1School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2College of Pharmacy, Yeungnam University, Gyeongsan 712-749
3Department of Pathology, Catholic University of Daegu School of Medicine, Daegu 705-718
4Department of Biological Science, Sungkyunkwan University, Suwon 440-746
5Research and Development Division, Korean Promotion Institute for Traditional Medicine Industry, Gyeongsan 712-210, Republic of Korea
The aim of this study was to determine whether britanin, isolated from the flowers of
Aggregation of Fc?RI results in the phosphorylation of two tyrosine residues within its immunoreceptor tyrosine-based activation motif (ITAM) by Lyn, or by another member of the Src family of tyrosine kinases that associates with the receptor. Phosphorylated ITAM then serves as a docking site for Syk, and this interaction leads to the downstream propagation of signals. Syk phosphorylates adapter proteins, such as, linker for the activation of T cells (LAT), and these phosphorylations result in the formation of a macromolecular signaling complex that allows the diversification of downstream signals required for the release of various pro-inflammatory mediators (Siraganian, 2003). These signaling pathways include phospholipase Cγ (PLCγ)-mediated Ca2+ mobilization, which is a prerequisite for mast cell degranulation and subsequent arachidonic acid (AA) release from membranes by cytosolic phospholipase A2 (cPLA2), which is activated by an increase in Ca2+ influx and phosphorylation by MAPKs (Clark
The flowers of
Britanin was isolated from
Mouse anti-dinitrophenyl (DNP) IgE and DNP-human serum albumin (HSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies used were used: rabbit polyclonal antibodies specific for phospho-IκB, IKKα/β, ERK1/2, JNK, PLCγ1, p38, β-actin, and total form for IκB, ERK1/2, JNK, p38, and 5-LO were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal antibodies against NF-κB p65, Syk, LAT, PLCγ1, phospho-cPLA2 (Ser505), IKKα/β and lamin B as well as secondary goat anti-rabbit IgG-HRP and rabbit anti-goat IgG-HRP antibodies, total Syk, total LAT, and Bay 61-3606 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and antibodies for phosphotyrosine was purchased from Millipore (Millipore, Billerica, MA, USA). The enzyme immnoassay (EIA) kits for PGD2 and LTC4 were purchased from Cayman Chemicals (Ann Arbor, MI, USA).
BMMCs were isolated from male Balb/cJ mice (Sam Taco, INC, Seoul, Korea) and were cultured at 37°C in RPMI 1640 media (Thermo Scientific, UT, USA) containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin (Thermo Scientific, Utah, USA), 10 mM HEPES buffer (Sigma-Aldrich, St. Louis, MO, USA), 100 μM MEM non-essential amino acid solution (Invitrogen, Carlsbad, CA, USA) and 20% PWM-SCM (poke-weed mitogen-spleen cell conditioned medium) as a source of IL-3. After 6 weeks, >98% of the cells was found to be BMMCs as determined by a previously described procedure, as described previously (Lu
After stimulating with DNP-HSA for 15 min with or without pretreatment with britanin or Bay 61-3606 for 1 h, degranulation was determined by measuring the release of β-hexosaminidase (β-Hex), a marker of mast cell degranulation, by a spectrophotometric method, as described previously (Lu
Cell viability was assessed by MTT (Sigma) assay. Briefly, BMMCs were seeded onto 96 well culture plates at 2×104 cells/200 ml/well. After incubation with various concentrations of britanin for 8 h, 20 μl of MTT (5 mg/ml) was added to each well. After 4 h incubation, 150 μl of culture medium was removed, and cells were dissolved in 0.4 N HCl/isopropyl alcohol. The optical densities (OD) at 570 nm and 630 nm were measured using a microplate reader (Sunrise, Tecan, Switzerland).
IgE sensitized BMMCs were pretreated with britanin or Bay 61-3606 for 1 h and stimulated with DNP-HSA (100 ng/ml). After 15 min of stimulation, the supernatants were isolated for further analysis by EIA. LTC4 was determined using an enzyme immunoassay kit. To assess COX-2-dependent PGD2 synthesis, BMMCs were preincubated with 1 μg/ml of aspirin for 2 h to irreversibly inactivate preexisting COX-1. After washing, BMMCs were activated with DNP-HSA (100 ng/ml) at 37°C for 7 h with britanin or Bay 61-3606. PGD2 in the supernatants were quantified using PGD2 EIA kit and cells were used for immunoblots analysis.
Intracellular Ca2+ level was determined with FluoForteTM Calcium Assay Kit (Enzo Life Sciences, Ann Arbor, MI, USA), as described previously (Hwang
The nuclear and cytoplasmic extracts were prepared as described previously (Lu
Immunoblotting was performed as described previously (Lu
Western blotting was performed as described previously (Lu
All experiments were performed three or more times. Average values are expressed as means ± S.D. Statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, IL, USA). The Student’s
Initially, the cytotoxicity of britanin on BMMCs was examined using the MTT assay. However, britanin did not affect cell viability up to a concentration of 20 μM (Fig. 1A). Therefore, britanin levels from 1 to 10 μM were used in subsequent experiments. Previously, we reported that PGD2 generation by BMMCs is biphasic. More specifically, after IgE/Ag stimulation, immediate (occurring within 2 h) PGD2 generation, which is regulated by constitutive COX-1, is followed by delayed (2?10 h) PGD2 generation, which is regulated by inducible COX-2 (Moon
The generation of LTC4 is regulated by two steps, namely, the liberation of AA from membrane phospholipids by cPLA2 and the oxygenation of free AA by 5-LO. Both 5-LO and cPLA2α translocate from the cytosol to the nuclear membrane in response to an increase in intracellular Ca2+ level (Fischer
Since NF-κB is an essential transcription factor for several inflammatory genes, such as, COX-2, iNOS, and tumor necrosis factor-α (TNF-α) (Reddy
Previously, we reported that the inhibition of COX-2 expression and of attendant PGD2 generation occurred after NF-κB inactivation and/or treating cells with either of three MAPKs inhibitors, that is, U0126 for extracellular regulated kinase1/2 (ERK1/2), SP600125 for c-jun N-terminal kinase (JNK), or SB03580 for p38 MAP kinase (Lu
The tyrosine kinase Syk plays an essential role in the initiation of the Fc?RI-dependent signaling pathway, and thus, we examined whether britanin affects the phosphorylation of Syk, and the phosphorylations of LAT and PLCγ1, which lie immediately downstream of Syk (Lu
To explore the biological activity of britanin, we investigated its effects on the generation of PGD2, LTC4, and degranulation in IgE/Ag-induced BMMCs. This study shows that britanin inhibits degranulation, PGD2 and LTC4 generation in IgE/Ag-induced BMMCs by suppressing the Syk pathway. Several sesquiterpenes, namely, britanin, tomentosin (Lu
When mast cells are activated by the cross-linking of IgE/Ag on their membranes, the Lyn/Syk/LAT axis phosphorylates PLCγ1 causing Ca2+ influx, which triggers degranulation and activates AA metabolizing enzymes (Cruse
The other AA metabolite PGD2 (the major prostaglandin) is produced by the COX pathway in mast cells (Fischer
Furthermore, as shown in Fig. 4, britanin dose-dependently inhibited the phosphorylations of ERK1/2, JNK, and p38 MAP kinase, which suggests that it prevents the phosphorylation and translocation of cPLA2 to the nuclear membrane. Since both britanin and Bay 61-3606 inhibited calcium influx (Fig. 1E), MAPKs phosphorylation (Fig. 3), and the activation of the NF-κB pathway located downstream of the Fc?RI proximal Syk pathway, these results suggest that Syk plays an important roles in the generation of eicosanoids (PGD2 and LTC4) and degranulation in IgE/Ag-induced BMMCs. Because, Syk plays an essential role in the initiation of Fc?RI-dependent signaling (Siraganian, 2003; Masuda and Schmitz, 2008), we examined whether britanin suppresses Syk phosphorylation and the expressions of its downstream signal molecules. As shown in Fig. 5, the phosphorylations of Syk, LAT, and PLCγ1 were found to be significantly inhibited by britanin and by Bay 61-3606 (a Src family kinase inhibitor and positive control).
In conclusion, the present study reveals that britanin inhibits IgE/Ag-induced degranulation and the generation of LTC4 and PGD2 in BMMCs. Furthermore, biochemical analyses showed that britanin inhibited IgE/Ag-induced degranulation via the PLCγ1-Ca2+ pathway, PGD2 production via the IKK/IκBα/NF-κB/COX-2 and MAPKs pathways, and LTC4 generation through the MAPKs/cPLA2/5-LO pathway. Furthermore, the observed almost identical effects of Bay 61-3606 suggest the inhibitory effects of britanin are controlled by the Syk pathway. In a previous report, it was suggested Syk plays a central role in the activations mediated by Fc receptors (mast cells, macrophages, neutrophils, eosinophils and basophils) and B cell receptors (Ruzza