Inhibition of long-chain acyl-CoA synthetase 4 facilitates production of 5, 11-dihydroxyeicosatetraenoic acid via the cyclooxygenase-2 pathway.

Long chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert free long chain fatty acids into their acyl-CoA forms. Among ACSL enzymes, ACSL4 prefers arachidonic acid (AA) as a substrate and plays an important role in re-esterification of free AA. We previously reported that the suppression of ACSL4 activity by treatment with an ACSL inhibitor or a small interfering RNA markedly enhanced interleukin-1β (IL-1β)-dependent prostaglandin (PG) biosynthesis in rat fibroblastic 3Y1 cells. We show here that in addition to these prostanoids, cytokine-dependent production of 5,11-dihydroxyeicosatetraenoic acid (5,11-diHETE), a cyclooxygenase product of 5-hydroxyeicosatetraenoic acid (5-HETE), was enhanced by the inhibition of ACSL4 activity. Treatment of several types of cells with an ACSL inhibitor, triacsin C, markedly enhanced IL-1β-dependent production of 5,11-diHETE. siRNA-mediated knockdown of ACSL4 also enhanced IL-1β-dependent production of 5,11-diHETE from 3Y1 cells. The production of 5,11-diHETE was significantly decreased by a cyclooxygenase (COX)-2 selective inhibitor, NS-398, but not by a 5-lipoxygenase activating protein (FLAP) inhibitor, MK-886. The inhibition of ACSL enzymes significantly facilitated release of not only 5-HETE but also 8-HETE, 9-HETE, 11-HETE, 12-HETE, and 15-HETE, independently of IL-1β stimulation. In vitro analysis showed that a recombinant COX-2 enzyme more effectively metabolized 5(S)-HETE to 5-11-diHETE compared to COX-1 enzyme. From these results, we proposed the following mechanism of 5,11-diHETE biosynthesis in these cells: 1) inhibition of ACSL4 causes accumulation of free AA; 2) the accumulated AA is nonspecifically converted into various HETEs; and 3) among these HETEs, 5-HETE is metabolized into 5,11-diHETE by cytokine-induced COX-2.