therefore, we project that the anti-inflammatory effect of SC-236, at least in part, might occur through induction of the MKP-1 regulation MAPK pathway (Fig. 6). However, further study is necessary to explain the precise role of SC-236 in relation between MKP-1 induction and the MAPK pathway in HMC-1.
In conclusion, we have shown that SC-236 can regulate the inflammatory response induced by PMACI in mast cells. SC-236 affected the expression of inflammatory genes through regulation of the NF-B/IB- pathway. In addition, SC-236 suppressed MAPK activation and induced MKP-1 expression. Induced MKP-1 would modulate MAPK activation; thus, inflammatory genes might be inhibited. These results provided new insight into the pharmacological actions of SC-236 as a potential molecule for therapy of mast cell-mediated inflammatory diseases.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PG, prostaglandin; TNF, tumor necrosis factor; IL, interleukin; VEGF, vascular endothelial growth factor; COX, cyclooxygenase; iNOS, inducible nitric-oxide synthase; HIF, hypoxia-inducible factor; NF, nuclear factor; MAPK, mitogen-activated protein kinase; ERK, extracellular-regulated kinase; JNK, c-Jun N-terminal kinase; AP, activated protein; MKP, MAPK phosphatase; SC-236, 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1-pyrazol-1-l] benzenesulfonamide; PMA, phorbol 12-myristate 13-acetate; PMACI, PMA plus calcium ionophore A23187 [GenBank] ; HMC-1, human mast cell line; MTT, 3-[4,5-dimetylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide; Ab, antibody; PBC, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; PCR, polymerase chain reaction; BSA, bovine serum albumin.
Address correspondence to: Dr. Hyung-Min Kim, Department of Pharmacology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-Dong, Dongdaemun-Gu, Seoul, 130-701, Republic of Korea. E-mail: hmkim{at}khu.ac.kr
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