Inhibitory effects of thunberginols A and B isolated from Hydrangeae Dulcis Folium on mRNA expression of cytokines and on activation of activator protein-1 in RBL-2H3 cells.
Mast cells and basophils play important roles in the pathogenesis of allergic diseases through the release of inflammatory mediators such as histamine and several cytokines. The aggregation of Fc[epsilon]RI by antigens results in tyrosine phosphorylation and [Ca.sup.2+] influx via [Ca.sup.2+] release-activated [Ca.sup.2+] channels. The elevation of intracellular free [Ca.sup.2+] levels plays an essential role in the degranulation process. Furthermore, mast cells and basophils concomitantly synthesize and release a variety of cytokines including interleukin (IL)-3, IL-4, granulocyte/macrophage-colony stimulating factor (GM-CSF), and tumor necrosis factor (TNF-[alpha]) and these cytokines induce late-phase reactions, the production of IgE, etc. The nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1), which is composed of c-fos and c-jun, induce the transcription of target genes of cytokines by binding at gene promoters and enhancers (Fischer et al., 1998; Goldsby et al., 2002; Lorentz et al., 2003; Matsubara et al., 2004; Novotny et al., 1998).
The 3-phenylisocoumarins, thunberginols A and B (Fig. 1), were isolated from Hydrangeae Dulcis Folium, the fermented leaves of Hydrangea macrophylla SERINGE var. thunbergii MAKINO, as antiallergic constituents (Matsuda et al., 1999a-c; Yoshikawa et al., 1992. 1994). Previously, we reported that the structural requirements of the 3-phenylisocoumarins for inhibition of degranulation and/or release of TNF-[alpha] and IL-4 in rat basophilic leukemia (RBL-2H3) cells (Wang et al., 2007). Among them, thunberginols A and B having the 3',4'-dihydroxyl group and 3,4-double bond potently inhibited the degranulation ([IC.sub.50] = 17 and 5.7 [micro]M) and the release of TNF-[alpha] ([IC.sub.50] = 23 and 11 [micro]M) and IL-4 ([IC.sub.50] = 43 and 19 [micro]M) into the medium from the cells stimulated with an antigen, and inhibited the increase in intracellular free [Ca.sup.2+] levels (Wang et al., 2007), which is essential for the degranulation and theproduction of cytokines such as IL-4, in RBL-2H3 cells induced by antigen (Matsubara et al., 2004).
The chemical structure of thunberginol B is similar to that of luteolin, a natural flavone that has been reported to show potential anti-allergic effects (Kimata et al., 2000; Hirano et al., 2006). Previously, we also reported that luteolin was one of the most potently active flavonoids in the same conditions (Matsuda et al., 2002).
Kimata et al. (2000) reported that luteolin inhibited IgE-mediated [Ca.sup.2+] influx, and the activation of protein kinase C, extracellular signal-regulated kinases (ERKs) and c-jun N[H.sub.2]-terminal kinase (JNK), but not the activation of p38 mitogen-activated protein kinase (p38 MAPK), and then inhibited the release of leukotrienes and prostaglandin [D.sub.2] and expression of GM-CSF mRNA in cultured human mast cells. Hirano et al. (2006) also showed that luteolin inhibited phosphorylation of c-jun and DNA binding activity of AP-1 in a human basophilic cell line. KU812, stimulated by A23187 and 12-myristate 13-acetate (PMA).
In our continuing study of the anti-allergic effects of thunberginols, we examined the effects of thunberginol B, which showed the most potent inhibitory activity for the release of TNF-[alpha] and IL-4 in RBL-2H3 cells (Wang et al., 2007), on the expression of cytokine mRNA using a cDNA microarray system and the reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, effects of thunberginols A and B on the expression of c-fos mRNA and on the phosphorylation of ERK1/2 and c-jun were examined to obtain information about their mechanism of action.
[FIGURE 1 OMITTED]
Materials and methods
Thunberginols A and B and luteolin were isolated and purified as described in our previous reports (Matsuda et al., 2002; Yoshikawa et al., 1994). Eagle's minimum essential medium (MEM) and anti-DNP IgE (monoclonal anti-DNP) were purchased from Sigma; fetal calf serum (FCS) was from Gibco; the cDNA microarray system (CodeLink[TM] Bioarray System) and PCR kit (illustra[TM] Ready-to-Go RT-PCR Beads[TM]) were from GE Healthcare; the total RNA extraction kit (RNeasy[TM] Mini) was from Qiagen; protease inhibitor cocktail (Complete Mini) and phosphatase inhibitor cocktail (phosSTOP) were from Roche; the BCA[TM] Protein Assay Kit was from Pierce; other chemicals were from Wako Pure Chemical Industries. Dinitrophenylated bovine albumin (DNP-BSA) was prepared as described previously (Tada and Okumura, 1971; Matsuda et al., 1999b). The 6-well and 24-well plates and 96-well microplates were from Sumitomo Bakelite. The culture plates (128 x 86mm, OmniTray) was from Nunc.
The following primary antibodies were used for the Western blotting: anti-ERK1/2 (anti-p44/42 MAP kinase), anti-phospho-ERK [anti-phospho-p44/42 MAP kinase (Thy202/Tyr204)], anti-phospho-c-jun (Ser73), and anti-[beta]-actin antibodies (Cell Signaling Technology). Horseradish peroxidase-linked donkey anti-rabbit IgG antibody from GE Healthcare was used as the secondary antibody.
cDNA microarray analysis
RBL-2H3 cells [Cell No. JCRB0023, obtained from Health Science Research Resources Bank (Osaka, Japan)] were grown in MEM containing 10% FCS and penicillin (100 units/ml) in a humidified incubator at 37 [degrees]C in an atmosphere of 5% C[O.sub.2] in air. RBL-2H3 cells (3 x [10.sup.6] cells/2 ml/well) in 6-well plates were sensitized with anti-DNP IgE antibody (0.45 [micro]g/ml) for 24 h. The cells were washed twice with Siraganian buffer [119mM NaCl, 5mM KCl, 0.4 mM Mg[Cl.sub.2], 25 mM piperazine-N', N-bis(2-ethanesulfonic acid) (PIPES), and 40 mM NaOH, pH 7.2] supplemented with 5.6 mM glucose, 1 mM Ca[Cl.sub.2] and 0.1% bovine serum albumin (BSA), and incubated in 800 [micro]l of the buffer for 10 min at 37 [degrees]C. Then, 100 [micro]l of test compound solution or vehicle (final conc. 30 [micro]M in 0.1% DMSO/Siraganian buffer) was added and the cells were incubated for 10 min before being stimulated with 100 [micro]l of DNP-BSA (final conc. 10 [micro]g/ml) for 30min or 2h. The cells were washed twice with ice-cold PBS, and total RNA was extracted using an RNeasy[TM] Mini kit (Qiagen) according to the manufacturer's instructions. The preparation of cRNA and hybridization were performed using a CodeLink[TM] Expression Assay Reagent Kit (GE Healthcare) according to the manufacturer's directions. Briefly, cDNA was prepared from 2 [micro]g of total RNA, and then cRNA was translated by in vitro transcription and labeled with biotin-11-UTP. The cRNA was fragmented at 94 [degrees]C for 20 min and hybridized with CodeLink[TM] Rat Whole Genome Bioarray. The hybridized cRNA was stained with streptavidin-Cy5, and then the array plate was scanned with arrayWoR[x.sup.e] (GE Healthcare). The scanned image was analyzed by Code Link Expression Analysis ver. 4.0 (GE Healthcare), and the normalized intensity of each gene expression was calculated.
RT-PCR analysis of the mRNA expression of c-fos and cytokines
Total RNA was extracted from RBL-2H3 cells as described above. RT-PCR was performed using inllustra[TM] Ready-to-Go[TM] RT-PCR Beads (GE Healthcare). Equal amounts of total RNA (2 [micro]g) corresponding to each priming dose were reverse transcribed using oligo(dT)[.sub.12-18](0.5 [micro]g/[micro]l) as a first-strand primer. Reverse transcription was performed at 42 [degrees]C for 30 min. The specific primers (Table 1, Invitrogen), thermocycling parameters and concentration of agarose gel are listed in Table 1. After PCR, 15 [micro]l of the reaction mixture and 3 [micro]l of 6x loading buffer (Wako) were subjected to electrophoresis on agarose gel, and visualized by ethidium bromide staining under ultraviolet light.
Western blot analysis of signal transduction factors
RBL-2H3 cells (1 x [10.sup.7] cells/10 ml/plate) in culture plates (OmniTray, Nunc) were sensitized with anti-DNP IgE antibody (0.45 [micro]g/ml) for 24 h. The cells were washed twice with Siraganian buffer supplemented with 5.6 mM glucose, 1 mM Ca[Cl.sub.2], and 0.1% BSA, and incubated in 8 ml of the buffer for 10 min at 37 [degrees]C. Then, 1 ml of test compound solution or vehicle (final conc. 30 [micro]M, 0.1% DMSO) was added and the cells were incubated for 10 min before being stimulated with 1 ml of DNP-BSA (final conc. 10 [micro]g/ml) for 0.5-2 h. The cells were washed twice with ice-cold PBS, then added 700 [micro]l of lysis buffer A (15mM NaCl, 2mM [beta]-glycerophosphate, 0.2 mM EGTA, Complete Mini 0.5 tab/25 ml, phosSTOP 1 tab/10 ml, 1 mM Tris, and 1.1% Triton-X pH 7.4) for collection of the cells. After sonication of the cells in 700 [micro]l of the buffer, 600 [micro]l of the buffer was added to 300 [micro]l of buffer B composed of 0.9 mM EGTA, 8% glycerol, 6% SDS, 3 mM Tris, 6% mercaptoethanol and 0.03% bromophenol blue (pH 6.8), and the mixture was boiled for 5min. After quantification of the total protein concentration in the lysate using a BCA Protein Assay Reagent Kit (Pierce), proteins (40 [micro]g) were separated by 10% SDS polyacrylamide gels (10% READY GELS J, Bio-Rad) and blotted onto nitrocellulose membranes (Bio-Rad). The membranes were blocked in 5% skim milk for 3h at room temperature. After washing with Tris-buffered saline containing 0.1% Tween 20 (T-TBS), the blots were probed with primary antibody (1:1000). The membranes were washed three times with T-TBS and incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000) for 1 h at room temperature. The detection of immunoreactive protein was performed using an enhance chemiluminescence kit (GE Healthcare) and X-ray film (Hyperfilm, GE Healthcare), according to the manufacturer's instructions.
[FIGURE 2 OMITTED]
Results and discussion
Effects of thunberginol B and luteolin on mRNA expression of cytokines
The activation of mast cells is regulated by aggregation of Fc[epsilon]RI, leading to an increase in intracellular [Ca.sup.2+] levels and activation of the members of the MAPK family. These signal transduction factors were identified to mediate the expression of cytokine mRNA, leading to the release of various cytokines (Hirasawa et al., 1998, 2000; Ishizuka et al., 1996; Jeong et al., 2002; Kimata et al., 2000; Lorentz et al., 2003; Oliver et al., 2000). However, the mechanism regulating cytokine gene expression in mast cells is not fully understood. To compare the effect of thunberginol B with that of luteolin on the IgE-antigen-mediated gene expression profiles of RBL-2H3 cells, we used a cDNA microarray system to analyze the activation of a large number of genes as a pre-examination. Table 2 shows several genes selected in RBL-2H3 cells. The expression of the mRNA of c-fos and various cytokines such as ILs-2, 3, 4 and 13, TNF-[alpha] and GM-CSF was up-regulated 0.5 or 2h after the stimulation, and these increases were inhibited by thunberginol B and luteolin. mRNA levels of TNF-[alpha] and c-fos were higher soon (0.5 h) after the stimulation, while those of ILs-2, 3, 4 and 13 and GM-CSF were higher at 2h. This finding suggests differences between the signal network for the early-expressed cytokine TNF-[alpha] and that for the later-expressed cytokines, although these signal networks have not been fully clarified. AP-1, composed of c-fos and c-jun, can activate the gene expression of cytokines by binding at the sites of cytokine promoters or enhancers (Fischer et al., 1998; Hirano et al., 2006; Hirasawa et al., 1998; Lorentz et al., 2003; Novotny et al., 1998). c-Fos mRNA was expressed by activation of ERK1/2, and c-jun was activated by phosphorylation of it by activation of JNK (Ishizuka et al., 1996, 1997; Kimata et al., 2000; Lorentz et al., 2003). Results of the cDNA microarray assay showed that c-fos mRNA expression was up-regulated 30 min after stimulation by the antigen in RBL-2H3 cells. Thunberginol B and luteolin markedly inhibited the increase in c-fos mRNA levels.
To confirm the mRNA expression of several cytokines, we examined the levels of gene expression using RT-PCR. The time-dependent expression of the genes by stimulation with the antigen showed that ILs-2, 3, 4 and 13 and GM-CSF mRNA levels increased at 30 min and reached a maximum at 1-2 h, then, decreased to near normal levels 8 h after stimulation, but TNF-[alpha] increased earlier than the other cytokines, reaching a maximum at 30min. Thunberginol B and luteolin inhibited the expression of TNF-[alpha], ILs-2, 3, 4 and 13, and GM-CSF mRNA 2h after the stimulation in a concentration-dependent manner (Fig. 2). These results of RT-PCR were similar to those of the cDNA maicroarray analysis.
Next, the time-dependent gene expression stimulated by the calcium ionophore A23187 was examined using RT-PCR. The results showed that the mRNA levels of ILs-3, 4 and 13, TNF-[alpha] and GM-CSF reached a maximum at 2-4 h and maximum expression tended to be delayed compared to that induced by the antigen. However, the expression of IL-2 mRNA was not increased by the ionophore. Thunberginol B and luteolin inhibited the mRNA expression of ILs-3, 4 and 13, TNF-[alpha] and GM-CSF in a concentration-dependent manner (Fig. 2).
[FIGURE 3 OMITTED]
The increase in intracellular free [Ca.sup.2+] was reported to induce activation of NFAT and c-fos (Ishizuka et al., 1996; Lorentz et al., 2003; Matsubara et al., 2004). The present results suggested that certain transcription factors which bind the promoter and enhancer areas of IL-2 mRNA could not be activated in A23187-stimulated RBL-2H3 cells, although the expression of c-fos mRNA was markedly increased. Furthermore, the mRNA levels of TNF-[alpha] increased earlier than that of the other cytokines, and reached a maximum at 30 min in the time-course experiments in accordance with the result of the cDNA microarray assay. This finding implies that the mechanism of TNF-[alpha] mRNA expression is also different.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
Effects of thunberginols A and B on expression of c-fos mRNA and phosphorylation of ERK1/2 in RBL-2H3 cells induced by antigen
Next, effects of thunberginols A and B on the expression of c-fos mRNA were re-examined using the RT-PCR method. As shown in Fig. 3. c-fos mRNA reached a maximum level 30 min after the stimulation by the antigen. Thunberginols A and B inhibited the expression of c-fos mRNA 30 min after the stimulation. Since it is known that activation of ERK1/2 induces expression of c-fos mRNA (Kimata et al., 2000; Lorentz et al., 2003), effects of thunberginols A and B on the phosphorylation of ERK1/2 were examined using Western blotting. As a result, the phosphorylation of ERK1/2 was inhibited by both compounds as well as luteolin at 30 [micro]M. These findings indicate that the ERK pathway is involved in the gene expression of cytokines induced by thunberginols A and B, at least in part, similar to luteolin.
Effects of thunberginols A and B on phosphorylation of c-jun
Next, effects of thunberginols A and B on the activation of c-jun were examined using Western blotting. Thunberginols A and B as well as luteolin (30 [micro]M) apparently inhibited the increase in phospho-c-jun (Fig. 4). Previously, luteolin was reported to inhibit the phosphorylation of JNK in human mast cells mediated by IgE (Kimata et al., 2000) and phosphorylation of c-jun in KU812 stimulated by A23187 and PMA (Hirano et al., 2006). Similar to luteolin, the suppression of cytokine production by thunberginols A and B is involved in the suppressive effects of the phosphorylation of c-jun (Fig. 5).
In conclusion, thunberginol B inhibited the expression of various cytokines such as ILs-2, 3, 4 and 13, TNF-[alpha] and GM-CSF as determined using a cDNA microarray and RT-PCR. Thunberginols A and B inhibited the phosphorylation of ERK1/2 and expression of c-fos mRNA and phosphorylation of c-jun, indicating the suppression of AP-1's activation similar to luteolin. Although the target proteins of thunberginols A and B as well as luteolin are yet to be clarified, the profile of gene expression by thunberginol B was very similar to that by luteolin, suggesting that the mechanism of action of tunberginol B is similar to that of luteolin. Recently, photolabile ligands have been investigated for drug discovery (Dorman and Prestwich, 2000). Since thunberginols A and B having a isocoumarin structure are fluorescence compounds, they should be available for the development of photolabile ligands to clarify the targets of thunberginols A and B and luteolin.
This research was supported by the 21st COE Program, Academic Frontier Project, and a Grant-in-aid for Scientific Research from the Ministry of Education Culture, Sports, Science and Technology of Japan.
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Hisashi Matsuda (a), Qilong Wang (a,b), Koudai Matsuhira (a), Seikou Nakamura (a), Dan Yuan (b), Masayuki Yoshikawa (a,*)
(a) Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
(b) Shenyang Pharmaceutical University, Shenyang 110016, The People's Republic of China
*Corresponding author. Tel.: +81 75 595 4633; fax: +81 75 595 4768.
E-mail address: firstname.lastname@example.org (M. Yoshikawa).
Table 1. PCR primers sets and thermocycling parameters Annealing Gene Forward PCR primer Reverse PCR primer Cycle temp. TNF-[alpha] CGGAATTCGGCT GCTCTAGACCC 27 60 CCCTCTCATCAGTTC TTGAAGAGAACCTGGG IL-2 CTGTGTTGCACT CTGAGTCATTG 32 60 GACGCTTGTC TTGAGATGATGC IL-3 GTATGCTGCTCC CATTCCACGGTC 22 60 CGCTCCTGATG ATAGGGCGAAAG IL-4 ACCTTGCTGTCA TTGTGAGCGTG 24 60 CCCTGTTC GACTCATTC IL-13 GCTCTCGCTTGCC CATCCGAGGCCTT 22 60 TTGGTGGTC TTGGTTAGAG GM-CSF GCATGTAGATGC CATTTCTGGACC 34 63 CATCAAAGAAGC GGCTTCCAGC c-fos AGCCGACTCCTT CAGATAGCTGCTC 23 60 CTCCAGCAT TACTTTGC GAPDH ACCACAGTCCAT TCCACCACCCTG 23 55 GCCATCAC TTGCTGTA Gene Gel conc. mRNA product TNF-[alpha] 2.0% 501 bp IL-2 2.0% 444 bp IL-3 2.0% 473 bp IL-4 2.0% 378 bp IL-13 2.2% 276 bp GM-CSF 2.2% 342 bp c-fos 2.8% 198 bp GAPDH 2.0% 440 bp Each cycle consists of denaturation at 95 [degrees]C for 1 min, annealing at each temp, for 1 min, and extension at 72 [degrees]C for 2 min. Table 2. Gene expression of several cytokines and c-fos in RBL-2H3 cells stimulated by antigen 0.5 h after stimulation Normal (N) Control (C) Test sample (T) Genes N.I. N.I. Ratio (C/N) N.I. Ratio (T/N) Thunberginol B (30 [micro]M) TNF-[alpha] 3.7 57.4 15.3 5.5 1.5 IFN-[gamma] 0.0 (L) 0.1 (L) -- 0.3 (L) -- IL-1[beta] 0.1 (L) 0.1 (L) -- 0.2 (L) -- IL-2 0.1 (L) 0.1 (L) -- 0.2 (L) -- IL-3 10.1 264.8 26.1 19.8 1.9 IL-4 4.8 30.7 6.4 8.2 1.7 IL-5 0.4 (L) 0.7 -- 0.8 -- IL-6 0.6 (L) 0.8 -- 0.9 -- IL-10 0.1 (L) 0.3 (L) -- 0.2 (L) -- IL-13 2.2 24.0 11.1 2.8 1.3 GM-CSF 0.4 (L) 0.8 -- 0.0 (L) -- c-fos 1.5 18.0 11.7 2.4 1.6 Luteolin (30 [micro]M) TNF-[alpha] 6.9 62.1 9.1 3.5 0.5 IFN-[gamma] 0.1 (L) 0.3 (L) -- 0.1 (L) -- IL-1[beta] 0.3 (L) 0.1 (L) -- 0.0 (L) -- IL-2 0.1 (L) 0.2 (L) -- 0.0 (L) -- IL-3 24.3 555.5 22.8 70.1 2.9 IL-4 16.0 73.3 4.6 19.9 1.2 IL-5 0.6 (L) 0.4 (L) -- 0.5 -- IL-6 0.7 1.18 1.7 0.8 1.2 IL-10 0.3 (L) 0.3 (L) -- 0.2 (L) -- IL-13 9.6 59.0 6.1 6.0 0.6 GM-CSF 0.1 (L) 0.5 (L) -- 0.1 (L) -- c-fos 2.6 15.6 5.9 3.4 1.3 2 h after stimulation Control (C) Test sample (T) Genes N.I. Ratio (C/N) N.I. Ratio (T/N) Thunberginol B (30 [micro]M) TNF-[alpha] 35.5 9.5 5.1 1.4 IFN-[gamma] 0.0 (L) -- 0.0 (L) -- IL-1[beta] 0.0 (L) -- 0.1 (L) -- IL-2 3.2 -- 0.1 -- IL-3 848.5 83.7 22.9 2.3 IL-4 121.4 25.2 5.0 1.0 IL-5 0.6 -- 0.4 (L) -- IL-6 1.1 -- 0.8 -- IL-10 0.4 (L) -- 0.2 (L) -- IL-13 74.3 34.3 3.6 1.6 GM-CSF 8.2 -- 0.0 (L) -- c-fos 5.4 3.5 2.2 1.4 Luteolin (30 [micro]M) TNF-[alpha] 30.5 4.5 3.5 0.5 IFN-[gamma] 0.2 (L) -- 0.6 -- IL-1[beta] 0.1 (L) -- 0.6 -- IL-2 0.7 -- 0.4 (L) -- IL-3 1115.4 45.9 107.5 4.4 IL-4 533.0 33.4 18.3 1.1 IL-5 0.6 -- 0.6 -- IL-6 3.4 4.9 0.7 1.0 IL-10 0.1 (L) -- 0.9 -- IL-13 209.6 21.8 4.0 0.4 GM-CSF 2.1 -- 0.5 -- c-fos 2.0 0.8 2.2 0.8 The values represent the normalized intensity (N.I.) and ratio vs. unstimulation (Normal). L indicates low level of fluorescent intensity near to back-ground; therefore, the ratio was not calculated. The results were obtained from one plate of cDNA microarray for each treatment.
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|Title Annotation:||messenger ribonucleic acid|
|Author:||Matsuda, Hisashi; Wang, Qilong; Matsuhira, Koudai; Nakamura, Seikou; Yuan, Dan; Yoshikawa, Masayuki|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Mar 1, 2008|
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