The mrf-1 gene has been isolated from microglia exposed to cultured cerebellar granule neurons undergoing apoptosis. We have shown that mrf-1 is upregulated in response to neuronal death and degeneration both in vitro and in vivo. However, the exact role of MRF-1 remains unknown. Here we show that MRF-1 is released from cultured rat microglia, and its release is greatly enhanced under inflammatory conditions. When microglia were treated with ATP, the amount of MRF-1 that was released increased 10-fold compared to the basal level of release. Enhanced MRF-1 release was induced within 10 min and peaked within 1 h; after approximately 4 h, the MRF-1 release had returned to normal. MRF-1 release was stimulated by 2-methyl-thio-ATP (five-fold) and a P2X(7) selective agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (ten-fold). Moreover, the ATP-stimulated MRF-1 release was inhibited by a P2X(7) selective antagonist, oxidized ATP (oATP), and also under a Ca(2+)-free condition. These results indicate that the effects of ATP are dependent on Ca(2+) influx through P2X(7) receptors. MRF-1 release was enhanced by Ca(2+)-ionophore A23187 (sixfold), thapsigargin (threefold); however, it was not enhanced by glutamate or lipopolysaccharide. Moreover, a platelet-activating factor enhanced microglial MRF-1 release in a dose-dependent manner. We also showed that a conditioned medium from cerebellar granule neurons undergoing apoptosis markedly increased MRF-1 release from microglia; that effect was significantly inhibited by oATP. These results indicate that selective inflammatory stimulations, including ATP and PAF, enhance MRF-1 release from microglia through a Ca(2+)-dependent mechanism and suggest that MRF-1 may play a role in cell-cell interactions under inflammatory conditions.
Copyright 2002 Wiley-Liss, Inc.