LEVELS AND LOCALIZATION OF GROUP-II PHOSPHOLIPASE-A(2) AND ANNEXIN-I IN INTERLEUKIN-TREATED AND DEXAMETHASONE-TREATED RAT MESANGIAL CELLS -EVIDENCE AGAINST ANNEXIN MEDIATION OF THE DEXAMETHASONE-INDUCED INHIBITION OF GROUP-II PHOSPHOLIPASES-A(2)

The mechanism by which glucocorticosteroids inhibit the synthesis and secretion of pro-inflammatory arachidonate metabolites is still contro versial. Initially it was postulated that glucocorticoids can induce t he formation of PLA(2) inhibitory proteins termed annexins. We have pr eviously shown that the cytokine-induced 14 kDa PLA(2) activity and th e synthesis of prostaglandin E(2) in rat mesangial cells is dose-depen dently blocked by pretreatment of the cells with dexamethasone (Schalk wijk et al. (1991) Biochem. Biophys. Res. Commun. 180, 46-52). Concurr ently, the synthesis of 14 kDa group II PLA(2) is suppressed. The regu lation of PLA(2) activity is complex and may well involve superimposab le mechanisms. Thus, although the decrease in PLA(2) protein levels co uld in itself explain the dexamethasone-induced decrease in PLA(2) act ivity, a contribution of the glucocorticoid-induced anti-phospholipase A(2) protein annexin cannot be ruled out a priori. To investigate thi s possibility we analyzed the level of annexin I by Western blotting a nd immunostaining in mesangial cells treated with interleukin-1 beta a nd/or dexamethasone. Under conditions where 14 kDa group II PLA(2) act ivity and protein levels were dramatically affected by interleukin-1 a nd dexamethasone, the level of annexin I in the cells remained constan t. Dexamethasone also did not induce the secretion of annexin I. In ad dition, no evidence for dexamethasone-induced translocation of annexin I from the cytosol to membranes, thereby possibly sequestering the su bstrates for PLA(2), was obtained. Immunofluorescence studies localize d the cytokine-induced PLA(2) to the Golgi area and punctate structure s in the cytoplasm. We have also studied the subcellular localization of annexin I in rat mesangial cells using confocal microscopy. These s tudies located annexin I mainly in the cytoplasma and the nucleus. We conclude from these experiments that the dexamethasone-induced inhibit ion of 14 kDa group II PLA(2) in rat mesangial cells is not mediated b y annexin I and is solely due to the suppression of PLA(2) gene expres sion.