Abscisic Acid Released by Human Monocytes ActivatesMonocytes and Vascular Smooth Muscle Cell ResponsesInvolved in Atherogenesis*Received for publication, December 19, 2008, and in revised form, March 18, 2009 Published, JBC Papers in Press, March 30, 2009, DOI 10.1074/jbc.M809546200

Mirko Magnone, Santina Bruzzone, Lucrezia Guida, Gianluca Damonte, Enrico Millo, Sonia Scarf,Cesare Usai, Laura Sturla, Domenico Palombo, Antonio De Flora, and Elena Zocchi1

From the Department of Experimental Medicine, Section of Biochemistry and Center of Excellence for Biomedical Research,University of Genova, Viale Benedetto XV 1, 16132 Genova, the Advanced Biotechnology Center, Largo Rosanna Benzi 1,16132 Genova, the Institute of Biophysics, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova, and the Vascularand Endovascular Surgery Unit, San Martino Hospital, 16132 Genova, Italy

Abscisic acid (ABA) is a phytohormone recently identified as anew endogenous pro-inflammatory hormone in human granu-locytes. Here we report the functional activation of humanmonocytes and vascular smooth muscle cells by ABA. Incuba-tion of monocytes with ABA evokes an intracellular Ca2 risethrough the second messenger cyclic ADP-ribose, leading toNF-B activation and consequent increase of cyclooxygenase-2expression and prostaglandin E2 production and enhancedrelease of MCP-1 (monocyte chemoattractant protein-1) and ofmetalloprotease-9, all events reportedly involved in atherogen-esis.Moreover,monocytes releaseABAwhenexposed to throm-bin-activated platelets, a condition occurring at the injured vas-cular endothelium; monocyte-derived ABA behaves as anautocrine and paracrine pro-inflammatory hormone-stimulat-ing monocyte migration and MCP-1 release, as well as vascularsmooth muscle cells migration and proliferation. These results,and the presence of ABA in human arterial plaques at a 10-foldhigher concentration compared with normal arterial tissue,identify ABA as a new signal molecule involved in the develop-ment of atherosclerosis and suggest a possible new target foranti-atherosclerotic therapy.

ABA2 is an isoprenoid phytohormone regulating severalphysiological functions in Metaphyta, including response toabiotic stress (1), and in lower Metazoa, where ABA mediates

temperature-induced oxygen consumption andwater filtrationin sponges (2) and light-stimulated tissue regeneration inhydroids (3). Recently, human granulocytes have been demon-strated to be activated by ABA through a signaling pathwaysequentially involving a pertussis toxin (PTX)-sensitive recep-tor-G protein complex, activation of adenylyl cyclase, cAMPoverproduction, PKA-mediated phosphorylation of the ADP-ribosyl cyclase (ADPRC) CD38, increase of the intracellularconcentration of cADPR ([cADPR]i), a Ca2-mobilizing sec-ond messenger (4), and increase of the intracellular calciumconcentration ([Ca2]i) (5). TheABA-induced [Ca2]i rise acti-vates reactive oxygen species and nitric oxide release, phagocy-tosis, and chemotaxis toward ABA (5). The increase of intracel-lular ABA in heat-stressed granulocytes and ABA release bythese cells during phagocytosis indicate that ABA behaves as anew human pro-inflammatory hormone.Monocytes play a key role in both inflammation and immu-

nity by performing antigen presentation, phagocytosis, andimmunomodulation through the production of various cyto-kines and growth factors (6). In atherosclerosis, a chronicimmune inflammatory disease, the interaction between mono-cytes, platelets, and injured luminal endothelium is a crucialevent leading to the atherosclerotic lesion of the arterial intima(7, 8). Platelets trigger atherogenesis through adhesion to dam-aged endothelial cells and release of pro-inflammatory media-tors, recruiting circulating monocytes to the site of the lesion(9). Monocytes attach to and penetrate the vessel wall, wherethey undergo transformation into macrophages and eventuallyinto foam cells (10). At the site of the atherosclerotic lesion,monocytes/macrophages, platelets, and endothelial cellsrelease chemoattractants, growth factors, and cytokines, whichstimulate VSMC proliferation and migration into the sub-en-dothelium. Thrombus organization, extracellular matrix syn-thesis, and VSMC proliferation eventually lead to the develop-ment of the atheromatous plaque (710). Here we investigatethe effect of ABA on human monocytes and VSMC and itspossible role in the activation of cell responses known to beresponsible for atherogenesis.

EXPERIMENTAL PROCEDURES

MaterialsFURA-3PE/AM, protein kinase inhibitors (pro-tein kinase A inhibitor peptide sequence 1422, cell-permeant,

* This work was supported in part by grants from Regione Liguria, ItalianMinistry for University and Research, University of Genova, and Fondazi-one CARIGE.

1 To whom correspondence should be addressed: Dept. of ExperimentalMedicine, Section of Biochemistry, University of Genova, Viale BenedettoXV, 1 16132 Genova, Italy. Tel.: 39-010-3538158; Fax: 39-010-354415;E-mail: ezocchi@unige.it.

2 The abbreviations used are: ABA, abscisic acid; ADPRC, ADP-ribosyl cyclase;[cADPR]i, intracellular concentration of cyclic ADP-ribose; [Ca

2]i, intracel-lular concentration of calcium; [cAMP]i, intracellular cyclic AMP; cGDPR,cyclic GDP-ribose; PKC, protein kinase C; PKA, cAMP-dependent proteinkinase; bio-ABA, biotinylated ABA; AoSMC, aortic smooth muscle cell;COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; MMP-9, metallopro-tease-9; PTX, pertussis toxin; WT, Wedelolactone; GDPRC, GDP-ribosylcyclase; mAb, monoclonal antibody; HBSS, Hanks balanced salt solution;DMEM, Dulbeccos modified Eagles medium; HPLC, high pressure liquidchromatography; PDGF, platelet-derived growth factor; 8-Br-cADPR,8-bromo-cADPR; PLC, phospholipase C; CI, chemotaxis index; KI, chemoki-nesis index; VSMC, vascular smooth muscle cell.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 26, pp. 17808 17818, June 26, 2009 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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myristoylated; protein kinase C inhibitor peptide sequence2028, cell-permeant, myristoylated), and Ro 20-1724 werepurchased from Calbiochem. SYTOX Green, Alexa-streptavi-din, and calcein-AM were obtained from Molecular Probes(Eugene, OR). Cell culture media were from Cambrex Bio Sci-ence (Walkersville, MD). Ficoll-Paque Plus, [3H]()-ABA (40Ci/mmol), MCP-1 Elisa kit, and cAMP radioimmunoassay kitwere fromGEHealthcare. All antibodies were from Santa CruzBiotechnology (Santa Cruz, CA), except for the anti-ABAmonoclonal antibody (mAb), which was from Agdia (Elkart,IN). PGE2 monoclonal EIA kit was from Cayman Chemical Co.(Ann Arbor, MI); chemotaxis chambers (ChemoTx systemmicroplates) were from NeuroProbe (Gaithersburg, MD). ()-cis,trans-ABA and all other chemicals were obtained fromSigma. Biotinylated ABA (bio-ABA) was prepared as described(5).Isolation of Human MonocytesMononuclear cells, isolated

by Ficoll-Paque sedimentation from fresh buffy coats, wereseeded in tissue culture dishes in Dulbeccos modified Eaglesmedium (DMEM). After 6 h, adherent monocytes were washedand cultured in DMEMwith 10% autologous plasma, penicillin(100 units/ml), and streptomycin (100 g/ml) (monocytemedium).Fluorimetric [Ca2]i Measurements[Ca2]imeasurements

were performed on FURA-3PE/AM-loaded monocytes seededon 20-mm glass coverslips as described in Ref. 11.Determination of Intracellular cAMP ([cAMP]i) and

[cADPR]iMonocytes (2 106/assay) were preincubated inHanks balanced salt solution (HBSS) for 5 min at 37 C with 10M cAMP phosphodiesterase inhibitor 4-(3-butoxy-4-me-thoxybenzyl)imidazolidin-2-one (Ro 20-1724) and, when indi-cated, for 45 min with 2 g/ml PTX. After incubation with orwithout 10MABA for 0, 30, 60, and 150 s, cells were scraped in300 l of distilled water, and the reaction was stopped with 20l of 9 M perchloric acid at 4 C. The [cAMP]i was determinedby radioimmunoassay. For determination of the [cADPR]i,monocytes (2 106/assay) were incubated for 0, 5, 15, and 60min at 25 C without (control) or with 10 M ()-, ()-, or()-cis,trans-ABA. At each time point, a 500-l aliquot of thecell suspension was withdrawn and centrifuged at 5,000 g for15 s; cell pellets were lysed in 300 l of 0.6 M perchloric acid at4 C and centrifuged, and the cADPR content was measured(12).Assay of GDP-ribosyl Cyclase (GDPRC) ActivityMonocytes

were resuspended in HBSS (2 106/ml), and ectocellularGDPRC activity wasmeasured in the presence or absence (con-trol) of 10 M ABA as described in Ref. 5.NF-B Nuclear TranslocationMonocytes (3 106/dish),

incubated without (control) or with ABA or cADPR for 30min,were resuspended in 400 l of ice-cold buffer A (20 mM Tris-HCl, pH 7.8, 50 mM KCl, 10 g/ml leupeptin, 0.1 M dithiothre-itol, 1mMphenylmethylsulfonyl fluoride) and 400l of buffer B(buffer A containing 1.2%Nonidet P-40), vortexed for 10 s, andcentrifuged at 14,000 g for 30 s at 4 C, and supernatants werediscarded. Pelleted nuclei were washed once with buffer A,resuspended in 100 l of buffer B, sonicated (10 s at 3 watts),and centrifuged at 14,000 g for 20 min at 4 C. Supernatantswere recovered, and 40 g of nuclear proteins per sample were

subjected to 10% SDS-PAGE andWestern blot. Densitometricanalysis was performed using the Chemi-Doc System (Bio-Rad). Values are expressed as a luminescence increase relativeto control, untreated cells, and normalized on RNApolymeraseII.COX-2 Expression, PGE2 Production, and MCP-1 Release in

MonocytesMonocytes (3 106/dish) were incubated for 6 hwithout (control) or with ABA or cADPR. Where indicated,cells were pretreated for 45 min with 50 M Wedelolactone(WT). After