Received for publication July 11, 2007.
Revised September 14, 2007.
Accepted for publication September 17, 2007.

DHA INDUCES INCREASES IN [Ca²⁺]_i IP₃ PRODUCTION AND ACTIVATES SELECTIVELY PKC AND VIA PHOSPHATIDYLSERINE BINDING SITE : IMPLICATION IN APOPTOSIS IN U937 CELLS

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 IP₃ and store-operated Ca²⁺ (SOC) influx, via opening of Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Chemical inhibition of PLC, and PKC and PKC, but not of PKCI/II, PKC or I, significantly diminished DHA-induced increases in [Ca²⁺]_i. In vitro PKC assays revealed that DHA induced a ~2-fold increase in PKC and activities which were temporally correlated with the DHA-induced increases in [Ca²⁺]_i. In cell-free assays, DHA, but not other structural analogues 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 PKC/ 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 PKC/ via its action on PS binding site, may be involved in apoptosis in U937 cells.

Key words: Protein Kinase C, Oxidative stress/antioxidants, Mechanisms of cell killing/apoptosis