TNF--induced Suppressor of cytokine signaling-3 (SOCS-3) protein expression mediated through the JNK1/2, ERK1/2/NADPH oxidase/AP-1 in Human tracheal smooth muscle cells

Suppressor of cytokine signaling-3 (SOCS-3) is an intracellular protein that involved in a wide range of biological processes. Recently, more and more study indicated that SOCS-3 not only can down-regulate the cytokine stimulated signaling, but also can anti-inflammatory response. However, whether cytokine can induce the expression of SOCS-3 and the mechanism in airway is still unclear. Here, we report that TNF- induced SOCS-3 expression mediated through the signaling pathway in human tracheal smooth muscle cells (HTSMCs). TNF--induced SOCS-3 protein, mRNA expression and promoter activity were attenuated by pretreatment with the inhibitor and si-RNA of JNK1/2 (SP600125), p38 (SB202190), MEK1/2 (U0126), NADPH oxidase (diphenylene iodonium chloride (DPI) and apocynin (APO)), and reactive oxygen species (ROS) scavenger (N-acetyl-L-cysteine). In transcription factors, we used the inhibitor and si-RNA of NF-B (Bay11-7082) and AP-1 (Tanshinone) can down-regulate the SOCS3 expression induced by TNF-, respectively. TNF--stimulated ROS production was attenuated by pretreatment with the inhibitor of JNK1/2, MEK1/2, and NADPH oxidase. Furthermore, we found that ROS production led to activation of AP-1 but not the NF-B. Taken together, our results suggested that TNF--induced ROS production was mediated through the JNK1/2 and ERK1/2/NADPH oxidase pathway, in turn activation of AP-1, and ultimately induces SOCS-3 expression in HTSMCs. Introduction Asthma is a chronic inflammatory airways disease characterized by early and late asthmatic reactions that are associated with infiltration and activation of inflammatory cells in the airways and airway hyperresponsiveness to a variety of stimuli, including neurotransmitters, inflammatory mediators, and inhaled contractile (Bousquet et al, 2000;Gosens et al, 2008;Pascual et al, 2003). TNF- is the most widely studied pro-inflammatory cytokine of the TNF superfamily. TNF-, one of the best characterized cytokines, was originally discovered in the mouse serum during endotoxemia and recognized for its anti-tumor activity (Carswell et al, 1975). Owing to its strong pro-inflammatory and immuno-stimulatory activities, TNF- is associated with a number of pathological events, like involved in the progression of rheumatoid arthritis and inflammatory bowel diseases. The possibility that TNF- contributes to inflammatory response seen in the asthmatic airway is supported by observations that TNF- mRNA and protein levels were increased in the airways of patients with asthma (Ying et al, 1991;Bradding et al, 1994). It is also plausible that TNF- in the development of AHR and other diseases. Until now, have a lot of articles mention that TNF- affects both acute bronchoconstriction and chronic changes in airway structure, termed remodeling that contribute to airway hyperresponsiveness (Hansbro et al, 2011;Moore and Pascual, 2010;Tritar et al, 2009). An inflammatory response consists of the sequential release of mediators including inflammatory cytokines and the recruitment of circulating leukocytes, which become activated at the inflammatory site and release further mediators. In most cases, however, an inflammatory response is resolved by the release of endogenous anti-inflammatory mediators as well as by the accumulation of intracellular negative regulatory factors. suppressor of cytokine signaling (SOCS) proteins and cytokine-inducible SRC homology 2 (SH2)-domain-containing proteins (CISs; also known as CISHs) comprise a family of intracellular proteins, several of which have been shown to down regulate the responses of immune cells to cytokines (Ilangumaran et al, 2004;Naka et al, 2005;Davey et al, 2006). There are eight CIS/SOCS family proteins: CIS, SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, SOCS-6 and SOCS-7, and most of SOCS proteins are induced by cytokines and therefore act in a classical negative-feedback loop to inhibit cytokine signal transduction (Ilangumaran et al, 2004;Kubo et al, 2003;Wajant et al, 2003). Therefore, SOCS proteins are involved in a wide range of biological processes. Inflammatory cytokines and chemokines have been shown to relay intracellular signals mediated via activation of MAPKs pathways. Previous study has demonstrated that MAPKs-cascade is required for TNF- mediated stabilization of SOCS-3 mRNA and results in enhanced SOCS-3 protein expression (Ehlting et al, 2007). Recently, some studies indicated that SOCS-3 induced by cytokines have the function of anti-inflammatory response (Qin et al, 2007;Qin et al, 2006;Lee et al, 2010). Reactive oxygen species (ROS) are molecules or ions formed by the incomplete one-electron reduction of oxygen. These reactive oxygen intermediates include singlet oxygen, superoxides, peroxides, hydroxyl radical, and hypochlorous acid. They contribute to the microbicidal activity of phagocytes, regulation of signal transduction and gene expression, and induce oxidative damage to nucleic acids, proteins, and lipids. During aerobic respiration, mammalian cells produce energy by reducing molecular oxygen (O2) to water (H2O). As a natural byproduct of normal metabolism, ROS play a regulatory part in cellular function (Lubos et al, 2008). Oxidative stresses are believed to play an important role in the induction of both of cell adhesion molecules and pro-inflammatory cytokines, a key event in a variety of inflammatory processes. Some studies have indicated that TNF- mediates through the c-Src to induce NADPH oxidase-dependent ROS generation in HTSMCs (Lee et al, 2009). Therefore, TNF- may play a potential role in the regulation of SOCS-3 expression, and thereby prevent inflammatory response. However, the mechanisms of intracellular signaling pathway involved in TNF--induced SOCS-3 expression in HTSMCs are unclear. Here, we found TNF--induced ROS production was mediated through the JNK1/2 and ERK1/2/NADPH oxidase pathway, in turn activation of AP-1, and ultimately induces SOCS-3 expression in HTSMCs. Materials and methods Materials DMEM/F-12 medium, FBS, TRIzol will be purchased from Gibco BRL (Gaithersburg, MD). Hybond C membrane, enhanced chemiluminescence (ECL) Western blotting detection system and Hyperfilms will be from GE Healthcare Biosciences (Buckinghamshire, England). U0126, SB202190, SP600125, PP1, Bay11-7082, Tanshinone IIA, Cycloheximide (CHI), actinomycin D (Act.D), N-acetyl-L-cystein (NAC), Apocynin (APO), and Diphenylene iodonium chloride (DPI) will be from Biomol (Plymouth Meeting, PA). Lipofectamine2000 transfection reagent was from Biontex (Munich, Germany). Luciferase assay kit was from Promega (Madison, WI, USA). CM-H2DCFDA was from Molecular Probes (Eugene, OR). TNF-, enzymes and other chemicals will be from Sigma (St. Louis, MO). Antibodies Anti-TNFR1 neutralizing antibody was from R&D system (Minneapolis, MN). Polyclonal antibody SOCS3, -actin, phospho-c-Src, phospho-p65, phospho-p38, phospho-JNK1/2, phospho-ERK1/2, phosphor-c-Jun and phospho-ATF2 will be from Santa Cruz (Santa Cruz, CA). Cell culture HTSMCs were purchased from ScienCell Research Lab (San Diego, CA) and grew in DMEM/F-12 containing 10% (v/v) FBS and antibiotics (100 U/ml penicillin G, 100 g/ml streptomycin, and 250 ng/ml fungizone) at 37°C in a humidified 5% CO2 atmosphere. When the cultures reached confluence (4 days), cells were treated with 0.05% (w/v) trypsin/0.53 mM EDTA for 5 min at 37°C. The cell suspension was diluted with DMEM/F-12 containing 10% FBS to a concentration of 2x10 cells/ml. Preparation of cell extracts and Western blotting analysis Growth-arrested cells were incubated with or without different concentrations of TNF- at 37°C for the indicated time. When inhibitors were used, they were added 2 h prior to the application of TNF-. The cell lysates were collected and the protein concentration was determined by the BCA reagents according to the instructions of the manufacture. Samples from these cell lysates (30 μg protein) were denatured and subjected to SDS-PAGE using a 10% running gel. Proteins were transferred to nitrocellulose membrane. Membrane was incubated overnight at 4°C with antibody used at a dilution of 1:1000 in TTBS. Membranes were washed with TTBS four times for 15 min each, and incubated with a 1:2000 dilution of anti-rabbit or anti-mouse horseradish peroxidase antibody for 1 h. Following each incubation, the membrane was washed extensively with TTBS. The immunoreactive bands detected by ECL reagents were developed by Hyperfilm-ECL. Total RNA extraction and RT-PCR analysis Total RNA was isolated from HTSMCs treated with TNF- for the indicated time in 10-cm culture dishes with TRIzol according to the protocol of the manufacturer. RNA concentration was spectrophotometrically determined at 260 nm. First-strand cDNA synthesis was performed with 2 g of total RNA using random hexamers as primers in a final volume of 20 l (5 g/l random hexamers, 1 mM dNTPs, 2 units/l RNase, and 10 units/l Moloney murine leukemia virus reverse transcriptase). The reaction was carried out at 37°C for 60 min. cDNAs encoding SOCS3 and -actin were amplified from 3 to 5 l of the cDNA reaction mixture using specific gene primers. Oligonucleotide primers for -actin and SOCS3 were as follows: -actin: Forward : 5-TGACGGGGTCACCCACACTGTGCCCATCT-3 Reverse : 5-CTAGAAGCATTTGCGGTGGACGATG-3 SOCS3: Forward : 5-TTCTTCACGCTCAGCGTCAAG-3 Reverse : 5-ATGTAATAGGCTCTTCTGGGG-3 DNA amplification profile includes 1 cycle of initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 1 min, primer annealing at 60°C (-actin), 55°C (SOCS3) for 1 min, and extension at 72°C for 1 min then 1 cycle of final extension at 72°C for 10 min. Determination of ROS production The intracellular H2O2 levels were determined by measuring flurorescence of DCFH-DA. The fluorescence for DCF staining was detected at 495/529 nm, using a fluorescence microscope (Zeiss, Axiovert 200M). HTSMCs were washed with warm HBSS and incubated in HBSS or cell medium containing 10 M DCFH-DA at 37°C for 30 min. Subsequently, HBSS or medium containing DCFH-DA was removed and replaced with fresh medium. HTSMCs were then incubated with TNF-. Cells were washed twice with PBS and detached with trypsin/EDTA, and the fluorescence intensity of cells was analyzed using ELISA and FACScan flow cytometer at 495 nm excitation and 529 nm emission. Transfection of si-RNA and luciferase reporter gene assays SMARTpool RNA duplexes corresponding to human NOX2, p47 and scrambled #2 siRNA were from Dharmacon Research Inc (Lafayette, CO, USA). Transient transfection of siRNAs was carried out using Metafectene transfection reagent. siRNA (100 nM) was formulated with Metafectene transfection reagent according to the manufacturer's instruction. Statistical analysis of data All the data will be analyzed by using the GraphPad Prizm Program (GraphPad, San Diego, CA, USA). Quantitative data were expressed as the mean ± SEM and analyzed with a one-way ANOVA to make comparisons with Bonferroni’s test at a P < 0.05 level of significance. Error bars were omitted when they fell within the dimensions of the symbols. Results TNF- induces SOCS-3 expression in HTSMCs To investigate the effect of TNF- on SOCS-3 expression, TNF--induced SOCS-3 protein expression in a concentration-dependent and time-dependent manner was determined by western blot (Fig. 1A). There was a concentration-dependent and significant increase with 12 h, which sustained up to 24 h. Then, TNF--induced SOCS3 mRNA expression in a time-dependent manner was detected by RT-PCR and peaked within 6 h (Fig. 1B). Furthermore, exposure to TNF- also increased SOCS-3 promoter activity in a time-dependent fashion and peaked within 4 h (Fig. 1C). Moreover, pretreatment with TNF receptor neutralize Ab (10 g/ml), markedly attenuated TNF--induced SOCS-3 expression, suggesting that TNF--induced SOCS-3 expression via TNF receptor in HTSMCs (Fig. 1D). TNF- induces SOCS-3 expression require ongoing transcription and translation in HTSMCs To further determine if TNF--induced SOCS-3 expression required transcription or translation, HTSMCs were stimulated with TNF- (5 ng/ml) in the absence or presence of a transcriptional level inhibitor, actinomycin D (Act. D) (Fig. 2A) and a translational level inhibitor, cycloheximide (CHI) (Fig. 2B) and SOCS-3 protein expression was determined by Western blot analysis. TNF--mediated induction of SOCS-3 expression was abolished by either actinomycin D or cycloheximide in a concentration-dependent manner. Taken together, these findings demonstrate that the induction of SOCS-3 by TNF- depends on de novo protein synthesis in HTSMCs. TNF- induces SOCS-3 expression mediated through MAPKs pathway in HTSMCs Previous study has demonstrated that MAPK-cascade is required for cytokine mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression (Ehlting et al, 2007). We investigated whether TNF-α-induced SOCS3 expression was mediated via p42/p44 MAPK, p38 MAPK, and JNK1/2 MAPK in HTSMCs. Our results show that pretreatment with the inhibitor of MEK1/2 (U0126), p38 (SB202190), or JNK (SP600125) for 2 h prior to exposure to TNF-α (5 ng/ml) for 24 h caused an attenuation of SOCS3 expression in a concentration-dependent manner revealed by Western blotting (Fig. 3A, B, C). To determine whether p42/p44 MAPK, p38 MAPK, and JNK1/2 MAPK phosphorylation were necessary for the induction of SOCS3 expression induced by TNF-, activation of these kinases was assayed by Western blotting using an anti-phospho-p42/p44 MAPK, -p38 MAPK, or -JNK1/2 MAPK antibody. TNF- stimulated p42/p44, p38 and JNK1/2 MAPK phosphorylation in a time-dependent fashion in HTSMCs (Fig. 3D, E, F). These results suggested that TNF-α-induced SOCS3 expression is mediated through p42/p44 MAPK, p38 MAPK, and JNK1/2 MAPK in HTSMCs. Involvement of NADPH oxidase and ROS generation in TNF--induced SOCS-3 expression in HTSMCs Some studies have indicated that TNF- mediates through the c-Src to induce NADPH oxidase-dependent ROS generation in HTSMCs (Lee et al, 2009b). However, whether TNF--induced SOCS-3 expression mediated through NADPH oxidase-dependent ROS generation and how regulated is still unknown. To determine whether c-Src induced NADPH oxidase dependent ROS generation participated in SOCS-3 expression induced by TNF-. HTSMCs were pretreatment with PP1 (c-Src kinase inhibitor), NAC (a ROS scavenger), APO, DPI (NADPH oxidase inhibitors) significantly inhibited TNF-α-induced SOCS3 expression in a concentration-dependent manner, revealed by Western blotting (Fig. 4A, B, C, D). To determine whether c-Src phosphorylation was necessary for the induction of SOCS3 expression induced by TNF-, activation of these kinases was assayed by Western blotting using an anti-phospho-Src antibody (Fig. 4E). To further determine NOX2 and p47 was involved in NADPH oxidase dependent ROS generation, NOX2 and p47 siRNA were used. Our data showed that knocked down the expression of NOX2 and p47 protein significantly attenuated TNF--induced SOCS-3 expression in HTSMCs (Fig 5A, B). To further ascertain that NADPH oxidase activity and generation of ROS were involved in TNF- induced SOCS-3 expression in HTSMCs. Cells were labeled with DCFH-DA, incubated with TNF- (30 ng/ml) for the indicated time intervals, and the fluorescence intensity was measured at 485 nm excitation and 530 nm emission. As illustrated in Fig. 5C, D and E. TNF- induced a significant increase in NADPH oxidase activity and ROS generation in HTSMCs. Involvement of MAPK-dependent NADPH oxidase/ROS generation pathway in the induction of SOCS-3 expression by TNF- Before, we have demonstrated that MAPKs and NADPH oxidase dependent ROS generation participate in the expression of SOCS-3 induced by TNF- in HTSMCs (Fig 3 and 4). To further investigate whether NADPH oxidase dependent ROS generation induced by TNF- via MAPKs. Our result showed that NADPH oxidase activity and ROS generation induced by TNF- was significantly attenuated by pretreated with U0126 (MEK inhibitor), p38 (SB202190), and JNK (SP600125) (Fig. 6A, B). Our results suggest that TNF--induced SOCS-3 expression via MAPK/NADPH oxidase dependent ROS cascade in HTSMCs. TNF-α induced SOCS3 expression via NF-κB and MAPK-dependent NADPH oxidase/ROS generation /AP-1 cascade Inflammatory responses following stimulation with TNF-α are highly dependent on activation of the NF-B and AP-1 transcription factors. Therefore, involvement of NF-B and AP-1 activation in SOCS3 expression induced by TNF-α was further confirmed using Bay11-7082 (a pharmacological inhibitor of NF-B) and Tanshinone (a pharmacological inhibitor of AP-1) in HTSMCs. As shown in Figs. 7A and B, pretreatment of HTSMCs with Bay11-7082 or Tanshinone caused an attenuation of TNF-α-induced SOCS3 expression in a concentration-dependent manner, revealed by Western blotting. To determine whether p65, c-Jun, and ATF2 phosphorylation were necessary for the induction of SOCS3 expression induced by TNF-, activation of these kinases was assayed by Western blotting using an anti-phospho-p65, anti-phospho-c-Jun, or anti-phospho-ATF2 antibody. As shown in Figs. 7C, D, and E, TNF- stimulated p65, c-Jun and ATF2 phosphorylation in a time-dependent fashion in HTSMCs. Furthermore, to investigate whether MAPK-dependent NADPH oxidase/ROS generation cascade were involved in NF-B and AP-1 activation. The result showed that TNF--stimulated activation of AP-1 was attenuated by pretreatment TNFR neutralize Ab (TNF receptor inhibitor), Tanshinone (AP-1 inhibitor), U0126 (MEK inhibitor), SB202190 (p38 inhibitor), SP600125 (JNK inhibitor), NAC (a ROS scavenger), APO and DPI (NADPH oxidase inhibitors) (Fig. 7F), but not NF-B pathway. These results suggested that TNF-α-induced SOCS3 expression is mediated through the activation of NF-B and MAPK/NADPH oxidase dependent ROS/AP-1 cascade in HTSMCs. Discussion Many reports have indicated that SOCS3 is induced by various inflammatory and anti-inflammatory cytokines, such as IL-6, IL-12, IFN- and IL-10, and that it negatively regulates the effects of cytokines as well as JAK-STAT functions (Stoiber et al, 1999;Cassatella et al, 1999). Moreover, SOCS3 is induced by IL-1 and TNF- as and LPS. Several lines of evidence suggest that SOCS3 may suppress inflammatory responses in various cell types (Shouda et al, 2001). SOCS3 exerts as a relatively specific inhibitor of gp130. STAT3 activation and elevated levels of SOCS3 expression have been found in epithelial and lamina propria cells in the colon of IBD (inflammatory bowel disease) model mice, as well as in human ulcerative colitis and patients with Crohn’s disease, and in synovial fibroblasts of patients with RA (Leroith and Nissley, 2005). Despite an obviously important role of SOCS-3 in diseases. However, whether SOCS3 can suppress the inflammatory responses in airway disease remains enigmatic. Therefore, we made an attempt to study the mechanisms and function of SOCS3 expression induced by TNF- in HTSMCs. In our results, we found that expression of SOCS3 protein, mRNA and promoter activity were induced by TNF- in a timeand concentration-dependent manner. Here, we demonstrated that TNF- will induce SOCS-3 in HTSMCs. Extracellular proteins bound to cell-surface receptors can change nuclear gene expression patterns in minutes, with far-reaching consequences for development, cell growth and homeostasis. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) proteins are among the most well studied of the latent cytoplasmic signal-dependent transcription-factor pathways. Many studies reported that SOCS3 expression through JAK-STAT pathway was induced by various cytokines and LPS (Levy and Darnell, Jr., 2002;Bromberg and Darnell, Jr., 2000). In our results, we found TNF--induced SOCS-3 expression via JAK2-STAT3 pathway in HTSMCs (data not shown). Previous studies have shown that TNF--induced SOCS3 expression by stabilizing SOCS3 mRNA (Ehlting et al, 2007a). Activation of the MAPK kinase 6 (MKK6)/p38 MAPK-cascade is required for TNF- mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38 MAPK cascade, basal SOCS3-expression is strongly reduced and TNF- induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38 MAPK-dependent signals (Ehlting et al, 2007). Another study indicated that LPS-induced MAPK-ERK1/2, JNK, and p38 pathways activation, the production of endogenous IL-10, and STAT-3 activation play critical roles in SOCS-3 expression, which provides for feedback attenuation of cytokine-induced immune and inflammatory responses in macrophages and microglia (Qin et al, 2007;Qin et al, 2006). However, whether MAPKs signaling pathways involved in SOCS3 expression were still unclear. Here, we studied the mechanisms of SOCS3 expression induced by TNF- in HTSMCs. In our results, we found that TNF- induced-SOCS3 expression was mediated through not only JAK-STAT but also MAPKs pathway. In our results, we pretreated with MEK1/2 inhibitor (U0126), p38 inhibitor (SB202190), JNK inhibitor (SP600125) significantly attenuated SOCS-3 protein expression induced by TNF- in HTSMCs. Furthermore, we demonstrated that p42/p44 MAPK, p38 MAPK, and JNK1/2 MAPK phosphorylation were necessary for the induction of SOCS3 expression induced by TNF-. According these results, we suggested that TNF--induced SOCS-3 expression via MAPKs pathway activation. ROS concentration-dependently exerts a key role in the normal physiological functions and inflammatory responses (Floyd, 1999;Kamata and Hirata, 1999;Valko et al, 2007). In inflammatory status, ROS will be generated by various inflammatory mediators. TNF- is an important cytokine that induces ROS generation. A previous study indicated that ROS can modulate the inflammatory response and smooth muscle relaxation (Li et al, 2010;Lee et al, 2010). However, whether overexpression of SOCS3 modulated ROS production induced by TNF- is still unclear in HTSMCs. In our results, we found that TNF--induced SOCS-3 expression attenuated by pretreatment with ROS scavenger (NAC) and NADPH oxidase inhibitor (APO and DPI) in HTSMCs. Another way, we determined that NADPH oxidase dependent ROS generation in time-dependent induced by TNF-. Furthermore, we demonstrated that TNF- induced ROS generation mediated through TNF receptor dependent MAPKs/NADPH oxidase cascade, because pretreatment with TNF receptor neutralize Ab (TNFR-Ab), MEK1/2 inhibitor (U0126), p38 inhibitor (SB202190), JNK inhibitor (SP600125), ROS scavenger (NAC) and NADPH oxidase inhibitor (APO and DPI) attenuated NADPH oxidase activity and ROS generation induced by TNF-. Here, we suggested that TNFR dependent MAPK/NADPH oxidase cascade involved in TNF--induced ROS generation. Transcription factors NF-B and AP-1 are closely correlated to inflammatory regulation. NF-B is a ubiquitously expressed transcription factor involving in immune, inflammatory and anti-apoptoic response. On the other hand, activator protein-1 (AP-1) is a dimeric protein, consisting either of dimmers composed of members of the Fos, Jun and ATF families of proteins. AP-1 has been shown to be implicated in a variety of biological processes including cell differentiation, proliferation, apoptosis and inflammatory (Velazquez and Gariglio, 2002). However, the mechanism underlying NF-B and AP-1 involved in the expression of SOCS3 induced by TNF- in HTSMCs is not clear. And which transcription factor is the most important or all of them are essentially in TNF--induced SOCS3 expression still unknown. Here, we also propose that induction of SOCS3 by TNF-α may be mediated through NF-B and AP-1. In addressing these questions, the experiments will be performed to investigate the roles of NF-B and AP-1 in TNF-α-induced SOCS3 expression in HTSMCs. We found TNF--induced SOCS-3 expression attenuated by pretreatment with NF-B inhibitor (Bay11-7082) and AP-1 inhibitor (Tanshinone IIA). Furthermore, we demonstrated that p65, c-Jun and ATF2 phosphorylation were necessary for the induction of SOCS3 expression induced by TNF-. Moreover, we pretreated with MEK1/2 inhibitor (U0126), p38 inhibitor (SB202190), JNK inhibitor (SP600125), ROS scavenger (NAC) and NADPH oxidase inhibitor (APO and DPI) significantly attenuated phosphorylation of c-Jun and ATF2, but not p65 induced by TNF- in HTSMCs. According these results, we demonstrated that TNF--induced SOCS-3 expression mediated activation of AP-1, but not mediated NF-B in HTSMCs. In conclusion, we have demonstrated that TNF- directly induces SOCS-3 expression via TNF receptor and MAPKs dependent NADPH oxidase/ROS generation pathway, in turn activation of AP-1. Pharmacological approaches suggest that targeting SOCS-3 and their upstream signaling components may provide useful therapeutic strategies for airway diseases.