Epidermal Growth Factor Facilitates Epinephrine Inhibition of P2X7-Receptor-Mediated Pore Formation and Apoptosis: A Novel Signaling Network
http://www.100md.com
内分泌学杂志 2005年第1期
Departments of Pharmacology (L.W., Y.-H.F.), Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814; and Departments of Reproductive Biology (G.I.G.), Physiology and Biophysics (G.I.G.), and Oncology (G.I.G.), Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Address all correspondence and requests for reprints to: George I. Gorodeski, M.D., Ph.D., University MacDonald Women’s Hospital, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: gig@cwru.edu.
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 ?2-adrenoceptor, and the epinephrine antiapoptotic effect could be mimicked by ?2-adrenoceptor agonists and by activators of adenylyl cyclase. Likewise, the effect could be blocked by ?2-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 ?2-adrenoceptor. In contrast, EGF, acting via the EGF receptor, augmented ?2-adrenoceptor recycling, and it inhibited ?2-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 ?2-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.
Introduction
PROPER FUNCTION OF the epithelia of the female genital tract depends on coordinated growth and differentiation of the epithelial cells, and deviations from these well-controlled functions can cause infertility and lead to dysplasia and cancer. The uterine cervical epithelium is maintained by a balance between proliferation of the basal layer of cells, and death of cells in upper layers. Our current state of knowledge suggests three levels of growth-control of cervical epithelial cells: proliferation, controlled by mitogenic signals [e.g. estrogen and epidermal growth factor (EGF)] vs. growth inhibitory factors (e.g. TGF?) (1); terminal differentiation, controlled mainly by estrogen (2); and senescence of cells evading growth control and terminal differentiation due to the erosion of telomeres (3). Recently it was reported that growth of human cervical epithelial cells is also regulated by apoptosis; the effect involves predominantly the P2X7 receptor as its proximal signaling arm, and it uses the mitochondrial apoptotic pathway (4). However, relatively little is known about regulation of these effects and the mechanisms involved. Because apoptosis functions to eliminate abnormal cells (5) and dysregulation of apoptotic cell-death has been implicated in the premature death of cells, loss of tissue, aging, states of disease, and neoplastic transformation (6), elucidation of these data could be important for our understanding of cervical cell biology and tumorigenesis.
Both EGF (7) and epinephrine (8) are added routinely to cultures of squamous epithelial cells to enhance cell-growth. EGF and members of the EGF receptor family influence cell survival predominantly by stimulating proliferation (9), including of human cervical epithelial cells (1). Until recently, no mechanistic explanation was given for the growth-promoting effect of epinephrine. Recently we found that epinephrine increases the number of human cervical epithelial cells in culture, and that EGF potentiates the epinephrine effect. Because epinephrine, in contrast to EGF alone, inhibited apoptosis of cervical cells, we hypothesized that EGF, in addition to its mitogenic role, also potentiates epinephrine antiapoptotic effect.
The specific objective of the present study was to understand the mechanisms of EGF and epinephrine antiapoptotic effects. The results suggest that the epinephrine effect is mediated by ?2-adrenoceptor-dependent inhibition of P2X7 receptor pore formation. The results also suggest a dual role for EGF: as mitogen, using the MAPK/MAPK kinase (MAPK/MEK) signaling pathway, and as facilitator of epinephrine effect. We also found that the effect of EGF involves facilitated, phosphoinositide 3-kinase (PI3K)-dependent inhibition of ?2-adrenoceptor internalization, and facilitated ?2-adrenoceptor recycling, thereby increasing the level of ?2-adrenoceptors in the plasma membrane. These findings underscore a novel signaling network that could be a general paradigm for functional communication between the receptor tyrosine kinases (RTKs), the G protein-coupled receptors (GPCRs), and the purinergic P2X7 receptor.
Materials and Methods
Cell cultures
The experiments used CaSki cells, a line of transformed cervical epithelial cells that were obtained from the ATCC (Manassas, VA) and were previously characterized as stably expressing phenotypic characteristics of human cervical epithelial cells (7). Cells were grown and subcultured in RPMI-1640 supplemented with 8% fetal calf serum, 0.2% NaHCO3, nonessential amino acids (0.1 mM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and gentamycin (50 μg/ml) at 37 C in a 91%O2-9%CO2 humidified incubator. Cultures were routinely tested for mycoplasma. Preliminary experiments were conducted on cells plated on culture plates, and definitive experiments were repeated on cells plated on filters. The latter method improves culturing conditions and promotes epithelial cell polarization and differentiation (7). Filters were either Anocell (Anocell-10, Oxon, UK, obtained through Sigma Chemicals, St. Louis, MO), Transwell (Costar Corporation, Cambridge, MA), or Millicell-CM (Millipore, Bedford, MA). Filters were coated on their upper (luminal) surface with 3–5 μg/cm2 collagen type IV and incubated at 37 C overnight. The remaining collagen solution was aspirated, and the filter was dried at 37 C. Before plating, both sides of the filters were rinsed three times with Hanks’ balanced salt solution (HBSS). Cells were plated on the upper surface of the filter at 3 x 105 cells/cm2. By plating at this high density, the cultures become confluent and polarized within 12 h after plating (7). All solutions and agents were added to both the luminal and subluminal bathing solutions.
DNA synthesis
DNA synthesis was measured by [3H]thymidine incorporation. Six hours before assays, cells attached on filters were labeled for 4 h with 1 μM methyl-[3H]-thymidine (1 μCi/ml; specific activity, 87 Ci/mmol; Amersham, Piscataway, NJ) and maintained for 2 additional hours in medium devoid of radioactive thymidine. After labeling, the cells were washed several times in ice-cold PBS, lysed in 200 μl 0.1 N NaOH, and precipitated in 2 ml ice-cold 10% trichloroacetic acid for 2 d at 4 C. The radioactivity [dpm/mg protein, determined by Bio-Rad (Hercules, CA) Protein Assay solution] of triplicated samples was determined by ?-scintillation counting (LS1801 scintillation counter; Beckman, Fullerton, CA).
DNA solubilization assay
Twenty-four hours before the end of the experiment, cells were labeled with [3H]thymidine (specific activity, 93 Ci/mmol; 5 μCi/1 x 106 cells) for 16 h. The medium was removed, and cells were washed three times with fresh medium lacking [3H]thymidine and were incubated in the same medium for an additional 6 h. At the end of incubation, the supernatant was stored, and cells were lysed in 0.5 ml lysis buffer (10 mM Tris-HCl, pH 7.5; plus 1 mM EDTA–0.2% Triton X-100) for 1 h at 4 C. The intact chromatin was separated from the fragmented DNA by 5 min centrifugation at 4 C in Eppendorf microcentrifuge at 12,000 x g. The supernatant (lysate) was stored, while the pellet was resuspended in 0.5 ml of 1% sodium dodecyl sulfate (SDS). The radioactivity contained in the supernatants, lysates, and pellets was counted in a scintillation counter, and DNA fragmentation was calculated as: [% solubilized DNA] = ([cpm supernatant + cpm lysates]/[cpm supernatant + cpm lysate + cpm pellet]) x 100.
Binding studies
Binding studies of the adrenoceptors and the EGF receptor used a similar method. Two hundred fifty microliters of binding medium (serum-free DMEM, 0.1% BSA, 10 mM HEPES) containing 10–1000 pM of the radioligand were added to the apical container of the filter, and 500 μl of the same solution to the basolateral container; parallel tubes ontained an additional 200-fold excess radioinert ligand. The cells were then incubated at 4 C for 3 h; at the completion of incubations cells were washed three times with ice-cold HBSS containing 0.1% BSA. Cells were solubilized in 0.2 N NaOH, and the bound ligand was separated on glass fiber filters (Whatman, GF/C, Tonawanda, NY) by vacuum filtration. The associated radioligand was counted either in a ?-scintillation counter (for [3H] associated ligands) or in a -counter (for [125I] associated ligands). The following radioligands were used: [3H]prazosin and [3H]tamsulosin (-adrenoceptor radioligand) and [125I]cyanopindolol [?-adrenoceptor radioligand (10)] (all at 1 μCi/ml; specific activity, about 90 Ci/mmol; Amersham), and [125I]EGF (5 μCi/ml; specific activity, about 100 Ci/mmol; Biomedical Technology, Stoughton, MA). The - and ?2-adrenoceptor radioinert ligands were phenylephrine and zinterol (Sigma Chemicals). Human recombinant EGF was from R&D Systems (Minneapolis, MN). Specific receptor binding was expressed per milligram DNA, and data were analyzed by Scatchard plot as described (11).
Fluorescence changes
Determinations of fluorescence changes in cells attached on filters were published (12). For measurements of cytosolic calcium, cells on filters were incubated with 7 μM fura-2/acetoxymethyl ester plus 0.25% pluronic F12 (12). Measurements of fluorescence were conducted in a custom-designed fluorescence chamber (12). In this apparatus, a filter with cells is placed horizontally in an enclosed dark chamber maintained at a fixed temperature, under conditions that permit selective perfusion of the luminal (0.2 ml) and subluminal compartments (0.5 ml) at rates of 1–1.5 ml/min. Cells were illuminated over the apical surface, and the intensity of the emitted light from the apical surface was measured. Agents were added to the luminal and subluminal solutions, and changes in cytosolic calcium were determined by switching the excitation filters to record the maximal (340/510 nm excitation/emission) and minimal (380/510 nm) fluorescence. Levels of cytosolic calcium were determined by the formula [Ca2+]i (nM) = [(R – Rmin)/(Rmax – R)]·Kd·(Sf2/Sb2), where [Ca2+]i is the level of cytosolic calcium, R is the ratio of fluorescence excitation measurements at 340 to 380 nm, Rmin and Rmax are the experimentally determined minimum and maximum calcium measurement ratios, 340 nm to 380 nm respectively, Kd is the dissociation constant for fura-2 (224 nM), and Sf2/Sb2 is the ratio of fluorescence value at 380 nm excitation determined at Rmin (0 calcium) and Rmax (maximal calcium). Maximal calcium fluorescence was obtained by adding 10 μM ionomycin in the presence of 10 mM CaCl2, and minimal calcium fluorescence was obtained by competing calcium from fura-2 with 2.5 mM MnCl2. This method was also used for experiments with the nuclear stain ethidium bromide (molecular mass, 394 Da). Ethidium bromide was added to the perfusing solutions from a concentrated stock (x100) at a final concentration of 5 μM. Upon influx into cells, the dye binds to nuclear chromatin and elicits specific fluorescence. Changes in fluorescence were measured on-line at wavelengths 518/605 nm (excitation/emission).
Western blot analysis
The postnuclear supernatant of cells was solubilized in lysis buffer (50 mM Tris-HCl, pH 6.8; 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate; 5 mM EDTA, pH 8.0) containing 50 μg/ml phenylmethylsulfonylfluoride, 10 μg/ml benzamidine, 10 μg/ml bacitracine, 10 μg/ml leupeptin, and 2 μg/ml aprotinine. Aliquots (about 45 μl), normalized to 15 μg protein, were loaded on 10% polyacrylamide SDS gel, and vertical electrophoresis was conducted at 50 mA for 1.5 h. Gels were transferred onto Immobilon membrane (Millipore) at 200 V for 1.5 h, membranes were blocked in 5% milk, and receptor polypeptides were visualized using 1.5 μg/ml rabbit anti-P2X7 antibody at 4 C overnight. Membranes were washed three times in PBS and fluorescent stained for 1 min using an enhanced chemiluminescence kit of antirabbit peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). For protein deglycosylation, 20 μg total lysate proteins containing 1% SDS was denatured by heating for 5 min at 100 C. The mixture was incubated overnight at room temperature after addition of 1 U fresh N-glycosidase F (GlycoTech, Rockville, MD) in 20 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 50 mM NaCl. Samples were analyzed by Western blot as described above.
Cellular cAMP
Cellular cAMP was assayed in total homogenates of cells attached on filters. For assays, cells were incubated in HEPES-buffered Krebs-Ringer’s bicarbonate buffer (pH 7.4) containing 1 mg/ml BSA and 2.5 mM glucose (Krebs bicarbonate Ringer) at 37 C for 30 min. The reactions were terminated by adding 12% trichloroacetic acid (1:1 vol/vol). Cells were scraped off the filters and centrifuged at 3,000 x g for 15 min. Supernatants were extracted with 2 ml water-saturated diethylether three times. The concentration of cAMP in the aqueous phase was determined in duplicate by RIA using a commercially available kit (Amersham).
?2-Adrenoceptor internalization (endocytosis) assays
Fifteen minutes before assays, cells were shifted to ice-cold medium and labeled with 1 μCi/ml [125I]cyanopindolol for 30 min at 4 C. After three washes with ice-cold medium lacking the radioligand, cells were shifted to fresh, warm (37 C) medium and treated with 1 μM zinterol. At time intervals of 0, 15, 30, and 60 min, cells were washed with ice-cold medium. Surface-bound and internalized ?2-adrenoceptors were distinguished according to Haigler et al. (13), with some modifications, as we have described (1). Briefly, cells were washed three times with ice-cold HBSS to remove unbound radioactivity. Cultures were then incubated for 10 min at 4 C in 0.2 M acetic acid plus 0.5 M NaCl (pH 2.5) (250 and 500 μl, respectively, in the apical and basolateral containers of the filter). The filters were then rinsed with 250 and 500 μl, respectively, of the same solution, and the washes (defined as the surface bound fraction) were collected. The remaining cell-associated radioactivity was removed by incubating the cells for 60 min at 37 C with 1 N NaOH. The percent membrane bound [125I]cyanopindolol was determined relative to membrane bound [125I]cyanopindolol in cells not exposed to zinterol.
?2-Adrenoceptor recycling assays
Fifteen min before assays, cells were shifted to ice-cold medium and labeled with 1 μCi/ml [125I]cyanopindolol for 30 min at 4 C. After three washes with ice-cold medium lacking the radioligand, cells were incubated at 4 C for 15 min with 1 μM zinterol, and then shifted to 37 C and incubated for an additional 60 min in the same medium. At the completion of incubation, cells were washed three times at 37 C with the same medium, lacking zinterol, to remove unbound zinterol, and internalized processed zinterol was released into the medium. After the wash, cells were incubated further at 37 C for 0, 0.5, 3, 4, 8, 16, and 24 h, and the amount of membrane bound [125I]cyanopindolol was determined as above. Data about recycled receptor are presented in terms of percent control bound [125I]cyanopindolol (compared with cells not exposed to zinterol).
Densitometry
Densitometry was done using AGFA Arcus II scanner (AGFA, New York, NY) and Un-Scan-It gel automated digital software (Silk Scientific, Orem, OR).
Statistical analysis of the data
Data are presented as means (±SD), and significance of differences among means was estimated by Student’s t test. Trends were calculated using GB-STAT V5.3, 1995 (Dynamic Microsystems Inc., Silver Spring, MD) and analyzed with ANOVA.
Chemicals and supplies
Ethidium bromide and fura-2/AM were obtained from Molecular Probes (Eugene, OR). Benzyloxy-valine-alanine-aspartate-O-methyl-fluoromethylketone (zVAD-FMK) was from Calbiochem (La Jolla, CA). All other chemicals, unless specified otherwise, were obtained from Sigma Chemicals. The anti-P2X7 receptor pAb was from Alomone Laboratories (Jerusalem, Israel).
Results
Effects of epinephrine and EGF on proliferation and apoptosis of cervical cells
Near-confluent cultures of CaSki cells on filters were shifted for 14 h to serum-free medium supplemented with 1% BSA and were treated in the same medium with 0.2 nM EGF, 2 nM epinephrine, or both for an additional 24 h. These concentrations were chosen because they are within the physiological range in vivo (14, 15, 16). At the completion of treatments, cells were harvested off the filters and counted. Treatments with EGF or with epinephrine alone increased the number of cells in culture (Fig. 1A). However, treatment with EGF plus epinephrine increased cell number more than did each drug alone (Fig. 1A), suggesting that EGF facilitates the effect of epinephrine.
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).
To better understand the effects of EGF and epinephrine on the growth of cervical cells, two additional experiments were done. First, the effects of EGF and epinephrine on proliferation were determined in terms of DNA synthesis. EGF, but not epinephrine, increased [3H]thymidine incorporation (Fig. 1B), confirming the mitogenic properties of EGF in human cervical epithelial cells (1). In the second experiment, effects of EGF and epinephrine on apoptosis were determined in terms of modulation of changes in DNA solubilization induced by the P2X7 receptor-specific agonist 2',3'-0-(4-benzoylbenzoyl)-ATP (BzATP). It was recently shown that human cervical epithelial cells undergo apoptosis constitutively and that the effect involves the purinergic P2X7 receptor mechanism (4). Because the degree of constitutive apoptosis is low and does not allow for experimental manipulations, BzATP was added to increase the apoptosis. As is shown in Fig. 1C, treatment with 100 μM BzATP for 9 h increased DNA solubilization significantly from the baseline level of 1.9% to 7.8%. Pretreatment for 24 h with 0.2 nM EGF alone had little effect on the BzATP-induced apoptosis (Fig. 1C). In contrast, pretreatment for 24 h with 2 nM epinephrine decreased DNA solubilization to 4.6%, and cotreatment with EGF facilitated the epinephrine effect (2.9%). The control for this experiment was zVAD-FMK, a pan-caspase inhibitor that was added 30 min before assays at a concentration of 50 μM. Treatment with zVAD-FMK decreased the BzATP-induced DNA solubilization to levels that were lower than baseline apoptosis (about 1.4%, Fig. 1C). Collectively, the data in Fig. 1 indicate that epinephrine increases cell number by attenuation of apoptosis, whereas EGF has a dual role: as mitogen, and as facilitator of epinephrine antiapoptotic effect.
Mechanism of epinephrine effect
Involvement of the ?2-adrenoceptor mechanism.
The low concentration of epinephrine that inhibited apoptosis (2 nM) suggested an effect mediated by adrenergic receptor(s). Epinephrine is a relatively nonspecific ?-adrenergic agonist with some -adrenergic potential. To understand the mechanism of epinephrine effect, three experiments were done. First, cells were tested for the expression of - and ?-adrenoceptors. Binding assays revealed no specific binding of the -adrenoceptor radioligands [3H]prazosin or [3H]tamsulosin (not shown), ruling out expression of functional -adrenoceptors. In contrast, CaSki cells bound, with high affinity, the ?-adrenoceptor radioligand [125I]cyanopindolol (Fig. 2A, Kd0.3 nM), and the binding activity was about 15 fmol/mg DNA (10,000 receptors/cell). The radiolabel could be displaced by coincubation with the ?2-adrenoceptor ligands zinterol and terbutaline (Fig. 2A) but not with the ?1-adrenoceptor ligand isoproterenol (not shown). These data indicate that CaSki cells express the ?2-adrenoceptor.
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 ?2-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).
The second and third experiments tested the degree of which activation and inhibition, respectively, of ?2-adrenoceptor modulate BzATP-induced apoptosis. Pretreatment for 24 h with either zinterol or terbutaline (1 μM) inhibited BzATP-induced apoptosis (Fig. 2B). Similarly, pretreatment for 14 h with 10 μM forskolin [in the presence of 0.5 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, to activate directly adenylyl cyclase] also inhibited BzATP-induced apoptosis (Fig. 2B). In contrast, pretreatments with 10 μM of the ?1-adrenoceptor ligand isoproterenol or with 100 μM of the 1-adrenoceptor ligand phenylephrine had no effect on BzATP-induced apoptosis (Fig. 2B).
Cotreatment for 24 h with either 10 μM propranolol (?-adrenoceptor antagonist), 100 nM ICI 118,551 (?2-adrenoceptor antagonist), or 30 μM of H-89 dihydrochloride (protein kinase A inhibitor) blocked the zinterol (1 μM)-induced inhibition of BzATP-induced apoptosis (Fig. 2C). Collectively, the data in Fig. 2 indicate that epinephrine-dependent attenuation of BzATP-induced apoptosis is mediated by the ?2-adrenoceptor. The data also suggest that the effect involves cAMP and protein kinase A as downstream mediators.
Modulation of the mitochondrial pathway.
Apoptosis can be induced by the cell-surface receptor and/or mitochondrial pathways. The former usually involves the Fas and TNF mechanisms followed by activation of caspase-8 (6, 17, 18). The mitochondrial pathway involves the release of cytochrome-c (19) and activation of caspase-9 (20). The cell-surface receptor and mitochondrial-dependent pathways integrate at the level of the effector caspases, so that activation of caspases 9 and 8 triggers the nonreversible execution of apoptosis by caspases 6, 7, and 3 (5, 6). In CaSki cells, apoptosis induced by ligation of the P2X7 receptor mechanism involves predominantly the mitochondrial pathway (4). This knowledge was used to dissect the effects of epinephrine on BzATP-induced apoptosis.
CaSki cells were treated with either 100 μM BzATP for 9 h or with 10 μM TNF for 14 h, and effects on DNA solubilization were determined. These concentrations produce a submaximal degree of apoptosis in human cervical epithelial cells, and their effects involve, respectively, the cell-surface receptor and mitochondrial pathways (4). Either of these two agents significantly increased DNA solubilization (Fig. 3), confirming our previous results (4) and the results in Figs. 1 and 2 regarding BzATP effects. Pretreatment for 24 h with 1 μM zinterol inhibited BzATP-induced increase in DNA solubilization, but it had no effect on the increase in DNA solubilization induced by TNF (Fig. 3A). Pretreatment for 24 h with 0.2 nM EGF alone had no significant effect on BzATP- or TNF-induced increase in DNA solubilization (Fig. 3B).
FIG. 3. Effects of zinterol (A) and EGF (B) on DNA solubilization induced by BzATP and by TNF. Data are means (±SD) of three independent experiments. *, P < 0.01 (BzATP group, Zinterol vs. Baseline).
Inhibition of P2X7 receptor pore formation.
One of the explanations for the results in Fig. 3 is that the ?2-adrenoceptor-dependent antiapoptotic signaling pathway interacts with proximal steps of the P2X7 receptor signaling cascade, possibly at the level of the P2X7 receptor. Activation of the P2X7 receptor can induce apoptosis by a number of known mechanisms, including IL-1? (21), TNF-TNF-related apoptosis-inducing ligand (22), and the p38, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (23) and NF-B pathways (24). However, the P2X7 receptor is unique in its ability to form pores in the plasma membrane in the continued presence of the ligand (25). Plasma membrane pore formation depends on the long C terminus of the receptor (26), and oligomerization of neighboring molecules is believed to cause progressive dilatation of the pore to a diameter of approximately 4 nm and an increase in the permeation path to molecules of molecular mass of 400–900 Da (27). In its final size, the pore is relatively permeable to Ca2+, but it remains selective to other cations and is impermeable to anions (28). Uncontrolled influx of Ca2+ and the sustained increases in cytosolic calcium are believed to trigger the apoptotic mitochondrial pathway (28). The objective of the following experiments was to test the hypothesis that epinephrine inhibits apoptosis by blocking P2X7 receptor pore formation.
The first experiment tested the degree of which pretreatment with epinephrine blocks BzATP-induced Ca2+ influx. We used fura-2-loaded CaSki cells attached on filters housed in a fluorescence chamber as described in Materials and Methods and in our previous publication (12). Under these conditions, treatment with BzATP induced 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62)-sensitive, time-dependent sustained Ca2+ influx that began about 10 min after adding the ligand (Fig. 4A). EGF alone had no significant effect, and pretreatment with epinephrine attenuated the BzATP-induced increase in cytosolic calcium (inset, Fig. 4A). Pretreatment with epinephrine plus EGF attenuated the BzATP-induced increase in cytosolic calcium to a degree that was observed in cells treated with the P2X7 receptor modifier KN-62 (inset, Fig. 4A).
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).
The second experiment used the same design, but it tested more directly P2X7 receptor pore formation. Cells attached on filters were treated with BzATP in the presence of 5 μM ethidium bromide (molecular mass, 394 Da). The rationale was that, in cells with intact plasma membrane, influx of the dye is minimal. As is shown in Fig. 4B, under baseline conditions, there were no changes in fluorescence at 518/605 nm (excitation/emission), indicating no influx of ethidium bromide. Treatment with BzATP resulted in time-related increase in the fluorescence (Fig. 4B), indicating nuclear binding of ethidium bromide and suggesting that the nucleotide induced influx of ethidium bromide. The increase in fluorescence began about 10 min after adding the agonist, and it correlated with the sustained increase in cytosolic calcium (Fig. 4, A and B). Similar to the effect on cytosolic calcium, the BzATP-induced influx of ethidium bromide could be blocked by pretreatment with KN-62 (inset, Fig. 4B). Pretreatment with EGF alone had no significant effect; pretreatment with epinephrine attenuated the BzATP-induced influx of ethidium bromide, and pretreatment with epinephrine plus EGF inhibited the BzATP-induced influx of ethidium bromide, similar to the effect of KN-62 (Fig. 4B and inset). Collectively, the results in Fig. 4 suggest that epinephrine inhibited ligation-induced P2X7 receptor pore formation; EGF alone had no significant effect, but it facilitated the effect of epinephrine.
Modulation of P2X7 receptor expression.
The objective of the experiments in this section was to determine epinephrine effect on the expression of the P2X7 receptor. Expression of the P2X7 receptor protein was analyzed using the Alomone polyclonal rabbit anti-P2X7 receptor antibody as the primary antibody. This antibody was raised against the purified peptide (C)KIRK EFPKT QGQYS GFKYP Y (corresponding to residues 576–595 of rat P2X7 with an additional N-terminal cysteine (29), and our data (see below) and data by others (30) support its usefulness for the objectives of the present study.
Western immunoblot analysis of total homogenates from CaSki cells revealed specific reactivities to 85-kDa, 65-kDa, and 18-kDa forms that were abolished in the presence of the antigen used to raise the antibody (Fig. 5A). The 65-kDa is most likely the native form of the P2X7 receptor corresponding to the reported P2X7 receptor 70-kDa isoform, whereas the 18-kDa is a degradation product (28, 31, 32, 33). To better understand the role of the 85-kDa form, two additional experiments were done. First, the effects of length in culture on the responses to BzATP were compared with the cellular expression of the receptor. The levels of BzATP-induced Ca2+ influx and of BzATP-induced influx of ethidium bromide were greater in cells cultured for 6 d than in cells cultured for 2 d (Fig. 5B). Length in culture also affected the levels of the P2X7 receptor forms: in d-2 cells, the predominant form was the 65-kDa; in contrast, in d-6 cells, the levels of the 85-kDa and 18-kDa forms were greater than in d-2 cells (Fig. 5D, left lanes). These results suggest that the mature and functional form of the P2X7 receptor protein is the 85-kDa form. This hypothesis was further supported by the results shown in Fig. 5C. Incubation in vitro of cell lysates with N-glycosidase F showed a decrease in the density of the 85-kDa form along with an increase in the density of the 65-kDa form (Fig. 5C). Densitometry analysis of two experiments indicates that the ratio of 65-kDa/85-kDa increased more than 3-fold after treatment in vitro with N-glycosidase F. These data are in general agreement with previous studies in the field (29, 30, 32) and indicate that the 85-kDa form is the glycosylated form of the P2X7 receptor.
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.
Pretreatment of CaSki cells with EGF alone had only mild effect on the levels of the P2X7 receptor forms, increasing mainly the 18-kDa form in d-6 cells (Fig. 5 and Table 1). Pretreatment of d-2 cells with epinephrine decreased the 65-kDa form; whereas in d-6 cells, it decreased the 85-kDa and 65-kDa forms and increased the 18-kDa form (Fig. 5 and Table 1). Cotreatment of d-2 cells with EGF plus epinephrine increased the 18-kDa form 50-fold; cotreatment of d-6 cells decreased the 85-kDa form and increased the 18-kDa isoform 35-fold. In addition, in d-6 cells, treatment with epinephrine plus EGF resulted in the appearance of an intermediate form of 75-kDa (Fig. 5, Table 1). Collectively, the data in Fig. 5 and Table 1 indicate that EGF facilitates epinephrine-induced decrease of the glycosylated 85-kDa form of the P2X7 receptor, and of P2X7 receptor degradation. The data also suggest that EGF, acting in concert with epinephrine, induces deglycosylation of the P2X7 receptor.
TABLE 1. Effects of day in culture and of treatments with EGF and epinephrine on the expression of the P2X7 receptor
Mechanism of EGF-dependent facilitation of epinephrine effect
EGF facilitated epinephrine antiapoptotic effect (Figs. 1, 4, and 5), but treatment with EGF alone had little effect on apoptosis or on the density level of the ?2-adrenoceptor (Fig. 2A). These data suggested that the EGF effect involves modulation of ?2-adrenoceptor function. To better understand the effect of EGF, we assayed the functionality of the EGF receptor in CaSki cells. CaSki cells bound EGF specifically and with high affinity (Fig. 6A). Scatchard analysis of the specific [125I]EGF binding suggested two populations of EGF receptors: high-affinity sites (Kd0.5 nM, binding activity of about 50 fmol/mg DNA, 50,000 receptors/cell); and low-affinity sites (Kd2.1 nM). These data confirm previous results in normal human cervical epithelial cells (1) and in human papillomavirus 16 (immortalization human ectocervical epithelial cells) (34, 35). Treatment with epinephrine did not affect EGF receptor binding capacity or binding affinity (Fig. 6A). The positive control for the experiment was estrogen, whereby treatment of cells with 100 nM 17?-estradiol for 24 h increased binding capacity of the high-affinity EGF receptor sites to about 85 fmol/mg DNA (Fig. 6A) without affecting binding affinity.
FIG. 6. A, Scatchard analysis of [125I]EGF binding to CaSki cells and the effects of epinephrine and 17?-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].
Functionality of the EGF receptor was determined by incubating CaSki cells with the EGF receptor inhibitor AG1478. In cells treated for 24 h with 0.2 nM EGF, cotreatment with 5 μM AG1478 blocked EGF-increase in [3H]thymidine incorporation (Fig. 6B). Similarly, cotreatment with 10 μM of the MAPK/MEK inhibitor PD98059 blocked EGF-induced increase in [3H]thymidine incorporation (Fig. 6B). In contrast, cotreatment with 10 μM of the PI3K inhibitor wortmannin had no significant effect on [3H]thymidine incorporation compared with that observed in cells treated only with EGF (Fig. 6B). These data indicate that the EGF-induced increase in [3H]thymidine incorporation (and probably in cell number) is mediated by the EGF receptor and the MAPK/MEK pathway.
The results in Fig. 2, B and C, suggested to us that epinephrine inhibition of BzATP-induced apoptosis involves activation of adenylyl cyclase and protein kinase A, and therefore that epinephrine uses cAMP as the downstream messenger of the ?2-adrenoceptor. These data were used to dissect the mechanism by which EGF facilitates epinephrine action. Treatments with 2 nM epinephrine or with 1 μM zinterol increased cellular cAMP by about 2-fold, and cotreatment with 0.2 nM EGF augmented the responses to both epinephrine and zinterol (Fig. 7A). These data suggest that EGF exerts its effect at a level proximal to the adenylyl cyclase.
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]cyanopindolol. B, Effects on [125I]cyanopindolol internalization. C, Effects on [125I]cyanopindolol recycling. The experiments are described in the text.
The next set of experiments tested the hypothesis that EGF modulates plasma membrane levels of ?2-adrenoceptor. The experiments looked into effects of EGF on plasma membrane ?2-adrenoceptor steady-state. To test the degree of which EGF modulates ?2-adrenoceptor internalization, CaSki cells were incubated at 4 C with [125I]cyanopindolol, shifted to 37 C, and treated with zinterol. As shown in Fig. 7B and summarized in Table 2, pretreatment for 24 h with 0.2 nM EGF attenuated the time-related zinterol-induced decrease in the membrane-bound [125I]cyanopindolol. One explanation for this result is that EGF inhibits internalization of the ?2-adrenoceptor. An additional explanation is that EGF accelerates recycling of the ligated ?2-adrenoceptor receptor and stimulates its restoration into the plasma membrane. To study this question more directly, CaSki cells were labeled with [125I]cyanopindolol and treated with zinterol at 4 C, and then shifted to 37 C. As shown in Fig. 7C and summarized in Table 2, the increase in the membrane-bound [125I]cyanopindolol was faster in cells pretreated with EGF than in control cells. Collectively, the data in Fig. 7, B and C, and in Table 2 suggest that treatment with EGF increases plasma membrane ?2-adrenoceptor levels, possibly by attenuating receptor internalization and facilitating recycling of the receptor.
TABLE 2. Modulation of EGF effects by wortmannin a and PD98059
To better understand the mechanism of EGF modulation of plasma membrane ?2-adrenoceptor levels, the above experiments were repeated in the presence of PD98059 or wortmannin. Cotreatment with PD98059 had no significant effect on EGF modulation of plasma membrane ?2-adrenoceptor steady-state (Table 2). Treatment with wortmannin alone had no significant effect on the internalization and recycling of the membrane-bound [125I]cyanopindolol or on DNA solubilization (not shown). Cotreatment with EGF plus wortmannin also had no significant effect on the EGF-induced facilitated ?2-adrenoceptor recycling (Table 2). In contrast, coincubation with wortmannin inhibited the EGF-induced attenuation of ?2-adrenoceptor internalization and the EGF-induced decrease in DNA solubilization (Table 2).
Discussion
The present study reports novel data about epinephrine- and EGF-regulation of apoptosis in human cervical epithelial cells. We discovered that EGF facilitates ?2-adrenoceptor-dependent inhibition of P2X7 receptor-mediated apoptosis. EGF mechanism of action involves up-regulation of plasma membrane ?2-adrenoceptor levels by acceleration of ?2-adrenoceptor recycling, and by PI3K-depedent inhibition of ?2-adrenoceptor internalization. ?2-Adrenoceptor-dependent modulation of apoptosis has been previously described, but effects vary and depend on cell type and on the experimental conditions. Previous studies in cardiac myocytes reported that ?2-adrenoceptors exert antiapoptotic effects via an effect mediated by the caspase 9 mitochondrial pathway (36, 37, 38). These data could be relevant to the present study because, in human cervical epithelial cells, activation of ?2-adrenoceptors blocks P2X7 receptor-dependent apoptosis (present results), which also uses a caspase 9 mitochondrial pathway (4).
In human cervical epithelial cells, activation of the P2X7 receptor depended on membrane expression of the glycosylated 85-kDa form (Fig. 5), which is the mature and functional form of the P2X7 receptor protein. Previous groups described the expression and function of the glycosylated P2X1 and P2X2 receptors (39, 40, 41), as well as the P2X4 (42) and P2X6 receptors (43). Our novel data suggest that membrane expression and functionality of the P2X7 receptor also depend on glycosylation of the receptor, because receptor deglycosylation was detrimental for receptor function and pore formation. The P2X7 receptor possesses five putative N-glycosylation sites: Asn187, Asn202, Asn213, Asn241, and Asn284 (29); but at present, little is known about the role of the attached N-glycans for the expression and function of the P2X7 receptor. The present data suggest that epinephrine, in an action mediated by ?2-adrenoceptor activation of protein kinase A, stimulates deglycosylation of the P2X7 receptor and receptor degradation. The molecular mechanism of this novel protein kinase A effect is unknown. One possibility is an effect at the level of the endoplasmic reticulum/Golgi, namely inhibition of receptor processing trafficking and membrane expression. Another explanation is an effect at the level of the plasma membrane, i.e. enhanced degradation of previously expressed receptor.
The effect of epinephrine was mediated by the GPCR ?2-adrenoceptor. The expression of ?2-adrenoceptors in uterine cervical and vaginal cells was previously reported, both in the myometrial and uterine smooth muscle cells (44, 45), as well as in uterine epithelial cells. Tolszczuk and Pelletier (46) described the expression of ?2-adrenoceptors in rat endometrial cells. Whitaker et al. (47) showed intense labeling of [125I]cyanopindolol over glands and surface columnar epithelium of the human cervix, and moderate labeling over the squamous cervical/vaginal epithelium, and surrounding the basal layer. In females, plasma levels of epinephrine range from 0.1–3 nM and change relatively little throughout life (14). In addition, plasma levels of epinephrine are within the range of the Kd of the cervical ?2-adrenoceptor (Fig. 2A). Collectively, these data suggest that epinephrine tonically exerts antiapoptotic effect in the cervix in vivo.
We also found that EGF facilitated epinephrine antiapoptotic effect. EGF is a multifunctional factor whose actions are mediated by a RTK linked to multiple downstream cascades such as protein kinase C, PI3K, JNK, and the MAPK signaling pathways (recently reviewed in Ref. 48). All cascades have been described in human cervical cells, and the MAPK signaling pathway is usually associated with mitogenic properties of EGF (Ref. 1 and Fig. 1B). Interactions between EGF-induced signals and GPCR signaling pathways have been reported (49, 50, 51, 52, 53, 54). In some systems, the early convergence of signals originating from GPCRs and RTKs reflects GPCR-mediated transactivation of an RTK (55). However, only limited observations have been related to effects of EGF on adenylyl cyclase activation via ?-adrenergic or other stimulatory GPCRs (56). EGF can increase transcription of ?2-adrenoceptor (57) and cause rapid changes of adenylyl cyclase activities in a variety of in vitro systems (50, 58). The present findings are novel, in that EGF blocked ?2-adrenoceptor internalization via a PI3K-dependent mechanism and facilitated receptor recycling via a PI3K-independent mechanism. The molecular mechanisms of EGF action are, at present, unknown; but based on studies in other systems, they could involve modulation of signaling along the classical GPCR cascade of endocytosis, resensitization, recycling, and degradation (59).
Based on these findings, our hypothesis is summarized in Fig. 8, and it depicts that, in the cervix, EGF has a dual role regarding the regulation of growth of cervical epithelial cells. Acting via the MAPK cascade pathway, EGF stimulates mitosis and proliferation, and it is probably the mediator of estrogen effect (11). EGF also facilitates ?2-adrenoceptor-dependent stimulation of deglycosylation and degradation of the P2X7 receptor. EGF up-regulates plasma membrane ?2-adrenoceptor levels via two possible mechanisms: facilitated recycling of the ?2-adrenoceptor, and attenuation of ?2-adrenoceptor internalization by a PI3K-dependent mechanism. These effects of EGF would tend to increase the pool of the receptor that is available for activation upon ligand binding. Our novel findings underscore a new signaling network that could be a general paradigm for functional communication among RTKs, GPCRs, and the purinergic P2X7 receptor.
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 ?2-adrenoceptor-dependent stimulation of deglycosylation and degradation of the P2X7 receptor. The effect of EGF involves attenuation of ?2-adrenoceptor internalization by a PI3K-dependent mechanism and facilitated recycling of the ?2-adrenoceptor by a PI3K-independent mechanism. The EGF effects on ?2-adrenoceptor endocytosis and recycling would tend to up-regulate ?2-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.
The present results are physiologically relevant because human cervical epithelial cells express the EGF receptor (Ref. 1 and Fig. 6A) and the ?2-adrenoceptor (Ref. 47 and Fig. 2A). Moreover, in women, plasma EGF concentrations range from 0.1–5 nM (15, 16) and are within the range of EGF binding by the EGF receptor (6A). Furthermore, in the cervix, EGF is released from stromal cells on which cells of the basal layer of the cervical epithelium rest (60). We conclude that, in the cervix, the observed EGF effects of stimulated epithelial growth are the combination of EGF mitogenic and EGF antiapoptotic effects, and the latter are the result of EGF-dependent facilitation of ?2-adrenoceptor inhibition of P2X7 receptor-mediated apoptosis.
Acknowledgments
The technical support of Kim Frieden, Brian De-Santis, and Dipika Pal is acknowledged.
References
Jacobberger JW, Sizemore N, Gorodeski G, Rorke EA 1995 Transforming growth factor ? regulation of epidermal growth factor receptor in ectocervical epithelial cells. Exp Cell Res 220:390–396
Vijayalakshmi V, Gupta PD 1994 Estradiol-regulated transamidation of keratins by vaginal epithelial cell transglutaminase. Exp Cell Res 214:358–366
Mandelblatt J 1993 Squamous cell cancer of the cervix, immune senescence and HPV: is cervical cancer an age-related neoplasm? Adv Exp Med Biol 330:13–26
Wang Q, Wang L, Feng YH, Li X, Zeng R, Gorodeski GI 2004 P2X7-receptor-mediated apoptosis of human cervical epithelial cells. Am J Physiol Cell Physiol 287:C1349–C1358
Ellis HM, Yuan J, Horvitz HR 1991 Mechanisms and functions of cell death. Annu Rev Cell Biol 7:663–698
Ashkenazi A, Dixit VM 1998 Death receptors: signaling and modulation. Science 281:1305–1308
Gorodeski GI, Romero MF, Hopfer U, Rorke E, Utian WH, Eckert RL 1994 Human uterine cervical epithelial cells grown on permeable support—a new model for the study of differentiation and transepithelial transport. Differentiation 56:107–118
Sime A, McKellar Q, Nolan A 1997 Method for the growth of equine airway epithelial cells in culture. Res Vet Sci 62:30–33
Wells A 1999 EGF receptor. Int J Biochem Cell Biol 31:637–643
Katz MS 1988 Food restriction modulates ?-adrenergic-sensitive adenylate cyclase in rat liver during aging. Am J Physiol Endocrinol Metab 254:E54–E62
Gorodeski GI, Eckert RL, Utian WH, Rorke EA 1990 Maintenance of in vivo-like keratin expression, sex steroid responsiveness and estrogen receptor expression in cultured human ectocervical epithelial cells. Endocrinology 126:399–406
Gorodeski GI, Whittembury J 1998 A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters. Res Vet Sci 29:307–332
Haigler HT, Maxfield FR, Willingham MC, Pastan I 1980 Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB-3T3 cells. J Biol Chem 255:1239–1241
Engstr?m BE, Karlsson FA, Wide L 1999 Gender differences in diurnal growth hormone and epinephrine values in young adults during ambulation. Clin Chem 45:1235–1239
Lev-Ran A, Hwang DL 1990 Epidermal growth factor in blood and urine of athyreotic adults. Clin Physiol Biochem 8:184–187
Hoffmann TK, Ballo H, Braunstein S, Van Lierop A, Wagenmann M, Bier H 2001 Serum level and tissue expression of c-erbB-1 and c-erbB-2 proto-oncogene products in patients with squamous cell carcinoma of the head and neck. Oral Oncol 37:50–56
Nagata S, Goldstein P 1995 The Fas death factor. Science 267:1449–1456
Rath PC, Aggarwal BB 1999 TNF-induced signaling in apoptosis. J Clin Immunol 19:350–364
Liu X, Kim CN, Yang J, Jemmerson R, Wang X 1996 Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147–157
Takahashi A 1999 Caspase: executioner and undertaker of apoptosis. Int J Hematol 70:226–232
Ferrari D, Chiozzi P, Falzoni S, Dal Susino M, Melchiorri L, Baricordi OR, Di Virgilio F 1997 Extracellular ATP triggers IL-1? release by activating the purinergic P2Z receptor of human macrophages. J Immunol 159:1451–1458
Guerra AN, Fisette PL, Pfeiffer ZA, Quinchia-Rios BH, Prabhu U, Aga M, Denlinger LC, Guadarrama AG, Abozeid S, Sommer JA, Proctor RA, Bertics PJ 2003 Purinergic receptor regulation of LPS-induced signaling and pathophysiology. J Endotoxin Res 9:256–263
Humphreys BJ, Rice J, Kertesy SB, Dubyak GR 2000 Stress-activated protein kinase/JNK activation and apoptotic induction by the macrophage P2X7 nucleotide receptor. J Biol Chem 275:26792–26798
Ferrari D, Wesselborg S, Bauer MK, Schulze-Osthoff K 1997 Extracellular ATP activates transcription factor NF-B through the P2Z purinoreceptor by selectively targeting NF-B p65. J Cell Biol 139:1635–1643
Virginio C, MacKenzie A, North RA, Surprenant A 1999 Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor. J Physiol (Lond) 519:335–346
Denlinger LC, Sommer JA, Parker K, Gudipaty L, Fisette PL, Watters JW, Proctor RA, Dubyak GR, Bertics PJ 2003 Mutation of a dibasic amino acid motif within the C terminus of the P2X7 nucleotide receptor results in trafficking defects and impaired function. J Immunol 171:1304–1311
Kim M, Spelta V, Sim J, North RA, Surprenant A 2001 Differential assembly of rat purinergic P2X7 receptor in immune cells of the brain and periphery. J Biol Chem 276:23262–23267
Ralevic V, Burnstock G 1998 Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492
Surprenant A, Rassendren F, Kawashima E, North RA, Buell G 1996 The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272:735–738
Ramirez AN, Kunze DL 2001 P2X purinergic receptor channel expression and function in bovine aortic endothelium. Am J Physiol 282:H2106–H2116
Bianchi BR, Lynch KJ, Touma E, Niforatos W, Burgard EC, Alexander KM, Park HS, Yu H, Metzger R, Kowaluk E, Jarvis MF, van Biesen T 1999 Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol 376:127–138
Torres GE, Egan TM, Voigt MM 1999 Hetero-oligomeric assembly of P2X receptor subunits. J Biol Chem 274:6653–6659
Glass R, Burnstock G 2001 Immunohistochemical identification of cells expressing ATP-gated cation channels (P2X receptors) in the adult rat thyroid. J Anat 198:569–579
Sizemore N, Rorke EA 1993 Human papillomavirus 16 immortalization of normal human ectocervical epithelial cells alters retinoic acid regulation of cell growth and epidermal growth factor receptor expression. Cancer Res 53:4511–4517
Sizemore N, Choo CK, Eckert RL, Rorke EA 1998 Transcriptional regulation of the EGF receptor promoter by HPV16 and retinoic acid in human ectocervical epithelial cells. Exp Cell Res 244:349–356
Communal C, Singh K, Sawyer DB, Colucci WS 1999 Opposing effects of ?1- and ?2-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive G protein. Circulation 100:2210–2212
Zaugg M, Xu W, Lucchinetti E, Shafiq SA, Jamali NZ, Siddiqui MAQ 2000 ?-Adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes. Circulation 102:344–350
Singh K, Xiao L, Remondino A, Sawyer DB, Colucci WS 2001 Adrenergic regulation of cardiac myocyte apoptosis. J Cell Physiol 189:257–265
Newbolt A, Stoop R, Virginio C, Surprenant A, North RA, Buell G, Rassendren F 1998 Membrane topology of an ATP-gated ion channel (P2X receptor). J Biol Chem 273:15177–15182
Torres GE, Egan TM, Voigt MM 1998 N-Linked glycosylation is essential for the functional expression of the recombinant P2X2 receptor. Biochemistry 37:14845–14851
Rettinger J, Aschrafi A, Schmalzing G 2000 Roles of individual N-glycans for ATP potency and expression of the rat P2X1 receptor. J Biol Chem 275:33542–33547
Hu B, Senkler C, Yang A, Soto F, Liang BT 2002 P2X4 receptor is a glycosylated cardiac receptor mediating a positive inotropic response to ATP. J Biol Chem 277:15752–15757
Jones CA, Vial C, Sellers LA, Humphrey PP, Evans RJ, Chessell IP 2004 Functional regulation of P2X6 receptors by N-linked glycosylation: identification of a novel?-methylene ATP-sensitive phenotype. Mol Pharmacol 65:979–985
Bryman I, Norstrom A, Lindblom B 1986 Influence of prostaglandins and adrenoceptor agonists on contractile activity in the human cervix at term. Obstet Gynecol 67:574–578
Kovacs L, Falkay G 1993 Changes in adrenergic receptors in the pregnant human uterine cervix following mifepristone or placebo treatment in the first trimester. Hum Reprod 8:119–121
Tolszczuk M, Pelletier G 1988 Autoradiographic localization of ?-adrenoreceptors in rat uterus. J Histochem Cytochem 36:1475–1479
Whitaker EM, Nimmo AJ, Morrison JF, Griffin NR, Wells M 1989 The distribution of ?-adrenoceptors in human cervix. Q J Exp Physiol 74:573–576
Carpenter G 2000 The EGF receptor: a nexus for trafficking and signaling. Bioessays 22:697–707
Gutierrez GE, Mundy GR, Derynck R, Hewlett EL, Katz MS 1987 Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factorand epidermal growth factor. J Biol Chem 262:15845–15850
Ramirez I, Tebar F, Grau M, Soley M 1995 Role of heterotrimeric G-proteins in epidermal growth factor signaling. Cell Signal 7:303–311
del Carmen Medina L, Vazquez-Prado J, Garcia-Sainz JA 2000 Cross-talk between receptors with intrinsic tyrosine kinase activity and 1b-adrenoceptors. Biochem J 350:413–419
Gschwind A, Zwick E, Prenzel N, Leserer M, Ullrich A 2001 Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene 20:1594–1600
Pierce KL, Luttrell LM, Lefkowitz RJ 2001 New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades. Oncogene 20:1532–1539
Saito Y, Berk BC 2001 Transactivation: a novel signaling pathway from angiotensin II to tyrosine kinase receptors. J Mol Cell Cardiol 33:3–7
Maudsley S, Pierce KL, Zamah M, Miller WE, Ahn S, Daakai Y, Lefkowitz RJ, Luttrell LM 2000 The ?2-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem 275:9572–9580
Knecht M, Catt KJ 1983 Modulation of cAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 258:2789–2794
Yeh CK, Hymer TK, Sousa AL, Zhang BX, Lifschitz MD, Katz MS 2003 Epidermal growth factor up-regulates ?-adrenergic receptor signaling in a human salivary cell line. Am J Physiol Cell Physiol 284:C1164–C1175
Nakagawa Y, Gammichia J, Purushotham KR, Schneyer CA, Humphreys-Beher MG 1991 Epidermal growth factor activation of rat parotid gland adenylate cyclase and mediation by a GTP-binding regulatory protein. Biochem Pharmacol 42:2333–2340
Ferguson SSG 2001 Evolving concepts in GPCR endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24
Hom YK, Young P, Wiesen JF, Miettinen PJ, Derynck R, Werb Z, Cunha GR 1998 Uterine and vaginal organ growth requires epidermal growth factor receptor signaling from stroma. Endocrinology 139:913–921(Liqin Wang, Ying-Hong Fen)
Address all correspondence and requests for reprints to: George I. Gorodeski, M.D., Ph.D., University MacDonald Women’s Hospital, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: gig@cwru.edu.
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 ?2-adrenoceptor, and the epinephrine antiapoptotic effect could be mimicked by ?2-adrenoceptor agonists and by activators of adenylyl cyclase. Likewise, the effect could be blocked by ?2-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 ?2-adrenoceptor. In contrast, EGF, acting via the EGF receptor, augmented ?2-adrenoceptor recycling, and it inhibited ?2-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 ?2-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.
Introduction
PROPER FUNCTION OF the epithelia of the female genital tract depends on coordinated growth and differentiation of the epithelial cells, and deviations from these well-controlled functions can cause infertility and lead to dysplasia and cancer. The uterine cervical epithelium is maintained by a balance between proliferation of the basal layer of cells, and death of cells in upper layers. Our current state of knowledge suggests three levels of growth-control of cervical epithelial cells: proliferation, controlled by mitogenic signals [e.g. estrogen and epidermal growth factor (EGF)] vs. growth inhibitory factors (e.g. TGF?) (1); terminal differentiation, controlled mainly by estrogen (2); and senescence of cells evading growth control and terminal differentiation due to the erosion of telomeres (3). Recently it was reported that growth of human cervical epithelial cells is also regulated by apoptosis; the effect involves predominantly the P2X7 receptor as its proximal signaling arm, and it uses the mitochondrial apoptotic pathway (4). However, relatively little is known about regulation of these effects and the mechanisms involved. Because apoptosis functions to eliminate abnormal cells (5) and dysregulation of apoptotic cell-death has been implicated in the premature death of cells, loss of tissue, aging, states of disease, and neoplastic transformation (6), elucidation of these data could be important for our understanding of cervical cell biology and tumorigenesis.
Both EGF (7) and epinephrine (8) are added routinely to cultures of squamous epithelial cells to enhance cell-growth. EGF and members of the EGF receptor family influence cell survival predominantly by stimulating proliferation (9), including of human cervical epithelial cells (1). Until recently, no mechanistic explanation was given for the growth-promoting effect of epinephrine. Recently we found that epinephrine increases the number of human cervical epithelial cells in culture, and that EGF potentiates the epinephrine effect. Because epinephrine, in contrast to EGF alone, inhibited apoptosis of cervical cells, we hypothesized that EGF, in addition to its mitogenic role, also potentiates epinephrine antiapoptotic effect.
The specific objective of the present study was to understand the mechanisms of EGF and epinephrine antiapoptotic effects. The results suggest that the epinephrine effect is mediated by ?2-adrenoceptor-dependent inhibition of P2X7 receptor pore formation. The results also suggest a dual role for EGF: as mitogen, using the MAPK/MAPK kinase (MAPK/MEK) signaling pathway, and as facilitator of epinephrine effect. We also found that the effect of EGF involves facilitated, phosphoinositide 3-kinase (PI3K)-dependent inhibition of ?2-adrenoceptor internalization, and facilitated ?2-adrenoceptor recycling, thereby increasing the level of ?2-adrenoceptors in the plasma membrane. These findings underscore a novel signaling network that could be a general paradigm for functional communication between the receptor tyrosine kinases (RTKs), the G protein-coupled receptors (GPCRs), and the purinergic P2X7 receptor.
Materials and Methods
Cell cultures
The experiments used CaSki cells, a line of transformed cervical epithelial cells that were obtained from the ATCC (Manassas, VA) and were previously characterized as stably expressing phenotypic characteristics of human cervical epithelial cells (7). Cells were grown and subcultured in RPMI-1640 supplemented with 8% fetal calf serum, 0.2% NaHCO3, nonessential amino acids (0.1 mM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and gentamycin (50 μg/ml) at 37 C in a 91%O2-9%CO2 humidified incubator. Cultures were routinely tested for mycoplasma. Preliminary experiments were conducted on cells plated on culture plates, and definitive experiments were repeated on cells plated on filters. The latter method improves culturing conditions and promotes epithelial cell polarization and differentiation (7). Filters were either Anocell (Anocell-10, Oxon, UK, obtained through Sigma Chemicals, St. Louis, MO), Transwell (Costar Corporation, Cambridge, MA), or Millicell-CM (Millipore, Bedford, MA). Filters were coated on their upper (luminal) surface with 3–5 μg/cm2 collagen type IV and incubated at 37 C overnight. The remaining collagen solution was aspirated, and the filter was dried at 37 C. Before plating, both sides of the filters were rinsed three times with Hanks’ balanced salt solution (HBSS). Cells were plated on the upper surface of the filter at 3 x 105 cells/cm2. By plating at this high density, the cultures become confluent and polarized within 12 h after plating (7). All solutions and agents were added to both the luminal and subluminal bathing solutions.
DNA synthesis
DNA synthesis was measured by [3H]thymidine incorporation. Six hours before assays, cells attached on filters were labeled for 4 h with 1 μM methyl-[3H]-thymidine (1 μCi/ml; specific activity, 87 Ci/mmol; Amersham, Piscataway, NJ) and maintained for 2 additional hours in medium devoid of radioactive thymidine. After labeling, the cells were washed several times in ice-cold PBS, lysed in 200 μl 0.1 N NaOH, and precipitated in 2 ml ice-cold 10% trichloroacetic acid for 2 d at 4 C. The radioactivity [dpm/mg protein, determined by Bio-Rad (Hercules, CA) Protein Assay solution] of triplicated samples was determined by ?-scintillation counting (LS1801 scintillation counter; Beckman, Fullerton, CA).
DNA solubilization assay
Twenty-four hours before the end of the experiment, cells were labeled with [3H]thymidine (specific activity, 93 Ci/mmol; 5 μCi/1 x 106 cells) for 16 h. The medium was removed, and cells were washed three times with fresh medium lacking [3H]thymidine and were incubated in the same medium for an additional 6 h. At the end of incubation, the supernatant was stored, and cells were lysed in 0.5 ml lysis buffer (10 mM Tris-HCl, pH 7.5; plus 1 mM EDTA–0.2% Triton X-100) for 1 h at 4 C. The intact chromatin was separated from the fragmented DNA by 5 min centrifugation at 4 C in Eppendorf microcentrifuge at 12,000 x g. The supernatant (lysate) was stored, while the pellet was resuspended in 0.5 ml of 1% sodium dodecyl sulfate (SDS). The radioactivity contained in the supernatants, lysates, and pellets was counted in a scintillation counter, and DNA fragmentation was calculated as: [% solubilized DNA] = ([cpm supernatant + cpm lysates]/[cpm supernatant + cpm lysate + cpm pellet]) x 100.
Binding studies
Binding studies of the adrenoceptors and the EGF receptor used a similar method. Two hundred fifty microliters of binding medium (serum-free DMEM, 0.1% BSA, 10 mM HEPES) containing 10–1000 pM of the radioligand were added to the apical container of the filter, and 500 μl of the same solution to the basolateral container; parallel tubes ontained an additional 200-fold excess radioinert ligand. The cells were then incubated at 4 C for 3 h; at the completion of incubations cells were washed three times with ice-cold HBSS containing 0.1% BSA. Cells were solubilized in 0.2 N NaOH, and the bound ligand was separated on glass fiber filters (Whatman, GF/C, Tonawanda, NY) by vacuum filtration. The associated radioligand was counted either in a ?-scintillation counter (for [3H] associated ligands) or in a -counter (for [125I] associated ligands). The following radioligands were used: [3H]prazosin and [3H]tamsulosin (-adrenoceptor radioligand) and [125I]cyanopindolol [?-adrenoceptor radioligand (10)] (all at 1 μCi/ml; specific activity, about 90 Ci/mmol; Amersham), and [125I]EGF (5 μCi/ml; specific activity, about 100 Ci/mmol; Biomedical Technology, Stoughton, MA). The - and ?2-adrenoceptor radioinert ligands were phenylephrine and zinterol (Sigma Chemicals). Human recombinant EGF was from R&D Systems (Minneapolis, MN). Specific receptor binding was expressed per milligram DNA, and data were analyzed by Scatchard plot as described (11).
Fluorescence changes
Determinations of fluorescence changes in cells attached on filters were published (12). For measurements of cytosolic calcium, cells on filters were incubated with 7 μM fura-2/acetoxymethyl ester plus 0.25% pluronic F12 (12). Measurements of fluorescence were conducted in a custom-designed fluorescence chamber (12). In this apparatus, a filter with cells is placed horizontally in an enclosed dark chamber maintained at a fixed temperature, under conditions that permit selective perfusion of the luminal (0.2 ml) and subluminal compartments (0.5 ml) at rates of 1–1.5 ml/min. Cells were illuminated over the apical surface, and the intensity of the emitted light from the apical surface was measured. Agents were added to the luminal and subluminal solutions, and changes in cytosolic calcium were determined by switching the excitation filters to record the maximal (340/510 nm excitation/emission) and minimal (380/510 nm) fluorescence. Levels of cytosolic calcium were determined by the formula [Ca2+]i (nM) = [(R – Rmin)/(Rmax – R)]·Kd·(Sf2/Sb2), where [Ca2+]i is the level of cytosolic calcium, R is the ratio of fluorescence excitation measurements at 340 to 380 nm, Rmin and Rmax are the experimentally determined minimum and maximum calcium measurement ratios, 340 nm to 380 nm respectively, Kd is the dissociation constant for fura-2 (224 nM), and Sf2/Sb2 is the ratio of fluorescence value at 380 nm excitation determined at Rmin (0 calcium) and Rmax (maximal calcium). Maximal calcium fluorescence was obtained by adding 10 μM ionomycin in the presence of 10 mM CaCl2, and minimal calcium fluorescence was obtained by competing calcium from fura-2 with 2.5 mM MnCl2. This method was also used for experiments with the nuclear stain ethidium bromide (molecular mass, 394 Da). Ethidium bromide was added to the perfusing solutions from a concentrated stock (x100) at a final concentration of 5 μM. Upon influx into cells, the dye binds to nuclear chromatin and elicits specific fluorescence. Changes in fluorescence were measured on-line at wavelengths 518/605 nm (excitation/emission).
Western blot analysis
The postnuclear supernatant of cells was solubilized in lysis buffer (50 mM Tris-HCl, pH 6.8; 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate; 5 mM EDTA, pH 8.0) containing 50 μg/ml phenylmethylsulfonylfluoride, 10 μg/ml benzamidine, 10 μg/ml bacitracine, 10 μg/ml leupeptin, and 2 μg/ml aprotinine. Aliquots (about 45 μl), normalized to 15 μg protein, were loaded on 10% polyacrylamide SDS gel, and vertical electrophoresis was conducted at 50 mA for 1.5 h. Gels were transferred onto Immobilon membrane (Millipore) at 200 V for 1.5 h, membranes were blocked in 5% milk, and receptor polypeptides were visualized using 1.5 μg/ml rabbit anti-P2X7 antibody at 4 C overnight. Membranes were washed three times in PBS and fluorescent stained for 1 min using an enhanced chemiluminescence kit of antirabbit peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). For protein deglycosylation, 20 μg total lysate proteins containing 1% SDS was denatured by heating for 5 min at 100 C. The mixture was incubated overnight at room temperature after addition of 1 U fresh N-glycosidase F (GlycoTech, Rockville, MD) in 20 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 50 mM NaCl. Samples were analyzed by Western blot as described above.
Cellular cAMP
Cellular cAMP was assayed in total homogenates of cells attached on filters. For assays, cells were incubated in HEPES-buffered Krebs-Ringer’s bicarbonate buffer (pH 7.4) containing 1 mg/ml BSA and 2.5 mM glucose (Krebs bicarbonate Ringer) at 37 C for 30 min. The reactions were terminated by adding 12% trichloroacetic acid (1:1 vol/vol). Cells were scraped off the filters and centrifuged at 3,000 x g for 15 min. Supernatants were extracted with 2 ml water-saturated diethylether three times. The concentration of cAMP in the aqueous phase was determined in duplicate by RIA using a commercially available kit (Amersham).
?2-Adrenoceptor internalization (endocytosis) assays
Fifteen minutes before assays, cells were shifted to ice-cold medium and labeled with 1 μCi/ml [125I]cyanopindolol for 30 min at 4 C. After three washes with ice-cold medium lacking the radioligand, cells were shifted to fresh, warm (37 C) medium and treated with 1 μM zinterol. At time intervals of 0, 15, 30, and 60 min, cells were washed with ice-cold medium. Surface-bound and internalized ?2-adrenoceptors were distinguished according to Haigler et al. (13), with some modifications, as we have described (1). Briefly, cells were washed three times with ice-cold HBSS to remove unbound radioactivity. Cultures were then incubated for 10 min at 4 C in 0.2 M acetic acid plus 0.5 M NaCl (pH 2.5) (250 and 500 μl, respectively, in the apical and basolateral containers of the filter). The filters were then rinsed with 250 and 500 μl, respectively, of the same solution, and the washes (defined as the surface bound fraction) were collected. The remaining cell-associated radioactivity was removed by incubating the cells for 60 min at 37 C with 1 N NaOH. The percent membrane bound [125I]cyanopindolol was determined relative to membrane bound [125I]cyanopindolol in cells not exposed to zinterol.
?2-Adrenoceptor recycling assays
Fifteen min before assays, cells were shifted to ice-cold medium and labeled with 1 μCi/ml [125I]cyanopindolol for 30 min at 4 C. After three washes with ice-cold medium lacking the radioligand, cells were incubated at 4 C for 15 min with 1 μM zinterol, and then shifted to 37 C and incubated for an additional 60 min in the same medium. At the completion of incubation, cells were washed three times at 37 C with the same medium, lacking zinterol, to remove unbound zinterol, and internalized processed zinterol was released into the medium. After the wash, cells were incubated further at 37 C for 0, 0.5, 3, 4, 8, 16, and 24 h, and the amount of membrane bound [125I]cyanopindolol was determined as above. Data about recycled receptor are presented in terms of percent control bound [125I]cyanopindolol (compared with cells not exposed to zinterol).
Densitometry
Densitometry was done using AGFA Arcus II scanner (AGFA, New York, NY) and Un-Scan-It gel automated digital software (Silk Scientific, Orem, OR).
Statistical analysis of the data
Data are presented as means (±SD), and significance of differences among means was estimated by Student’s t test. Trends were calculated using GB-STAT V5.3, 1995 (Dynamic Microsystems Inc., Silver Spring, MD) and analyzed with ANOVA.
Chemicals and supplies
Ethidium bromide and fura-2/AM were obtained from Molecular Probes (Eugene, OR). Benzyloxy-valine-alanine-aspartate-O-methyl-fluoromethylketone (zVAD-FMK) was from Calbiochem (La Jolla, CA). All other chemicals, unless specified otherwise, were obtained from Sigma Chemicals. The anti-P2X7 receptor pAb was from Alomone Laboratories (Jerusalem, Israel).
Results
Effects of epinephrine and EGF on proliferation and apoptosis of cervical cells
Near-confluent cultures of CaSki cells on filters were shifted for 14 h to serum-free medium supplemented with 1% BSA and were treated in the same medium with 0.2 nM EGF, 2 nM epinephrine, or both for an additional 24 h. These concentrations were chosen because they are within the physiological range in vivo (14, 15, 16). At the completion of treatments, cells were harvested off the filters and counted. Treatments with EGF or with epinephrine alone increased the number of cells in culture (Fig. 1A). However, treatment with EGF plus epinephrine increased cell number more than did each drug alone (Fig. 1A), suggesting that EGF facilitates the effect of epinephrine.
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).
To better understand the effects of EGF and epinephrine on the growth of cervical cells, two additional experiments were done. First, the effects of EGF and epinephrine on proliferation were determined in terms of DNA synthesis. EGF, but not epinephrine, increased [3H]thymidine incorporation (Fig. 1B), confirming the mitogenic properties of EGF in human cervical epithelial cells (1). In the second experiment, effects of EGF and epinephrine on apoptosis were determined in terms of modulation of changes in DNA solubilization induced by the P2X7 receptor-specific agonist 2',3'-0-(4-benzoylbenzoyl)-ATP (BzATP). It was recently shown that human cervical epithelial cells undergo apoptosis constitutively and that the effect involves the purinergic P2X7 receptor mechanism (4). Because the degree of constitutive apoptosis is low and does not allow for experimental manipulations, BzATP was added to increase the apoptosis. As is shown in Fig. 1C, treatment with 100 μM BzATP for 9 h increased DNA solubilization significantly from the baseline level of 1.9% to 7.8%. Pretreatment for 24 h with 0.2 nM EGF alone had little effect on the BzATP-induced apoptosis (Fig. 1C). In contrast, pretreatment for 24 h with 2 nM epinephrine decreased DNA solubilization to 4.6%, and cotreatment with EGF facilitated the epinephrine effect (2.9%). The control for this experiment was zVAD-FMK, a pan-caspase inhibitor that was added 30 min before assays at a concentration of 50 μM. Treatment with zVAD-FMK decreased the BzATP-induced DNA solubilization to levels that were lower than baseline apoptosis (about 1.4%, Fig. 1C). Collectively, the data in Fig. 1 indicate that epinephrine increases cell number by attenuation of apoptosis, whereas EGF has a dual role: as mitogen, and as facilitator of epinephrine antiapoptotic effect.
Mechanism of epinephrine effect
Involvement of the ?2-adrenoceptor mechanism.
The low concentration of epinephrine that inhibited apoptosis (2 nM) suggested an effect mediated by adrenergic receptor(s). Epinephrine is a relatively nonspecific ?-adrenergic agonist with some -adrenergic potential. To understand the mechanism of epinephrine effect, three experiments were done. First, cells were tested for the expression of - and ?-adrenoceptors. Binding assays revealed no specific binding of the -adrenoceptor radioligands [3H]prazosin or [3H]tamsulosin (not shown), ruling out expression of functional -adrenoceptors. In contrast, CaSki cells bound, with high affinity, the ?-adrenoceptor radioligand [125I]cyanopindolol (Fig. 2A, Kd0.3 nM), and the binding activity was about 15 fmol/mg DNA (10,000 receptors/cell). The radiolabel could be displaced by coincubation with the ?2-adrenoceptor ligands zinterol and terbutaline (Fig. 2A) but not with the ?1-adrenoceptor ligand isoproterenol (not shown). These data indicate that CaSki cells express the ?2-adrenoceptor.
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 ?2-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).
The second and third experiments tested the degree of which activation and inhibition, respectively, of ?2-adrenoceptor modulate BzATP-induced apoptosis. Pretreatment for 24 h with either zinterol or terbutaline (1 μM) inhibited BzATP-induced apoptosis (Fig. 2B). Similarly, pretreatment for 14 h with 10 μM forskolin [in the presence of 0.5 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, to activate directly adenylyl cyclase] also inhibited BzATP-induced apoptosis (Fig. 2B). In contrast, pretreatments with 10 μM of the ?1-adrenoceptor ligand isoproterenol or with 100 μM of the 1-adrenoceptor ligand phenylephrine had no effect on BzATP-induced apoptosis (Fig. 2B).
Cotreatment for 24 h with either 10 μM propranolol (?-adrenoceptor antagonist), 100 nM ICI 118,551 (?2-adrenoceptor antagonist), or 30 μM of H-89 dihydrochloride (protein kinase A inhibitor) blocked the zinterol (1 μM)-induced inhibition of BzATP-induced apoptosis (Fig. 2C). Collectively, the data in Fig. 2 indicate that epinephrine-dependent attenuation of BzATP-induced apoptosis is mediated by the ?2-adrenoceptor. The data also suggest that the effect involves cAMP and protein kinase A as downstream mediators.
Modulation of the mitochondrial pathway.
Apoptosis can be induced by the cell-surface receptor and/or mitochondrial pathways. The former usually involves the Fas and TNF mechanisms followed by activation of caspase-8 (6, 17, 18). The mitochondrial pathway involves the release of cytochrome-c (19) and activation of caspase-9 (20). The cell-surface receptor and mitochondrial-dependent pathways integrate at the level of the effector caspases, so that activation of caspases 9 and 8 triggers the nonreversible execution of apoptosis by caspases 6, 7, and 3 (5, 6). In CaSki cells, apoptosis induced by ligation of the P2X7 receptor mechanism involves predominantly the mitochondrial pathway (4). This knowledge was used to dissect the effects of epinephrine on BzATP-induced apoptosis.
CaSki cells were treated with either 100 μM BzATP for 9 h or with 10 μM TNF for 14 h, and effects on DNA solubilization were determined. These concentrations produce a submaximal degree of apoptosis in human cervical epithelial cells, and their effects involve, respectively, the cell-surface receptor and mitochondrial pathways (4). Either of these two agents significantly increased DNA solubilization (Fig. 3), confirming our previous results (4) and the results in Figs. 1 and 2 regarding BzATP effects. Pretreatment for 24 h with 1 μM zinterol inhibited BzATP-induced increase in DNA solubilization, but it had no effect on the increase in DNA solubilization induced by TNF (Fig. 3A). Pretreatment for 24 h with 0.2 nM EGF alone had no significant effect on BzATP- or TNF-induced increase in DNA solubilization (Fig. 3B).
FIG. 3. Effects of zinterol (A) and EGF (B) on DNA solubilization induced by BzATP and by TNF. Data are means (±SD) of three independent experiments. *, P < 0.01 (BzATP group, Zinterol vs. Baseline).
Inhibition of P2X7 receptor pore formation.
One of the explanations for the results in Fig. 3 is that the ?2-adrenoceptor-dependent antiapoptotic signaling pathway interacts with proximal steps of the P2X7 receptor signaling cascade, possibly at the level of the P2X7 receptor. Activation of the P2X7 receptor can induce apoptosis by a number of known mechanisms, including IL-1? (21), TNF-TNF-related apoptosis-inducing ligand (22), and the p38, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (23) and NF-B pathways (24). However, the P2X7 receptor is unique in its ability to form pores in the plasma membrane in the continued presence of the ligand (25). Plasma membrane pore formation depends on the long C terminus of the receptor (26), and oligomerization of neighboring molecules is believed to cause progressive dilatation of the pore to a diameter of approximately 4 nm and an increase in the permeation path to molecules of molecular mass of 400–900 Da (27). In its final size, the pore is relatively permeable to Ca2+, but it remains selective to other cations and is impermeable to anions (28). Uncontrolled influx of Ca2+ and the sustained increases in cytosolic calcium are believed to trigger the apoptotic mitochondrial pathway (28). The objective of the following experiments was to test the hypothesis that epinephrine inhibits apoptosis by blocking P2X7 receptor pore formation.
The first experiment tested the degree of which pretreatment with epinephrine blocks BzATP-induced Ca2+ influx. We used fura-2-loaded CaSki cells attached on filters housed in a fluorescence chamber as described in Materials and Methods and in our previous publication (12). Under these conditions, treatment with BzATP induced 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62)-sensitive, time-dependent sustained Ca2+ influx that began about 10 min after adding the ligand (Fig. 4A). EGF alone had no significant effect, and pretreatment with epinephrine attenuated the BzATP-induced increase in cytosolic calcium (inset, Fig. 4A). Pretreatment with epinephrine plus EGF attenuated the BzATP-induced increase in cytosolic calcium to a degree that was observed in cells treated with the P2X7 receptor modifier KN-62 (inset, Fig. 4A).
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).
The second experiment used the same design, but it tested more directly P2X7 receptor pore formation. Cells attached on filters were treated with BzATP in the presence of 5 μM ethidium bromide (molecular mass, 394 Da). The rationale was that, in cells with intact plasma membrane, influx of the dye is minimal. As is shown in Fig. 4B, under baseline conditions, there were no changes in fluorescence at 518/605 nm (excitation/emission), indicating no influx of ethidium bromide. Treatment with BzATP resulted in time-related increase in the fluorescence (Fig. 4B), indicating nuclear binding of ethidium bromide and suggesting that the nucleotide induced influx of ethidium bromide. The increase in fluorescence began about 10 min after adding the agonist, and it correlated with the sustained increase in cytosolic calcium (Fig. 4, A and B). Similar to the effect on cytosolic calcium, the BzATP-induced influx of ethidium bromide could be blocked by pretreatment with KN-62 (inset, Fig. 4B). Pretreatment with EGF alone had no significant effect; pretreatment with epinephrine attenuated the BzATP-induced influx of ethidium bromide, and pretreatment with epinephrine plus EGF inhibited the BzATP-induced influx of ethidium bromide, similar to the effect of KN-62 (Fig. 4B and inset). Collectively, the results in Fig. 4 suggest that epinephrine inhibited ligation-induced P2X7 receptor pore formation; EGF alone had no significant effect, but it facilitated the effect of epinephrine.
Modulation of P2X7 receptor expression.
The objective of the experiments in this section was to determine epinephrine effect on the expression of the P2X7 receptor. Expression of the P2X7 receptor protein was analyzed using the Alomone polyclonal rabbit anti-P2X7 receptor antibody as the primary antibody. This antibody was raised against the purified peptide (C)KIRK EFPKT QGQYS GFKYP Y (corresponding to residues 576–595 of rat P2X7 with an additional N-terminal cysteine (29), and our data (see below) and data by others (30) support its usefulness for the objectives of the present study.
Western immunoblot analysis of total homogenates from CaSki cells revealed specific reactivities to 85-kDa, 65-kDa, and 18-kDa forms that were abolished in the presence of the antigen used to raise the antibody (Fig. 5A). The 65-kDa is most likely the native form of the P2X7 receptor corresponding to the reported P2X7 receptor 70-kDa isoform, whereas the 18-kDa is a degradation product (28, 31, 32, 33). To better understand the role of the 85-kDa form, two additional experiments were done. First, the effects of length in culture on the responses to BzATP were compared with the cellular expression of the receptor. The levels of BzATP-induced Ca2+ influx and of BzATP-induced influx of ethidium bromide were greater in cells cultured for 6 d than in cells cultured for 2 d (Fig. 5B). Length in culture also affected the levels of the P2X7 receptor forms: in d-2 cells, the predominant form was the 65-kDa; in contrast, in d-6 cells, the levels of the 85-kDa and 18-kDa forms were greater than in d-2 cells (Fig. 5D, left lanes). These results suggest that the mature and functional form of the P2X7 receptor protein is the 85-kDa form. This hypothesis was further supported by the results shown in Fig. 5C. Incubation in vitro of cell lysates with N-glycosidase F showed a decrease in the density of the 85-kDa form along with an increase in the density of the 65-kDa form (Fig. 5C). Densitometry analysis of two experiments indicates that the ratio of 65-kDa/85-kDa increased more than 3-fold after treatment in vitro with N-glycosidase F. These data are in general agreement with previous studies in the field (29, 30, 32) and indicate that the 85-kDa form is the glycosylated form of the P2X7 receptor.
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.
Pretreatment of CaSki cells with EGF alone had only mild effect on the levels of the P2X7 receptor forms, increasing mainly the 18-kDa form in d-6 cells (Fig. 5 and Table 1). Pretreatment of d-2 cells with epinephrine decreased the 65-kDa form; whereas in d-6 cells, it decreased the 85-kDa and 65-kDa forms and increased the 18-kDa form (Fig. 5 and Table 1). Cotreatment of d-2 cells with EGF plus epinephrine increased the 18-kDa form 50-fold; cotreatment of d-6 cells decreased the 85-kDa form and increased the 18-kDa isoform 35-fold. In addition, in d-6 cells, treatment with epinephrine plus EGF resulted in the appearance of an intermediate form of 75-kDa (Fig. 5, Table 1). Collectively, the data in Fig. 5 and Table 1 indicate that EGF facilitates epinephrine-induced decrease of the glycosylated 85-kDa form of the P2X7 receptor, and of P2X7 receptor degradation. The data also suggest that EGF, acting in concert with epinephrine, induces deglycosylation of the P2X7 receptor.
TABLE 1. Effects of day in culture and of treatments with EGF and epinephrine on the expression of the P2X7 receptor
Mechanism of EGF-dependent facilitation of epinephrine effect
EGF facilitated epinephrine antiapoptotic effect (Figs. 1, 4, and 5), but treatment with EGF alone had little effect on apoptosis or on the density level of the ?2-adrenoceptor (Fig. 2A). These data suggested that the EGF effect involves modulation of ?2-adrenoceptor function. To better understand the effect of EGF, we assayed the functionality of the EGF receptor in CaSki cells. CaSki cells bound EGF specifically and with high affinity (Fig. 6A). Scatchard analysis of the specific [125I]EGF binding suggested two populations of EGF receptors: high-affinity sites (Kd0.5 nM, binding activity of about 50 fmol/mg DNA, 50,000 receptors/cell); and low-affinity sites (Kd2.1 nM). These data confirm previous results in normal human cervical epithelial cells (1) and in human papillomavirus 16 (immortalization human ectocervical epithelial cells) (34, 35). Treatment with epinephrine did not affect EGF receptor binding capacity or binding affinity (Fig. 6A). The positive control for the experiment was estrogen, whereby treatment of cells with 100 nM 17?-estradiol for 24 h increased binding capacity of the high-affinity EGF receptor sites to about 85 fmol/mg DNA (Fig. 6A) without affecting binding affinity.
FIG. 6. A, Scatchard analysis of [125I]EGF binding to CaSki cells and the effects of epinephrine and 17?-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].
Functionality of the EGF receptor was determined by incubating CaSki cells with the EGF receptor inhibitor AG1478. In cells treated for 24 h with 0.2 nM EGF, cotreatment with 5 μM AG1478 blocked EGF-increase in [3H]thymidine incorporation (Fig. 6B). Similarly, cotreatment with 10 μM of the MAPK/MEK inhibitor PD98059 blocked EGF-induced increase in [3H]thymidine incorporation (Fig. 6B). In contrast, cotreatment with 10 μM of the PI3K inhibitor wortmannin had no significant effect on [3H]thymidine incorporation compared with that observed in cells treated only with EGF (Fig. 6B). These data indicate that the EGF-induced increase in [3H]thymidine incorporation (and probably in cell number) is mediated by the EGF receptor and the MAPK/MEK pathway.
The results in Fig. 2, B and C, suggested to us that epinephrine inhibition of BzATP-induced apoptosis involves activation of adenylyl cyclase and protein kinase A, and therefore that epinephrine uses cAMP as the downstream messenger of the ?2-adrenoceptor. These data were used to dissect the mechanism by which EGF facilitates epinephrine action. Treatments with 2 nM epinephrine or with 1 μM zinterol increased cellular cAMP by about 2-fold, and cotreatment with 0.2 nM EGF augmented the responses to both epinephrine and zinterol (Fig. 7A). These data suggest that EGF exerts its effect at a level proximal to the adenylyl cyclase.
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]cyanopindolol. B, Effects on [125I]cyanopindolol internalization. C, Effects on [125I]cyanopindolol recycling. The experiments are described in the text.
The next set of experiments tested the hypothesis that EGF modulates plasma membrane levels of ?2-adrenoceptor. The experiments looked into effects of EGF on plasma membrane ?2-adrenoceptor steady-state. To test the degree of which EGF modulates ?2-adrenoceptor internalization, CaSki cells were incubated at 4 C with [125I]cyanopindolol, shifted to 37 C, and treated with zinterol. As shown in Fig. 7B and summarized in Table 2, pretreatment for 24 h with 0.2 nM EGF attenuated the time-related zinterol-induced decrease in the membrane-bound [125I]cyanopindolol. One explanation for this result is that EGF inhibits internalization of the ?2-adrenoceptor. An additional explanation is that EGF accelerates recycling of the ligated ?2-adrenoceptor receptor and stimulates its restoration into the plasma membrane. To study this question more directly, CaSki cells were labeled with [125I]cyanopindolol and treated with zinterol at 4 C, and then shifted to 37 C. As shown in Fig. 7C and summarized in Table 2, the increase in the membrane-bound [125I]cyanopindolol was faster in cells pretreated with EGF than in control cells. Collectively, the data in Fig. 7, B and C, and in Table 2 suggest that treatment with EGF increases plasma membrane ?2-adrenoceptor levels, possibly by attenuating receptor internalization and facilitating recycling of the receptor.
TABLE 2. Modulation of EGF effects by wortmannin a and PD98059
To better understand the mechanism of EGF modulation of plasma membrane ?2-adrenoceptor levels, the above experiments were repeated in the presence of PD98059 or wortmannin. Cotreatment with PD98059 had no significant effect on EGF modulation of plasma membrane ?2-adrenoceptor steady-state (Table 2). Treatment with wortmannin alone had no significant effect on the internalization and recycling of the membrane-bound [125I]cyanopindolol or on DNA solubilization (not shown). Cotreatment with EGF plus wortmannin also had no significant effect on the EGF-induced facilitated ?2-adrenoceptor recycling (Table 2). In contrast, coincubation with wortmannin inhibited the EGF-induced attenuation of ?2-adrenoceptor internalization and the EGF-induced decrease in DNA solubilization (Table 2).
Discussion
The present study reports novel data about epinephrine- and EGF-regulation of apoptosis in human cervical epithelial cells. We discovered that EGF facilitates ?2-adrenoceptor-dependent inhibition of P2X7 receptor-mediated apoptosis. EGF mechanism of action involves up-regulation of plasma membrane ?2-adrenoceptor levels by acceleration of ?2-adrenoceptor recycling, and by PI3K-depedent inhibition of ?2-adrenoceptor internalization. ?2-Adrenoceptor-dependent modulation of apoptosis has been previously described, but effects vary and depend on cell type and on the experimental conditions. Previous studies in cardiac myocytes reported that ?2-adrenoceptors exert antiapoptotic effects via an effect mediated by the caspase 9 mitochondrial pathway (36, 37, 38). These data could be relevant to the present study because, in human cervical epithelial cells, activation of ?2-adrenoceptors blocks P2X7 receptor-dependent apoptosis (present results), which also uses a caspase 9 mitochondrial pathway (4).
In human cervical epithelial cells, activation of the P2X7 receptor depended on membrane expression of the glycosylated 85-kDa form (Fig. 5), which is the mature and functional form of the P2X7 receptor protein. Previous groups described the expression and function of the glycosylated P2X1 and P2X2 receptors (39, 40, 41), as well as the P2X4 (42) and P2X6 receptors (43). Our novel data suggest that membrane expression and functionality of the P2X7 receptor also depend on glycosylation of the receptor, because receptor deglycosylation was detrimental for receptor function and pore formation. The P2X7 receptor possesses five putative N-glycosylation sites: Asn187, Asn202, Asn213, Asn241, and Asn284 (29); but at present, little is known about the role of the attached N-glycans for the expression and function of the P2X7 receptor. The present data suggest that epinephrine, in an action mediated by ?2-adrenoceptor activation of protein kinase A, stimulates deglycosylation of the P2X7 receptor and receptor degradation. The molecular mechanism of this novel protein kinase A effect is unknown. One possibility is an effect at the level of the endoplasmic reticulum/Golgi, namely inhibition of receptor processing trafficking and membrane expression. Another explanation is an effect at the level of the plasma membrane, i.e. enhanced degradation of previously expressed receptor.
The effect of epinephrine was mediated by the GPCR ?2-adrenoceptor. The expression of ?2-adrenoceptors in uterine cervical and vaginal cells was previously reported, both in the myometrial and uterine smooth muscle cells (44, 45), as well as in uterine epithelial cells. Tolszczuk and Pelletier (46) described the expression of ?2-adrenoceptors in rat endometrial cells. Whitaker et al. (47) showed intense labeling of [125I]cyanopindolol over glands and surface columnar epithelium of the human cervix, and moderate labeling over the squamous cervical/vaginal epithelium, and surrounding the basal layer. In females, plasma levels of epinephrine range from 0.1–3 nM and change relatively little throughout life (14). In addition, plasma levels of epinephrine are within the range of the Kd of the cervical ?2-adrenoceptor (Fig. 2A). Collectively, these data suggest that epinephrine tonically exerts antiapoptotic effect in the cervix in vivo.
We also found that EGF facilitated epinephrine antiapoptotic effect. EGF is a multifunctional factor whose actions are mediated by a RTK linked to multiple downstream cascades such as protein kinase C, PI3K, JNK, and the MAPK signaling pathways (recently reviewed in Ref. 48). All cascades have been described in human cervical cells, and the MAPK signaling pathway is usually associated with mitogenic properties of EGF (Ref. 1 and Fig. 1B). Interactions between EGF-induced signals and GPCR signaling pathways have been reported (49, 50, 51, 52, 53, 54). In some systems, the early convergence of signals originating from GPCRs and RTKs reflects GPCR-mediated transactivation of an RTK (55). However, only limited observations have been related to effects of EGF on adenylyl cyclase activation via ?-adrenergic or other stimulatory GPCRs (56). EGF can increase transcription of ?2-adrenoceptor (57) and cause rapid changes of adenylyl cyclase activities in a variety of in vitro systems (50, 58). The present findings are novel, in that EGF blocked ?2-adrenoceptor internalization via a PI3K-dependent mechanism and facilitated receptor recycling via a PI3K-independent mechanism. The molecular mechanisms of EGF action are, at present, unknown; but based on studies in other systems, they could involve modulation of signaling along the classical GPCR cascade of endocytosis, resensitization, recycling, and degradation (59).
Based on these findings, our hypothesis is summarized in Fig. 8, and it depicts that, in the cervix, EGF has a dual role regarding the regulation of growth of cervical epithelial cells. Acting via the MAPK cascade pathway, EGF stimulates mitosis and proliferation, and it is probably the mediator of estrogen effect (11). EGF also facilitates ?2-adrenoceptor-dependent stimulation of deglycosylation and degradation of the P2X7 receptor. EGF up-regulates plasma membrane ?2-adrenoceptor levels via two possible mechanisms: facilitated recycling of the ?2-adrenoceptor, and attenuation of ?2-adrenoceptor internalization by a PI3K-dependent mechanism. These effects of EGF would tend to increase the pool of the receptor that is available for activation upon ligand binding. Our novel findings underscore a new signaling network that could be a general paradigm for functional communication among RTKs, GPCRs, and the purinergic P2X7 receptor.
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 ?2-adrenoceptor-dependent stimulation of deglycosylation and degradation of the P2X7 receptor. The effect of EGF involves attenuation of ?2-adrenoceptor internalization by a PI3K-dependent mechanism and facilitated recycling of the ?2-adrenoceptor by a PI3K-independent mechanism. The EGF effects on ?2-adrenoceptor endocytosis and recycling would tend to up-regulate ?2-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.
The present results are physiologically relevant because human cervical epithelial cells express the EGF receptor (Ref. 1 and Fig. 6A) and the ?2-adrenoceptor (Ref. 47 and Fig. 2A). Moreover, in women, plasma EGF concentrations range from 0.1–5 nM (15, 16) and are within the range of EGF binding by the EGF receptor (6A). Furthermore, in the cervix, EGF is released from stromal cells on which cells of the basal layer of the cervical epithelium rest (60). We conclude that, in the cervix, the observed EGF effects of stimulated epithelial growth are the combination of EGF mitogenic and EGF antiapoptotic effects, and the latter are the result of EGF-dependent facilitation of ?2-adrenoceptor inhibition of P2X7 receptor-mediated apoptosis.
Acknowledgments
The technical support of Kim Frieden, Brian De-Santis, and Dipika Pal is acknowledged.
References
Jacobberger JW, Sizemore N, Gorodeski G, Rorke EA 1995 Transforming growth factor ? regulation of epidermal growth factor receptor in ectocervical epithelial cells. Exp Cell Res 220:390–396
Vijayalakshmi V, Gupta PD 1994 Estradiol-regulated transamidation of keratins by vaginal epithelial cell transglutaminase. Exp Cell Res 214:358–366
Mandelblatt J 1993 Squamous cell cancer of the cervix, immune senescence and HPV: is cervical cancer an age-related neoplasm? Adv Exp Med Biol 330:13–26
Wang Q, Wang L, Feng YH, Li X, Zeng R, Gorodeski GI 2004 P2X7-receptor-mediated apoptosis of human cervical epithelial cells. Am J Physiol Cell Physiol 287:C1349–C1358
Ellis HM, Yuan J, Horvitz HR 1991 Mechanisms and functions of cell death. Annu Rev Cell Biol 7:663–698
Ashkenazi A, Dixit VM 1998 Death receptors: signaling and modulation. Science 281:1305–1308
Gorodeski GI, Romero MF, Hopfer U, Rorke E, Utian WH, Eckert RL 1994 Human uterine cervical epithelial cells grown on permeable support—a new model for the study of differentiation and transepithelial transport. Differentiation 56:107–118
Sime A, McKellar Q, Nolan A 1997 Method for the growth of equine airway epithelial cells in culture. Res Vet Sci 62:30–33
Wells A 1999 EGF receptor. Int J Biochem Cell Biol 31:637–643
Katz MS 1988 Food restriction modulates ?-adrenergic-sensitive adenylate cyclase in rat liver during aging. Am J Physiol Endocrinol Metab 254:E54–E62
Gorodeski GI, Eckert RL, Utian WH, Rorke EA 1990 Maintenance of in vivo-like keratin expression, sex steroid responsiveness and estrogen receptor expression in cultured human ectocervical epithelial cells. Endocrinology 126:399–406
Gorodeski GI, Whittembury J 1998 A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters. Res Vet Sci 29:307–332
Haigler HT, Maxfield FR, Willingham MC, Pastan I 1980 Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB-3T3 cells. J Biol Chem 255:1239–1241
Engstr?m BE, Karlsson FA, Wide L 1999 Gender differences in diurnal growth hormone and epinephrine values in young adults during ambulation. Clin Chem 45:1235–1239
Lev-Ran A, Hwang DL 1990 Epidermal growth factor in blood and urine of athyreotic adults. Clin Physiol Biochem 8:184–187
Hoffmann TK, Ballo H, Braunstein S, Van Lierop A, Wagenmann M, Bier H 2001 Serum level and tissue expression of c-erbB-1 and c-erbB-2 proto-oncogene products in patients with squamous cell carcinoma of the head and neck. Oral Oncol 37:50–56
Nagata S, Goldstein P 1995 The Fas death factor. Science 267:1449–1456
Rath PC, Aggarwal BB 1999 TNF-induced signaling in apoptosis. J Clin Immunol 19:350–364
Liu X, Kim CN, Yang J, Jemmerson R, Wang X 1996 Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147–157
Takahashi A 1999 Caspase: executioner and undertaker of apoptosis. Int J Hematol 70:226–232
Ferrari D, Chiozzi P, Falzoni S, Dal Susino M, Melchiorri L, Baricordi OR, Di Virgilio F 1997 Extracellular ATP triggers IL-1? release by activating the purinergic P2Z receptor of human macrophages. J Immunol 159:1451–1458
Guerra AN, Fisette PL, Pfeiffer ZA, Quinchia-Rios BH, Prabhu U, Aga M, Denlinger LC, Guadarrama AG, Abozeid S, Sommer JA, Proctor RA, Bertics PJ 2003 Purinergic receptor regulation of LPS-induced signaling and pathophysiology. J Endotoxin Res 9:256–263
Humphreys BJ, Rice J, Kertesy SB, Dubyak GR 2000 Stress-activated protein kinase/JNK activation and apoptotic induction by the macrophage P2X7 nucleotide receptor. J Biol Chem 275:26792–26798
Ferrari D, Wesselborg S, Bauer MK, Schulze-Osthoff K 1997 Extracellular ATP activates transcription factor NF-B through the P2Z purinoreceptor by selectively targeting NF-B p65. J Cell Biol 139:1635–1643
Virginio C, MacKenzie A, North RA, Surprenant A 1999 Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor. J Physiol (Lond) 519:335–346
Denlinger LC, Sommer JA, Parker K, Gudipaty L, Fisette PL, Watters JW, Proctor RA, Dubyak GR, Bertics PJ 2003 Mutation of a dibasic amino acid motif within the C terminus of the P2X7 nucleotide receptor results in trafficking defects and impaired function. J Immunol 171:1304–1311
Kim M, Spelta V, Sim J, North RA, Surprenant A 2001 Differential assembly of rat purinergic P2X7 receptor in immune cells of the brain and periphery. J Biol Chem 276:23262–23267
Ralevic V, Burnstock G 1998 Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492
Surprenant A, Rassendren F, Kawashima E, North RA, Buell G 1996 The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272:735–738
Ramirez AN, Kunze DL 2001 P2X purinergic receptor channel expression and function in bovine aortic endothelium. Am J Physiol 282:H2106–H2116
Bianchi BR, Lynch KJ, Touma E, Niforatos W, Burgard EC, Alexander KM, Park HS, Yu H, Metzger R, Kowaluk E, Jarvis MF, van Biesen T 1999 Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol 376:127–138
Torres GE, Egan TM, Voigt MM 1999 Hetero-oligomeric assembly of P2X receptor subunits. J Biol Chem 274:6653–6659
Glass R, Burnstock G 2001 Immunohistochemical identification of cells expressing ATP-gated cation channels (P2X receptors) in the adult rat thyroid. J Anat 198:569–579
Sizemore N, Rorke EA 1993 Human papillomavirus 16 immortalization of normal human ectocervical epithelial cells alters retinoic acid regulation of cell growth and epidermal growth factor receptor expression. Cancer Res 53:4511–4517
Sizemore N, Choo CK, Eckert RL, Rorke EA 1998 Transcriptional regulation of the EGF receptor promoter by HPV16 and retinoic acid in human ectocervical epithelial cells. Exp Cell Res 244:349–356
Communal C, Singh K, Sawyer DB, Colucci WS 1999 Opposing effects of ?1- and ?2-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive G protein. Circulation 100:2210–2212
Zaugg M, Xu W, Lucchinetti E, Shafiq SA, Jamali NZ, Siddiqui MAQ 2000 ?-Adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes. Circulation 102:344–350
Singh K, Xiao L, Remondino A, Sawyer DB, Colucci WS 2001 Adrenergic regulation of cardiac myocyte apoptosis. J Cell Physiol 189:257–265
Newbolt A, Stoop R, Virginio C, Surprenant A, North RA, Buell G, Rassendren F 1998 Membrane topology of an ATP-gated ion channel (P2X receptor). J Biol Chem 273:15177–15182
Torres GE, Egan TM, Voigt MM 1998 N-Linked glycosylation is essential for the functional expression of the recombinant P2X2 receptor. Biochemistry 37:14845–14851
Rettinger J, Aschrafi A, Schmalzing G 2000 Roles of individual N-glycans for ATP potency and expression of the rat P2X1 receptor. J Biol Chem 275:33542–33547
Hu B, Senkler C, Yang A, Soto F, Liang BT 2002 P2X4 receptor is a glycosylated cardiac receptor mediating a positive inotropic response to ATP. J Biol Chem 277:15752–15757
Jones CA, Vial C, Sellers LA, Humphrey PP, Evans RJ, Chessell IP 2004 Functional regulation of P2X6 receptors by N-linked glycosylation: identification of a novel?-methylene ATP-sensitive phenotype. Mol Pharmacol 65:979–985
Bryman I, Norstrom A, Lindblom B 1986 Influence of prostaglandins and adrenoceptor agonists on contractile activity in the human cervix at term. Obstet Gynecol 67:574–578
Kovacs L, Falkay G 1993 Changes in adrenergic receptors in the pregnant human uterine cervix following mifepristone or placebo treatment in the first trimester. Hum Reprod 8:119–121
Tolszczuk M, Pelletier G 1988 Autoradiographic localization of ?-adrenoreceptors in rat uterus. J Histochem Cytochem 36:1475–1479
Whitaker EM, Nimmo AJ, Morrison JF, Griffin NR, Wells M 1989 The distribution of ?-adrenoceptors in human cervix. Q J Exp Physiol 74:573–576
Carpenter G 2000 The EGF receptor: a nexus for trafficking and signaling. Bioessays 22:697–707
Gutierrez GE, Mundy GR, Derynck R, Hewlett EL, Katz MS 1987 Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factorand epidermal growth factor. J Biol Chem 262:15845–15850
Ramirez I, Tebar F, Grau M, Soley M 1995 Role of heterotrimeric G-proteins in epidermal growth factor signaling. Cell Signal 7:303–311
del Carmen Medina L, Vazquez-Prado J, Garcia-Sainz JA 2000 Cross-talk between receptors with intrinsic tyrosine kinase activity and 1b-adrenoceptors. Biochem J 350:413–419
Gschwind A, Zwick E, Prenzel N, Leserer M, Ullrich A 2001 Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene 20:1594–1600
Pierce KL, Luttrell LM, Lefkowitz RJ 2001 New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades. Oncogene 20:1532–1539
Saito Y, Berk BC 2001 Transactivation: a novel signaling pathway from angiotensin II to tyrosine kinase receptors. J Mol Cell Cardiol 33:3–7
Maudsley S, Pierce KL, Zamah M, Miller WE, Ahn S, Daakai Y, Lefkowitz RJ, Luttrell LM 2000 The ?2-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem 275:9572–9580
Knecht M, Catt KJ 1983 Modulation of cAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 258:2789–2794
Yeh CK, Hymer TK, Sousa AL, Zhang BX, Lifschitz MD, Katz MS 2003 Epidermal growth factor up-regulates ?-adrenergic receptor signaling in a human salivary cell line. Am J Physiol Cell Physiol 284:C1164–C1175
Nakagawa Y, Gammichia J, Purushotham KR, Schneyer CA, Humphreys-Beher MG 1991 Epidermal growth factor activation of rat parotid gland adenylate cyclase and mediation by a GTP-binding regulatory protein. Biochem Pharmacol 42:2333–2340
Ferguson SSG 2001 Evolving concepts in GPCR endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24
Hom YK, Young P, Wiesen JF, Miettinen PJ, Derynck R, Werb Z, Cunha GR 1998 Uterine and vaginal organ growth requires epidermal growth factor receptor signaling from stroma. Endocrinology 139:913–921(Liqin Wang, Ying-Hong Fen)