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. 2008 Oct;84(4):1159-71.
doi: 10.1189/jlb.0907612. Epub 2008 Jul 14.

The nucleotide receptor P2RX7 mediates ATP-induced CREB activation in human and murine monocytic cells

Monica L Gavala  1 , Zachary A Pfeiffer, Paul J Bertics
Affiliations

The nucleotide receptor P2RX7 mediates ATP-induced CREB activation in human and murine monocytic cells

Monica L Gavala et al. J Leukoc Biol. 2008 Oct.

Abstract

Nucleotide receptors serve as sensors of extracellular ATP and are important for immune function. The nucleotide receptor P2RX7 is a cell-surface, ligand-gated cation channel that has been implicated in many diseases, including arthritis, granuloma formation, sepsis, and tuberculosis. These disorders are often exacerbated by excessive mediator release from activated macrophages in the inflammatory microenvironment. Although P2RX7 activation can modulate monocyte/macrophage-induced inflammatory events, the relevant molecular mechanisms are poorly understood. Previous studies suggest that MAPK cascades and transcriptional control via CREB-linked pathways regulate the inflammatory capacity of monocytic cells. As P2RX7 promotes MAPK activation and inflammatory mediator production, we examined the involvement MAPK-induced CREB activation in P2RX7 action. Our data reveal that stimulation of multiple monocytic cell lines with P2RX7 agonists induces rapid CREB phosphorylation. In addition, we observed a lack of nucleotide-induced CREB phosphorylation in RAW 264.7 cells expressing nonfunctional P2RX7 and a gain of nucleotide-induced CREB phosphorylation in human embryonic kidney-293 cells that heterologously express human P2RX7. Furthermore, our results indicate that P2RX7 agonist-induced CREB phosphorylation is partly mediated via Ca(2+) fluxes and the MEK/ERK system. Mechanistic analyses revealed that macrophage stimulation with a P2RX7 agonist induces CREB/CREB-binding protein complex formation, which is necessary for CREB transcriptional activation. Also, we demonstrate that P2RX7 activation induces a known CREB-dependent gene (c-fos) and that dominant-negative CREB constructs attenuate this response. These studies support the idea that P2RX7 stimulation can directly regulate protein expression that is not dependent on costimulation with other immune modulators such as LPS.

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Figures

Fig. 1.
Fig. 1.
Effect of P2RX7 agonists on pCREB in human monocytic cells. (A) Isolated peripheral human blood monocytes were treated with vehicle (2.5 mM HEPES), 100 μM BzATP, or 100 ng/mL LPS for the indicated times. A representative immunoblot displaying pCREB is shown (n=3). The pCREB antibody recognizes pCREB and phosphorylated ATF-1 (pATF1); thus, it is also shown. Immunoblotting was performed with anti-Grb2 antibody as a loading control. (B) THP-1 human monocytes were treated with vehicle (2.5 mM HEPES), 100 μM PMA, 1 mM ATP, or 250 μM BzATP for 15 min. A representative immunoblot displaying pCREB/pATF1 is shown (n=3). Immunoblotting was performed with anti-Grb2 antibody as a loading control. (C) Murine RAW 264.7 macrophages were stimulated with vehicle (2.5 mM HEPES), 250 μM BzATP, or 1 μg/mL LPS for the indicated time-points. Immunoblotting was performed with anti-pCREB/pATF1 and anti-β-tubulin (loading control) antibodies. Results of the three independent experiments were combined and graphed ± sem.
Fig. 2.
Fig. 2.
Dependence of nucleotide-induced pCREB on P2RX7 function/expression. (A) Wild-type (WT) and serine to phenylalanine mutation (SF) mutant RAW 264.7 macrophages were treated with vehicle (2.5 mM HEPES), 10 μg/mL anisomycin, 1 μg/mL LPS, 250 μM BzATP, or the indicated concentrations of ATP for the time-points shown. A representative immunoblot displaying pCREB/pATF1 is shown (n≥3). Immunoblotting was performed with anti-pan-ERK1/2 antibody as a loading control. (B) Untransfected HEK-293 cells and HEK-293 cells transfected with the pIREShyg (Hyg) or pIRES/hP2RX7 (P2RX7) expression vectors were stimulated with vehicle (2.5 mM HEPES), 10 μg/mL anisomycin, 250 μM BzATP, or 100 μM forskolin for the indicated times and immunoblotted for pCREB/pATF1. Immunoblotting was performed with anti-pan-ERK1/2 antibody as a loading control. Results of the three independent experiments were collated, and BzATP-induced pCREB was graphed ± sem.
Fig. 3.
Fig. 3.
P2RX7 agonist-induced ERK1/2 phosphorylation. RAW 264.7 macrophages, untransfected HEK-293 cells, and HEK-293 cells that were transfected with the pIREShyg or pIRES/hP2RX7 expression vectors were stimulated with vehicle (2.5 mM HEPES), 10 μg/mL anisomycin, 250 μM BzATP, or 100 μM forskolin for the indicated times and immunoblotted to detect ERK1/2 phosphorylation. Immunoblotting was performed with the anti-Grb2 antibody as a loading control. The results of the three independent experiments were combined, and BzATP-induced ERK1/2 phosphorylation was graphed (mean±sem).
Fig. 4.
Fig. 4.
Effect of an ERK1/2 kinase inhibitor on P2RX7-induced CREB phosphorylation in murine macrophages. (A) RAW 264.7 macrophages were pretreated for 15 min with vehicle (DMSO), the MEK1/2 inhibitor UO126 (10 μM), or the MEK1/2 inactive inhibitor analog UO124 (10 μM). The cells were subsequently treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for 15 min. A representative immunoblot displaying pCREB/pATF1 is shown. Immunoblotting was performed with anti-pan-ERK1/2 antibody as a loading control. The results of five independent experiments were collated and are represented as normalized CREB phosphorylation (mean±sem); *, P < 0.00001. (B) RAW 264.7 macrophages were pretreated for 15 min with vehicle (DMSO) or UO126 at concentrations from 1 μM to 30 μM. The cells were subsequently treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for 15 min. A representative immunoblot displaying pCREB/pATF1 is shown (n=3). (C) RAW 264.7 macrophages were pretreated for 15 min with vehicle (DMSO) or 10 μM UO126. The cells were subsequently treated with vehicle (2.5 mM HEPES) or 3 mM ATP for 15 min. The results of three independent experiments were combined and are represented as a percent of ATP-induced CREB phosphorylation (mean±sem); **, P < 0.05.
Fig. 5.
Fig. 5.
Effect of an ERK1/2 kinase inhibitor on P2RX7-induced CREB phosphorylation in primary human monocytes. Isolated human peripheral blood monocytes were pretreated for 15 min with vehicle (DMSO) or 10 μM UO126 and were subsequently stimulated with vehicle (2.5 mM HEPES), 250 μM BzATP, or 10 μM PMA for the indicated time-points. A representative immunoblot displaying pCREB/pATF1 is shown. Immunoblotting was performed with an anti-β-tubulin antibody as a loading control. Analogous results were observed in two separate experiments.
Fig. 6.
Fig. 6.
P2RX7 agonist-induced p90Rsk phosphorylation in RAW 264.7 macrophages, which were pretreated for 15 min with vehicle (DMSO) or the MEK1/2 inhibitor UO126 at concentrations from 1 μM to 30 μM. The cells were subsequently treated with vehicle (2.5 mM HEPES), 250 μM BzATP, or 3 mM ATP for 15 min. A representative immunoblot displaying p90Rsk phosphorylation (pp90Rsk) is shown. Immunoblotting was performed with anti-β-tubulin antibody as a loading control. The results of three independent experiments were collated and are represented as a percent of nucleotide-induced p90Rsk phosphorylation (mean±sem); *, P < 0.05, as compared with DMSO-pretreated, BzATP-induced p90Rsk phosphorylation; Ψ, P < 0.05, as compared with DMSO-pretreated, ATP-induced p90Rsk phosphorylation.
Fig. 7.
Fig. 7.
Effect of calcium chelator BAPTA on P2RX7 agonist-induced ERK/CREB phosphorylation. (A) RAW 264.7 macrophages were loaded with vehicle (DMSO) or BAPTA-AM at increasing concentrations (1 μM–30 μM) for 30 min at 37°C. Following inhibitor incubation, the cells were washed twice and treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for 5 min and immunoblotted for ERK1/2 phosphorylation. (Note: BzATP-induced ERK1/2 phosphorylation without BAPTA-AM was done in duplicate.) Immunoblotting was performed with anti-Grb2 antibody as a loading control. The results of three independent experiments were combined and are represented as a percent of BzATP-induced ERK1 (left panel) and ERK2 (right panel) phosphorylation (mean±sem). (B) RAW 264.7 macrophages were loaded with vehicle (DMSO) or BAPTA-AM at increasing concentrations (1 μM–30 μM) for 30 min at 37°C. Following inhibitor incubation, the cells were washed twice and treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for 5 min and immunoblotted for pCREB/pATF1. Immunoblotting was performed with anti-Grb2 antibody as a loading control. The results of three independent experiments were collated and are represented as a percent of BzATP-induced CREB phosphorylation (mean±sem); *, P < 0.01; **, P < 0.05; ***, P < 0.003.
Fig. 8.
Fig. 8.
P2RX7 agonist-induced CREB/CBP complex formation. (A) RAW 264.7 macrophages were treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for the indicated time-points. Cells were lysed with 3× GST lysis buffer, and equal amounts of protein were incubated with GST-KIX beads for 18 h. The proteins that were pulled down with the GST-KIX beads were immunoblotted for CREB. A representative blot from three independent experiments is shown. (B) RAW 264.7 macrophages were pretreated for 15 min with vehicle (DMSO) or 10 μM UO126 and then were treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for the indicated time-points at 37°C. Cells were lysed with 3× GST lysis buffer, and equal amounts of protein were incubated with GST-KIX beads for 18 h. Pulled-down proteins were immunoblotted for CREB. The results of independent experiments (n=3- to 5-min treatment; n=4 for 15-min treatment) were collated and are represented as a percent of BzATP-induced CREB phosphorylation (mean±sem); *, P < 0.0005.
Fig. 9.
Fig. 9.
Attenuation of CREB activation leads to down-regulation of P2RX7 agonist-induced c-Fos protein expression. (A) HEK-293 cells transfected with the pIREShyg or pIRES/hP2RX7 expression vectors were stimulated with vehicle (2.5 mM HEPES), 250 μM BzATP, or 1 μg/mL PMA for the indicated times and immunoblotted for total c-Fos protein. Immunoblotting was performed with an anti-Grb2 antibody as a loading control. Analogous results were observed in three separate experiments. (B) HEK-293 cells stably transfected with the pIRES/hP2RX7 expression vector were transiently transfected with MT, S133A (S), or KCREB (K) CREB dn vectors. Twenty-four hours post-transfection, cells were treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for 2 h and immunoblotted for total c-Fos protein expression. Immunoblotting was performed with an anti-Grb2 antibody as a loading control. The results of four independent experiments were collated and are represented as a percent of BzATP-induced c-Fos expression over basal c-Fos expression (mean±sem); *, P < 0.05; **, P < 0.005, as compared with MT-transfected, BzATP-treated c-Fos expression.
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