Boswellic acids stimulate arachidonic acid release and 12-lipoxygenase activity in human platelets independent of Ca and differentially interact with platelet-type 12- lipoxygenase

Boswellic acids inhibit the transformation of arachidonic acid to leukotrienes via 5-lipoxygenase but can also enhance the liberation of arachidonic acid in human leukocytes and platelets. Using human platelets, we explored the molecular mechanisms underlying the boswellic acid-induced release of arachidonic acid and the subsequent metabolism by platelet-type 12-lipoxygenase (p12-LO). Both -boswellic acid and 3-O-acetyl11-keto-boswellic acid (AKBA) markedly enhanced the release of arachidonic acid via cytosolic phospholipase A2 (cPLA2), whereas for generation of 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], AKBA was less potent than -boswellic acid and was without effect at higher concentrations ( 30 M). In contrast to thrombin, -boswellic acid-induced release of arachidonic acid and formation of 12-H(P)ETE was more rapid and occurred in the absence of Ca . The Ca -independent release of arachidonic acid and 12-H(P)ETE production elicited by -boswellic acid was not affected by pharmacological inhibitors of signaling molecules relevant for agonist-induced arachidonic acid liberation and metabolism. It is noteworthy that in cell-free assays, -boswellic acid increased p12-LO catalysis approximately 2-fold in the absence but not in the presence of Ca , whereas AKBA inhibited p12-LO activity. No direct modulatory effects of boswellic acids on cPLA2 activity in cell-free assays were evident. Therefore, immobilized KBA (linked to Sepharose beads) selectively precipitated p12-LO from platelet lysates but failed to bind cPLA2. Taken together, we show that boswellic acids induce the release of arachidonic acid and the synthesis of 12-H(P)ETE in human platelets by unique Ca -independent routes, and we identified p12-LO as a selective molecular target of boswellic acids. The pentacyclic triterpenes boswellic acids (Fig. 1) are regarded as the active pharmacological principles of ethanolic extracts of Boswellia serrata, and there is accumulating evidence for an anti-inflammatory and antitumorigenic potential of boswellic acids based on experimental cellular and animal models (Safayhi et al., 1992; Winking et al., 2000; Syrovets et al., 2005a,b; Anthoni et al., 2006; Poeckel et al., 2006). Attempts to identify the responsible molecular mechanisms and/or receptors revealed a number of proteins that may be targeted by boswellic acids, including 5-lipoxygenase, human leukocyte elastase, topoisomerases, and I B kinases (Safayhi et al., 1995, 1997; Syrovets et al., 2000, 2005b). Interaction with these targets may indeed provide a molecular basis for the pharmacological effects observed in animals and human subjects. In particular, suppression of leukotriene biosynthesis from arachidonic acid by inhibition of 5-lipoxygenase is generally reThis study was supported by the Deutsche Forschungsgemeinschaft (WE 2260/4-1 and GRK 757). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.024836. ABBREVIATIONS: cPLA2, cytosolic phospholipase A2; A -BA, 3-O-acetyl-boswellic acid; AKBA, 3-O-acetyl-11-keto-boswellic acid; CDC, cinnamyl-3,4-dihydroxy-cyanocinnamate; 12-H(P)ETE, 12-hydro(pero)xyeicosatetraenoic acid; KBA, 11-keto-boswellic acid; MAFP, methylarachidonyl-fluorophosphonate; MAPK, mitogen-activated protein kinase; p12-LO, platelet-type 12-lipoxygenase; PG buffer, phosphate-buffered saline and glucose; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PMNL, polymorphonuclear leukocytes; SDS-b, 2 SDS-polyacrylamide gel electrophoresis sample loading buffer; PAGE, polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid; AM, acetoxymethyl ester; Seph, Sepharose beads without ligand; Seph-KBA, Sepharose beads linked with 11-keto-boswellic acid; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)5-(4-pyridyl)1H-imidazole; SU6656, 2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; A23187, calcimycin; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine. 0026-895X/06/7003-1071–1078$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 3 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 24836/3136678 Mol Pharmacol 70:1071–1078, 2006 Printed in U.S.A. 1071 at A PE T Jornals on A ril 5, 2017 m oharm .aspeurnals.org D ow nladed from garded as the most important pharmacological action of boswellic acids accounting for their anti-inflammatory properties (Safayhi et al., 1995, 1997). Many cell types are able to release arachidonic acid from phospholipids within cellular membranes by the action of specific phospholipases A2 (Six and Dennis, 2000). Arachidonic acid is an important precursor for a number of highly bioactive metabolites formed by various oxygenases, including cyclooxygenases, lipoxygenases, and monooxygenases of the cytochrome P450 family. The 85-kDa cytosolic PLA2 (cPLA2) has been accounted as a responsible enzyme providing free arachidonic acid as substrate for cyclooxygenases and lipoxygenases in leukocytes and platelets (Leslie, 2004). This soluble enzyme is distributed within the cytosol of resting cells and associates with membranes upon elevation of intracellular Ca and/or serine phosphorylations by members of the mitogen-activated protein kinase (MAPK) family (Gijon and Leslie, 1999), occurring in response to a number of agonists. In addition, binding to phosphatidylinositol-4,5bisphosphate (PIP2) (Balsinde et al., 2000) or ceramide(1phosphate) (Huwiler et al., 2001; Pettus et al., 2004; Subramanian et al., 2005) via specific binding-site(s) may promote cPLA2 catalysis. Exposure of leukocytes or platelets to boswellic acids differentially affects signaling pathways and functional responses including Ca mobilization, MAPK activation, formation of reactive oxygen species, release of arachidonic acid, and stimulation of 5-lipoxygenase product formation. Thus, stimulating properties (Safayhi et al., 2000; Altmann et al., 2002, 2004; Poeckel et al., 2005) and inhibitory effects (Safayhi et al., 1992, 1995; Werz et al., 1998; Poeckel et al., 2006) of boswellic acids have been reported for these functions, depending on the cell type and the respective experimental settings. For example, for inhibition of 5-lipoxygenase by AKBA, IC50 values in the range of 1.5 M (Safayhi et al., 1995) up to 50 M (Werz et al., 1997, 1998) were determined, but also 5-lipoxygenase stimulatory effects in this concentration range were described previously (Safayhi et al., 2000; Altmann et al., 2004). We observed recently that boswellic acids are capable of elevating the release of arachidonic acid in human isolated polymorphonuclear leukocytes (PMNL) (Altmann et al., 2004) and platelets (Poeckel et al., 2005). Platelets do not express 5-lipoxygenase but contain the closely related p12-LO that converts arachidonic acid to 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE] (Yoshimoto and Takahashi, 2002). Here we characterized the liberation of arachidonic acid by boswellic acids and the subsequent conversion by p12-LO, and we investigated the underlying molecular mechanisms. Materials and Methods Materials. Boswellic acids were synthesized and prepared as described previously (Jauch and Bergmann, 2003). Antibodies against human p12-LO were kindly provided by Dr. Colin D. Funk (Queen’s University, Kingston, ON, Canada). SB203580, PP2, PP3, SU6656, methyl-arachidonyl-fluorophosphonate (MAFP), bromoenol lactone, the cPLA2 inhibitor, and U0126 were from Calbiochem (Bad Soden, Germany); BAPTA/AM and Fura-2/AM were from Alexis (Grünberg, Germany); wortmannin was from Biotrend (Köln, Germany); cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) was from BIOMOL Research Laboratories (Plymouth Meeting, PA); EAHSepharose 4B was from GE Healthcare (Freiburg, Germany); and all other chemicals were obtained from Sigma (Deisenhofen, Germany). Cells. Platelets were freshly isolated from human venous blood of healthy adult donors (St. Markus Hospital, Frankfurt, Germany) as described previously (Poeckel et al., 2005). Washed platelets were finally resuspended in PBS, pH 7.4, and 1 mg/ml glucose (PG buffer) or in PBS, pH 7.4, and 1 mg/ml glucose plus 1 mM CaCl2. For incubations with solubilized compounds, ethanol or DMSO was used as vehicle, never exceeding 1% (v/v). For the measurement of [H]arachidonic acid release, platelet-rich plasma was prepared from freshly drawn blood (in 3.13% citrate) from healthy adult donors by centrifugation for 10 min at 750g. Determination of Release of H-Labeled Arachidonic Acid from Intact Platelets. Platelet rich plasma was labeled with 19.2 nM [H]arachidonic acid (1 Ci/ml; specific activity, 200 Ci/mmol) for 2 h at 37°C in the presence of 100 M aspirin to avoid clotting. Then, cells were washed twice with PBS, pH 5.9, plus 1 mM MgCl2, 11.5 mM NaHCO3, 1 g/l glucose, and 1 mg/ml fatty acid-free bovine serum albumin and finally resuspended in PG buffer (10/ml). Preparation of cells at pH 5.9 is believed to minimize temperature-induced activation. Platelets were incubated at 37°C with 1 mM EDTA plus 30 M BAPTA/AM for 15 min or incubated with CaCl2 (1 mM) for 2.5 min before stimulation with the indicated agents. After the indicated times, incubations were put on ice for 10 min, followed by centrifugation (5000g, 15 min). Aliquots (300 l) of the supernatants were measured (Wallac MicroBeta TriLux; PerkinElmer Life and Analytical Sciences, Boston, MA) to detect the amounts of H-labeled arachidonic acid released into the medium. Determination of 12-Lipoxygenase Formation. To determine p12-LO product formation in intact cells, freshly isolated platelets (10/ml PG buffer) were supplemented with either 1 mM CaCl2, 1 mM EDTA, or 1 mM EDTA plus 30 M BAPTA/AM. Platelets were preincubated with the indicated agents for 15 min at 37°C. After the addition of stimuli and further incubation at 37°C for the times indicated, p12-LO products [12(S)-hydro(pero)xy-6-trans-8,11,14cis-eicosatetraenoic acid (12-H(P)ETE)] were extracted and then analyzed by high-performance liquid chromatography as described previously (Albert et al., 2002). 12-HETE and 12-H(P)ETE elute as one major peak, and integration of this peak represents p12-LO product formation, expressed as nanograms of metabolites per 10 cells. For the determination of p12-LO product formation in broken cell preparations, platelets (10/ml PG buffer plus 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride) were sonicated (3 10 s) and centrifuged (100,000g/70 min/4°C). To the resulting 100,000g supernatant, boswellic acids were added, and samples were prewarmed at 37°C for 30 s. CaCl2 (2 mM) was added as indicated, and p12-LO product formation was started by the addition of arachidonic acid (10 M). After 10 min at 37°C, the formation of 12-H(P)ETE was determined as described for intact cells. Immobilization of Boswellic Acids and Protein Pull-Down Assays. For immobilization of KBA at EAH Sepharose 4B beads, the free 3-OH group of KBA was used (N. Kather, L. Tausch, D. Poeckel, O. Werz, E. Herdtweck, and J. Jauch, unpublished data). In brief, KBA was treated with glutaric anhydride to form the half-ester glutaroyl-KBA and analyzed by H and C NMR and by mass spectrometry. This substance was ready for immobilization at EAH Fig. 1. Chemical structures of -boswellic acid and AKBA. AKBA lacking the 3-O-acetyl group yields KBA; 3-O-acetylation of -boswellic acid results in A -BA. 1072 Poeckel et al. at A PE T Jornals on A ril 5, 2017 m oharm .aspeurnals.org D ow nladed from Sepharose 4B by standard amide coupling procedures. The carboxylic acid of the KBA core was unlikely to react under standard conditions because of steric crowding. The success of the coupling reaction was determined by two methods: 1) glutaroyl-KBA was used in defined excess (2 mol of glutaroyl-KBA per 1 mol NH2 groups of the EAH Sepharose 4B), and after the coupling reaction, the hypothetical excess of glutaroyl-KBA (1 mol) could be indeed recovered; and 2) treatment of glutaroyl-KBA with KOH in isopropanol under reflux for approximately 3 h, cleaved the ester bond, and gave KBA, which was then analyzed by thin-layer chromatography. For protein fishing experiments, 10 platelets were lysed in 1 ml of lysis buffer (50 mM HEPES, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, and 120 g/ml soybean trypsin inhibitor). After sonication (3 8 s) and centrifugation for 10 min at 12,000g, 50 l of the Sepharose slurries[50% (v/v)] was added to supernatants and incubated at 4°C overnight under continuous rotation. The Sepharose beads were washed three times with binding buffer (HEPES, pH 7.4, 200 mM NaCl, and 1 mM EDTA), and precipitated proteins were finally separated and denatured by the addition of 2 SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer [SDS-b; 20 mM Tris/HCl, pH 8, 2 mM EDTA, 5% SDS (w/v), and 10% -mercaptoethanol]. After boiling (95°C, 6 min), Sepharose beads were removed by centrifugation and proteins in the supernatant were analyzed by SDS-PAGE as described previously (Poeckel et al., 2005). Proteins were visualized by Western blotting (Poeckel et al., 2005) or Coomassie staining, respectively. Statistics. Statistical evaluation of the data was performed by one-way analyses of variance for independent or correlated samples followed by Tukey honestly significantly different post hoc tests. Where appropriate, Student’s t test for paired and correlated samples was applied. A p value of 0.05 ( ) or 0.01 ( , ##) was considered significant.