A truncated naturally occurring variant of the human receptor P2X 7 was identified in cancer cervical cells. The novel protein (P2X 7-j ), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X 7 receptor. The P2X 7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X 7 receptor agonist benzoyl-ATP. The P2X 7-j interacted with the full-length P2X 7 in a manner suggesting heterooligomerization and blocked the P2X 7 -mediated actions. Interestingly, P2X 7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but fulllength P2X 7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X 7 immunoreactivity suggesting lack of P2X 7 homo(tri)-oligomerization. These results identify a novel P2X 7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.The receptor P2X 7 belongs to the P2X subfamily of P2 nucleotide receptors (1, 2), which are membrane-bound, ligand-operated channels (3-5). ATP is the naturally occurring ligand for the P2X 7 and activation of the receptor by brief exposure to extracellular ATP opens cation channels that allow Ca 2ϩ , Na ϩ , and K ϩ influx (6). Longer exposure to ATP allows passage of cations with progressively larger diameters, up to 900 Da, through formation of pores (7). The mechanism of pore formation is unclear, and opinions vary between decreased filter selectivity of existing channels (8) to rearrangement of receptor molecules (9). P2X 7 receptors function in a cell-specific manner and effects of receptor activation are determined by receptor expression (10), trafficking and plasma membrane localization (11-13), oligomerization (5), and postactivation internalization, recycling, and degradation (14). Expression of P2X 7 can be regulated hormonally; in human cervical epithelial cells epinephrine down-regulates expression of the glycosylated form of the P2X 7 and increases receptor degradation, and the effects can be potentiated by epidermal growth factor (15). Evidence for the physiological role of the P2X 7 comes from studies of P2X 7 -deficient mice, indicating its role in inflammatory (16) and immune processes (17). Epithelial cells of the female lower reproductive tract express the P2X 7 (18), and in human cervical epithelial cells ligand binding induces apoptosis by a mechanism that involves pore formation, augmented calcium influx, and calcium-dependent activation of the apoptotic mitochondrial pathway (19,20). Because human cervical epithelial cells secrete ATP into the extra...
Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X7 receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 microM in hECE cells and approximately 1 microM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-alpha, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-alpha, it had only a mild effect on caspase-8. Both BzATP and TNF-alpha activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was approximately 0.5 microM, which is in the range of values that suffice to activate the P2X7 receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X7 receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway.
The P2X 7 receptor regulates cell growth through mediation of apoptosis. P2X 7 levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X 7 was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X 7 mRNA stability. Overexpression of a reporter containing the full-length human P2X 7 3-untranslated region (3-UTR) or reporters containing parts of the 3-UTR-P2X 7 were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3-UTR-P2X 7 , which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X 7 transcript. Overexpression in cancer cells of mutated miR-186 and miR-150 target sites was associated with lower levels of the reporter genes. In normal cells overexpression of the mutated miR-186 target site was associated with marked increased concentration, but overexpression of the miR-150 target site reporters, wild-type and mutant, did not change over time. Levels of miR-186 and miR-150 were higher in cancer than in normal cells, and treatment with miR-186 and miR-150 inhibitors increased P2X 7 mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3-UTR-P2X 7 luciferase reporter, miR-186 and miR-150 inhibitors increased luciferase activity, whereas miR-186 and miR-150 mimics decreased luciferase activity after actinomycin D treatment. These data suggest that increased expression of miR-186 and miR-150 in cancer epithelial cells decreases P2X 7 mRNA by activation of miR-186 and miR-150 instability target sites located at the 3-UTR-P2X 7 .The receptor P2X 7 is a membrane-bound, ligand-operated channel (1-6). ATP is the naturally occurring ligand for the P2X 7 , and extracellular levels of ATP may reach low micromolar levels (7-12), which are sufficient to activate the receptor (13). Activation of the receptor may induce formation of pores in the plasma membrane (14), which in epithelial cells mediate apoptosis via the caspase-9 mitochondrial pathway (15, 16). The P2X 7 apoptosis effects can be regulated by receptor glycosylation (16), trafficking, plasma membrane expression (17-20), oligomerization (7, 21), and by receptor post-activation internalization, recycling, and degradation (14,21).In epithelial tissues the P2X 7 receptor is expressed predominantly by proliferative (germinative) epithelial cells (21-23), and it controls the growth of the epithelial cells. Previous studies in human uterine epithelial cells showed that base-line and P2X 7 -mediated apoptosis are lower in cancer cells than in normal cells (12,22,23). The differences were not the result of ligand availability because steady-state levels of ATP in conditioned media of cancer epithelial cells were similar to those of normal epithelial cells (12). Similarly, there were no significant differences in P2X 7 receptor activation, oligomerization, or cycling betwe...
Objective: To determine expression of the P2X 7 receptor in normal and in cancer uterine tissues. The rationale was that the receptor P2X 7 regulates constitutive apoptosis in uterine epithelial cells, and previous studies showed diminished P2X 7 -mediated apoptosis in cancer uterine cells compared with normal cells. Methods: A clinical, experimental feasibility study. Normal (n = 42) and cancer uterine tissues (n = 47) were obtained from a total of 72 women ages 25 to 75. End points for P2X 7 mRNA were quantitative PCR and in situ hybridization, and end points for P2X 7 protein were Western blots and immunostaining using anti-P2X 7 antibody. Results: (a) In normal uteri, P2X 7 mRNA and protein were expressed predominantly in the epithelial (endometrial, endocervical, and ectocervical) cells. (b) Expression of the P2X 7 mRNA and protein was absent from endometrial and endocervical adenocarcinoma tissues and from cervical squamous cell carcinoma tissues. (c) In cervical dysplasia, P2X 7 protein was absent in the dysplastic lesions. (d) Semiquantitative analysis using P2X 7 mRNA (normalized in each tissue to the constitutive glyceraldehyde-3-phosphate dehydrogenase) and P2X 7 protein levels (normalized in each tissue to the constitutive tubulin) revealed that P2X 7 mRNA and/or protein levels can distinguish uterine normal from cancer tissues at high degrees of sensitivity (92%, 100%) and specificity (100%, 90%). Summary and Conclusions: (a) Levels of the P2X 7 are lower in uterine epithelial cancer tissues than in the corresponding normal tissues. (b) The data suggest that tissue P2X 7 mRNA and protein levels could be used as a novel biomarker to differentiate normal and cancer uterine epithelial tissues. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1906 -13)
The objective of this study was to understand the mechanisms involved in P2X 7 receptor activation. Treatments with ATP or with the P2X 7 receptor-specific ligand 2′,3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X 7 receptor than in human embryonic kidney (HEK)-293 cells expressing exogenous P2X 7 receptor (HEK-293-hP2X 7 -R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85-kDa form present mainly in the membrane and 65-and 18-kDa forms expressed in both the plasma membrane and the cytosol. Treatments with ATP transiently decreased the 85-kDa form and increased the 18-kDa form in the membrane, suggesting internalization, degradation, and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85-kDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP requirements for phosphorylation on tyrosine and serine residues, suggesting a dominant-negative effect. In both CaSki and in HEK-293-hP2X 7 -R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, β-arrestin-2, and dynamin, and it stimulated β-arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X 7 receptor action, whereby activation involves a GRK-3-, β-arrestin-2-, and dynamin-dependent internalization of the receptor into clathrin domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.Keywords purinergic receptor; recycling; dynamin; clathrin; cervix; epithelium The P2X 7 Receptor Belongs to the P2X receptor subfamily of P2 nucleotide receptors (6,45), which are membrane-bound, ligand-operated K + -, Na + -, and Ca 2+ -permeable channels that function as homo-or heteromultimeric complexes (38,48). Activation of the P2X 7 receptor can stimulate various cell-specific signaling cascades (11,40). Effects unique to the receptor are the induction of membrane fusion and blebbing associated with microvesicle generation (9,10), interleukin-1β processing and secretion (11,24,33), and opening of membrane pores (11,37,40,46). Epithelial cells of the female lower reproductive tract express the P2X 7 receptor Address for reprint requests and other correspondence: G. I. Gorodeski, University MacDonald Women's Hospital, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail: gig@cwru.edu). NIH Public AccessAuthor Manuscript Am J Physiol Cell Physiol. Author manuscript; available in PMC 2008 December 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (3), and in human cervical epithelial cells activation of the receptor induces apoptosis by a mechanism that involves calcium-dependent activation of the mitochondrial pathway (51). Apoptosis plays an important role in the regulation of cell cycle and control of neoplas...
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