Introduction

As an adaptive response to systemic stress, the hypothalamic–pituitary–adrenal (HPA) axis is activated (Chrousos, 1995). The process starts with the hypothalamic release of corticotropin-releasing hormone (CRH) which, in turn stimulates production of adrenocorticotropic hormone (ACTH) and other proopiomelanocortin (POMC) peptides via CRH receptor type 1 (CRH-R1). ACTH, a 39-amino-acid peptide, regulates glucocorticoid production through its effect on the adrenal cortex. Steroids are powerful anti-inflammatory agents that counteract stressors and buffer tissue damage (Chrousos, 1995). Recent evidence suggests that ACTH is produced outside the pituitary tissue, which includes the skin (Chakraborty et al., 1996; Wakamatsu et al., 1997; Slominski et al., 2000a). Slominski et al. have acknowledged the skin as a cutaneous equivalent of the central HPA (Slominski and Mihm, 1996; Slominski et al., 2000b, 2001, 2004a). Although similar to its systemic equivalent, the cutaneous HPA axis is responsive to local stressors (solar, thermal, chemical, biological, etc.) and resultantly activates the neuronal, endocrine, and immune systems in the skin. A variety of cytokines (e.g., IL-1, IL-6, and TNF-) are upregulated by stress, suggesting their role in mediating stress responses (Dugue et al., 1993). However, the molecular mechanism of stress-induced cytokine in impairing host defenses is not well understood.

IL-18 was originally discovered as IFN--inducing factor (Okamura et al., 1995; Ushio et al., 1996). It is normally expressed in its precursor form (24 kDa), but can be activated (18 kDa) by caspase-1 (IL-1-converting enzyme) (Ghayur et al., 1997; Gu et al., 1997). IL-18 performs multiple biological activities, which include Fas (CD95, APO-1) ligand induction, enhancement of T-cell cytolytic activity, and production of T helper type 2 (Th2) cytokines (Hoshino et al., 1999; Yoshimoto et al., 2000; Nakanishi et al., 2001). IL-18 is expressed in a wide range of cells, including Kupffer cells, activated macrophages, keratinocytes, intestinal epithelial cells, osteoblasts, and adrenal cortex cells. It plays an important role in the Th1 response to toxic shock and shares functional similarities with IL-12 (Dinarello, 1999). As a proinflammatory cytokine, overproduction of IL-18 induces severe inflammatory disorders. Thus, it is important to know how IL-18 expression is regulated in inflammatory response and in maintaining the balance of immune system function. Recently, IL-18 by keratinocytes was reported to play an important role in response to cutaneous inflammation (Mee et al., 2000).

In this study, we investigated the effect of ACTH on IL-18 expression of human HaCaT keratinocytes, and also examined its regulatory mechanism.

Results

Effect of ACTH on IL-18 production in HaCaT cells

To measure the effect of ACTH on IL-18 expression in HaCaT cells, we treated the cells with the fixed concentrations of ACTH, and then assessed IL-18 mRNA and its protein expression by reverse transcriptase-PCR and ELISA, respectively. IL-18 mRNA expression was increased in a dose-dependent manner by ACTH, with the peak response at 10^-9 M (Figure 1a). IL-18 assessed by ELISA was also increased in a dose-dependent manner (Figure 1b). As shown in Figure 1, IL-18 mRNA expression and its production were significantly stimulated by ACTH in a dose-dependent manner.

Figure 1.

Effects of ACTH on the mRNA expression and production of IL-18 in HaCaT keratinocytes. (a) HaCaT cells were harvested after ACTH treatment for 6 hours. Total RNA was extracted, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to -actin by densitometry. (b) Cells were treated with indicated concentration of ACTH for 24 hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are meanSD. ^*P<0.05 versus control.

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Induction of caspase-1 by ACTH

Because caspase-1 is required for cleaving pro-IL-18 into its mature form, its involvement in IL-18 production was examined by pretreating HaCaT cells for 2 hours with the caspase-1-specific inhibitor Ac-YVAD-CHO before stimulation with ACTH. We observed Ac-YVAD-CHO to suppress the production of IL-18 (Figure 2a). Next, we investigated the molecular mechanism underlying ACTH-induced IL-18 production by performing caspase-1 colorimetric assay. Caspase-1 activity in HaCaT cells were elevated 10-fold by ACTH (Figure 2b). The results indicate that ACTH is involved in IL-18 production of HaCaT cells probably via a caspase-1 activation pathway. In a previous report, caspase-1 was activated by caspase-11 (Wang et al., 1998). It has also been reported that caspase-11 can be activated by cathepsins B (Schotte et al., 2001). To understand the involvement of caspase-11 in the ACTH-induced caspase-1 activation, we examined it by using Z-FA-FMK, an inhibitor of cathepsins B. In our observation, Z-FA-FMK suppressed the production of IL-18 induced by ACTH, indicating that caspase-11 is also involved in the ACTH-induced IL-18 production in HaCaT cells (Figure 2c).

Figure 2.

Caspase-1 is essential for the ACTH-induced IL-18 production in HaCaT keratinocytes. (a) HaCaT cells were pretreated with 40 M of the caspase-1 inhibitor Ac-YVAD-CHO for 1 hour, and then challenged with 1 nM ACTH. The IL-18 concentration in the culture supernatants was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1 nM ACTH for 6 hours. Caspase-1 enzymatic activity was assayed with a caspase-1 colorimetric assay kit according to the manufacturer's instruction. The data normalized to the negative control are shown as fold increases in caspase activity. (c) Effects of Z-FA-fmk on ATCH-induced IL-18 production. HaCaT cells were pretreated with 20 M of Z-FA-FMK for 2 hours, and then stimulated with 1 nM ACTH. The IL-18 concentration in the supernatant was measured by ELISA, each performed in triplicate. Data are meanSD. ^*P<0.05 versus control, ^§P<0.05 versus ACTH treated group.

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Effect of mitogen-activated protein kinase inhibitors on IL-18 expression

The mitogen-activated protein kinases (MAPKs) control a wide variety of cellular events, which include very complex cellular programs such as cell differentiation, proliferation and apoptosis to processes involved in immune responses. To identify its involvement in ACTH-induced IL-18 expression, HaCaT cells were treated with 10 M of p38 MAPK inhibitor (SB203580) or extracellular signal-regulated kinase (ERK) inhibitor (PD98059) for 2 hours before adding ACTH (1 nM). We then assessed the expression of IL-18 mRNA transcripts and its products. As shown in Figure 3, both SB203580 and PD98059 had an inhibitory effect over ACTH on IL-18 mRNA expression and its production.

Figure 3.

Effects of the p38 MAPK inhibitor SB203580, or the ERK inhibitor PD98059 on ACTH-induced IL-18 expression in HaCaT cells. HaCaT cells were pretreated with SB203580 (10 M) or PD98059 (10 M) for 2 hours before addition of 1 nM of ACTH. (a) Total RNA was extracted at 6 hours following ACTH treatment, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to -actin by densitometry. (b) After cells were harvested for 24 hours, IL-18 concentration was measured by ELISA, each performed in triplicate. Data are meanSD. ^*P<0.05 versus control, ^§P<0.05 versus ACTH treated group.

Full figure and legend (57K)

To further identify the role of MAP kinase signaling pathways in ACTH-induced IL-18 expression, HaCaT cells were treated with ACTH, and the phosphorylation status of p38 kinase and ERK were analyzed by immunoprecipitation and Western blotting using commercially available antibodies specific for the active forms of the two kinases. ACTH induced transient phosphorylation of both p38 MAP kinase and ERK. The maximal activation of p38 kinase and ERK by ACTH was observed after 5 minutes (Figure 4a and b). These data demonstrate that ACTH stimulates IL-18 production through p38 kinase and ERK signaling pathways.

Figure 4.

Effects of ACTH on activation of MAPKs in HaCaT cells. HaCaT cells were treated with 1 nM of ACTH for indicated time periods. Phosphorylation of (a) p38 kinase and (b) ERK were analyzed by immunoprecipitation and Western blot as described in Materials and methods. After cells were harvested for 5 minutes, phosphorylation of p38 kinase and ERK were analyzed by immunoprecipitation and Western blot. The band intensities were quantitated. Data are meanSD. ^*P<0.05 versus control, ^§P<0.05 versus ACTH treated group.

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Melanocortin receptor 1 and MC2R expression in HaCaT cells

To study whether melanocortin receptor 1 (MC1R) and/or MC2R are involved in the mediating ACTH-induced IL-18 production in HaCaT cells, the cells were treated with ACTH and then MC1R and MC2R were analyzed by Western blotting. We determined that MC1R and MC2R were both expressed on HaCaT cells (Figure 5a). Next, we used anti-MC1R and anti-MC2R antibody to examine whether they could neutralize the ACTH-induced IL-18 production. Anti-MC2R antibody was more effective and almost reduced it to the control level (Figure 5b). These results suggest that the induction of IL-18 by ACTH may be mainly mediated by MC2R.

Figure 5.

Involvement of MC1R and MC2R in ACTH-induced IL-18 production. (a) HaCaT cells were treated with 1 nM of ACTH. After that, cells were harvested for 24 hours. The expression level of MC1R and MC2R were analyzed by Western blot. (b) Cells were pretreated with anti-MC1R (2 g/mL) or anti-MC2R antibody (2 g/mL) for 1 hour and then incubated with 1 nM of ACTH for 24 hours. The supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are meanSD. ^*P<0.05 versus control, ^§P<0.05 versus ACTH treated group.

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Effect of ACTH on IL-18 production in human primary keratinocytes

To see whether ACTH regulates IL-18 production in primary human epidermal keratinocytes, we assessed IL-18 production by ELISA. As shown in Figure 6a, IL-18 production was significantly stimulated by ACTH treatment in human primary keratinocytes. Next, we observed that caspase-1 activity in primary human epidermal keratinocytes was stimulated by ACTH (Figure 6b). Ac-YVAD-CHO, a caspase-1 inhibitor, effectively suppresses IL-18 production by ACTH in primary human epidermal keratinocytes, as shown in the result from human keratinocyte cell line HaCaT (Figure 6a). In the end, we showed that SB203580 and PD98059 could suppress the ACTH-induced IL-18 production, suggesting that p38 kinase and ERK were both involved in the signaling pathway in primary human epidermal keratinocytes (Figure 6c).

Figure 6.

Effects of ACTH on the production of IL-18 in primary human epidermal keratinocytes. (a) Primary human epidermal keratinocytes were pretreated with or without Ac-YVAD-CHO (40 M) for 2 hours before addition of 1 nM of ACTH for 24 hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1 nM ACTH for 6 hours. Caspase-1 enzymatic activity was assayed with a caspase-1 colorimetric assay kit according to the manufacturer's instruction. The data, normalized to the negative control, are shown as fold increases in caspase activity. (c) Cells were treated with or without SB203580 (10 M) or PD98059 (10 M) for 2 hours before addition of 1 nM of ACTH for 24 hours. Supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are meanSD. ^*P<0.05 versus control, ^§P<0.05 versus ACTH treated group.

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Discussion

In this study, we investigated the regulatory effect of ACTH on expression of IL-18 in HaCaT and normal keratinocytes. Our results show that ACTH stimulates human keratinocytes to secrete IL-18 through MC1R and MC2R. Furthermore, ACTH activates the MAPK p38 and ERK pathways that are further required for IL-18 expression.

In response to systemic stress, CRH stimulates the pituitary CRH-R, which leads to production and secretion of POMC-derived peptides, including melanocyte-stimulating hormone (MSH) and ACTH (Chrousos, 1995). Recent evidence suggests that CRH stimulation of corticosteroids production in melanocytes and dermal fibroblasts is mediated by ACTH (Slominski et al., 2005a, 2005b). CRH, CRH-R, and POMC genes are expressed in human skin as well as in cultured keratinocytes and melanocytes (Slominski et al., 1993, 1995, 2001, 2004a, 2004b). Slominski et al. (2000b, 2001) proposed that an equivalent of HPA axis, composed of the CRH-CRHR-POMC loop, is conserved in the skin that may be activated in a skin stress response system. Recently, MSH and ACTH act as regulators of skin pigmentation and inhibitors of the skin immune systems (Luger et al., 1998; Slominski et al., 2004a, 2004b). In this study, ACTH induced IL-18 production, which is known as an inflammatory cytokine. However, it is also known that the closely related POMC peptide alpha-MSH exhibits anti-inflammatory effects through MC1R in keratinocytes (Pavey and Gabrielli, 2002). To date, five melanocortin receptors are known, and they have a variety of important biological functions (Bohm et al., 2006). To understand the difference between ACTH and alpha-MSH, we detected the expression of MC receptors in HaCaT cells. Figure 5 showed that ACTH signals through MC1R and MC2R. This might be an explanation for the differential effects of ACTH and alpha-MSH in inflammation as MC1R and MC2R have different signal pathways (Bohm et al., 2006). IL-18 has multiple and complicated functions as it participates in innate immunity and in Th1- and Th2-mediated responses. During inflammation, the activation of the stress system, through induction of a Th2 shift, protects the organism from systemic "overshooting" with Th1/pro-inflammatory cytokines. Interestingly, it has been reported that the expression of IL-18 is markedly increased in suprabasal keratinocytes in psoriatic lesions (Naik et al., 1999; Ohta et al., 2001), raising the possibility that IL-18 expression is upregulated in differentiated keratinocytes. Therefore, ACTH induced IL-18 expression by keratinocytes may provide new insights into the mechanism underlying the pathogenesis of skin disorders.

In a previous report, cytokines IL-1 and IL-6 were able to stimulate CRH secretion from the hypothalamus (Navarra et al., 1991), and IL-18 was originally identified as a member of the IL-1 family (Dinarello, 1999) and expected to be able to stimulate CRH production. Interestingly, recent evidence documented that IL-18 inhibited basal and stimulated CRH secretion from isolated rat hypothalami, and reduced CRH gene expression (Tringali et al., 2005). We have recently reported that CRH inhibits IL-18 expression in HaCaT cells, suggesting that IL-18 and CRH act antagonistically to each other (Park et al., 2005). Conti et al. (2000) have shown that the production of IL-18 in the adrenal cortex is stimulated by ACTH and cannot be inhibited by the direct action of corticosterone. In this study, we also showed that IL-18 mRNA expression and its production were significantly stimulated by ACTH in a dose-dependent manner in HaCaT keratinocytes (Figure 1). The above findings suggest that IL-18 may play an important role on the negative feedback loop, CRH upregulating POMC and, subsequently, ACTH leads to the induction of IL-18, and then the downregulation of CRH.

Sugama et al. (2000) have reported differential IL-18 promoter usage in the adrenal gland and immune cells with adrenal gland-specific expression of IL-18 mRNA by ACTH, suggesting that IL-18 may be induced in the adrenal gland during stress. Besides, Sekiyama et al. (2005) demonstrated that ACTH can enhance IL-18 production through superoxide-mediated caspase-1 activation and p38 MAPK kinase. In these studies, animal models were used to show the systematic effects of ACTH by injecting ACTH into the animals. In this study, the aim was to test the direct effect of ACTH in keratinocytes and to compare the difference between the adrenal glands and keratinocytes. The results showed for the first time that ACTH can directly regulate immune functions of keratinocytes, and that the mechanism was the same as that of the adrenal gland. These findings suggest that caspase-1 activation and p38 MAPK kinase pathways are commonly involved in ACTH-induced IL-18 production.

In conclusion, we demonstrated that ACTH stimulates IL-18 expression by phosphorylation of p38 and ERK MAPK signaling pathway. Therefore, our findings may provide an insight into cellular interactions between ACTH and inflammatory mediators. Because IL-18 is involved in both Th1 and Th2 functions, the ability of ACTH to induce the production of inflammatory mediators by keratinocytes provides a new mechanism for the involvement of ACTH in innate and adaptive immunity and its role in the pathogenesis of skin disorders.

Materials and Methods

Cell culture

The human keratinocyte cell line HaCaT was cultured in RPMI 1640 (Gibco-BRL, Gaithersburg, MD) medium supplemented with 2mM L-glutamine, antibiotics (100 U/ml penicillin G and 100 g/ml streptomycin) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL). This cell line was used for experiments while in their log phase of growth. Primary cultures of normal human epidermal keratinocytes were prepared from foreskin of three children who underwent circumcision. The ethical committee of the Catholic University of Korea approved the study, and all patients provided an informed consent. Our experiments adhered to the Declaration of Helsinki Principles. Skin specimens were cut into small pieces and incubated overnight in dispase II (2.4 U/ml; Roche Applied Science, Mannheim, Germany) at 4°C. The epidermis was detached from the dermis with fine forceps and incubated in Hank's solution with 0.25% trypsin (Gibco-BRL) for 20 minutes at 37°C. Trypsin activity was stopped by adding FBS. The cell suspension passed through a 40 m sterile gauze and the keratinocytes obtained were washed twice. Freshly isolated keratinocytes were cultured in keratinocyte basal medium (BioWhittaker, Heidelberg, Germany) with full supplements (0.1 ng/ml human epidermal growth factor, 0.5 g/ml hydrocortisone, 5 mg/ml insulin, 7.5 mg/ml bovine pituitary extract, 50 g/ml gentamicin, 50 ng/ml amphotericin-B, and 0.15 mM calcium) according to the manufacturer's instruction. All cells were incubated in a humidified atmosphere containing 5% CO₂ at 37°C and were used at the passage two to five.

Cell treatment with inhibitors

HaCaT cells were seeded at a density of 1 10⁶ cells/100-mm dishes. After 48 hours, the cells were washed with a serum-free medium and then placed at least 16 hours before experiments in a media without FBS. The cells were pretreated with 10 M SB203580 (Sigma, St Louis, MO), a p38 MAPK inhibitor, 10 M PD98059 (Sigma), an ERK inhibitor, 40 M Ac-YVAD-CHO (SantaCruz Biotechnology, Santa Cruz, CA), an IL-1-converting enzyme/caspase-1 inhibitor, or 20 M Z-FA-FMK (R&D systems, Minneapolis, MN), a cathepsin-B inhibitor for 2 hours in a serum-free medium. We added human ACTH (10^-11 M10^-8 M) to the cells, which was purchased from Sigma.

Treatment of cells with Anti-MC1R and Anti-MC2R antibody

HaCaT cells were pretreated with anti-MC1R or anti-MC2R antibodies (SantaCruz Biotechnology) for 1 hour in serum-free medium. The cells were washed with serum-free medium and then ACTH (1 nM) was added to the cells and incubated for 24 hours.

Reverse transcription-PCR

After treatment, total RNA was extracted from HaCaT cells using the EasyBlue reagent (iNtRON, Inc., Sungnam, Korea). The reverse transcription was performed using a power cDNA synthesis kit (iNtRON, Inc.). IL-18 primers were: forward, 5'-AGGAATAAA GATGGCTGCTGAAC-3'; reverse, 5'-GCTCACCACAACCTCTACC TCC-3'. The PCR amplification process consisted of 32 cycles of 95°C for 1 minute; 60°C for 1 minute; and 72°C for 45 seconds. Human -actin primers were: forward, 5'-GGCCATCTCTTGCTC GAAGT-3'; reverse, 5'-GCCCAGAGCAAG AGAGGCAT-3'. The PCR amplification process consisted of 30 cycles of 94°C for 30 seconds; 56°C for 30 seconds; and 72°C for 1 minute. Reaction products subjected to electrophoresis on 2.0% agarose gel were visualized with ethidium bromide. Signal strengths were quantified using a densitometric program (Quantity One 1-D Analysis software, Bio-Rad Laboratories, Hercules, CA). After normalizing versus -actin intensity, the increase or decrease in percentage was determined for each gene. Each experiment was repeated at least three times.

IL-18 ELISA

The concentration of IL-18 in the culture supernatant was measured by a commercially available ELISA (MBL Medical & Biological Laboratories Co., Ltd, Nagoya, Japan). After stimulation, cell culture supernatants were collected, concentrated ( 30) by a microcon (Millipore, Bedford, MA), and then added to coated wells. After adding diluted biotinylated anti-IL-18 antibody to the wells, they were incubated for 3 hours at room temperature. After washing for four times with phosphate-buffered saline-Tween-20 (pH 7.4), streptavidin-horseradish peroxidase conjugated anti-human IL-18 antibody was added, which was incubated for an hour. The wells were washed four times with PBS-Tween-20, and substrate solution was added, which was incubated for an hour. Relative absorbance was measured at 450 nm, and the IL-18 concentration was calculated using a standard curve. Each supernatant was analyzed in triplicate.

Measurement of caspase-1 activity

The caspase-1 activity assay was performed with an enzyme activity assay kit (Caspase-1 Colorimetric Assay kit, R&D systems) according to the manufacturer's instructions. After stimulation, the cell pellets were lysed in a cell lysis buffer. The protein concentration was determined using a BCA kit (Pierce, Rockford, IL). To 50 l of cell lysate (200 g protein), 50 l of 2 reaction buffer (containing DTT 10 mmol/l) and 5 l of the 4 mol/l caspase-1 colorimetric substrate (WEHD-pNA) were added, and the mixture was incubated at 37°C for 2 hours. The samples were read at 405 nm with a spectrophotometer.

Immunoprecipitation and Western blot analysis

Whole-cell lysates were prepared by extracting proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM Na₃VO₄, 1 g/ml leupeptin, and 1 mM freshly added phenylmethylsulfonyl fluoride. To detect the phosphorylated form of p38 MAPK, the lysates were then immunoprecipitated with 2 g of anti-phosphotyrosine antibody and 25 l of protein-A agarose for 3 hours at 4°C. After washing three times (20 minutes at 4°C) with lysis buffer, the proteins bound to the beads were solubilized in elution buffer consisting of 1 M Tris (pH 6.8) and 0.1% SDS. The protein concentration was measured using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories). Equal amounts of protein were resolved by 10% SDS-PAGE, blotted onto nitrocellulose membranes and immunostained with anti-p38 MAPK antibody. Blots were processed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL Detection Kit, Amersham Biosciences, Piscataway, NJ). To detect MC1R, MC2R, and the phosphorylation level of ERK and p38 MAPK, ACTH-treated and untreated cells were lysed with lysis buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 1.0% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride in PBS (pH 7.4). Whole-cell lysates were directly resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour in PBS containing 0.1% (v/v) Tween-20 and 5% (w/v) non-fat milk proteins. Blocked membranes were then incubated with primary antibodies (MC1R, MC2R rabbit polyclonal IgG antibodies at the final concentration of 1:50, Santa Cruz Biotechnology; phospho-ERK, total ERK or p38 rabbit polyclonal antibodies at the final concentration of 1:1,000, Cell Signaling, Beverly, MA) overnight at 4°C, washed three times (5 minutes each) with PBS containing 0.1% Tween-20, and incubated with a horseradish peroxidase-conjugated secondary antibody (1:2,000) for 1 hour at room temperature. The bands were visualized by an enhanced chemiluminescence detection system (Amersham Biosciences). The band intensities were quantified using the Bio-Rad imaging system, and the quantity of the phosphorylated proteins was expressed as ratio of the phosphorylated over total protein in each case.

Statistical analysis

The statistical significance was performed using Student's t-test. Mean differences were considered to be significant when P<0.05. The results are shown as the meanSD.

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

This work was supported by the SRC/ERC program of MOST/KOSEF (No. R11-2005-017) and the Korea Research Foundation Grant funded by Korea Government (MOEHRD, Basic Research Promotion Fund, KRF-2005-204-E00062).