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 controversial. Initially it was postulated that glucocorticoids can induce the formation of PLA(2) inhibitory proteins termed annexins. We have previously shown that the cytokine-induced 14 kDa PLA(2) activity and the synthesis of prostaglandin E(2) in rat mesangial cells is dose-dependently blocked by pretreatment of the cells with dexamethasone (Schalkwijk et al. (1991) Biochem. Biophys. Res. Commun. 180, 46-52). Concurrently, the synthesis of 14 kDa group II PLA(2) is suppressed. The regulation of PLA(2) activity is complex and may well involve superimposable mechanisms. Thus, although the decrease in PLA(2) protein levels could in itself explain the dexamethasone-induced decrease in PLA(2) activity, a contribution of the glucocorticoid-induced anti-phospholipase A(2) protein annexin cannot be ruled out a priori. To investigate this possibility we analyzed the level of annexin I by Western blotting and immunostaining in mesangial cells treated with interleukin-1 beta and/or dexamethasone. Under conditions where 14 kDa group II PLA(2) activity and protein levels were dramatically affected by interleukin-1 and dexamethasone, the level of annexin I in the cells remained constant. Dexamethasone also did not induce the secretion of annexin I. In addition, no evidence for dexamethasone-induced translocation of annexin I from the cytosol to membranes, thereby possibly sequestering the substrates for PLA(2), was obtained. Immunofluorescence studies localized the cytokine-induced PLA(2) to the Golgi area and punctate structures in the cytoplasm. We have also studied the subcellular localization of annexin I in rat mesangial cells using confocal microscopy. These studies located annexin I mainly in the cytoplasma and the nucleus. We conclude from these experiments that the dexamethasone-induced inhibition of 14 kDa group II PLA(2) in rat mesangial cells is not mediated by annexin I and is solely due to the suppression of PLA(2) gene expression.