Activation of P2X7 purinoceptor-stimulated TGF-beta 1 mRNA expression involves PKC/MAPK signalling pathway in a rat brain-derived type-2 astrocyte cell line, RBA-2

The present study investigates the receptor and mechanisms involved in ATP-stimulated transforming growth factor-beta 1 (TGF-β1) mRNA expression of a type-2 astrocyte cell line, RBA-2. RT-PCR analysis revealed that RBA-2 type-2 astrocytes possess abundant P2X₄ and P2X₇ receptors. ATP and P2X₇ receptor-sensitive agonist, BzATP, both stimulated TGF-β1 mRNA expression in a time and dose-dependent manner. The stimulation required a minimum of 500 μM ATP; BzATP was much more potent that ATP, and P2X₇-selective antagonist, oATP, inhibited the effects. In addition, ATP metabolites ADP, AMP and adenosine were ineffective in stimulation of TGF-β1 mRNA expression. Thus, the effect of ATP was mediated through the P2X₇ receptors. To investigate further the mechanisms by which the P2X₇ receptor mediated the TGF-β1 mRNA expression, the cells were treated with inhibitors for mitogen-activated kinase (MAPK) or protein kinase C (PKC), PD98059 or GF109203X, respectively. Both PD98059 and GF109203X inhibited the ATP-stimulated TGF-β1 mRNA expression. Furthermore, ATP and BzATP stimulated ERK1/2 activation and the activation was inhibited by PKC inhibitors, GF109203X and Gö6976. In conclusion, activation of P2X₇ receptors enhanced TGF-β1 mRNA expression and the effect involved PKC/MAPK signalling pathway in RBA-2 type-2 astrocytes.     

Involvement of de novo ceramide biosynthesis in macrophage death induced by activation of ATP-sensitive P2X7 receptor

Macrophage ionotropic P2X7 receptors regulate cell-death through ill-defined signaling pathways. Here, we investigated the role of ceramide, an apoptogenic sphingolipid and showed that ATP stimulated ceramide accumulation in macrophages. Benzoylbenzoyl-ATP, a potent P2X7 agonist, was able to mimic the effects of ATP on ceramide accumulation while oxidized ATP had the opposite effect. Ceramide accumulation was blocked by de novo ceramide biosynthesis inhibitors. Interestingly, ATP-induced caspase-3/7 activation was dependent on ceramide generation. Finally, we showed that de novo ceramide biosynthesis is involved in ATP-induced macrophage death in a caspase-dependent manner. Our results indicate a novel role of ceramide in P2X7-regulated cell-death.