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CELLULAR AND MOLECULAR

3-O-Acetyl-11-keto-boswellic Acid Decreases Basal Intracellular Ca²⁺ Levels and Inhibits Agonist-Induced Ca²⁺ Mobilization and Mitogen-Activated Protein Kinase Activation in Human Monocytic Cells

Institute of Pharmaceutical Chemistry, University of Frankfurt, Frankfurt, Germany (D.P., L.T., S.G.); Institute of Organic Chemistry, University of Saarland, Saarbrücken, Germany (J.J.); and Department of Pharmaceutical Analysis, Institute of Pharmacy, Eberhard-Karls-University Tubingen, Tubingen, Germany (O.W.)

Received May 12, 2005; accepted September 15, 2005.

	Abstract

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Previously, we showed that 11-keto-boswellic acid and 3-O-acetyl-11-keto-BA (AKBA) stimulate Ca²⁺ mobilization and activate mitogen-activated protein kinases (MAPKs) in human polymorphonuclear leukocytes (PMNLs). Here, we addressed the effects of boswellic acids on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and on the activation of p38^MAPK and extracellular signal-regulated kinase (ERK) in the human monocytic cell line Mono Mac (MM) 6. In contrast to PMNLs, AKBA concentration dependently (1–30 µM) decreased the basal [Ca²⁺]_i in resting MM6 cells but also in cells where [Ca²⁺]_i had been elevated by stimulation with platelet-activating factor (PAF). AKBA also strongly suppressed the subsequent elevation of [Ca²⁺]_i induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), PAF, or by the direct phospholipase C activator 2,4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide, but AKBA failed to prevent Ca²⁺ signals induced by thapsigargin or ionomycin. Suppression of Ca²⁺ homeostasis by AKBA was also observed in primary monocytes, isolated from human blood. Moreover, AKBA inhibited the activation of p38^MAPK and ERKs in fMLP-stimulated MM6 cells. Although the effects of AKBA could be mimicked by the putative phospholipase C (PLC) inhibitor U-73122 (1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), AKBA appears to operate independent of PLC activity since the release of intracellular inositol-1,4,5-trisphosphate evoked by 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide was hardly diminished by AKBA. Inhibitor studies indicate that AKBA may decrease [Ca²⁺]_i by blocking store-operated Ca²⁺ and/or nonselective cation channels. Together, AKBA interferes with pivotal signaling events in monocytic cells that are usually required for monocyte activation by proinflammatory stimuli. Interruption of these events may represent a possible mechanism underlying the reported anti-inflammatory properties of AKBA.

Stimulation of inflammatory cells by an adequate agonist may evoke a number of functional responses including chemotaxis, phagocytosis, degranulation, formation of reactive oxygen species, release of cytokines and chemokines, and liberation of lipid mediators. The transduction and mediation of such agonist-induced responses requires appropriate intracellular signaling systems that operate at multiple levels. Elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is one central signaling event for cell activation (Li et al., 2002), being involved in the regulation of functional responses such as degranulation or the generation of reactive oxygen species in agonist-challenged leukocytes (Bernardo et al., 1988). Extracellular stimuli, including the platelet-activating factor (PAF) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), increase the [Ca²⁺]_i in monocytes/macrophages, which is composed of a rapid release of Ca²⁺ from intracellular stores and a Ca²⁺ influx through plasma membrane Ca²⁺ channels (Randriamampita and Trautmann, 1989). Besides Ca²⁺, protein phosphorylation is a common signal transduction mechanism integrating extracellular inflammatory signals into leukocyte functions; in particular, MAPK pathways have been shown to play important roles in this respect (Herlaar and Brown, 1999; Johnson and Druey, 2002). Accordingly, inhibitors of these kinases have been developed to intervene with inflammatory disorders.

We have recently shown that 11-keto-BAs can activate MAPK and induce Ca²⁺ mobilization in human isolated polymorphonuclear leukocytes (PMNLs) and granulocytic HL-60 cells, which could be linked to various functional responses, including release of arachidonic acid, increased formation of leukotrienes, and generation of reactive oxygen species (Altmann et al., 2002, 2004). Since monocytes play key roles in the course of inflammatory processes, we examined the effect of BAs on the Ca²⁺ homeostasis and MAPK pathways in human monocytic Mono Mac (MM) 6 cells. Interestingly, in contrast to PMNLs or HL-60 cells, AKBA exerted opposite effects in MM6 cells, inasmuch as it decreased basal [Ca²⁺]_i, inhibited agonist-induced Ca²⁺ mobilization, and blocked agonist-induced activation of p38^MAPK and ERKs. These findings support an anti-inflammatory potential of AKBA.

	Materials and Methods

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Materials. BAs were prepared as described previously (Jauch and Bergmann, 2003). 2,4,6-Trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS) was a generous gift from Dr. T.G. Lee (SIGMOL, Pohang, Korea). U-73122 and SK&F96365 were purchased from Calbiochem (San Diego, CA), Fura-2/AM was from Alexis Corporation (Läufelfingen, Switzerland), PAF was from Cayman Chemical (Ann Arbor, MI), 2-aminoethoxydiphenylborate (2-APB) was from Tocris Cookson Inc. (Bristol, UK), and all other chemicals were obtained from Sigma Chemie (Deisenhofen, Germany).

Cells. MM6 cells were maintained in RPMI 1640 medium with glutamine supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, 100 U/ml penicillin, 1 mM sodium pyruvate, 1x nonessential amino acids, 1 mM oxalacetic acid, and 10 µg/ml bovine insulin. All cultures were seeded at a density of 2 x 10⁵ cells/ml. MM6 cells were treated with 2 ng/ml transforming growth factor and 50 nM calcitriol for 4 days. Cells were harvested by centrifugation (200g, 10 min at room temperature) and washed once in phosphate-buffered saline (PBS), pH 7.4. To exclude toxic effects of BAs during various incubation periods, the viability of MM6 cells was analyzed by trypan blue exclusion. Incubation with 30 µM AKBA or 3 µM U-73122 at 37°C for up to 30 min caused no significant change in the number of viable cells.

Human PMNLs were freshly isolated from leukocyte concentrates obtained at St. Markus Hospital (Frankfurt, Germany) as described (Werz et al., 2002). In brief, venous blood was taken from healthy adult donors, and leukocyte concentrates were prepared by centrifugation at 4000g/20 min/20°C. PMNLs were immediately isolated by dextran sedimentation, centrifugation on Nycoprep cushions (PAA Laboratories GmbH, Linz, Austria), and hypotonic lysis of erythrocytes. Monocytes were obtained from the same leukocyte concentrates after dextran sedimentation and centrifugation on Nycoprep cushions. The mononuclear cells including lymphocytes and monocytes appear as a layer on Nycoprep cushion after centrifugation. The cells were washed twice with PBS, pH 5.9, containing 2 mM EDTA, resuspended in RPMI-1640 supplemented with 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, and 20% human plasma, and spread in cell culture flasks at 37°C and 5% CO₂. After 3 h, lymphocytes in suspension were removed, and adhered monocytes were gently detached and resuspended in PBS plus 1 mg/ml glucose and 1 mM CaCl₂ (PGC buffer).

Measurement of Intracellular Ca²⁺ Levels. MM6 cells or blood monocytes (3 x 10⁷/ml PG buffer) were incubated with 2 µM Fura-2/AM for 30 min at 37°C. After washing, 3 x 10⁶ cells/ml PG buffer were incubated in a thermally controlled (37°C) fluorometer cuvette in a spectrofluorometer (Aminco-Bowman series 2; Thermo Electron Corporation, Waltham, MA) with continuous stirring. Two min prior to stimulation, 1 mM CaCl₂ or 1 mM EDTA was added. The fluorescence emission at 510 nm was measured after excitation at 340 and 380 nm, respectively, and [Ca²⁺]_i was calculated according to Grynkiewicz et al. (1985). F_max (maximal fluorescence) was obtained by lysing the cells with 1% Triton X-100 and F_min by chelating Ca²⁺ with 10 mM EDTA.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. Prewarmed (37°C) MM6 cells were preincubated with the indicated concentrations of AKBA or vehicle [DMSO, final concentration 1% (v/v)] for 5 min prior to stimulation with fMLP (1 µM) for 1 min at 37°C. The reaction was stopped by addition of the same volume of ice-cold 2x SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer (SDS-b), samples for SDS-PAGE (aliquots corresponding to 2 x 10⁶ cells in 20 µl SDS-b) were prepared, and proteins were separated as described (Werz et al., 2002). Correct loading of the gel and transfer of proteins to the nitrocellulose membrane was confirmed by Ponceau staining. Western blotting using phosphospecific antibodies (1:1000 dilution, each; New England Biolabs, Beverly, MA) against pERK1/2 (Thr202/Tyr204) and pp38^MAPK (Thr180/Tyr182) was performed using a Li-Cor Odyssey two-color Western detection system (Li-Cor, Lincoln, NE), according to the instructions of the manufacturer. Alternatively, detection of immunoreactive proteins was performed as described previously using alkaline phosphatase-conjugated secondary antibody (Werz et al., 2002).

Determination of IP₃ Formation. Prewarmed (37°C) MM6 cells (1.2 x 10⁷/ml PGC buffer) were either preincubated with the indicated compounds [or vehicle, DMSO 1% (v/v)] for 20 s and then subsequently stimulated with m-3M3FBS (100 µM) for 15 s or directly stimulated with the indicated compounds for 15 s at 37°C. Incubations were stopped by the addition of 0.2 volumes of ice-cold HClO₄ [20% (v/v)] and kept on ice for 20 min. Further extraction and evaluation of IP₃ released into the medium was determined using an [³H]IP₃ Biotrak Assay System (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instructions. Data are expressed as percentage of vehicle-treated controls ± S.E., n = 3 to 4. Statistical analysis was performed prior to normalization, p < 0.05 (*) or <0.01 (**).

Statistics. Statistical evaluation of the data were performed by one-way analysis of variance for independent or correlated samples followed by Tukey honestly significant difference post hoc tests. Where appropriate, Student's t test for paired observations was applied. p < 0.05 (*) or <0.01 (**) was considered significant.

	Results

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AKBA Decreases Basal [Ca²⁺]_i of Resting MM6 Cells. In our previous reports, we showed that AKBA and KBA, but not ABA and BA (lacking the 11-keto group), cause a marked mobilization of Ca²⁺ in human isolated PMNLs or in the granulocytic cell line HL-60 (Altmann et al., 2002, 2004). Accordingly, 30 µM AKBA induced a rapid and pronounced elevation of [Ca²⁺]_i in PMNLs, whereas ABA was hardly effective (Fig. 1A, left panel). In contrast, exposure of differentiated MM6 cells to AKBA (30 µM) resulted in a sudden drop of resting [Ca²⁺]_i from 155 ± 8 to 73 ± 7 nM (Fig. 1A, right panel). This decrease in [Ca²⁺]_i was sustained, and markedly reduced levels of basal [Ca²⁺]_i were still detectable 20 min after exposure to AKBA (see below). In a previous study using MM6 cells (Feisst and Werz, 2004), we observed a similar drop of [Ca²⁺]_i, when the PLC inhibitor U-73122 (3 µM) was added to the cells, which was confirmed in the present experiments (Fig. 1A, right panel). Of interest, in PMNLs, U-73122 (3 µM) caused no decrease in [Ca²⁺]_i (Fig. 1A, left panel). The effect of AKBA on resting [Ca²⁺]_i was concentration-dependent and was clearly detectable already at 1 µM (Fig. 1B). Combined addition of 30 µM AKBA plus 3 µM U-73122 gave no additive effects versus AKBA or U-73122 alone (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 1. Effects of BAs and U-73122 on basal [Ca2+]i in resting PMNLs and MM6 cells. Fura-2-loaded PMNLs (107/ml PG buffer, A, left panel) or MM6 cells (3 x 106/ml PG buffer, A, right panel, B, and C), were supplemented with 1 mM CaCl2, 2 min prior to stimulation, and [Ca2+]i was determined. A, addition of AKBA, ABA (30 µM, each), U-73122 (3 µM), or vehicle (DMSO) to resting PMNLs (left panel) or MM6 cells (right panel) is indicated by the arrow. Traces are representative for three to six independent recordings. B, concentration-response curves of AKBA on [Ca2+]i in resting MM6 cells. The minimum [Ca2+]i within 30 s after addition was determined. C, structure-activity relationship of BAs on [Ca2+]i of resting MM6 cells. Vehicle (veh, DMSO), AKBA, KBA, ABA, BA (30 µM, each), or U-73122 (3 µM) was added as described above. The amplitude or an average value of the Ca2+ decrease (in nanomolar) was determined. Values in (B and C) are given as mean ± S.E., n = 4 to 5. Statistically different values compared with vehicle-treated controls are marked (**, p < 0.01).

In analogy to PMNLs, the effectiveness of the BAs to affect [Ca²⁺]_i in MM6 cells depended on the presence of the 11-keto group and the 3-O-acetyl moiety. Thus, the 11-keto-free counterpart of AKBA, namely ABA (30 µM), hardly decreased [Ca²⁺]_i, and KBA, lacking the 3-O-acetyl moiety, was less efficient than AKBA with respect to this response (Fig. 1, A and C). Finally, no effect was detectable for BA (30 µM).

AKBA Decreases Elevated [Ca²⁺]_i in PAF-Activated MM6 Cells. To evaluate whether AKBA also affects elevated [Ca²⁺]_i induced by an agonist, MM6 cells were first treated with 100 nM PAF that raises [Ca²⁺]_i, and AKBA, ABA, or U-73122 was added 50 s later. Addition of AKBA or U-73122 evoked an immediate drop of [Ca²⁺]_i in a concentration-dependent manner (Fig. 2). Notably, the minimum [Ca²⁺]_i attained after AKBA or U-73122 addition (78 ± 10 and 99 ± 9 nM, respectively) was lower than the basal Ca²⁺ levels prior to stimulation with PAF (157 ± 14 nM) and approached similar levels as found for cells exposed only to AKBA (73 ± 7 nM) or U-73122 (88 ± 8 nM), respectively (Fig. 2, compare with Fig. 1C). Thus, AKBA decreases [Ca²⁺]_i in MM6 cells to a comparable extent as U-73122, apparently regardless of the activation state of the cell.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Effects of BAs and U-73122 on elevated [Ca2+]i in stimulated MM6 cells. MM6 cells were prepared as described in Fig. 1. PAF (0.1 µM) was added as indicated, followed by vehicle (veh) or AKBA (3–30 µM, left panel); ABA (30 µM) or U-73122 (1–10 µM, right panel) after another 50 s. Curves are representative for three to five independent determinations.

AKBA Attenuates Agonist-Induced Elevation of [Ca²⁺]_i.. We sought to investigate whether AKBA could also prevent agonist-induced elevations of [Ca²⁺]_i. Agents that elevate [Ca²⁺]_i involving PLC/IP₃ signaling (e.g., PAF, fMLP, and m-3M3FBS) but also stimuli that raise [Ca²⁺]_i independent of the PLC/IP₃ pathway like ionomycin or thapsigargin (TG) were added to MM6 cells that received BAs or U-73122, 20 s prior to agonist addition. As shown in Fig. 3A, AKBA and U-73122, but not ABA, potently inhibited the subsequent Ca²⁺ mobilization induced by the physiological agonists PAF or fMLP as well as by the direct PLC activator m-3M3FBS (Bae et al., 2003). The IC₅₀ value for AKBA was in the range of 10 to 30 µM, depending on the stimulus. Representative [Ca²⁺]_i traces of PAF-stimulated samples are displayed in Fig. 3B, left panel. In contrast, initial elevation of [Ca²⁺]_i induced by the ER/SR-Ca²⁺-ATPase inhibitor TG or by the Ca²⁺-ionophore ionomycin were not affected (Fig. 3A). Closer examination revealed that AKBA transforms the sustained elevation of [Ca²⁺]_i evoked by TG to a transient signal (Fig. 3B, right panel).

View larger version (41K): [in this window] [in a new window]   Fig. 3. AKBA and U-73122 antagonize agonist-induced Ca2+ mobilization. MM6 cells were prepared as described in Fig. 1. A, cells were treated with vehicle (v), AKBA (3, 10, and 30 µM), ABA (A, 30 µM), or U-73122 (U, 3 µM) followed by the addition of PAF (0.1 µM), fMLP (0.1 µM), m-3M3FBS (50 µM), thapsigargin (TG, 0.1 µM), or ionomycin (0.2 µM) after 20 s as indicated. The amplitude of the agonist-induced elevation of [Ca2+]i was determined. Values are given as mean ± S.E., n = 4 to 5, and compared with the positive controls, p < 0.05 (*) or <0.01 (**). B, original Ca2+ recordings of measurements conducted for Fig. 3A. Left, cells were pretreated with vehicle (veh), AKBA (3, 10, and 30 µM), ABA (30 µM), or U-73122 (3 µM) for 20 s, and PAF (0.1 µM) was added as indicated by the arrows. Right, cells were pretreated with vehicle (veh), AKBA (30 µM), ABA (30 µM), or U-73122 (3 µM) for 20 s, and thapsigargin (TG, 0.1 µM) was added as indicated by the arrows. Curves are representative for three to five independent determinations. C, efficacy of AKBA and U-73122 to inhibit agonist-induced Ca2+ mobilization depends on the preincubation period. Cells were incubated with AKBA (30 µM) or U-73122 (3 µM). Then, PAF or fMLP (0.1 µM, each) was added as indicated either 20 s (black bars) or 20 min (white bars) after AKBA or U-73122. The resulting maximum increase in [Ca2+]i was determined and compared with vehicle-treated controls, given as percentage of control ± S.E., n = 3 to 5. Statistical analysis was performed prior to normalization, p < 0.05 (*) or p < 0.01 (**). D, Fura-2-loaded primary monocytes (3 x 106/ml PGC buffer), freshly isolated from human blood, were treated with vehicle (v) or AKBA (3 or 10 µM), followed by the addition of PAF (0.1 µM) after 20 s, as indicated by the arrows. Curves are representative for three independent determinations.

It appeared possible that the suppressive effects of AKBA observed in MM6 could be related to the fact that MM6 is a human leukemia cell line. Therefore, we used primary monocytes isolated from human blood to investigate effects of AKBA on [Ca²⁺]_i. As shown in Fig. 3D, AKBA (3 or 10 µM) rapidly decreased basal [Ca²⁺]_i and prevented PAF-induced Ca²⁺ mobilization in the same manner as observed for MM6 cells. ABA was without effect, and higher AKBA concentrations ( 30 µM) caused a rather slow but continuous increase in [Ca²⁺]_i seemingly related to cell lysis or unspecific toxic effects of the compound (data not shown).

AKBA Attenuates Ca²⁺ Mobilization from Intracellular Stores. Next, we investigated if AKBA may also affect the PAF-induced release of Ca²⁺ from intracellular stores, a process that is typically PLC/IP₃-dependent. MM6 cells were resuspended in Ca²⁺-free buffer containing 1 mM EDTA and treated with AKBA (ABA or U-73122), followed by the addition of PAF after another 20 s. Neither AKBA (or ABA) nor U-73122 exhibited an effect on basal [Ca²⁺]_i in resting cells under these conditions. However, AKBA or U-73122 reduced the release of Ca²⁺ from internal stores elicited by PAF (Fig. 4, A and B, left panel), although slightly higher concentrations of AKBA and U-73122 were required as compared with those needed to suppress total Ca²⁺ mobilization in the presence of extracellular Ca²⁺. Surprisingly, also, Ca²⁺ mobilization from internal storage sites induced by TG (Fig. 4B, right panel) was partly antagonized by AKBA, implying that PLC inhibition may not be the sole mechanism by which AKBA affects [Ca²⁺]_i since TG-mediated Ca²⁺ mobilization circumvents the PLC/IP₃ route.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Effects of BAs on Ca2+ release from internal stores. MM6 cells were prepared as described in Fig. 1, except that 1 mM EDTA was added instead of 1 mM Ca2+. A, original Ca2+ recordings of samples stimulated by PAF (0.1 µM) after preincubation with vehicle (veh), AKBA (3, 10, and 30 µM), ABA (30 µM), or U-73122 (3 µM) for 20 s. Curves are representative for three to four independent determinations. B, cells were treated with vehicle (v), AKBA (3, 10, and 30 µM), ABA (A, 30 µM), or U-73122 (U, 3 µM) followed by the addition of PAF (0.1 µM, left panel), or thapsigargin (TG, 0.1 µM, right panel). The amplitude of the stimulus-induced elevation of [Ca2+]i was determined. Values are given as mean ± S.E., n = 4, p < 0.05 (*) or <0.01 (**).

View larger version (48K): [in this window] [in a new window]   Fig. 5. AKBA attenuates the activation of ERK1/2 and p38MAPK. A, prewarmed MM6 cells (2 x 106/100 µl PGC buffer) were incubated with the indicated concentrations of AKBA or vehicle (DMSO) for 5 min. B, prewarmed MM6 cells were first incubated with the indicated concentrations of AKBA or vehicle (DMSO) for 15 min prior to stimulation with fMLP (1 µM) at 37°C for 1 min. Reactions were terminated by addition of equal volumes of SDS-b. Samples were subjected to SDS-PAGE and Western blotting using phosphospecific antibodies against the dually phosphorylated form of the MAPKs. Samples were concurrently analyzed for p38MAPK and ERK1/2 activation using the 2-color Western detection system of Li-Cor (Odyssey), hence, loading control (L.C.) bands refer to both antibodies. The results shown are representative of at least three independent experiments.

Effects of Boswellic Acids on Cellular PLC Activity. To test whether AKBA (in analogy to U-73122) inhibits cellular PLC activity, we assayed the effects of AKBA on the IP₃ formation in intact MM6 cells. Cellular PLC was directly activated using 100 µM m-3M3FBS to obtain a prominent increase in IP₃ production (7.9-fold elevation, Fig. 6B). In agreement with its ability to block total Ca²⁺ mobilization, U-73122 (5 µM) inhibited m-3M3FBS-induced IP₃ formation (80%, Fig. 6A). In contrast to its ability to decrease [Ca²⁺]_i, AKBA failed to significantly suppress IP₃ formation. Intriguingly, ABA, which hardly affected Ca²⁺ homeostasis, inhibited m-3M3FBS-induced IP₃ generation (50%, Fig. 6A).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Effects of BAs on IP3 formation. A, prewarmed MM6 cells (1.2 x 107/ml PGC buffer) were treated with vehicle (DMSO), AKBA (30 µM), ABA (30 µM), or U-73122 (5 µM) for 20 s prior to stimulation with m-3M3FBS (100 µM) for 15 s. Incubations were stopped by the addition of 0.2 volumes of ice-cold HClO4 (20%, v/v), and extraction and evaluation of IP3 released were determined according to the manufacturer's instructions (IP3 [3H] Biotrak Assay System; GE Healthcare). Data are expressed as percentage of vehicle-treated (m-3M3FBS-stimulated) control ± S.E., n = 3 to 4. Statistical analysis was performed prior to normalization, p < 0.05 (*) or <0.01 (**). B, prewarmed MM6 cells (1.2 x 107/ml PGC buffer) were incubated with vehicle (DMSO), AKBA (3, 10, and 30 µM), ABA (30 µM), U-73122 (5 µM), or m-3M3FBS (100 µM) for 15 s. Incubations were stopped by the addition of 0.2 volumes of ice-cold HClO4 (20%, v/v). The subsequent extraction procedure is identical to the description in A. Data are expressed as percentage of vehicle-treated (unstimulated) control ± S.E., n = 3 to 4. Statistical analysis was performed prior to normalization, p < 0.05 (*) or <0.01 (**).

Inhibitors of Plasma Membrane Ca²⁺ Channels Mimic the Effects of AKBA and Abolish AKBA-Induced Decrease of [Ca²⁺]_i. The fact that AKBA attenuates the secondary phase of TG-induced Ca²⁺ mobilization, which represents Ca²⁺ influx from the extracellular space, prompted us to elucidate if the AKBA-induced loss of intracellular (cytoplasmic) Ca²⁺ may be due to inhibition of plasma membrane Ca²⁺ influx channels such as store-operated Ca²⁺ channels (SOCCs), nonselective cation channels (NSCCs), or voltage-gated Ca²⁺ channels. SK&F96365, an inhibitor of NSCC and SOCC, reduced basal [Ca²⁺]_i in MM6 cells and was able to prevent the subsequent decrease of [Ca²⁺]_i induced by AKBA (Fig. 7). To distinguish between NSCC and SOCC, we applied 2-APB (50 µM), which blocks SOCCs and, on the other hand, LOE908 (10 µM), which selectively inhibits NSCC. Both 2-APB and LOE908 decreased basal [Ca²⁺]_i and prevented the effects of AKBA (Fig. 7). In contrast, inhibitors of voltage-gated Ca²⁺ channels (300 nM -conotoxin MVIIA or 1 µM verapamil, which block L- or N-type channels, respectively), SR-Ca²⁺ release channels (10 µM neomycin), or blockers of the Na⁺-Ca²⁺ exchanger (SEA0400 or KB-R7943, 10 µM each) failed to significantly decrease basal [Ca²⁺]_i and to prevent effects of AKBA (data not shown). Together, these results indicate that AKBA may mediate the decrease of [Ca²⁺]_i by blocking Ca²⁺ influx from the extracellular space via inhibition of SOCC/NSCC.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effects of plasma membrane Ca2+ channel inhibitors on Ca2+ homeostasis and on the actions of AKBA. Fura-2-loaded MM6 cells (3 x 106/ml PG buffer) were preincubated with vehicle (veh, DMSO), SK&F96365 (10 or 30 µM), 2-APB (50 µM), or LOE908 (10 µM) for 2 min at 37°C in the presence of 1 mM CaCl2. Then, cells were stimulated with AKBA (30 µM), and [Ca2+]i was determined. Traces are representative for three to four independent determinations.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
AKBA and KBA were shown to induce Ca²⁺ mobilization and activation of MAPK in primary PMNLs and granulocytic HL-60 cells, involving pertussis toxin-sensitive proximal signaling pathways (Altmann et al., 2002, 2004). Activation of these central signaling events were linked to typical functional responses of granulocytes, including peroxide formation and enhanced metabolism of arachidonic acid; in particular, an increased activity of 5-LO was evident (Altmann et al., 2004). Such an activation of granulocytes opposes the general observation that extracts of B. serrata or isolated BAs exert anti-inflammatory properties in several cellular experimental settings (Krieglstein et al., 2001; Syrovets et al., 2005) or animal models (Sharma et al., 1989; Gupta et al., 1994) and finally also in studies on human subjects (Gupta et al., 1998; Gerhardt et al., 2001).

Recently, Syrovets et al. (2005) showed that in activated human monocytes, BAs down-regulate TNF expression via a direct inhibition of IB kinases, providing a molecular basis for the anti-inflammatory properties of BAs. The result from the present investigation focusing on central signaling pathways in monocytes provides additional evidence for an anti-inflammatory implication of AKBA at the cellular level. Thus, AKBA decreased the basal [Ca²⁺]_i, prevented agonist-induced Ca²⁺ mobilization, and blocked the activation of ERK1/2 and p38^MAPK, signaling events that are determinants for typical functional monocyte/macrophage responses (Gijon and Leslie, 1999; Chen et al., 2001). Interestingly, TNF generation and NF-B activation in monocytic cells may depend on Ca²⁺ (Pollaud-Cherion et al., 1998; See et al., 2004), providing a possible link between interference with Ca²⁺ and down-regulation of NF-B and TNF.

Apparently, in view of the opposite, agonistic effects on PMNLs and HL-60 cells, AKBA and KBA exert disparate effects on certain cellular processes, depending on the cell type. Thus, in monocytic cells, AKBA may be regarded as pharmacologically active compound that suppresses important signaling events, implying anti-inflammatory functionality. PMNLs that are terminally differentiated are involved in acute inflammatory responses, whereas monocytes act more in chronic inflammation and can undergo differentiation prior to function. Indeed, opposite effects on Ca²⁺ homeostasis in analogy to AKBA are obvious in leukocytes exposed to arachidonic acid that decreases [Ca²⁺]_i in peritoneal macrophages (Randriamampita and Trautmann, 1990) and in MM6 cells (D. Poeckel and O. Werz, unpublished data) but on the other hand evokes Ca²⁺ mobilization in PMNLs (Naccache et al., 1989).

Many effector enzymes like phospholipases, 5-LO, and protein kinases respond to and are regulated by an elevation of [Ca²⁺]_i, leading to functional monocyte responses including lipid mediator and superoxide release, degranulation, and cytokine generation (Bernardo et al., 1988; Pollaud-Cherion et al., 1998). Among the four major -configurated BAs present in ethanolic extracts of B. serrata gum, AKBA was most potent, whereas the 11-methylene derivatives were hardly active; also, the absence of the 3-O-acetyl group led to a loss of efficacy. Similarly, for interference with so far all defined molecular pharmacological targets, i.e., 5-LO (Safayhi et al., 1992), human leukocyte elastase (Safayhi et al., 1997), topoisomerases I and II (Syrovets et al., 2000), as well as IB kinases (Syrovets et al., 2005), AKBA possesses the highest potency, being of considerable pharmacological interest (Ammon, 2002).

The effects of AKBA in MM6 cells showed similar characteristics as the PLC inhibitor U-73122 (Bleasdale et al., 1990) that was found to block acute and chronic inflammatory responses in vivo (Hou et al., 2004). Indeed, both U-73122 and AKBA rapidly decreased the basal [Ca²⁺]_i of resting cells but also caused an immediate drop of the elevated [Ca²⁺]_i after challenge with PAF, displaying comparable kinetics. Moreover, both agents reduced agonist-evoked Ca²⁺ mobilization, which in fact is a characteristic for monocyte activation by external stimuli (Kim et al., 1992; Bernardo et al., 1997; Li et al., 2002). Such Ca²⁺-antagonizing activity of AKBA or U-73122 was evident for agonists (fMLP, PAF, or m-3M3FBS) that act via the PLC/IP₃ pathway. In contrast, initial Ca²⁺ fluxes induced by the ER/SR-Ca²⁺-ATPase inhibitor TG or by the Ca²⁺-ionophore ionomycin, which both circumvent PLC/IP₃ for Ca²⁺ mobilization (Gouy et al., 1990), were unaffected by either U-73122 or AKBA. Experiments conducted to determine the duration of the Ca²⁺ suppressing effects, either in resting or in agonist-challenged cells, revealed rather transient efficacy of U-73122, whereas AKBA-mediated antagonism was sustained and long-lasting, implying that the compounds most likely operate through differing mechanisms.

Another common feature of U-73122 and AKBA was their ability to inhibit the PAF-induced release of Ca²⁺ from internal storage sites. Hence, based on the inhibitory profile and characteristics to affect Ca²⁺ homeostasis, it first appeared reasonable that the Ca²⁺-modulating effects of AKBA could be due to interference with PLC, which is a defined molecular target of U-73122 (Bleasdale et al., 1990). On the other hand, interference of AKBA with the IP₃ receptor could be a plausible explanation. Surprisingly, however, AKBA significantly inhibited TG-induced Ca²⁺ mobilization from internal stores and also the sustained elevation of [Ca²⁺]_i of TG-treated cells in Ca²⁺-containing buffer, suggesting that AKBA may influence Ca²⁺ homeostasis, at least in part, independent of PLC or IP₃. An important finding that favors a PLC-independent mechanism is the failure of AKBA to efficiently suppress the release of intracellular IP₃. Strikingly, in contrast to AKBA, ABA clearly failed to counteract m-3M3FBS-induced Ca²⁺ mobilization, even though it was more efficient than AKBA in inhibiting m-3M3FBS-evoked IP₃ formation. Based on these discrepancies, inhibition of PLC is no satisfying explanation for the potent impairment of [Ca²⁺]_i induced by AKBA. This hypothesis is further supported by the fact that AKBA on one hand even slightly increased basal IP₃ levels about 2-fold, which should actually lead to Ca²⁺ release from internal storage sites. However, in contrast, there is a strong decrease in the basal [Ca²⁺]_i under these conditions.

The AKBA-induced loss of intracellular (cytoplasmic) Ca²⁺ may result from different processes such as extrusion of intracellular Ca²⁺ to the extracellular space, stimulation of Ca²⁺ storage (uptake) into intracellular sites (e.g., by activation of a Ca²⁺/ATPase), or interference with ion channels allowing Ca²⁺ influx. Our studies using selective inhibitors of various plasma membrane Ca²⁺ influx channels imply that AKBA might act (at least in part) by inhibition of SOCC and/or NSCC. Thus, inhibitors of SOCC and/or NSCC mimicked the loss of [Ca²⁺]_i observed with AKBA and were able to inhibit the subsequent decrease of [Ca²⁺]_i induced by AKBA. Of interest, also for U-73122, inhibition of plasma membrane Ca²⁺ channels has been accounted for reduced [Ca²⁺]_i (see Feisst et al., 2005, and references therein). In contrast, voltage-gated N- and L-type Ca²⁺ channels, SR-Ca²⁺ release channels (neomycin), or the Na⁺-Ca²⁺ exchanger do not seem to mediate the effects of AKBA. It is conceivable that a block of SOCC/NSCC may shift the balance between Ca²⁺ influx and Ca²⁺ extrusion toward predominant extrusion that, as a result, leads to impaired [Ca²⁺]_i. However, more detailed experiments are required to elucidate the molecular targets and mechanisms underlying the complex regulation of Ca²⁺ homeostasis by AKBA in MM6 cells, which would go beyond the scope of this study.

Besides antagonizing Ca²⁺, AKBA potently prevented fMLP-induced activation of p38^MAPK and ERKs. These MAPK pathways play pivotal roles in the transduction of external mediators to many cellular processes and are strongly implicated in inflammatory disorders (Herlaar and Brown, 1999; Johnson and Druey, 2002). Among the pharmacological strategies for intervention with inflammation, inhibitors of ERKs may possess potential for the treatment of inflammatory and neuropathic pain (Ji, 2004). p38^MAPK inhibitors have been developed to treat for example rheumatoid arthritis (Pargellis and Regan, 2003) and Crohn's disease (Hommes et al., 2002), inflammatory disorders that in fact have been successfully treated with B. serrata extracts (Gerhardt et al., 2001). Of interest, recently also U-73122 was shown to reduce lysophosphatidylcholine-induced p38^MAPK activation in monocytic THP-1 cells (Jing et al., 2000).

In summary, our data show that AKBA is capable to suppress central signaling events in human monocytic cells, typically important for functional monocyte responses at inflammatory sites. These findings may be added to the list of pharmacological actions of BAs assumed to contribute to the effects of B. serrata extracts observed in animal models and in clinical studies of humans and may be another step forward to the elucidation of the cellular and molecular basis of the anti-inflammatory properties of BAs.

	Footnotes

 
This work was supported by Deutsche Forschungsgemeinschaft Grants WE 2260/4-1 and GRK 757.

doi:10.1124/jpet.105.089466.

ABBREVIATIONS: BA, boswellic acid; 5-LO, 5-lipoxygenase; NF, nuclear factor; TNF, tumor necrosis factor; PAF, platelet-activating factor; fMLP, N-formyl-methionyl-leucyl-phenylalanine; MAPK, mitogen-activated protein kinase; PMNL, polymorphonuclear leukocyte; MM, Mono Mac; AKBA, 3-O-acetyl-11-keto-boswellic acid; ERK, extracellular signal-regulated kinase; m-3M3FBS, 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide; U-73122, 1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; 2-APB, 2-aminoethoxydiphenylborate; PBS, phosphate-buffered saline; PGC buffer, PBS containing 1 mg/ml glucose and 1 mM CaCl₂; PG, PBS plus 1 mg/ml glucose; PAGE, polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; SDS-b, 2x SDS-PAGE sample loading buffer; IP₃, inositol-1,4,5-trisphosphate; KBA, 11-keto-boswellic acid; ABA, 3-O-acetyl-boswellic acid; PLC, phospholipase C; TG, thapsigargin; SOCC, store-operated Ca²⁺ channel; LOE908, (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl) ethyl]-acetamide; SK&F96365, 1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride; NSCC, nonselective cation channel; SEA0400, 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy-5-ethoxyaniline; KB-R7943, 2-[2-[4-(4-dinitrobenzyoxy)phenyl]ethyl]isothiourea.

Address correspondence to: Dr. Oliver Werz, Department of Pharmaceutical Analysis, Institute of Pharmacy, Eberhard-Karls-University Tubingen, Auf der Morgenstelle 8, 72076 Tubingen, Germany. E-mail: o.werz{at}pharmchem.uni-frankfurt.de

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