Abstract
We investigated, in monocytic leukemia U937 cells, the effects of docosahexaenoic acid (DHA; 22:6 n-3) on calcium signaling and determined the implication of phospholipase C (PLC) and protein kinase C (PKC) in this pathway. DHA induced dose-dependent increases in [Ca2+]i, which were contributed by intracellular pool, via the production of inositol-1,4,5-triphosphate (IP3) and store-operated Ca2+ (SOC) influx, via opening of Ca2+ release-activated Ca2+ (CRAC) channels. Chemical inhibition of PLC, PKCγ, and PKCδ, but not of PKCβ I/II, PKCα, or PKCβI, significantly diminished DHA-induced increases in [Ca2+]i. In vitro PKC assays revealed that DHA induced a ∼2-fold increase in PKCγ and -δ activities, which were temporally correlated with the DHA-induced increases in [Ca2+]i. In cell-free assays, DHA, but not other structural analogs of fatty acids, activated these PKC isoforms. Competition experiments revealed that DHA-induced activation of both the PKCs was dose-dependently inhibited by phosphatidylserine (PS). Furthermore, DHA induced apoptosis via reactive oxygen species (ROS) production, followed by caspase-3 activation. Chemical inhibition of PKCγ/δ and of SOC/CRAC channels significantly attenuated both DHA-stimulated ROS production and caspase-3 activity. Our study suggests that DHA-induced activation of PLC/IP3 pathway and activation of PKCγ/δ, via its action on PS binding site, may be involved in apoptosis in U937 cells.
Footnotes
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This work was supported by a contingent grant from Ministry of Higher Education and Research, France.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.039792.
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ABBREVIATIONS: PUFA, polyunsaturated fatty acid; AA, arachidonic acid; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; CRAC, Ca2+ release-activated Ca2+ channels; DAG, diacylglycerol; DHA, docosahexaenoic acid; DHA-meth, DHA methyl ester; DHE, dihydroethidine; Dic8, 1,2-dioctanoyl-sn-glycerol; DPEA, cis-7,10,13,16,19-docosapentaenoic acid; DTT, dithiothreitol; EPA, eicosapentaenoic acid; GF 109203X, bisindolylmaleimide I; Gö-6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; HBDDE, 2,2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether; IP3, inositol-1,4,5-trisphosphate; LSD, least-significant difference; MBP, myelin basic protein; MMP, mitochondrial membrane potential; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidyl-l-serine; ROS, reactive oxygen species; SKF-96365, 1-(β-[3-(4-methoxyphenyl-)propoxy]-4-methoxyphenethyl)-1H-imidazole; SOC, store-operated Ca2+ channels; TA9, tyrphostin A9; TG, thapsigargin; U-73122, 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione.
- Received July 9, 2007.
- Accepted September 17, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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