P2X(7) receptor stimulation upregulates Egr-1 biosynthesis involving a cytosolic Ca(2+) rise, transactivation of the EGF receptor and phosphorylation of ERK and Elk-1.

The P2X(7) receptor is an ATP-gated ionotropic receptor that is permeable for small cations including Ca(2+) ions. Using 293 cells expressing P2X(7) receptors, we show that the P2X(7) receptor-specific ligand 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) induces a signaling cascade leading to the biosynthesis of biologically active Egr-1, a zinc finger transcription factor. BzATP-triggered Egr-1 biosynthesis was attenuated by the mitogen-activated protein kinase kinase inhibitor PD98059, by BAPTA-AM, the acetoxymethylester of the cytosolic Ca(2+) chelator BAPTA, and by an epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor (AG1478). These results indicate that phosphorylation and activation of extracellular signal-regulated protein kinase ERK, elevated levels of intracellular Ca(2+) and the transactivation of the EGF receptor are essential for BzATP-induced upregulation of Egr-1. The requirement of Ca(2+) within the signaling cascade was upstream of Raf kinase activation. Lentiviral-mediated expression of MAP kinase phosphatase-1 (MKP-1), a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, inhibited Egr-1 biosynthesis following BzATP stimulation, indicating that MKP-1 functions as a nuclear shut-off device. Furthermore, the ternary complex factor Elk-1 was phosphorylated and the transcriptional activation potential of Elk-1 was enhanced following P2X(7) receptor stimulation. Expression of a dominant-negative mutant of Elk-1 impaired BzATP-induced upregulation of Egr-1 biosynthesis. Thus, Elk-1 connects the intracellular signaling cascade elicited by activation of P2X(7) receptors with the transcription of the Egr-1 gene.