Stefano Luisa, Rössler Oliver G, Griesemer Désirée, Hoth Markus, Thiel Gerald
Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, Homburg, Germany.
J Cell Physiol. 2007 Oct;213(1):36-44. doi: 10.1002/jcp.21085.
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.
P2X(7)受体是一种ATP门控离子型受体，对包括Ca(2+)离子在内的小阳离子具有通透性。利用表达P2X(7)受体的293细胞，我们发现P2X(7)受体特异性配体2',3'-O-(4-苯甲酰基-苯甲酰基)-ATP（BzATP）可诱导一个信号级联反应，导致具有生物活性的锌指转录因子Egr-1的生物合成。BzATP触发的Egr-1生物合成可被丝裂原活化蛋白激酶激酶抑制剂PD98059、胞质Ca(2+)螯合剂BAPTA的乙酰氧甲酯BAPTA-AM以及表皮生长因子（EGF）受体特异性酪氨酸激酶抑制剂（AG1478）所减弱。这些结果表明，细胞外信号调节蛋白激酶ERK的磷酸化和激活、细胞内Ca(2+)水平的升高以及EGF受体的反式激活对于BzATP诱导的Egr-1上调至关重要。信号级联反应中对Ca(2+)的需求在Raf激酶激活的上游。慢病毒介导的双特异性磷酸酶MAP激酶磷酸酶-1（MKP-1）的表达可使细胞核中的ERK去磷酸化并使其失活，从而抑制BzATP刺激后的Egr-1生物合成，这表明MKP-1起到核关闭装置的作用。此外，三元复合因子Elk-1在P2X(7)受体刺激后发生磷酸化，其转录激活潜能增强。Elk-1显性负突变体的表达损害了BzATP诱导的Egr-1生物合成上调。因此，Elk-1将P2X(7)受体激活引发的细胞内信号级联反应与Egr-1基因的转录联系起来。
