Ceramide accelerates dephosphorylation of extracellular signal-regulated kinase 1/2 to decrease prostaglandin D₂ production in RBL-2H3 cells

In the present study, the effect of ceramide on antigen-stimulated phosphorylation of extracellular signal-regulated kinase (ERK) in the mechanism responsible for regulating production of prostaglandin (PG) D₂ was investigated in the mast cell line, RBL-2H3 cells. Cell-permeable C₆-ceramide (N-hexanoylsphingosine) suppressed antigen-stimulated phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase. Ceramide also inhibited production of PGD₂ and an increase in the activity of cytosolic phospholipase A₂ (cPLA₂), whereas it did not influence the tyrosine phosphorylation of major cellular proteins in response to antigen. The ceramide-induced inhibition of ERK1/2 phosphorylation and of cPLA₂ activation was suppressed by orthovanadate, a tyrosine phosphatase inhibitor, but not by okadaic acid, a serine/threonine phosphatase inhibitor. Addition of ceramide to the lysate prepared from antigen-stimulated cells reduced the phosphorylated ERK1/2, and orthovanadate effectively prevented the reduction. These results suggest that ceramide accelerates the dephosphorylation of phosphorylated ERK1/2 via activation of a protein tyrosine phosphatase, thus preventing activation of cPLA₂ and production of PGD₂.