Secretory phospholipase A2 activates the cascade of mitogen-activated protein kinases and cytosolic phospholipase A2 in the human astrocytoma cell line 1321N1

The biological effects of type IIA 14-kDa phospholipase A(2) (sPLA(2)) on 1321N1 astrocytoma cells were studied. sPLA(2) induced a release of [H-3]arachidonic acid ([H-3]AA) similar to that elicited by lysophosphatidic acid (LPA), a messenger acting via a G-protein-coupled receptor and a product of sPLA(2) on lipid microvesicles. In contrast, no release of [1-C-14]oleate could be detected in cells labeled with this fatty acid. As these findings pointed to a selective mechanism of [H-3]AA release, it was hypothesized that sPLA(2) could act by a signaling mechanism involving the activation of cytosolic PLA, (cPLA(2)), i.e. the type of PLA(2) involved in the release of [H-3]AA elicited by agonists. In keeping with this view, stimulation of 1321N1 cells with sPLA(2) elicited the decrease in electrophoretic mobility that is characteristic of the phosphorylation of cPLA(2), as well as activation of p42 mitogen-activated protein (MAP) kinase, c-Jun kinase, and p38 MAP kinase. Incubation with sPLA(2), of quiescent 1321N1 cells elicited a mitogenic response as judged from an increased incorporation of [H-3]thymidine. Attempts to correlate the effect of extracellular PLA(2) with the generation of LPA were negative, Incubation with pertussis toxin prior to the addition of either sPLA(2) or LPA only showed abrogation of the response to LPA, thus suggesting the involvement of pertussis-sensitive G(i)-proteins in the case of LPA(2) Treatments with inhibitors of the catalytic effect of sPLA(2) such as p-bromophenacyl bromide and dithiothreitol did not prevent the effect on cPLA(2) activation. In contrast, preincubation of 1321N1 cells with the antagonist of the sPLA(2) receptor p-aminophenyl-alpha-D-mannopyranoside-bovine serum albumin, blocked cPLA(2) activation with a EC50 similar to that described for the inhibition of binding of sPLA(2) to its receptor. Moreover, treatment of 1321N1 cells with the MAP kinase kinase inhibitor PD-98059 inhibited the activation of both cPLA(2) and p42 MAP kinase produced by sPLA(2). In summary, these data indicate the existence in astrocytoma cells of a signaling pathway triggered by engagement of a sPLA(2)-binding structure, that produces the release of [H-3]AA by activating the MAP kinase cascade and cPLA(2), and leads to a mitogenic response after longer periods of incubation.