Epidermal growth factor facilitates epinephrine inhibition of P2X7-receptor-mediated pore formation and apoptosis: a novel signaling network

Abstract

Epidermal growth factor (EGF), epinephrine, and the P2X7 receptor system regulate growth of human uterine cervical epithelial cells, but little is known about how these systems intercommunicate in exerting their actions. The objective of this study was to understand the mechanisms of EGF and epinephrine regulation of growth of cervical cells. Treatment of cultured CaSki cells with 0.2 nM EGF increased cell number via a PD98059-sensitive pathway. Treatment with 2 nM epinephrine increased cell number, and the effect was facilitated by cotreatment with EGF. Whereas the effect of EGF alone involved up-regulation of [3H]-thymidine incorporation and an increase in cell proliferation, the effect of epinephrine was mediated by inhibition of apoptosis. Epinephrine inhibited apoptosis induced by the P2X7 receptor ligand 2',3'-0-(4-benzoylbenzoyl)-ATP, by attenuation of P2X7 receptor plasma membrane pore formation. Cotreatment with EGF facilitated epinephrine effect via a phosphoinositide 3-kinase-dependent mechanism. CaSki cells express the beta2-adrenoceptor, and the epinephrine antiapoptotic effect could be mimicked by beta2-adrenoceptor agonists and by activators of adenylyl cyclase. Likewise, the effect could be blocked by beta2-adrenoceptor blockers and by the inhibitor of protein kinase-A H-89. Western immunoblot analysis revealed that epinephrine decreased the levels of the glycosylated 85-kDa form of the P2X7 receptor and increased receptor degradation, and that EGF potentiated these effects of epinephrine. EGF did not affect cellular levels of the beta2-adrenoceptor. In contrast, EGF, acting via the EGF receptor, augmented beta2-adrenoceptor recycling, and it inhibited beta2-adrenoceptor internalization via a phosphoinositide 3-kinase-dependent mechanism. We conclude that, in cervical epithelial cells, EGF has a dual role: as mitogen, acting via the MAPK/MAPK kinase pathway, and as an antiapoptotic factor by facilitating epinephrine effect and resulting in greater expression of beta2-adrenoceptors in the plasma membrane. These findings underscore a novel signaling network of communication between the receptor tyrosine kinases, the G protein-coupled receptors, and the purinergic P2X7 receptor.

Figures

Fig. 1

Means (± SD, three to five independent experiments) of the effects of EGF and epinephrine (Epi) on number of CaSki cells in culture (panel A), DNA synthesis (panel B), and BzATP-induced DNA solubilization (panel C). In A: *, P < 0.01 (EGF vs. Baseline); **, P < 0.03 [Baseline, Epi vs. C (control)]; ***, P < 0.03 (EGF, Epi vs. C). In B: *, P < 0.01 (EGF vs. Baseline, in both C and Epi groups). In panel C: *, P < 0.01 (BzATP vs. Baseline, Control and EGF groups); **, P < 0.01 (BzATP, Epi vs. Control and EGF groups); ***, P < 0.03 (BzATP, EGF+EPI vs. Epi groups).

Fig. 2

A, Scatchard analysis of [125I]cyanopindolol binding to CaSki cells, and the effects of EGF, zinterol, and terbutaline. B, Modulation of BzATP-induced DNA solubilization by adrenoceptor agonists and by activators of adenylyl cyclase forskolin (Forsk., in the presence of 3-isobutyl-1-methylxanthine). C, Modulation of zinterol-dependent inhibition of BzATP-induced DNA solubilization by β2-adrenoceptor antagonists and by the protein kinase A inhibitor H-89 dihydrochloride (H-89-dHCl). Data in B and C are means (± SD) of three to four independent experiments. *, P < 0.01 (BzATP+Zinterol vs. BzATP).

Fig. 3

Effects of zinterol (A) and EGF (B) on DNA solubilization induced by BzATP and by TNFα. Data are means (± SD) of three independent experiments. *, P < 0.01 (BzATP group, Zinterol vs. Baseline).

Fig. 4

EGF and Epi modulation of BzATP-induced changes in cytosolic calcium (Cai) (A) and of BzATP-induced influx of ethidium bromide (B) in CaSki cells attached on filters. AU, Arbitrary units of fluorescence (Flu.) at 518/605 nm (excitation/emission). Insets, Means (± SD, three to five independent experiments) of the respective increases in Cai and in intracellular ethidium bromide Flu., 30 min after adding the BzATP. KN-62 was added at 100 nM, 15 min before adding BzATP. In both insets: *, P < 0.01 [Epi vs. C (control) and EGF groups]; **, P < 0.03 (EGF+Epi and KN-62 vs. Epi).

Fig. 5

A, Western immunoblot analysis of P2X7 receptor protein using total homogenates from d-6 cultured CaSki cells. + Ag denotes coincubation with the P2X7 antigen. B, Effects of length in culture on BzATP (100 μM)-induced influx of Ca2+ (solid lines, determined in terms of the increase in Cai, and on BzATP-induced influx of ethidium bromide (broken lines). C, Effects of incubation of CaSki-cell lysates in vitro with N-glycosidase F. The filled and empty bars represent the densitometric ratio of the 65-kDa/85-kDa forms before and after treatment with N-glycosidase F, respectively. D, Effects of day in culture, and of treatments with EGF and Epi on the cellular expression of the P2X7 receptor. Western immunoblot analysis was done on total homogenates of CaSki cells grown on filters for 2 or 6 d. Where indicated, cells were treated with EGF, epinephrine, or both as described in the text. Results of three experiments are summarized in Table 1.

Fig. 6

A, Scatchard analysis of [125I]EGF binding to CaSki cells and the effects of epinephrine and 17β-estradiol. B, Modulation of EGF-induced DNA synthesis by AG1478, PD98059, and wortmannin. Data are means ± SD of three independent experiments. *, P < 0.01 [EGF category, for both AG1478 vs. C (control) and PD98059 vs. C].

Fig. 7

A, EGF-dependent modulation of epinephrine- and zinterol-induced increase in cellular cAMP. *, P < 0.01 (Baseline category, for both epinephrine vs. control, and zinterol vs. control); **, P < 0.01 (EGF category, for both epinephrine vs. control, and zinterol vs. control). B and C, EGF and wortmannin modulation of zinterol-induced changes in membrane-bound [125I]cyanopindo-lol. B, Effects on [125I]cyanopindolol internalization. C, Effects on [125I]cyanopindolol recycling. The experiments are described in the text.

Fig. 8

A novel signaling network communication among RTKs, GPCRs, and the purinergic P2X7 receptor. In human cervical epithelial cells, EGF, acting via the MAPK cascade pathway, stimulates mitosis and proliferation. EGF also facilitates β2-adrenoceptor-dependent stimulation of deglycosylation and degradation of the P2X7 receptor. The effect of EGF involves attenuation of β2-adrenoceptor internalization by a PI3K-dependent mechanism and facilitated recycling of the β2-adrenoceptor by a PI3K-independent mechanism. The EGF effects on β2-adrenoceptor endocytosis and recycling would tend to up-regulate β2-adrenoceptor responsiveness by increasing the pool of the receptor that is available for activation upon ligand binding. The composite effects of EGF and epinephrine would tend to facilitate inhibition of P2X7 receptor-dependent pore formation and apoptosis. The combined EGF mitogenic and antiapoptotic effects would lead to an increase in cell number and to epithelial growth. PKA, Protein kinase A.