Interleukin-1b stimulates IL-8 expression through MAP kinase ... - Nature

Oncogene (2004) 23, 6603–6611

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Interleukin-1b stimulates IL-8 expression through MAP kinase and ROS signaling in human gastric carcinoma cells Young S Hwang1, Min Jeong1, Jung S Park1, Mi H Kim1, Dae B Lee1, Boo A Shin1, Naofumi Mukaida2, Lee M Ellis3, Hyeong R Kim1, Bong W Ahn1 and Young D Jung*,1 1 Department of Biochemistry, Chonnam University Research Institute of Medical Sciences, Chonnam National University Medical School, 5 Hakdong, Kwangju, 501-190, Korea; 2Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, 920-0934, Japan; 3Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Box 444, Houston, TX, 77030-4009, USA

Recent studies have suggested that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. In this study, the effect of IL-1b on IL-8 expression in human gastric cancer TMK-1 cells and the underlying signal transduction pathways were investigated. IL-1b induced the IL-8 expression in a time- and concentration-dependent manner. IL-1b induced the activation of extracellular signalregulated kinases-1/2 and P38 mitogen-activated protein kinase (MAPK), but not the activation of c-jun aminoterminal kinse and Akt. Specific inhibitors of MEK-1 (PD980590) and P38 MAPK (SB203580) were found to suppress the IL-8 expression and the IL-8 promoter activity. Expression of vectors encoding a mutated-type MEK-1 and P38 MAPK resulted in decrease in the IL-8 promoter activity. IL-1b also induced the production of reactive oxygen species (ROS). N-acetyl cysteine (NAC) prevented the IL-1b-induced ROS production and IL-8 expression. In addition, exogenous H2O2 could induce the IL-8 expression. Deletional and site-directed mutagenesis studies on the IL-8 promoter revealed that activator protein-1 (AP-1) and nuclear factor (NF)-jB sites were required for the IL-1b-induced IL-8 transcription. Electrophoretic mobility shift assay confirmed that IL-1b increased the DNA-binding activity of AP-1 and NF-jB. Inhibitor (PD980590, SB203580) and ROS scavenger (NAC) studies revealed that the upstream signalings for the transcription factors AP-1 and NF-jB were MAPK and ROS, respectively. Conditioned media from the TMK-1 cells pretreated with IL-1b could remarkably stimulate the in vitro growth of HUVEC and this effect was partially abrogated by IL-8-neutralizing antibodies. The above results suggest that MAPK-AP-1 and ROSNF-jB signaling pathways are involved in the IL-1binduced IL-8 expression and that these paracrine signaling pathways induce endothelial cell proliferation. Oncogene (2004) 23, 6603–6611. doi:10.1038/sj.onc.1207867 Published online 21 June 2004 Keywords: IL-8; MAPK; ROS; gastric cancer; IL-1b *Correspondence: YD Jung; E-mail: [email protected] Received 15 February 2004; revised 20 April 2004; accepted 22 April 2004; published online 21 June 2004

Introduction Although the incidence of gastric cancer has decreased over the last decades, it is still the most frequent digestive tract cancer with a poor prognosis and a high mortality. About 80% of the gastric cancers in Western countries are of an advanced stage at presentation (Ganesh et al., 1996). Owing to local invasion and metastasis, radiation therapy or chemotherapy does not significantly affect the length or quality of life of patients with advanced gastric cancer. The understanding of the detailed mechanisms of invasion and metastasis in gastric cancer would be therefore helpful in improving treatment. Recent studies have demonstrated that human gastric carcinomas overexpress interleukin-8 (IL-8) as compared to corresponding normal mucosa and that the IL8 mRNA level directly correlates with the vascularity of the tumors. IL-8 is multifunctional (Kitadai et al., 1998, 1999). In addition to its potent chemotactic activity, it can induce proliferation and migration of keratinocytes, endothelial cells, and melanoma cells (Rennekampff et al., 2000; Singh and Varney, 2000; Heidemann et al., 2003). IL-8 is expressed in many different cell types, including monocytes and macrophages, endothelial cells, keratinocytes, mesangial cells, and several human tumor cell lines (Schwarz et al., 1997; Klezovitch et al., 2001; Maeda et al., 2001). Although constitutive production of IL-8 appears to be infrequent in most cell types examined, its expression can be induced in many cell types by specific stimuli such as granulocyte colony-stimulating factor, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate, viruses, and doublestranded RNA (Kasahara et al., 1991). Analysis of the genomic structure of IL-8 reveals many potential targets for regulation at both the transcriptional and post-transcriptional levels. Within its 30 -flanking region, the IL-8 gene contains the repetitive ATTTA motif, which is responsible for destabilization of various cytokine mRNAs (Chen and Shyu, 1995). Within its 50 -flanking region, the IL-8 gene contains multiple cis elements, including a CCAAT box, a steroid-responsive element, a hepatocyte nuclear factor-1 element, two interferon regulatory factor-1

IL-8 expression via MAPK and ROS in gastric cancer YS Hwang et al

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elements, and the binding sites for an activator protein-1 (AP-1), CCAAT/enhancer binding protein (C/EBP), and nuclear factor-kB (NF-kB), all of which have been implicated in the induction of IL-8 gene transcription by the stimuli listed above (Kunsch et al., 1994). As demonstrated by mutational and deletional analyses, these promoter elements are regulated in highly cell type-specific fashions (Yasumoto et al., 1992). Although it is well known that IL-1b upregulates IL-8 expression in various cells such as endothelial cells, epithelial cells, and smooth muscle cells (Baggiolini et al., 1994; Jung et al., 2002), the molecular mechanism for the IL-1b-induced IL-8 expression in gastric cancer is not known. In particular, the pluripotent pro-inflammatory cytokine IL-1b has a central role in the pathogenesis of Helicobacter pylori-induced mucosal inflammation (Guiraldes et al., 2001; Maeda et al., 2001; Straubinger et al., 2003). IL-1b gene expression and protein production are increased in H. pylori infection and reduced with successful eradication (Wang et al., 1999). A myriad of intracellular signals have been suggested to mediate the effects of IL-1b, including activation of mitogenactivated protein kinase (MAPK), release of arachidonic acid, hydrolysis of sphingomyelin, and production of reactive oxygen species (ROS) (Welsh, 1996; Hofmeister et al., 1997; Anthonsen et al., 2001; Wolf et al., 2001). Activation of MAPK by IL-1b may subsequently induce the NF-kB and AP-1 DNA-binding activity, which promotes expression of the genes involved in cell survival, proliferation, and angiogenesis (Jung et al., 2002). The MAPK cascades are well-characterized pathways transducing signals from the cell surface to the nucleus. The family includes distinct subgroups: extracellular signal-related kinases (Erk-1/2), c-Jun NH2-terminal kinases (JNKs), and p38 MAPK (Shin et al., 2001). In addition, IL-1b has been shown to stimulate the production of ROS, a class of highly reactive, diffusible, and ubiquitous molecules, in various cell types. ROS are involved in aging and many diseases as follows: cancer, diabetes mellitus, atherosclerosis, neurological degeneration, angiogenesis, and tumor invasion (Harris and Shi, 2003; Wu et al., 2004). Also, a variety of evidences now support the idea that ROS can modulate various cellular events from gene expression to cellular proliferation (Duval et al., 2003). We hypothesized that cytokine paracrine signaling in gastric cancer induces IL-8 expression, thereby enhancing the proliferation of endothelial cells, which are essential for tumor growth and metatasis. In this study, we demonstrate that IL-1b upregulates IL-8 in human gastric cancer cells via activation of Erk-1/2, P38 MAPK, AP-1, and NF-kB, and production of ROS.

determined. Control cells (not treated with IL-1b) were harvested at each time point to exclude any effect of increasing cell confluence on IL-8 expression. The results showed that the level of IL-8 mRNA increased in a time-dependent manner after incubation of cells with IL-1b, with the highest level seen at 8 h (Figure 1a). We also found that IL-1b induced the IL-8 mRNA expression in a dose-dependent manner at 0.02–10 ng/ml (Figure 1b). Involvement of Erk-1/2 and P38 MAPK in IL-1b-induced IL-8 mRNA expression To determine the signaling pathways involved in IL-8 induction by IL-1b, TMK-1 cells exposed to IL-1b for various periods were determined for the levels of phosphorylated and total Erk-1/2, JNK, P38 MAPK, and Akt. IL-1b treatment led to remarkable increases in Erk-1/2 and P38 MAPK phosphorylation within 15 min and the increased levels were maintained for 90–120 min. The levels of total Erk-1/2 and P38 MAPK were not significantly altered after IL-1b treatment (Figure 2a). In contrast, however, IL-1b did not induce the phosphorylation of JNK and Akt (data not shown). To examine the specific roles of Erk-1/2 and P38 MAPK in IL-1binduced IL-8 expression, TMK-1 cells were pretreated with 50 mM PD98059 (a MEK inhibitor) and 2 mM SB203580 (a specific P38 MAPK inhibitor) before IL1b treatment. As shown in Figure 2b, PD98059 and SB203580 partially blocked the IL-1b-induced IL-8 expression. Furthermore, co-treatment with PD98059 and SB203580 almost completely blocked the IL-8 induction by IL-1b (Figure 2b). Next, we sought to examine the effect of IL-1b on transcriptional regulation of the IL-8 gene. To this end, TMK-1 cells were transiently transfected with the promoter–reporter construct (pGL2-IL-8) containing region 546 to þ 44 of the human IL-8 gene and the

Results Effect of IL-1b on IL-8 mRNA expression in TMK-1 cells TMK-1 cells were incubated with IL-1b for various periods and the level of IL-8 mRNA in the cells was Oncogene

Figure 1 Induction of IL-8 by IL-1b in human gastric TMK-1 cells. Northern blot analysis was performed to determine the effect of IL-1b on IL-8 mRNA expression in TMK-1 cells. (a) The cells were incubated with 10 ng/ml IL-1b for 0–24 h. (b) The cells were incubated with the indicated concentration of IL-1b for 8 h


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Figure 2 Involvement of Erk-1/2 and P38 MAPK in IL-1b-induced IL-8 expression. (a) TMK-1 cells were incubated with 10 ng/ml IL-1b for 0–120 min, and cell lysates were determined for the phosphorylated and total Erk-1/2 and P38 MAPK by Western blot analysis. (b) TMK-1 cells pretreated with PD98059 (P) and/or SB203580 (S) for 1 h were incubated with 10 ng/ml IL-1b for 8 h. After incubation, the cell lysates were determined for the IL-8 mRNA by Northern blot analysis. (c) TMK-1 cells were transiently transfected with pGL2-IL-8. The transfected cells, after being pretreated with SB203580 (S) and/or PD98059 (P) for 1 h, were incubated with 10 ng/ml IL-1b for 8 h. Where indicated, dominant-negative mutants of MEK-1 (K97M) and P38 MAPK (Mut.38) were cotransfected with pGL2-IL-8 into TMK-1 cells. After incubation with IL-1b, the cell lysates were determined for the IL-8 promoter activity. Data represent the means7s.d. from triplicate measurements

luciferase gene. TMK-1 cells transfected with pGL2-IL8 showed a sixfold increase in the promoter activity after IL-1b treatment (Figure 2c). When the transfected cells were pretreated with PD98059 and SB203580 before IL1b treatment, the induction of IL-8 promoter activity by IL-1b was remarkably inhibited (Figure 2c). When the mutant form of MEK-1 (K97M) and/or P38 MAPK (Mut38) was cotransfected with pGL-2-IL-8 into TMK1 cells, the induction of IL-8 promoter activity by IL-1b was significantly inhibited. In particular, double mutation of MEK-1 and P38 MAPK resulted in a decrease of IL-8 promoter activity below the basal level (Figure 2c). These results strongly suggest that both ERK-1/2 and P38 MAPK signaling pathways are involved in the IL1b-induced activation of IL-8 transcription.

Involvement of ROS in IL-8 induction by IL-1b First, the change in the level of ROS in TMK-1 cells treated with IL-1b was examined using the H2O2sensitive fluorophore DCFDA. The results showed that IL-1b induced ROS production in the cells (Figure 3a and b). The level of intracellular ROS increased progressively after incubation of cells with 10 ng/ml IL-1b, reaching the peak at B15 min, and thereafter the level declined slowly. Pretreatment of cells with 5 mM Nacetylcysteine (NAC) inhibited almost completely the ROS production (Figure 3a and b). Next, we examined the effects of NAC and H2O2 on IL-8 induction by IL1b. As shown in Figure 3c, NAC pretreatment blocked partially the IL-1b-induced IL-8 expression. In contrast, Oncogene


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exogenous H2O2 could alone induce the expression of IL-8 (Figure 3d). Effect of IL-1b on the activation of transcriptional factors AP-1 and NF-kB during IL-8 induction

Figure 3 Involvement of ROS in IL-1b-induced IL-8 expression. (a, b) Production of dichlorofluorescein (DCF)-sensitive ROS in TMK-1 cells by IL-1b treatment. Synchronized quiescent TMK-1 cells, after being pretreated or not with 5 mM NAC for 1 h, were incubated with 10 ng/ml IL-1b for 0–60 min. The cells were then incubated in the dark for 10 min with 5 mg/ml DCFDA. DCF fluorescence was imaged with a confocal laser-scanning fluorescence microscope (a) and then quantified (b). (c) TMK-1 cells, after being pretreated with 5 mM NAC for 1 h, were incubated with 10 ng/ml IL-1b for 8 h. After incubation, the cell lysates were determined for the IL-8 mRNA by Northern blot analysis. (d) TMK-1 cells were incubated with 0–100 mM H2O2 for 6 h, and the cell lysates were determined for the IL-8 mRNA by Northern blot analysis. Data represent the means7s.d. from triplicate measurements Oncogene

As shown in Figure 2c and d, IL-1b treatment could increase the activity of IL-8 promoter in TMK-1 cells. Deletion study was performed to explore the sequence– activity characteristics of IL-8 promoter. Deletion of the upstream (50 ) region of position 133 bp had little effect on IL-1b-induced IL-8 promoter activation. In contrast, elimination of the region between positions 133 and 98 bp resulted in a substantial decrease in promoter activity, and another IL-1b-inducible element was identified between nucleotides 85 and 50 (Figure 4a). The IL-8 gene fragments spanning positions 133 to 98 bp and 85 to 50 bp contain DNA– protein interaction sites for the transcription factors AP1 (126/120) and NF-kB (80/71), respectively. To study the importance of the AP-1 and NF-kB sites in IL8 induction by IL-1b, TMK-1 cells were transfected with site-specific mutant forms of the IL-8 promoter linked to the luciferase gene. As shown in Figure 4b, mutation of either the AP-1 or the NF-kB binding site significantly decreased the IL-8 promoter activity, suggesting the importance of both AP-1 and NF-kB in IL-8 upregulation by IL-1b in TMK-1 cells. The above suggestion was further supported by EMSA and inhibitor studies. In EMSA, IL-1b treatment caused a remarkable increase in the amount of AP-1, which could form a complex with the radiolabeled oligonucleotide probe (Figure 5a). Both PD98059 and SB203580 partially inhibited the IL-1b-induced formation of AP-1-probe complex (Figure 5b). However, 5 mM NAC did not significantly alter the AP-1-probe complex formation induced by IL-1b (data not shown). In consistent with the EMSA result, IL-1b treatment caused a increase in the AP-1-dependent transcriptional activity and both PD98059 and SB203580 partially blocked the IL-1b-induced AP-1-dependent transcriptional activity, as revealed by the transient transfection study using the pAP-1-Luc reporter construct (Figure 5c). Also, the amount of NF-kB which could form a complex with the radiolabeled oligonuclotide probe increased in the cells treated with IL-1b in a timedependent manner (Figure 6a). In contrast with the case of AP-1, only NAC, but not PD98059 and SB203580, partially inhibited the NF-kB-probe complex formation (Figure 6b and c). The activation of NF-kB is usually associated with induction of phosphorylation of I-kB, followed by its degradation by proteasome and NF-kB nuclear translocation (Katsuyama and Hirata, 2001). Therefore, change in the amount of I-kB in TMK-1 cells during induction of IL-8 was determined by Western blotting using an antibody to I-kB. The result showed that IL-1b treatment led to a decrease in the total I-kB level within 2 h (Figure 6d). In addition, pretreatment of the cells with the NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC, 50 mM) caused a marked decrease in


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Figure 4 Effects of sequential deletion and site-specific mutations in the IL-8 promoter region on IL-1b-induced IL-8 promoter activity. (a) IL-8 promoter was sequentially deleted in the 50 flanking region and the promoter-luciferase construct was transiently transfected into TMK-1 cells. (b) IL-8 promoter was mutated in the AP-1 or NF-B site and the promoter-luciferase construct was transiently transfected into TMK-1 cells. The transfected cells were incubated with 10 ng/ml IL-1b for 8 h. After incubation, the cells were lysed and the luciferase activity was measured using a luminometer. Data represent the means7s.d. from triplicate measurements (*Po0.05, **Po0.01)

IL-1b-induced IL-8 expression (Figure 6e). These results also strongly suggest the involvement of NF-kB in the IL-8 expression induced by IL-1b. Effect of conditioned media (CM) derived from IL-1btreated TMK-1 cells on proliferation of HUVEC It has been reported that IL-8 is a paracrine growth factor for endothelial cells. We investigated whether CM derived from IL-1b-treated TMK-1 cells could stimulate the growth of endothelial HUVEC. HUVECs were cultured in the presence or absence of CM and cell proliferation was determined 48 h later by MTT assay.

Figure 5 Effects of Erk-1/2 and P38 MAPK inhibitors on IL-1binduced AP-1 activation. (a) TMK-1 cells were incubated with 10 ng/ml IL-1b for the indicated time, and nuclear extracts from the cells were analysed by EMSA for the activated AP-1 using a radiolabeled oligonucleotide probe. (b) TMK-1 cells, after being pretreated with 25 mM PD98059 and 2 mM SB203580 for 1 h, were incubated with 10 ng/ml IL-1b for 4 h and then determined for the activated AP-1 by EMSA. (c) TMK-1 cells were transiently transfected with the pAP-1-Luc reporter construct. The transfected cells, after being pretreated with 25 mM PD98059 (P) and/or 2 mM SB203580 (S) for 1 h, were incubated with 10 ng/ml IL-1b for 8 h. After incubation, the cells were lysed and the luciferase activity was determined using a luminometer. Data represent the means7s.d. from triplicate measurements

As shown in Figure 7, CM remarkably stimulate the in vitro growth of HUVEC. When CM was pretreated with anti-IL-8 antibody, the proliferation-stimulatory effect was significantly abolished. The IL-1b and anti-IL-8 antibody itself did not influence the cell growth. These results suggest that IL-8 induced by IL-1b in cancer cells may stimulate the endothelial cell proliferation.

Discussion Recently, much effort has been directed at defining the signal transduction pathways induced by IL-1b. Several studies have documented that the MAPKs have roles in IL-1b-induced signal transduction, but the profiles of IL-1b-induced kinase activation appear to vary in a cell type-dependent fashion (Guan et al., 1997). In this study, we found that IL-1b induced the IL-8 mRNA expression and promoted the activation of Erk-1/2 and P38 MAPK in human gastric TMK-1 cells. Increased Oncogene


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Figure 7 Effect of CM derived from IL-1b-treated cells on HUVEC proliferation. HUVECs were incubated with 10 ng/ml IL-1b or CM for 24 h in the presence or absence of 1 mg/ml anti-IL8 antibody, and the number of cells were counted using MTT. Data represent the means7s.d. from triplicate measurements (*Po0.05)

Figure 6 Activation of NF-kB is involved in IL-1b-induced IL-8 expression. (a) TMK-1 cells were treated with IL-1b for the indicated time, and nuclear extracts from the cells were analysed by EMSA for the activated NF-kB using a radiolabeled oligonucleotide probe. (b, c) TMK-1 cells, after being pretreated with 5 mM NAC, 25 mM PD98059 (P), and 2 mM SB203580 (S) for 1 h, were incubated with 10 ng/ml IL-1b for 4 h and then determined for the activated NF-kB by EMSA. (d) TMK-1 cells were incubated with 10 ng/ml IL-1b for 0–4 h and then determined for the I-kB subunit of NF-kB by Western blot analysis. b-Actin was used as internal control for loading. (e) TMK-1 cells, after being pretreated with 50 mM PDTC for 1 h, were incubated with 10 ng/ml IL-1b for 8 h and then determined for the IL-8 mRNA by Northern blot analysis

Erk-1/2 and P38 MAPK phosphorylation was detectable within 15 min after exposure of the cells to 10 ng/ml of IL-1b. In contrast, JNK was not activated by the cytokine. Activation of Erk-1/2 and P38 MAPK preceded the induction of IL-8 mRNA expression, and this upregulation was attenuated by selective inhibitors of Erk-1/2 and P38 MAPK. In addition, transfection of kinase mutant forms of Erk-1/2 and P38 MAPK decreased the IL-8 promoter activity. All these results Oncogene

suggest that both ERK-1/2 and P38 MAPK signaling pathways are implicated in the activation of the IL-8 gene by IL-1b. The following observations in our study suggest that ROS are also involved in the signaling pathway for IL-8 induction by IL-1b in TMK-1 cells: (1) IL-1b stimulated the intracellular ROS production as determined with H2O2-sensitive fluorophore DCFDA. (2) NAC, an ROS scavenger, could suppress the IL-8 expression by IL-1b. (3) Treatment with H2O2 increased the IL-8 expression. The molecular mechanism for the ROS production by IL-1b remains to be elucidated. In previous studies, IL-1 was found to stimulate phospholipase A2, promoting release of arachidonic acid. Since arachidonic acid can activate NAPDH oxidase to produce superoxide, it is possible that this fatty acid may serve as an intermediate in the IL-1-induced activation of enzymes, leading to the production of ROS (Lo et al., 1998). The observations that NADPH oxidase inhibitors suppressed the IL-1band TNF-a-induced ROS production also indicated the involvement of NADPH oxidase in the generation of ROS induced by cytokines (Thannickal and Fanburg, 1995). However, several non-NADPH oxidase-dependent sources, including mitochondrial electron transport and arachidonate metabolism, may also be importantly involved in the cytokine-induced ROS generation. In the subsequent experiments, we characterized the sites in the IL-8 promoter that were required for IL-1binduced IL-8 gene expression in TMK-1 cells. As shown in Figure 4, regions of the promoter containing candidate-binding sites for AP-1 at 126 to 120 and NF-kB at 80 to 71 were required for IL-1b-mediated activation of the full-length (546-bp) IL-8 promoter. Site-directed mutagenesis study indicated that the AP-1and NF-kB-binding sites were required for activation of the minimal (133-bp) IL-8 promoter. Gel shift assays confirmed that IL-1b increased the DNA-binding activities of AP-1 and NF-kB. Our results are consistent


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with those of earlier studies implicating the involvement of AP-1 and NF-kB in the regulation of IL-8 expression (Jung et al., 2002), although the relative contributions of these transcription factors appear to vary depending on the cell line and the stimulus used. Interestingly, treatment with Erk-1/2 and P38 MAPK inhibitors prevented the activation of transcription factor AP-1, but not that of NF-kB. Therefore, it is suggested that AP-1 is a downstream target of MAPK pathways and that Erk-1/2 and P38 MAPK may mediate the IL-1b-induced IL-8 expression through AP-1 activation. This may be the first report to provide evidence that Erk-1/2 and P38 MAPK play an important role in the regulation of IL-1b-stimulated AP-1 activation in human gastric TMK-1 cells. The signaling pathway leading to NF-kB activation by IL-1b receptor binding remains controversial. Despite the use of a simple model for NF-kB activation by IL-1b (IL-1 type 1 receptor-IL-1 receptor-associated kinase-TNF receptor-associated factor-NF-kB-inducing kinase-I-kB kinase) (Guan et al., 1997), other intermediates like PI-3 kinase may also be important (Sizemore et al., 1999). In this study, NAC, an ROS scavenger, inhibited the IL-1b-induced NF-kB activation and IL-8 expression, indicating that ROS might also be a downstream effector of IL-1b in TMK-1 cells. Recently, Kitadai et al. (1998, 1999) demonstrated that human gastric carcinomas overexpressed IL-8 and the IL-8 mRNA level directly correlated with the vascularity of the gastric tumors. The process of angiogenesis is essential for tumor growth (Folkman, 1995). In the search for a better understanding of the process of tumor angiogenesis, it is needed to appreciate that the development of new blood vessels is dependent upon not only the activity of endothelial cells but also the function and activity of tumor cells in the vascular microenvironment (Hirschi and D’Amore, 1997; Goede et al., 1998). It is conceivable that a cytokine network exists between inflammatory cells producing cytokines that can initiate signaling in tumor cells. Tumor cells, in turn, may express factors including IL-8 that stimulate endothelial cell proliferation, an essential process in neovascularization.

Materials and methods Cell culture and culture conditions Human gastric carcinoma TMK-1 cells (Ochiai et al., 1985) were cultured at 371C in a 5% CO2 atmosphere in RPMI-1640 medium (Nissui, Tokyo, Japan) containing 10% fetal bovine serum (MA Bioproducts, Walkersville, MD, USA). Human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 15% FBS and 5 ng/ml basic fibroblast growth factor (bFGF). To determine the effects of IL-1b (R&D Systems, Inc., Minneapolis, MN, USA) on Erk-1/2, JNK, P38 MAPK, and Akt activation, cells were harvested at various intervals and phosphorylated, and total protein levels were determined by Western blot analysis. To

examine the role of specific signaling pathways in IL-8 induction by IL-1b, TMK-1 cells were pretreated with 2 mM SB203580 (a specific P38 MAPK inhibitor; Calbiochem, San Diego, CA, USA), and 25 mM PD98059 (a MEK inhibitor, New England Biolabs Inc., Beverly, MA, USA) for 1 h prior to exposure to IL-1b. To investigate the role of ROS in IL-1binduced IL-8 expression, the cells were pretreated with 5 mM NAC (Sigma, St Louis, MO, USA) for 1 h prior to IL-1b treatment. The levels of IL-8 mRNA were then measured by Northern blot analysis. Western blot anlaysis Protein extraction and Western blot analysis were performed as previously described (Jung et al., 2002). The primary antibody preparations used in this study were 1 : 1000 dilutions of rabbit polyclonal anti-phosphospecific p44/42 MAPK (Erk1/2), anti-phosphospecific P38 MAPK, anti-phosphospecific Akt, anti-phosphospecific JNK (New England Biolabs Inc.), anti-IkB, and anti-actin antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The secondary antibody was horseradish peroxidase-labeled anti-rabbit immunoglobulins from donkey (Amersham Corp., Arlington Heights, IL, USA) used at a 1 : 3000 dilution. Protein bands were visualized using a commercially available chemiluminescence kit (Amersham Corp.). Total protein levels were assayed by washing the blotted membrane with stripping solution (100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate, and 62.5 mM Tris–HCl (pH 6.7)) for 30 min at 501C and then reprobing the membrane with rabbit polyclonal anti-p44/42, anti-P38 and anti-actin antibodies diluted at 1 : 1000. Northern blot analysis Total RNA extraction and Northern blot hybridization were performed as previously described (Jung et al., 2002). The cDNA used in the study was a 0.5-kb EcoR1 cDNA fragment corresponding to human IL-8 (Mastronarde et al., 1998). A glyceraldehyde-phosphate dehydrogenase (GAPDH) probe was purchased from the American Type Culture Collection. Each cDNA probe was radiolabeled with [a-32P]deoxyribonucleoside triphosphate by the random-priming technique using the Rediprime labeling system (Amersham Corp.). The probed nylon membranes were exposed to radiographic films (Life Technologies, Inc., Grand Island, NY, USA). Measurement of intracellular H2O2 Intracellular H2O2 was measured using 5-(and 6)-carboxyl20 ,70 -dichlorodihydrofluorescein diacetate (DCFDA, Molecular Probes, Eugene, OR, USA) by the procedure of Ohba et al. (1994). Briefly, cells were grown in serum-starved RPMI-1640 medium supplemented with 0.5% FBS for an additional 2 days. The cells were then stabilized in serum-free RPMI-1640 medium without phenol red for at least 30 min before exposure to IL-Ib for 0–60 min. To assess the effect of NAC, the cells were pretreated with NAC for 30 min. The cells were then incubated with the H2O2-sensitive fluorophore DCFDA (5 ng/ ml) for 10 min, and immediately observed under a laserscanning confocal microscope. DCF fluorescence was excited at 488 nm using an argon laser, and the evoked emission was filtered with a 515 nm long pass filter. Measurement of IL-8 transcriptional activity The transcriptional regulation of IL-8 by IL-1b was examined using transient transfection with an IL-8 promoter-luciferase reporter construct (pGL2-IL-8) (Mukaida et al., 1994). Oncogene


6610 TMK-1 cells (5 105) were seeded and grown to 60–70% confluence, and then pRLTK (an internal control plasmid containing the herpes simplex thymidine kinase promoter linked to the constitutively active Renilla luciferase reporter gene) and pGL2-IL-8 were cotransfected into cells using FuGENE (Boehringer Mannheim, Indianapolis, IN, USA) according to the manufacturer’s protocol. pRLTK and pGL2 were cotransfected as a negative control. Cells were incubated in the transfection medium for 20 h and treated with IL-1b for 8 h. The effects of signaling inhibitors on IL-8 promoter activity were determined by pretreating cells with the inhibitors for 1 h prior to addition of IL-1b. Cotransfection studies were performed in the presence or absence of 2 mg dominantnegative mutants of MEK-1 (K97M, pMCL HA-hMKK1K97M) and P38 MAPK. Dominant-negative mutants of MEK-1 (Emrick et al., 2001) and P38 MAPK (Ge et al., 2002) were kindly provided by Dr Natalie Ahn (University of Colorado, USA) and Dr Jiahuai Han (Scripps Research Institute, USA), respectively. Cells were harvested with passive lysis buffer (Dual-Luciferase Reporter Assay System; Promega, Madison, WI, USA), and luciferase activity was determined using a single sample luminometer according to the manufacturer’s protocol. Transient transfection of AP-1-reporter The AP-1-reporter construct was purchased from Clonetech (Palo Alto, CA, USA). At 80–90% confluency, cells were washed with RPMI 1640 and incubated with RPMI 1640 without serum and antibiotics for 5 h. The cells were then transfected with 1 mg AP-1-reporter containing pGL3 vector using FuGENE6 (Boehringer Mannheim) for 24 h. To determine the roles of specific signaling pathways in AP-1 activation by IL-1b, the transfected cells were pretreated with 50 mM PD98059 and 2 mM SB203580 for 1 h and incubated with 10 ng/ ml IL-1b for 8 h. After incubation, cells were lysed and luciferase activity was measured using a luminometer. Extraction of nuclear proteins In all, 80–90% confluent TMK-1 cells were incubated overnight in medium containing 5% FBS and then treated with 10 ng/ml IL-1b for various periods. To determine the effect of SB203580 and PD98059 on IL-1b-induced AP-1-DNA and NF-kB-DNA complex formation, cells were pretreated with the inhibitors for 1 h prior to exposure to IL-1b. The cells were then resuspended in 500 ml cold buffer A (50 mM Tris (pH 7.4), 150 mM NaCl, 0.2 mM EDTA, 3% (v/v) glycerol, and 1.5 mM MgCl2). After the cells were allowed to swell for 5 min on ice, they were lysed with 500 ml buffer B (identical to buffer A, except containing 0.05% Nonidet P-40 (Sigma)). The homogenate was gently layered onto an equal volume cushion of buffer C (10 mM Tris (pH 7.4), 25% (v/v) glycerol, and 1.5 mM MgCl2) and centrifuged for 5 min at 200 g. The white nuclear pellet was resuspended in 75 ml cold high-salt lysis buffer (20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). This suspension was agitated for 30 min at 41C and then microcentrifuged for 15 min at 41C. The resulting supernatant was stored in aliquots at 801C. Protein was quantitated spectrophotometrically using the BCA assay (Pierce) with bovine serum albumin as a standard.

Electrophoretic mobility shift assay (EMSA) EMSA was performed with the Gel Shift Assay System (Promega). Briefly, oligonucleotides containing the consensus sequences for AP-1 (50 -CGC TTG ATG AGT CAG CCG GAA-30 ) and NF-kB (50 -AGT TGA GGG GAC TTT CCC AGG-30 ) were end-labeled with [a-32P]adenosine triphosphate (3000 Ci/mmol; Amersham Pharmacia Biotech, Buckinghamshire, UK) using T4 polynucleotide kinase and then purified in Microspin G-25 columns (Sigma) and used as probes for EMSA. Nuclear extract proteins (6 mg) were pre-incubated with the binding buffer (10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM dithiothreitol, 4% (v/v) glycerol, and 0.05 mg/ml poly(deoxyinosine-deoxycytosine)) for 5 min and then incubated with the labeled probe for 15 min at 371C. Each sample was electrophoresed in a 5% nondenaturing polyacrylamide gel in 0.5 Tris borate–EDTA buffer at 150 V for 4 h. The gel was dried and subjected to autoradiography. In competition studies, a 50-fold excess of unlabeled oligonucleotide was included in the reaction mixture along with the radiolabeled probe. Determination of the effect of TMK-1-derived CM on proliferation of HUVEC CM derived from TMK-1 cells were prepared as follows. Cells were grown to 95–100% confluence and incubated for 48 h in RPMI-1640 medium with 1% FBS and 10 ng/ml IL-1b. After incubation, the supernatants (CM) were collected, centrifuged, filtered, and stored at 201C. To determine the effect of CM on endothelial cell proliferation, HUVECs (5 103) were plated on 96-well plates (Falcon Laboratories, McLean, VA, USA) and incubated for 24 h with DMEM containing 15% FBS and 10 ng/ml bFGF. The medium was replaced with CM, and the cells were incubated for 24 h. The neutralizing effect of anti-IL-8 antibody on the proliferative activity of CM was determined by incubating the cells with CM after CM were treated for 1 h with 1 mg/ml neutralizing antibody to IL-8 or nonspecific IgG (R&D Systems). The cell proliferation was determined by the MTT assay.

Abbreviations IL-8, Interleukin-8; AP-1, activator protein-1; C/EBP, CCAAT/enhancer binding protein; NF-kB, nuclear factorkB; MAPK, mitogen-activated protein kinase; ROS, reactive oxygen species; Erk, extracellular signal-related kinases; JNK, c-Jun NH2-terminal kinases; NAC, N-acetylcysteine; DCFDA, 5-(and 6)-carboxyl-20 ,70 -dichlorodihydrofluorescein diacetate; CM, conditioned media. Acknowledgements We are very grateful to Dr Eiichi Tahara (Hiroshima University, Japan) for TMK-1 cells, to Dr Natalie Ahn (University of Colorado, USA) for the MEK-1 construct and to Dr Jiahuai Han (Scripps Research Institute, USA) for the P38 MAPK construct. This work was supported by a grant (PF0320504-00) from the Plant Diversity Research Center of the 21st Century Frontier Program Funded by the Ministry of Science and Technology of Korean government.

References Anthonsen MW, Andersen S, Solhaug A and Johansen B. (2001). J. Biol. Chem., 276, 35344–35351. Oncogene

Baggiolini M, Moser B and Clark-Lewis I. (1994). Adv. Immunol., 55, 97–179.


6611 Chen CY and Shyu AB. (1995). Trends Biochem. Sci., 20, 465–470. Duval C, Cantero AV, Auge N, Mabile L, Thiers JC, NegreSalvayre A and Salvayre R. (2003). Free Radic. Biol. Med., 35, 1589–1598. Emrick MA, Hoofnagle AN, Miller AS, TenEyck LF and Ahn NG. (2001). J. Biol. Chem., 276, 46469–46479. Folkman J. (1995). Nat. Med., 1, 27–31. Ganesh S, Sier CF, Heerding MM, Van Krieken JH, Griffioen G, Welvaart K, Van de Velde CJ, Verheijen JH, Lamers CB and Verspaget HW. (1996). Cancer, 77, 1035–1043. Ge B, Gram H, Di Padova F, Huang B, New L, Ulevitch RJ, Luo Y and Han J. (2002). Science, 295, 1291–1294. Goede V, Schmidt T, Kimmina S, Kozian D and Augustin HG. (1998). Lab. Invest., 78, 1385–1394. Guan Z, Baier LD and Morrison AR. (1997). J. Biol. Chem., 272, 8083–8089. Guiraldes E, Duarte I, Pena A, Godoy A, Espinosa MN, Bravo R, Larrain F, Schultz M and Harris P. (2001). J. Pediatr. Gastroenterol. Nutr., 33, 127–132. Harris GK and Shi X. (2003). Mutat. Res., 533, 183–200. Heidemann J, Ogawa H, Dwinell MB, Rafiee P, Maaser C, Gockel HR, Otterson MF, Ota DM, Lugering N, Domschke W and Binion DG. (2003). J. Biol. Chem., 278, 8508–8515. Hirschi KK and D’Amore PA. (1997). EXS, 79, 419–428. Hofmeister R, Wiegmann K, Korherr C, Bernardo K, Kronke M and Falk W. (1997). J. Biol. Chem., 272, 27730–27736. Jung YD, Fan F, McConkey DJ, Jean ME, Liu W, Reinmuth N, Stoeltzing O, Ahmad SA, Parikh AA, Mukaida N and Ellis LM. (2002). Cytokine, 18, 206–213. Kasahara T, Mukaida N, Yamashita K, Yagisawa H, Akahoshi T and Matsushima K. (1991). Immunology, 74, 60–67. Katsuyama K and Hirata Y. (2001). J. Biochem., 129, 585–591. Kitadai Y, Haruma K, Sumii K, Yamamoto S, Ue T, Yokozaki H, Yasui W, Ohmoto Y, Kajiyama G, Fidler IJ and Tahara E. (1998). Am. J. Pathol., 152, 93–100. Kitadai Y, Takahashi Y, Haruma K, Naka K, Sumii K, Yokozaki H, Yasui W, Mukaida N, Ohmoto Y, Kajiyama G, Fidler IJ and Tahara E. (1999). Br. J. Cancer, 81, 647–653. Klezovitch O, Edelstein C and Scanu AM. (2001). J. Biol. Chem., 276, 46864–46869.

Kunsch C, Lang RK, Rosen CA and Shannon MF. (1994). J. Immunol., 153, 153–164. Lo YY, Conquer JA, Grinstein S and Cruz TF. (1998). J. Cell. Biochem., 69, 19–29. Maeda S, Akanuma M, Mitsuno Y, Hirata Y, Ogura K, Yoshida H, Shiratori Y and Omata M. (2001). J. Biol. Chem., 276, 44856–44864. Mastronarde JG, Monick MM, Mukaida N, Matsushima K and Hunninghake GW. (1998). J. Infect. Dis., 177, 1275–1281. Mukaida N, Morita M, Ishikawa Y, Rice N, Okamoto S, Kasahara T and Matsushima K. (1994). J. Biol. Chem., 269, 13289–13295. Ochiai A, Yasui W and Tahara E. (1985). Jpn. J.Cancer Res., 76, 1064–1071. Ohba M, Shibanuma M, Kuroki T and Nose K. (1994). J. Cell Biol., 126, 1079–1088. Rennekampff HO, Hansbrough JF, Kiessig V, Dore C, Sticherling M and Schroder JM. (2000). J. Surg. Res., 93, 41–54. Schwarz M, Radeke HH, Resch K and Uciechowski P. (1997). Kidney Int., 52, 1521–1531. Shin EY, Kim SY and Kim EG. (2001). Exp. Mol. Med., 33, 276–283. Singh RK and Varney ML. (2000). Histol. Histopatho., 15, 843–849. Sizemore N, Leung S and Stark GR. (1999). Mol. Cell. Biol., 19, 4798–4805. Straubinger RK, Greiter A, McDonough SP, Gerold A, Scanziani E, Soldati S, Dailidiene D, Dailide G, Berg DE and Simpson KW. (2003). Infect. Immun., 71, 2693–2703. Thannickal VJ and Fanburg BL. (1995). J. Biol. Chem., 270, 30334–30338. Wang M, Furuta T, Takashima M, Futami H, Shirai N, Hanai H and Kaneko E. (1999). J. Gastroenterol., 34, 10–17. Welsh N. (1996). J. Biol. Chem., 271, 8307–8312. Wolf JS, Chen Z, Dong G, Sunwoo JB, Bancroft CC, Capo DE, Yeh NT, Mukaida N and Van Waes C. (2001). Clin. Cancer Res., 7, 1812–1820. Wu LL, Chiou CC, Chang PY and Wu JT. (2004). Clin. Chim. Acta., 339, 1–9. Yasumoto K, Okamoto S, Mukaida N, Murakami S, Mai M and Matsushima K. (1992). J. Biol. Chem., 267, 22506–22511.

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