RESULTS

ATRA increases the production of IL-8 in keratinocytes

IL-1 is the prominent inducer of IL-8 (^{Kristensen et al, 1991;Hoffmann et al, 2002}). Moreover, retinoids reduce IL-1 induction of IL-8 production in human monocytes (^{Gross et al, 1993}). Therefore, we initially investigated the effect of ATRA on IL-1-induced IL-8 production. Keratinocytes were treated with IL-1 (1 ng per mL) or IL-1 plus ATRA (10^-6 M), and the IL-8 levels in the culture supernatants were measured after 36 h of incubation. Marked induction of IL-8 was observed in IL-1-stimulated cells, and ATRA increased IL-8 production in a synergistic manner (Figure 1a).

Figure 1.

All-trans-retinoic acid (ATRA) simulates the production of interleukin (IL)-8 by human keratinocytes. (A) Subconfluent keratinocyte cultures were treated with IL-1 (1 ng per mL), ATRA (10^-6 M), or vehicle (dimethyl sulfoxide). After 36 h of incubation, the culture supernatants were collected and subjected to ELISA for the detection of IL-8. (B) Keratinocytes were stimulated with ATRA (10^-9–10^-5 M). IL-8 mRNA expression was monitored by RT-PCR after a 24-h incubation. (C) Keratinocytes were stimulated with ATRA for 36 h, and the level of secreted IL-8 was measured by ELISA.

Full figure and legend (30K)

To assess the effect of ATRA on IL-8 production in keratinocytes, we stimulated the keratinocytes with different concentrations of ATRA. IL-8 mRNA expression was scarcely detectable by the ribonuclease protection assay (RPA) in samples that contained the vehicle (dimethyl sulfoxide (DMSO)) alone (Figure 1b). After 24 h of treatment with various doses of ATRA (10^-9–10^-5 M), IL-8 mRNA expression was increased in a concentration-dependent manner (Figure 1b). The release of IL-8 into the culture medium was upregulated in a concentration-dependent manner, and was consistent with the increase in IL-8 mRNA (Figure 1c).

ATRA increases the expression of the NF-B family members in human keratinocytes

NF-B binding is essential for the activation of IL-8 gene transcription (^{Kunsch et al, 1994}). Several members of the NF-B family, such as p50 (NF-B1), p65 (RelA), c-Rel, and p52 (NF-B2), have been shown to bind to the NF-B motif of the IL-8 promoter (^{Kunsch and Rosen, 1993;Stein et al, 1993;Harant et al, 1996a}). To characterize the molecular mechanisms underlying ATRA-induced IL-8 production in keratinocytes, the expression of NF-B family members in ATRA-treated keratinocytes was examined using the RPA. The expression levels of p65, RelB, p52, and p50 mRNA started to increase 1 h after the addition of ATRA, and persisted for more than 24 h (Figure 2). Although c-Rel was detected faintly 12–48 h after ATRA treatment, the basal level of expression of c-Rel mRNA was very low in the RPA.

Figure 2.

All-trans-retinoic acid (ATRA) increases the expression of nuclear factor (NF)-B subunit mRNA. Keratinocytes were exposed to 10^-6 M ATRA, and total RNA samples were collected at the indicated time points. The expression levels of the NF-B subunit mRNAs were examined using the ribonuclease protection assay.

Full figure and legend (44K)

ATRA increases the DNA-binding activity of p65 in human keratinocytes

We also examined the DNA-binding activities of the NF-B family members in the nuclear extracts of ATRA-stimulated keratinocytes. The DNA-binding activity of p65 increased between 6 and 24 h post-treatment with ATRA (Figure 3a). In addition, ATRA upregulated the DNA-binding activity of p65 in a concentration-dependent manner (Figure 3b). In contrast, the DNA-binding activity of p50 was not influenced by ATRA. The DNA-binding activity of the c-Rel protein was not detected in either ATRA-treated or untreated keratinocytes (data not shown).

Figure 3.

Nuclear translocation of p65 is increased by all-trans-retinoic acid (ATRA) treatment. (A) Keratinocytes were stimulated with ATRA (10^-6 M), and the nuclear proteins were extracted at the indicated time points. An enzyme-linked immunoassay was performed to monitor the DNA-binding activities of the nuclear factor (NF)-B subunits in the nucleus. The relative values for the optical density at 655 nm are normalized to the value of 0 h as 1 U, and are plotted on the graph. (B) ATRA (10^-9–10^-5 M) was added to subconfluent-conditioned keratinocytes, and the cultures were incubated for 24 h. The DNA-binding activities of the NF-B subunits in the nucleus were examined, and the relative values are normalized to the value for 0 M, which is designated as 1 U. ^*p<0.05; ^**p<0.01.

Full figure and legend (37K)

ATRA phosphorylates IB in cultured human keratinocytes

The inhibitory molecule IB anchors NF-B in the inactive form in the cytosol (^{Baeuerle and Henkel, 1994}). A crucial step in NF-B signal transduction involves the dissociation of NF-B from IB, which is followed by NF-B translocation to the nucleus (^{Baeuerle and Henkel, 1994}). The dissociation of NF-B from IB requires the phosphorylation of IB, which results in rapid and ubiquitous degradation of IB. Thus, the effect of ATRA on the expression and phosphorylation of IB was investigated. ATRA increased the level of phosphorylation of IB, although ATRA had no significant effect on the total level of IB protein (Figure 4a and b). The relative level of expression of phosphorylated IB to total IB was increased significantly, 6 h post-ATRA treatment, and had increased 1.9-fold at 24 h (Figure 4a). In our concentration-dependency study, 24 h of ATRA treatment increased the level of phosphorylated IB 1.3-fold at 10^-7 M, 1.9-fold at 10^-6 and 2.2-fold at 10^-5 M, as compared with vehicle-treated keratinocytes (Figure 4b).

Figure 4.

All-trans-retinoic acid (ATRA) increases the level of phosphorylated IB in human keratinocytes. (A) Keratinocytes were stimulated with ATRA (10^-6 M), and the cellular proteins were extracted at the indicated time points. Immunoblotting was performed with anti-IB and anti-phospho-specific IB antibodies. The relative levels of phosphorylated IB are estimated using the total level of IB protein as the reference, and these values are normalized against the value at 0 min, which is designated as 1 U. (B) ATRA (10^-9–10^-5 M) was added and incubated for 24 h. The relative levels of phosphorylated IB protein expression are estimated from the immunoblots. ^*p<0.05; ^**p<0.01; ^***p<0.001.

Full figure and legend (53K)

Mutant IB abrogates the production of IL-8 by ATRA in human keratinocytes

To determine whether ATRA-induced IL-8 production in keratinocytes was dependent on NF-B, we introduced mutated-IB (IBM) gene using an adenovirus vector (AxIBM), which results in the blockade of NF-B signals by unphosphorylated IB (^{Brown et al, 1995;Chen et al, 1995}). Keratinocytes were transfected with AxIBM or AxLacZ, and stimulated with ATRA. The culture supernatants were collected at 12, 24, and 48 h after the addition of ATRA. The ELISA demonstrated that IBM not only abrogated ATRA-dependent production of IL-8, but also drastically suppressed the basal production of IL-8 (Figure 5a). IBM also suppressed IL-8 mRNA expression in both ATRA-treated and untreated keratinocytes (Figure 5b). The increased expression of IB protein in IBM-transfected keratinocytes confirms that mutated IB persists in the cell and suppresses the NF-B signal (Figure 5c).

Figure 5.

Blockade of nuclear factor (NF)-B signaling abrogates the induction of interleukin (IL)-8 by all-trans-retinoic acid (ATRA). (A) Keratinocytes were transfected with AxIBM or AxLacZ at a multiplicity of infection (MOI)=2 for 12 h before the addition of ATRA. ATRA (10^-6 M) or vehicle was then added, and the cultures were incubated for the indicated time periods. The culture supernatants were collected, and the levels of IL-8 protein were measured. ^*p<0.05; ^**p<0.01. (B) Keratinocytes were transfected with adenovirus vector and stimulated with ATRA (10^-6 M) or vehicle. The levels of IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression 24 h post-treatment with ATRA were analyzed by RT-PCR. (C) Keratinocytes were transfected with adenovirus vector and stimulated with ATRA (10^-6 M) or vehicle. The levels of IB protein expression 24 h after ATRA treatment were analyzed by western blotting.

Full figure and legend (36K)

The p38 MAPK inhibitor SB202180 partially suppresses ATRA-induced IL-8 expression in human keratinocytes

We investigated whether the p38 pathway was also involved in ATRA-induced IL-8 gene expression in keratinocytes. After the addition of ATRA, the phosphorylation of p38 increased, whereas the total level of p38 protein was unchanged (Figure 6a). The phosphorylation of p38 started at 5 min, peaked at 1 h, and persisted for 24 h post-treatment with ATRA (Figure 6a). ATRA-dependent p38 phosphorylation was inhibited by SB202180 (Figure 6b). The cells were treated with 5, 10, or 20 M of SB202180 for 2 h before the addition of ATRA, and the keratinocytes were then incubated for 36 h. ATRA-induced IL-8 production was inhibited in a concentration-dependent manner by SB202180 (Figure 6c). Consistent with the decrease in IL-8 production, the levels of ATRA-induced IL-8 mRNA expression were also decreased in a concentration-dependent manner by SB202180 (Figure 6d). In this study, SB202180 did not affect the basal level of IL-8 production (data not shown).

Figure 6.

The p38 pathway is involved in the induction of interleukin (IL)-8. (A) Keratinocytes were stimulated with all-trans-retinoic acid (ATRA) (10^-6 M), and the cellular proteins were extracted at the indicated time points. Immunoblotting was performed with the anti-p38 and anti-phospho-specific p38 antibodies. (B) Keratinocytes were treated with SB202180 (20 M) for 2 h prior to the addition of ATRA. ATRA (10^-6 M) or vehicle was then added and the cultures were incubated for 1 h. The expression levels of p38 and phospho-p38 were examined by western blotting. The intensity of each band was quantified as described in Materials and Methods, and the graph was made to show the significant effect of p38 inhibitor on ATRA-induced phospho-p38. (C) Keratinocytes were treated with various concentrations of SB202180 (0–20 M) for 2 h. ATRA (10^-6 M) or vehicle was then added and the cultures were incubated for 36 h. The levels of IL-8 in the culture supernatants were determined by ELISA. ^*p<0.05; ^**p<0.01. (D) Keratinocytes were treated with various concentrations of SB202180 (0–20 M) for 2 h. ATRA (10^-6 M) or vehicle was then added and the cultures were incubated for 18 h. The relative IL-8 mRNA expression levels were assessed by real-time quantitative RT-PCR.

Full figure and legend (40K)

DISCUSSION

In this study, we demonstrated that ATRA induces IL-8 production in cultured normal human keratinocytes. Skin irritation following topical application of retinoic acid at least partially explained by the local induction of IL-8 production by epidermal keratinocytes (^{Kim et al, 2003}). IL-1 increases IL-8 production in normal human keratinocytes (^{Kristensen et al, 1991;Hoffmann et al, 2002}), and ATRA increased IL-8 production with IL-1 synergistically (Figure 1a). This result is contrary to that of a previous report, in which IL-1-induced IL-8 production by human monocytes was markedly reduced by retinoids (^{Gross et al, 1993}). Nevertheless, LPS-induced IL-8 production was increased by ATRA in a same cell system (^{Gross et al, 1993}). In addition, the synergistic effects of ATRA and TNF- and 12-O-tetradecanoylphorbol-13-acetate on IL-8 gene activation have been reported for the human melanoma cell line G-361 (^{Harant et al, 1996b}) and the human acute promyelocytic leukemia cell line (^{Atkins and Troen, 1995}), respectively. Therefore, the effects of ATRA on IL-8 production vary, according to both cell type and the co-administered reagent.

Retinoic acid exerts its pleiotropic effects through binding to two groups of nuclear receptors, the retinoic acid receptors (RAR-, -, -) and the retinoid X receptors (RXR-, -, -) (^{Giguere et al, 1987;Petkovich et al, 1987;Benbrook et al, 1988;Brand et al, 1988;Krust et al, 1989}). RAR usually form heterodimers with RXR (^{Chambon, 1996}), and these dimers are able to bind to specific DNA sequences, which are known as retinoic acid-responsive elements (RARE). RAR–RXR complexes act as ligand-inducible transcription factors. The consensus sequence of RARE contains hexanucleotide half-sites that are arranged as inverted or direct repeats, spaced by various numbers of nt (^{de The et al, 1990}); however, the IL-8 gene does not contain the classical ATRA response elements (^{Baggiolini and Clark-Lewis, 1992}). Therefore, IL-8 induction by ATRA may not depend on this "classical pathway". In certain cases, retinoic acid has been shown to regulate genes that do not contain classical RARE motifs (^{Gudas et al, 1994}). These genes can be regulated by retinoic acid via secondary events, such as the induction and activation of NF-B (^{Segars et al, 1993}). It has been shown that the induction of major histocompatibility class I genes by retinoic acid in human embryonal carcinoma NTera2 (NT-2) cells involves both the activation of RAR–RXR heterodimers and the induction of the NF-B molecules p50 and p65 (^{Segars et al, 1993}). The binding of NF-B to the IL-8 promoter is essential for constitutive activation of IL-8 gene transcription (^{Hoffmann et al, 2002}), and p65 is one of the major components responsible for B binding to the IL-8 promoter (^{Kunsch and Rosen, 1993}). ATRA is able to induce IL-8 gene expression by increasing NF-B transcriptional activity in some cells (^{Harant et al, 1996a;Chang et al, 2000}). In airway epithelium cells, ATRA-enhanced NF-B binding to the promoter region involves p65 and p50 (^{Chang et al, 2000}). In the human melanoma cell line G-361, the synergistic effect of ATRA and TNF- on IL-8 expression is dependent on their combined activation of the IL-8 promoter, which requires an intact NF-B-binding site (^{Harant et al, 1996b}). In this study, we show nuclear translocation of p65 and IB phosphorylation in ATRA-treated keratinocytes. Therefore, ATRA-dependent IL-8 induction probably involves increased levels of p65 (^{Segars et al, 1993}). Similar effects of ATRA on NF-B subunits have been observed in other cell types (^{Segars et al, 1993;Feng and Porter, 1999;Farina et al, 2002}). ATRA-mediated increases in the levels of p65 and p50 transcripts are dependent on the "classical pathway".

An alternative mechanism for IL-8 induction via NF-B signaling is the phosphorylation of IB. Unfortunately, the molecular mechanism by which ATRA phosphorylates IB is unclear. The introduction of IBM completely abolished IL-8 production and reduced the basal level of IL-8 production. These findings suggest that NF-B activation is essential in ATRA-induced IL-8 production in normal keratinocytes.

Activation of p38 MAPK was elicited by ATRA in keratinocytes. The finding that the p38 inhibitor SB202180 specifically decreases ATRA-induced IL-8 production suggests that the p38 MAPK is also involved in ATRA-induced IL-8 production. A previous report has demonstrated that the p38 MAPK pathway contributes to cytokine/stress-induced IL-8 gene expression by stabilizing mRNA through an ARE-targeted mechanism at the post-transcriptional level (^{Hoffmann et al, 2002}). Therefore, ATRA may stabilize IL-8 mRNA by activating the p38 pathway, thereby enhancing IL-8 production in keratinocytes. In summary, ATRA induces IL-8 production in normal human keratinocytes via NF-B and the p38 MAPK signal pathways.

MATERIALS AND METHODS

Cell culture

Normal human keratinocytes were cultured in MCDB153 medium that was supplemented with insulin (5 g per mL), hydrocortisone (5 10^-7 M), ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), bovine hypothalamic extract (50 g per mL), and Ca²⁺ (0.03 mM), as described previously (^{Yamasaki et al, 2003a}). The study was conducted according to Declaration of Helsinki principles. All of the procedures that involved human subjects received prior approval from the Ethical Committee of Ehime University School of Medicine, and all the subjects provided written informed consent.

Reagents

ATRA was purchased from Sigma Chemical Co (St Louis, Missouri), and was dissolved in DMSO. Recombinant human IL-1 was a generous gift from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan). The anti-IB and anti-phospho-IB antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, Massachusetts). The p38 MAPK inhibitor SB202180 was purchased from Calbiochem–Novabiochem International Co. (San Diego, California), dissolved in DMSO at a concentration of 2 mM, and stored at -20°C.

Adenovirus construction and infection

The cosmid cassette pAxCAw (^{Miyake et al, 1996}) and the parental virus Ad5-dlX (^{Miyake et al, 1996}) were kind gifts from Dr Izumu Saito (Tokyo University, Japan). The full-length coding region of the IBM cDNA was obtained from the HindIII- and BamHI-digested fragments of pCMV-IBM (BD Biosciences Clontech, Palo Alto, California), and subcloned into the cosmid cassette pAxCAw. IBM encodes the dominant negative mutant form of IB, which is a non-degradable form of human IB, in which serines 32 and 36 are replaced by alanine residues (S32A/S36A), thereby blocking its phosphorylation and degradation (^{Brown et al, 1995;Chen et al, 1995}). The NF-B signal cannot be activated in cells that over-express IBM, regardless of the presence of extracellular stimuli that normally induce the phosphorylation of IB and activation of the NF-B signal (^{Brown et al, 1995;Feig et al, 1999}). An adenovirus vector that contains the CAG promoter and IBM (AxIBM) was generated using the cosmid cassettes and Ad DNA-TPC (COS-TPC) method, and virus stocks were prepared using the standard procedure (^{Miyake et al, 1996}). Concentrated, purified virus stocks were prepared by CsCl gradient centrifugation, and the virus titer was estimated in a plaque formation assay.

Subconfluent cultures of normal human keratinocytes were infected with AxIBM at a multiplicity of infection (MOI) of 2, and AxLacZ was used as the control vector. The vectors were cultured with the cells for 60 min, with a brief period of agitation every 15 min. This was followed by the addition of fresh culture medium, and re-incubation for 12 h, before treatment with ATRA. The expression of these genes was confirmed by RT-PCR using specific probes.

Oligonucleotide probe preparation

PCR-amplified human cDNAs were inserted into the EcoRI and HindIII sites of the pPMG vector (BD Pharmingen, San Diego, California). The inserted cDNA corresponded to the following sequences: nt 412–746 of RelA (p65) (GenBank/EBI accession no. NM021975); nt 709–1013 of RelB (NM006509); nt 804–1074 of NF-B2 (p52/p100) (NM002502); nt 877–1122 of NF-B1 (p50/p105) (NM003998); nt 1758–1982 of c-Rel (X75042). The pPMG vector (Pharmingen), which incorporates the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, was used as the internal standard.

RPA

Total RNA was isolated from cultured human keratinocytes using Isogen (Nippon Gene, Toyama, Japan). Single-stranded antisense riboprobes were prepared by in vitro transcription of human cDNA fragments using the RiboQuant In Vitro Transcription Kit (Pharmingen) in the presence of [-³²P] UTP. The hCK-5 probe (Pharmingen) for IL-8 detection and an oligonucleotide probe for NF-B were used as the templates for the in vitro transcription reaction. The hybridization products were separated on a gel, and exposed to photographic film, as described previously (^{Yamasaki et al, 2003b}). At least three independent studies were performed, with similar results. One representative experiment is shown in each figure. The GAPDH bands appear as a doublet, as reported previously (^{Dai et al, 2004}), which may be because of the fact that the end of the GAPDH mRNA is highly susceptible to RNase digestion, even though it is double stranded.

RT-PCR

Total RNA samples from cultured cells were isolated at the indicated time points using Isogen (Nippon Gene Co.). RT-PCR was performed using RT-PCR High Plus (Toyobo, Osaka, Japan), according to the manufacturer's instructions. The cDNA was generated from reverse transcription of total RNA for 30 min at 60°C, and then heated to 94°C for 2 min. The PCR cycle consisted of 1 min at 94°C for denaturation, and 1.5 min at 60°C for annealing and primer extension. The IL-8 sequences were amplified for 34 cycles, and the GAPDH sequences for 22 cycles. The following primer pairs were used: human IL-8, 5'-CTTCTCTGCAGCACATCC-3' and 5'-AAGACCTCTCAAGGCTTTG-3'; and GAPDH, 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. The PCR products were visualized on 2% agarose gels that contained ethidium bromide. The PCR products were sequenced, to confirm the accuracy of amplification.

Real-time quantitative PCR

Total RNA samples from cultured cells were isolated using Isogen (Nippon Gene Co.). Real-time RT-PCR was performed in the ABI PRISM 7700 sequence detector (PE Applied Biosystems, Branchburg, New Jersey). The Pre-Developed TaqMan assay reagents (set of primers and probe) for human IL-8 (Accession Numbers: NM_000584) and for human GAPDH endogenous control (Accession Numbers: NM_002046) were purchased from Applied Biosystems (Foster City, California). The RNA analysis was undertaken using the TaqMan One-Step RT-PCR Master Mix reagents kit (PE Applied Biosystems) and 500 ng of total RNA. Thermal cycling was initiated at 48°C for 30 min for reverse transcription reaction, followed by a first denaturation step at 95°C for 10 min, and then 40 cycles PCR at 95°C for 15 s and at 60°C for 1 min according to the manufacturer's instructions using 2 AmpliTaq Gold DNA Polymerase mix (25 L), 40 RT enzyme mix (1.25 L), 20 Pre-Developed TaqMan assay reagent (2.5 L). All one-step RT-PCR reactions were performed in 50 L reaction volumes, and carried out in a 96-well plate. This experiment used two dye layers to detect the presence of target and control sequences. The 6-FAM dye layer yielded the results for quantification of IL-8 mRNA, and the VIC dye layer yielded the results for quantification of GAPDH. Quantification of gene expression was performed using the comparative cycle threshold (CT) method (Sequence Detector User Bulletin 2; Applied Biosystems) and reported as the fold difference relative to GAPDH gene. To calculate the fold change (increase or decrease), the CT of the housekeeping gene (GAPDH) was subtracted from the CT of the target gene (IL-8) to yield the CT. Change in expression of the normalized target gene as a result of an experimental manipulation was expressed as 2^-CT where CT=CT sample—CT control as described previously (^{Babu and Nutman, 2003}). In this study, each assay was performed in triplicate, and the fold change of each sample was normalized against that of the vehicle as 1 U.

ELISA

Culture supernatants were collected at the indicated time points after treatment, and stored at -70°C until used for ELISA. The IL-8 ELISA kit was purchased from R&D Systems (Minneapolis, Minnesota), and used according to the manufacturer's instructions. The optical density at 450 nm was measured with an Immuno Mini NJ-2300 microplate reader (Nalge Nunc International K.K., Tokyo, Japan). All of the assays were performed at least three times.

Western blot analysis

The cells were harvested by scraping into extraction buffer that contained 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 7.4), and protease inhibitors. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The analysis was performed using the Vistra ECF Kit (Amersham Biosciences K.K., Tokyo, Japan) and the FluoroImager (Molecular Dynamics Inc., Sunnyvale, California), as described previously (^{Yamasaki et al, 2003b}). At least three independent studies were performed, with similar results. One representative experiment is shown in each of the figures. The intensity of each band was quantified using ImageQuant (Molecular Dynamics Inc.), with reference to the control signal, which was assigned the value of 1 U. Reproducibility was confirmed by performing three independent experiments.

Nuclear protein preparation and detection of transcription factor activity

Nuclear proteins were prepared according to the instructions provided in the TransFactor Extraction Kit (BD Biosciences Clontech, Palo Alto, California). Briefly, cells were harvested in lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 1 mM DTT) that contained protease inhibitors. Nuclear pellets were collected by centrifugation, and resuspended in extraction buffer (20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol, 1 mM DTT) that contained protease inhibitors. Finally, the protein concentrations of the nuclear extracts were determined, and the samples were stored at -70°C until used.

DNA-binding activities of the NF-B family members were determined using the BD Mercury Transfactor Kits (BD Biosciences Clontech), according to the manufacturer's instructions. The relative optical density values at 655 nm were normalized to the control values (at 0 h or for 0 M), which were designated as 1 U, and plotted on a graph. All of the assays were performed on at least three separate occasions.

Statistical analyses

The data were collected from at least three independent experiments. Quantitative data are expressed as the meanSE. Statistical significance was determined by the paired Student's t test. Differences were considered to be statistically significant for p<0.05. The levels of statistical significance are indicated, as follows, in the figures: ^*p<0.05; ^**p<0.01; and ^***p<0.001.

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Acknowledgments

We thank Teruko Tsuda and Eriko Tan for technical assistance, and Nobuko Nagai for secretarial assistance. This study was supported by INCS, Ehime University, and by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K. Y. and K. H.).