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The P2X7A receptor is the main physiological pro-apoptotic mechanism in epithelia in vivo. The natural ligand of the P2X7A is ATP. The P2X7A is expressed abundantly in epithelial reserve (stem) cells, and it regulates epithelial-cell terminal differentiation. The high expression of P2X7A in reserve cells renders the cells sensitive to conditions of increased extracellular ATP, in particular upon neoplastic transformation, and to the induction of apoptosis. Cellular expression of the P2X7A is reduced in cancer cells of epithelia derived from the ectoderm, the urogenital sinus, and the distal paramesonephric duct. Low levels of P2X7A have been linked to cancer development because reserve cells harboring a defective P2X7A mechanism can escape P2X7A pro-apoptotic control. Mechanisms that induce reduced expression of P2X7A in cancer epithelial cells involve hypermethylation of the P2X7 gene and decreased transcription; enhanced degradation of the P2X7 transcript through the action of micro-RNAs miR-186 and miR-150; and epidermal growth factor (EGF) regulated, protein kinase-A (PKA)-mediated de-glycosylation of the P2X7A. Reduced cellular P2X7A content has been proposed as a biomarker for human breast, bladder, ectocervix, endocervix, and endometrial cancers. The chemotherapeutic potential of augmented P2X7A-dependent apoptosis of neoplastic epithelial cells was recently demonstrated in the mouse in vivo. Local treatment on the skin with the P2X7A-specific agonist benzoyl-ATP (BzATP) decreased the development of 7,12-dimethyl-benz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin papillomas and skin cancers by 50%, by an effect that involves augmentation of keratinocytes reserve cells apoptosis. The P2X7A mechanism represents a novel pharmaceutical target for the prevention and treatment of epithelial neoplasia through the induction of apoptosis. WIREs Membr Transp Signal 2012,1:349–371. doi: 10.1002/wmts.33 For further resources related to this article, please visit the WIREs website.

The pro-apoptotic P2X(7) receptor regulates growth of epithelial cells. The objectives of the study were to understand P2X(7) gene transcription; to identify the active promoter and the transcription initiation site (TpIS); and to begin understanding regulation of P2X(7) gene transcription. Experiments in vitro utilized normal and cancerous cultured human uterine cervical epithelial cells, and HEK293 cells overexpressing P2X(7)-luciferase reporters. Experiments in vivo used surgical specimen of normal and cancerous uterine cervix. Assays involved DNA, RNA, and protein techniques. (a) The P2X(7) TpIS was localized to adenine (+1) at nt 1683 of the human P2X(7) gene [GenBank Y12851]), with a TTAAA sequence at nt -32/-28 and an active promoter region within nt -158/+32. (b) P2X(7) transcription was found to be regulated by two enhancers located at nt + 222/+232 and +401/+573 regions downstream of the active P2X(7) promoter. (c) The putative enhancer regions formed four DNA-protein complexes. (d) P2X(7) transcription was found to be controlled by hypermethylated cytosines at cytosine-phosphodiester-guanosines (CpG) that cluster or co-localize with the enhancers' sites. (e) We identified nine CpGs as inhibitory cis elements, and three CpG sites that are hypermethylated in cultured cervical epithelial cells and in cervix epithelia in vivo. (f) In cancer cervical cells, the degree of hypermethylation of the CpG sites was greater than in the normal cervical cells. Expression of the P2X(7) receptor is controlled by hypermethylated CpGs that flank transcription enhancers located within a 547-nt region downstream of the promoter.

The P2X7 receptor regulates cell growth through mediation of apoptosis. P2X7 levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X7 was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X7 mRNA stability. Overexpression of a reporter containing the full-length human P2X7 3′-untranslated region (3′-UTR) or reporters containing parts of the 3′-UTR-P2X7 were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3′-UTR-P2X7, which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X7 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 P2X7 mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3′-UTR-P2X7 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 P2X7 mRNA by activation of miR-186 and miR-150 instability target sites located at the 3′-UTR-P2X7.

An important role of estrogen is regulation of epithelial permeability and the control of fluid transudation into the cervical and vaginal lumens. Our present understanding of transepithelial transport in relatively leaky types of epithelia, such as the cervical and vaginal epithelia, is based on the pioneering works of Ussing and Zerahn1 and Spring and Hope.2 The Ussing-Zerahn model predicts that luminal fluids are derived from the blood compartment; that plasma moves through the epithelial paracellular (intercellular) space (Fig. 1B), and that paracellular transport of the fluid is controlled by the resistance of the epithelial tight junctions (RTJ) and of the epithelial lateral intercellular space (RLIS). The RTJ and the RLIS contribute in series to the total resistance (RTE) so that RTE ≈ RTJ + RLIS. The RTJ is a relatively high resistance and is determined by occlusion of the intercellular space through the interactions of extracellular loops of transcellular tight junctional proteins such as the claudins3 and occludin4 (Fig. 1D-E). The RLIS is considered a low transepithelial resistance and is determined by the proximity of neighboring epithelial cells (Fig. 1A). The Spring-Hope model2 predicts that the driving force moving the fluid from the blood into the lumen is the capillary blood pressure, which creates a subluminal to luminal hydrostatic gradient (Fig. 1A and B). Subluminal to luminal hydrostatic gradients dilate the lateral intercellular space, thereby decreasing the RLIS (Fig. 1B). Dilation is restricted to the basolateral intercellular space because of the apical location of adherens intercellular junctions, which prevent lateral separation near the apical regions of the cells (Fig. 1B). FIG. 1 Models of estrogen regulation of transepithelial permeability. A: The main resistances for free diffusion of fluid from the capillary bed into the lumen through the paracellular (intercellular) space are the tight junctional resistance (RTJ) and the resistance ... Estrogen increases fluid transudation by a number of known mechanisms. Estrogen decrease in capillary resistance increases blood flow, which would tend to increase the subluminal to luminal hydrostatic gradient (Fig. 1C).5 However, this effect contributes relatively little to net fluid transudation,6,7 and the main estrogenic control of transepithelial transport is by modulation of epithelial permeability through regulation of the RTJ (Fig. 1C-E) and the RLIS (Fig. 1C, F, G). Estrogen decrease in RTJ is thought to be mediated by matrix metalloproteinase-7-dependent modulation of occludin,8-10 and the decrease in RTJ is explained by loss of gating of the intercellular space due to degradation of occludin extracellular loops.10 Estrogen decrease in the RLIS involves at least three known molecular mechanisms: actin depolymerization and the formation of monomeric-dominated G-actin cytoskeleton11; decreased dimerization of nonmuscle myosin type II-B (NMMII-B), the predominant cytoskeletal epithelial myosin12,13; and increased magnesium-adenosine triphosphatase (MgATPase) activity of the NMMII-B.14 Collectively the actin and NMMII-B effects of estrogen would tend to fragment the cortical actomyosin ring and induce formation of a dynamic cytoskeleton (Fig. 1E-G). This would facilitate dilation of the basolateral intercellular space (ie, decrease the RLIS) in response to the capillary subluminal to luminal hydrostatic gradients (Fig. 1C). The objectives of the present study were to better understand estrogen modulation of the RTJ and RLIS and how menopause affects estrogen actions. The results revealed that estrogen increases epithelial permeability by inducing an early transient decrease in RLIS followed by a slower persistent decrease in RTJ. The potency of estrogen actions was similar in cells of premenopausal and postmenopausal women, but the effects on NMMII-B filamentation and MgATPase activity were shorter in cells of postmenopausal women, resulting in faster reversal of the decrease in RLIS.

Objectives. To understand the potential role of P2X7 as biomarker of endometrial cancer, and the molecular mechanisms by which cancerous epithelial cells maintain low expression of P2X7. Methods. Feasibility clinical experimental study. Normal (28), simple or complex hyperplasia (7), complex hyperplasia with atypia (6) and cancer endometrial discarded tissues (40) were obtained from a total of 81 women, ages 25–75. Endpoint for P2X7 protein was average pixel signal density of tissue immunoreactivity with anti-P2X7 antibody. Endpoint for P2X7 mRNA was one-step quantitative Real-Time PCR. Experiments in-vitro included normal (hEVEC) and cancerous cervical epithelial cells (HeLa) transfected with reporter plasmid containing luciferase-3′ untranslated region (3′UTR)-P2X7 cDNA, using as endpoint steady-state luciferase mRNA levels. Results. Levels of P2X7 protein and mRNA were significantly lower in vivo, in tissues of complex hyperplasia with atypia or endometrial adenocarcinoma, than in tissues of normal endometrium, simple hyperplasia or complex hyperplasia tissues (sensitivity and specificity of 89– 100%, p

Estrogen modulates tight junctional resistance through estrogen receptor-alpha-mediated remodeling of occludin. The objective of the study was to understand the mechanisms involved. Experiments using human normal vaginal-cervical epithelial cells showed that human normal vaginal-cervical epithelial cells secrete constitutively matrix-metalloproteinase-7 (MMP-7) into the luminal solution and that MMP-7 is necessary and sufficient to produce estrogen decrease of tight junctional resistance and remodeling of occludin. Treatment with estrogen stimulated activation of the pro-MMP-7 intracellularly and augmented secretion of the activated MMP-7 form. Steady-state levels of MMP-7 mRNA and protein were not affected by estrogen. Estrogen modulated phosphorylation of the MMP-7, but the changes were most likely secondary to changes in cellular MMP-7 mass. Estrogen increased coimmunoreactivity of MMP-7 with the Golgi protein GPP130. Tunicamycin and brefeldin-A had no effect on cellular MMP-7 but monensin (inhibitor of Golgi traffic) blocked estrogen effects, suggesting estrogen site of action is at the Golgi system. Estrogen increased generalized secretory activity, including of luminal exocytosis of polycarbohydrates. However, estrogen increased coimmunoreactivity of MMP-7 with synaptosomal-associated protein of 25 kDa in apical membranes, suggesting soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor-facilitated exocytosis of MMP-7. Treatment with the vesicular-ATPase inhibitor bafilomycin A(1) inhibited activation of MMP-7. These data suggest that estrogen up-regulates activation of the MMP-7 intracellularly, at the level of Golgi, and augments secretion of activated MMP-7 through soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor-dependent exocytosis. On the other hand, estrogen acidification of the luminal solution would tend to alkalinize exocytotic vesicles and may lead to decreased activation of the MMP-7. These mechanisms acting in concert could be important for regulation and control of estrogen modulation of paracellular permeability in vivo.

The study tested the hypothesis that estrogen controls epithelial paracellular resistance through modulation of myosin. The objective was to understand how estrogen modulates nonmuscle myosin-II-B (NMM-II-B), the main component of the cortical actomyosin in human epithelial cervical cells. Experiments used human cervical epithelial cells CaSki as a model, and end points were NMM-II-B phosphorylation, filamentation, and MgATPase activity. The results were as follows: 1) treatment with estrogen increased phosphorylation and MgATPase activity and decreased NMM-II-B filamentation; 2) estrogen effects could be blocked by antisense nucleotides for the estrogen receptor-alpha and by ICI-182,780, tamoxifen, and the casein kinase-II (CK2) inhibitor, 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole and attenuated by AG1478 and PD98059 (inhibitors of epithelial growth factor receptor and ERK/MAPK) but not staurosporine [blocker of protein kinase C (PKC)]; 3) treatments with the PKC activator sn-1,2-dioctanoyl diglyceride induced biphasic effect on NMM-II-B MgATPase activity: an increase at 1 nm to 1 microM and a decrease in activity at more than 1 microM; 4) sn-1,2-dioctanoyl diglyceride also decreased NMM-II-B filamentation in a monophasic and saturable dose dependence (EC(50) 1-10 microM); 5) when coincubated directly with purified NMM-II-B filaments, both CK2 and PKC decreased filamentation and increased MgATPase activity; 6) assays done on disassembled NMM-II-B filaments showed MgATPase activity in filaments obtained from estrogen-treated cells but not estrogen-depleted cells; and 7) incubations in vitro with CK2, but not PKC, facilitated MgATPase activity, even in disassembled NMM-II-B filaments. The results suggest that estrogen, in an effect mediated by estrogen receptor-alpha and CK2 and involving the epithelial growth factor receptor and ERK/MAPK cascades, increases NMM-II-B MgATPase activity independent of NMM-II-B filamentation status.

To understand myosin regulation of epithelial permeability.This was an experimental study, using human cervical epithelial cells CaSki. End points were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B-actin interaction (determined in vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in vitro using purified NMM-II-B filaments in terms of filaments disassembly/assembly ratios.Treatment of cells with the Rho-associated kinase (ROCK) inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the resistance of the lateral intercellular space (R(LIS)), and increased phosphorylation of NMM-II-B on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, a casein kinase-II (CK2) inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole (DRB) did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the R(LIS) was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in vivo with Y-27632, okadaic acid, or DRB.The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the R(LIS) and increase the paracellular permeability.

Objective: To determine expression of the P2X7 receptor in normal and in cancer uterine tissues. The rationale was that the receptor P2X7 regulates constitutive apoptosis in uterine epithelial cells, and previous studies showed diminished P2X7-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 P2X7 mRNA were quantitative PCR and in situ hybridization, and end points for P2X7 protein were Western blots and immunostaining using anti-P2X7 antibody. Results: (a) In normal uteri, P2X7 mRNA and protein were expressed predominantly in the epithelial (endometrial, endocervical, and ectocervical) cells. (b) Expression of the P2X7 mRNA and protein was absent from endometrial and endocervical adenocarcinoma tissues and from cervical squamous cell carcinoma tissues. (c) In cervical dysplasia, P2X7 protein was absent in the dysplastic lesions. (d) Semiquantitative analysis using P2X7 mRNA (normalized in each tissue to the constitutive glyceraldehyde-3-phosphate dehydrogenase) and P2X7 protein levels (normalized in each tissue to the constitutive tubulin) revealed that P2X7 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 P2X7 are lower in uterine epithelial cancer tissues than in the corresponding normal tissues. (b) The data suggest that tissue P2X7 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 P2X7receptor activation. Treatments with ATP or with the P2X7receptor-specific ligand 2′,3′- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X7receptor than in human embryonic kidney (HEK)-293 cells expressing exogenous P2X7receptor (HEK-293-hP2X7-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-hP2X7-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 P2X7receptor 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.

The objective of the study was to understand age-related contributions of the resistance of the intercellular tight junctions (R(TJ)) and the resistance of the lateral intercellular space (R(LIS)) to the transcervical-transvaginal permeability. The experiments used normal human ectocervical epithelial cells obtained from women aged 36-65 yr. Twenty-four hours of treatment of cells with 10 nm 17beta-estradiol decreased the R(LIS), whereas longer treatments were required to decrease The R(TJ). Aging had no effect on baseline or estrogen decrease in R(TJ), but it blocked both baseline and the estrogen decrease in R(LIS). Actin assays showed age-related decrease in the fraction of monomeric G-actin and attenuation of sodium-nitroprusside-induced increase in G-actin. These results suggest that the aging-related diminished capacity of human ectocervical epithelial cells to remodel cellular actin from polymerized actin toward monomeric G-actin confers increased cell rigidity and therefore increased R(LIS). Therefore, the hypoestrogenism-related decrease in R(TJ) and the hypoestrogenism- and aging-related increase in R(LIS) could be the cellular mechanisms of decreased permeability that lead to decreased fluid transport and decreased lubrication of the lower genital tract in older postmenopausal women.

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

The present study investigated the antiapoptotic effects of estrogen in normal and cancer human cervical cells and the mechanisms involved. Baseline apoptosis in human cervical epithelial cells is mediated predominantly by P2X7-receptor-induced, Ca2+-dependent activation of the mitochondrial (caspase-9) pathway. Treatment with 10 nm 17β-estradiol blocked apoptosis induced by the P2X7-receptor ligands ATP and 2′,3′-0-(4-benzoylbenzoyl)-ATP in normal human cervical epithelial cells (hECEs) and attenuated the effect in hECEs immortalized with human papillomavirus-16 (ECE16–1) and the cancer cervical cells HT3 and CaSki. Diethylstilbestrol and to a lesser degree estrone could mimic the effects of 17β-estradiol, whereas actinomycin-D and cycloheximide attenuated the response. The antiapoptotic effect of estrogen did not depend on cell cycle phase, and in both normal and cancer cervical cells, it involved attenuation of activation of caspase-9 and the terminal caspase-3. However, involvement of cascades upstream to the caspase-9 differed in normal vs. cancer cervical cells. In the normal hECEs estrogen blocked P2X7-receptor-induced calcium influx. In contrast, in the cancer CaSki cells, estrogen up-regulated expression of Bcl-2 and attenuated Ca2+-induced mitochondrial swelling (i.e. formation of mitochondrial permeability transition pores). Estrogen had no effect on P2X7-receptor-induced apoptosis in the anaplastic SiHa and Hela cells. These results point to a novel antiapoptotic effect of estrogen in the cervix that is independent of its mitogenic function. The results also suggest that cancer cervical cells evolved antiapoptotic mechanisms that enable the cells to evade apoptosis and could therefore promote tumor progression.

Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures, an acute increase (phase I response) followed by a slower decrease ( phase II response). Phase I and phase II responses involve two distinct calcium-dependent pathways, calcium mobilization and calcium influx. To test the hypothesis that phase I and phase II responses are mediated by distinct P2 purinergic receptors, changes in permeability were uncoupled by blocking calcium mobilization with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA) or by lowering extracellular calcium, respectively. Under these conditions ATP EC50was 25 μM for phase Iresponse and 2 μM for phase II response. The respective agonist profiles were ATP > UTP > adenosine 5′- O-(3-thiotriphosphate) (ATP-γS) = N6-([6-aminohexyl]carbamoylmethyl)adenosine 5′-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-γS. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course and pharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and a saturable concentration-dependent effect (EC50of 2–3 μM). RT-PCR experiments revealed expression of mRNA for both the P2Y2and P2X4receptors. These results suggest that the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.

The objective of the study was to understand effects of estrogen and aging on paracellular permeability of human vaginal-cervical epithelia. Assays included determinations of transepithelial electrical conductance across cultures of normal human ectocervical epithelial cells on filters. Baseline transepithelial electrical conductance across steroid-deprived cells from postmenopausal women was lower than across cells of premenopausal women. Short-term (24-48 h) treatment with 10 nM 17beta-estradiol increased transepithelial electrical conductance in both groups of cells. In cells of premenopausal women longer-term treatment with estrogen for up to 14 d had no additional effect on permeability, but in cells of postmenopausal women it caused a secondary increase in transepithelial electrical conductance that continued for the duration of the 2-wk treatment. Binding assays of 17beta-[(3)H]estradiol revealed saturable binding to high affinity (1.2-1.3 nM), low capacity sites (0.2-1.2 pmol/mg DNA) in cells of both premenopausal and postmenopausal women. In both types of cells treatment with 17beta-estradiol increased 17beta-[(3)H]estradiol binding activity in a time- and dose-related manner (EC(50) 1 nM; maximal effect within 9-12 h), and increased estrogen receptor-alpha and -beta mRNA. 8-Br-cGMP, a stable cell-permeant analog of cGMP, could mimic the estrogen first phase increase in transepithelial electrical conductance, but not the secondary increase. Treatment with estrogen augmented the increase in transepithelial electrical conductance that was induced by hydrostatic gradients (modulator of the resistance of the lateral intercellular space), and the effect was independent of woman's age or baseline transepithelial electrical conductance. In contrast, the effect of low extracellular calcium (modulator of the tight junctional resistance) was more potent in cells of premenopausal women than in cells of postmenopausal women and was independent of treatment with estrogen. These results suggest that changes in vaginal-cervical epithelial permeability after menopause are determined by aging-related increase in tight junctional resistance, and by low estrogen-dependent increase in lateral intercellular space that lead to net increase in total paracellular resistance and decreased permeability and result in reduced lubrication of the lower genital canal.

Estrogen increases baseline transepithelial permeability across CaSki cultures and augments the increase in permeability in response to hypertonic gradients. In estrogen-treated cells, lowering cytosolic calcium abrogated the hypertonicity-induced augmented increase in permeability and decreased baseline permeability to a greater degree than in estrogen-deprived cells. Steady-state levels of cytosolic calcium in estrogen-deprived cells were higher than in estrogen-treated cells. Increases in extracellular calcium increased cytosolic calcium more in estrogen-deprived cells than in estrogen-treated cells. However, in estrogen-treated cells, increasing cytosolic calcium was associated with greater increases in permeability in response to hypertonic gradients than in estrogen-deprived cells. Lowering cytosolic calcium blocked the estrogen-induced increase in nitric oxide (NO) release and in the in vitro conversion of l-[3H]arginine to l-[3H]citrulline. Treatment with estrogen upregulated mRNA of the NO synthase isoform endothelial nitric oxide synthase (eNOS). These results indicate that cytosolic calcium mediates the responses to estrogen and suggest that the estrogen increase in permeability and the augmented increase in permeability in response to hypertonicity involve an increase in NO synthesis by upregulation of the calcium-dependent eNOS.

The objective of the study was to characterize the effect and explore the mechanisms by which menopause affects paracellular permeability of cultured human cervical epithelium. The experimental system was cultures of human ectocervical epithelial (hECE) cells on filters. Assays included flux measurements of cell-impermeable molecules and determinations of transepithelial electrical conductance. hECE cells of postmenopausal women formed epithelia with lower paracellular permeability than hECE cells of premenopausal women. Treatment with estrogen increased paracellular permeability, but levels remained lower compared to cultures from premenopausal women. Lowering extracellular calcium or treatment with cytochalasin-D, conditions that decrease the tight junctional resistance (R(TJ)), increased the permeability, and the relative effects were greater in cells of premenopausal women than in postmenopausal women. Treatment of cells with sn-1,2-dioctanoyl diglyceride, an agent that increases the R(TJ), decreased the permeability, and the relative effect was greater in cells of postmenopausal women than in cells of premenopausal women. Estrogen had no significant effect on the responses to low calcium, cytochalasin-D, or sn-1,2-dioctanoyl diglyceride. Hydrostatic and hypertonic gradients in the subluminal to luminal direction, conditions that decrease the resistance of the lateral intercellular space increased the permeability to a similar degree in cultures of cells from premenopausal and from postmenopausal women. Pretreatment with estrogen augmented the increases in permeability in response to hydrostatic and hypertonic gradients. In cells exposed to low extracellular calcium, hydrostatic gradients had an additive increase in permeability. By extrapolation it was determined that in cultures of postmenopausal women R(TJ) contributes 97% to the total paracellular resistance, whereas in hECE cultures of premenopausal women the R(TJ) contributes only 84%. These results indicate that after menopause the transcervical paracellular permeability decreases significantly; this can explain the decrease in lubrication of the cervix and vagina in postmenopausal women. Part of the effect is due to lack of estrogen, and it can be reversed by treatment with the hormone. However, most of the effect is unrelated to estrogen and is caused by an increase in R(TJ).

Human cervical epithelial cells express mRNA for the nitric oxide (NO) synthase (NOS) isoforms ecNOS, bNOS, and iNOS and release NO into the extracellular medium. NG-nitro-l-arginine methyl ester (l-NAME), an NOS inhibitor, and Hb, an NO scavenger, decreased paracellular permeability; in contrast, the NO donors sodium nitroprusside (SNP) and N-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increased paracellular permeability across cultured human cervical epithelia on filters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect was blocked by KT-5823 (a blocker of cGMP-dependent protein kinase), but not by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583 and KT-5823 blocked the SNP-induced increase in permeability. Treatment with SNP increased cellular cGMP, and the effect was blocked by Hb and LY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulated cervical cation selectivity. In contrast, both agents increased fluorescence from fura 2-loaded cells in the Ca2+-insensitive wavelengths, indicating that SNP and 8-BrcGMP stimulate a decrease in cell size and in the resistance of the lateral intercellular space. Neither SNP nor 8-BrcGMP had an effect on total cellular actin, but both agents increased the fraction of G-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823 blocked the 8-BrcGMP-induced increase in G-actin. On the basis of these results, it is suggested that NO acts on guanylate cyclase and stimulates an increase in cGMP; cGMP, acting via cGMP-dependent protein kinase, shifts actin steady-state toward G-actin; this fragments the cytoskeleton and renders cells more sensitive to decreases in cell size and resistance of the lateral intercellular space and, hence, to increases in permeability. These results may be important for understanding NO regulation of transcervical paracellular permeability and secretion of cervical mucus in the woman.

Treatment of cultured human cervical epithelia on filters with 17beta-estradiol increases paracellular permeability in a time- and dose-related manner (EC50, 1.1 nM). The objective of the present study was to understand the molecular mechanisms of estrogen action. In cultured human cervical epithelial cells the nitric oxide (NO) donors sodium nitroprusside (SNP) and N-[ethoxycarbonyl]-3-[4-morpholinyl]sydnoneimine (SIN-I) and the cell-permeable cGMP analog 8-bromo-cGMP (8-Br-cGMP) increased paracellular permeability. In estrogen-treated cells SNP and 8-Br-cGMP increased permeability to a lesser degree than in estrogen-deprived cells, suggesting that NO and cGMP mediate the effect of estrogen on permeability. Tamoxifen blocked the estrogen-induced increase in permeability, but it had no effect on increases in permeability that were induced by SNP or by 8-Br-cGMP. LY-83583 (blocker of guanylate cyclase) attenuated the effect of SNP, whereas KT-5823 (blocker of cGMP-dependent protein kinase) abrogated the effects of both SNP and 8-Br-cGMP. Treatment with 17beta-estradiol increased NO release and cellular cGMP in a dose-related manner (EC50, approximately 1 nM), and the effects were inhibited by tamoxifen. Treatment with SNP increased cGMP maximally, even in estrogen-deficient cells. LY-83583 blocked the estrogen-induced increase in cGMP, but neither LY-83583 nor KT-5823 had a significant effect on the estrogen-induced increases in NO release and cellular cGMP. The NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester decreased NO release, and pretreatment of cells with L-arginine reversed the effect. Cultured human cervical epithelial cells express messenger RNA for the NOS isoforms endothelial NOS (ecNOS), brain NOS, and inducible NOS. 17beta-Estradiol up-regulated ecNOS messenger RNA, and tamoxifen blocked the effect. Based on these results we suggest that the effect of estradiol on permeability involves four signaling steps: 1) activation of estrogen receptors, 2) increase in ecNOS transcription and up-regulation of NO activity, 3) NO activation of guanylate cyclase and increase in cGMP, and 4) cGMP activation of cGMP-dependent protein kinase.

Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888-C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of approximately 0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time approximately 4 h, and EC(50) approximately 1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor alpha (alphaER) and estrogen receptor beta (betaER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of alphaER and betaER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased betaER mRNA. In cells treated with estrogen, neither ICI-182780 nor tamoxifen had modulated significantly the increase in alphaER or betaER mRNA. The transcription inhibitor actinomycin D blocked the estrogen-induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an alphaER-dependent increase in transcription.

In human cervical cells, extracellular ATP induces an acute decrease in the resistance of the lateral intercellular space, the phase I response, followed by a delayed increase in tight junctional resistance, the phase II response. These responses depend on vitamin A because incubation of cells in retinoid-free medium (RFM) abolished both responses. Treatment with retinoic acid restored the phase I response in full, but the amplitude of the phase II response was restored only partly. Shorter incubations and lower concentrations of retinoic acid [half-maximal effective concentration ( K½) = 0.1 μM] were required for restoring the phase I response than were required for reversing the phase II response ( K½= 1 μM). The phase I response could be restored by ligands that bind to either retinoic acid receptors (RARs) or retinoid X receptors, but only RAR agonists had an effect on phase II response. RFM had no effect on decreases in resistance induced by ionomycin, but it attenuated phase II-like increases in resistance induced by KCl or by 1,2-dioctanoyl- sn-diglycerol (diC8). Actinomycin D blocked phase IIresponse but not phase I response or the responses to ionomycin, KCl, or diC8. These results suggest that retinoids act on cervical cells via distinct retinoid receptor mechanisms and modulate phase I and phase II changes in resistance by regulating distinct signal mechanisms.

Extracellular ATP stimulates a biphasic change in transepithelial electrical resistance (R TE ) across cultures of human cervical epithelial cells: an acute decrease (phase I), followed by a delayed increase in resistance (phase II). The objective of this study was to determine the contributions of changes in the lateral intercellular space resistance (R LIS ) and the tight junctional resistance (R TJ ) to the changes in R TE . Phase I and phase II effects were uncoupled by treatment with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester, which blocks the ATP-induced increases in cytosolic Ca 2+ and abolishes phase I. BAPTA-loaded cells differed from control cells in that 1) phase I began when ATP was added, in contrast to a delay of 1.5-3.5 min in phase II, 2) phase I decreases in R LIS followed a simple exponential pattern, in contrast to the complex kinetics of phase II, and 3) the magnitude of phase II varied between 20 and 100% for increases of R TJ in day 2-6 cultures; the phase I decrease of 50% in R LIS was unrelated to different experimental conditions. These results indicate that phase I and phase II are induced simultaneously and independently by ATP, and they contribute to the total changes in R TE . We conclude that ATP regulation of R LIS and R TJ may be important mechanisms of modulating cervical mucus production in vivo.

The P2X(7) receptor is an important regulator of epithelial cell growth. The aim of the present study was to better understand the biological significance of P2X(7) receptor expression in normal and cancer human epithelial tissues. P2X(7) receptor and messenger RNA (mRNA) levels were determined in human tissues containing epithelial dysplastic, pre- or early cancerous, and cancer cells, and the levels were compared to those in the corresponding normal epithelial cells within the same tissue of the same case. P2X(7) receptor levels were determined by quantification of immunoreactivity specific to the functional (full-length) P2X(7) receptor, and P2X(7) mRNA levels were determined by real-time polymerase chain reaction. P2X(7) receptor levels in cancer cells were similar (colon adenocarcinoma) or greater (thyroid papillary carcinoma) than those in the corresponding normal cells. In contrast, in cancer cells of the ectocervix (squamous), endocervix and endometrium (adenocarcinoma), urinary bladder (transitional cell carcinoma), and breast (ductal and lobular adenocarcinomas), P2X(7) receptor levels were lower by about twofold than those in the corresponding normal epithelial cells. Similarly, P2X(7) mRNA levels were lower in uterine, bladder, and breast cancer epithelial tissues by about fourfold than those in the corresponding normal tissues. In addition, P2X(7) receptor levels were decreased already in dysplastic ectocervical cells and pre- or early cancerous endometrial and bladder cells. The data suggest that in epithelia originating from the ectoderm, the uro-genital sinus, and the distal paramesonephric duct, decreased expression of the P2X(7) receptor precedes or coincides with neoplastic changes in those tissues.

The objective of the study was to understand how estrogen modulates the rigidity of the cytoskeleton in epithelial cells. Estrogen depletion decreased, and treatment with 17beta-estradiol increased deformability of cervical-vaginal epithelial cells. Estrogen also induced redistribution of nonmuscle myosin II-B (NMM-II-B); lesser interaction of NMM-II-B with actin; increased phosphorylation of NMM-II-B-heavy chains at threonine and serine residues; and decreased filamentation of NMM-II-B in vitro. The effects of 17beta-estradiol were time and dose related and could be mimicked by diethylstilbestrol. The effects of estrogen were blocked by cotreatment with antisense oligonucleotide for the estrogen receptor-alpha and inhibited by ICI-182,780 and tamoxifen; omission of epithelial growth factor (EGF) from the culture medium; and cotreatments with the EGF receptor inhibitor AG1478, the ERK-MAPK inhibitor PD98059, the casein kinase-II (CK2) inhibitor 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole, the Rho-associated kinase inhibitor Y-27632, and the nonspecific phosphatase inhibitor okadaic acid. Coadministration of 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole plus okadaic acid blocked the 17beta-estradiol effect. H-89 or LY294002 did not significantly affect estrogen effects. Treatment with estrogen increased activation of ERK1/2 and CK2 activity. These data suggest a novel pathway of estrogen regulation of the cytoskeleton in epithelial cells. The effect is mediated by estrogen receptor-alpha and involves in part the EGF-EGF receptor and ERK-MAPK cascades as proximal signaling networks and the CK2 and Rho-associated kinase-regulated myosin heavy chain phosphatase as terminal effectors. Augmented phosphorylation of NMM-II-B can block filamentation and induce disassociation of the myosin from the cortical actin, and disruption of the actomyosin ring can increase cell deformability. This mechanism can explain estrogen regulation of paracellular permeability in cervical-vaginal epithelia in vivo.

A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-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 P2X7 receptor. The P2X7-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 P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting hetero-oligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 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 main function of the acidic environment of the vaginal lumen is to control infections of the lower genital tract. The vaginal acid-base environment is tightly regulated, and during premenopausal years vaginal-fluid pH ranges from 4.5 to 5.5 with mild alkalinization to about 6.0 before ovulation. Despite its important role, relatively little is known about mechanisms that regulate vaginal-fluid pH in women. Until recently, it was believed that the acidic milieu is determined by cohabituating Doderlein lactobacilli.1 We recently presented data that refute the Doderlein theory by showing that human normal vaginal-ectocervical epithelial cells grown on filters under sterile conditions acidify their luminal solution.2 The effect was estrogen-dependent, and could be blocked by low micromolar concentrations of bafilomycin-A1, indicating active proton (H+) secretion by V-type H+-ATPase3 that is located in the apical membrane (Fig. 1). In this regard, vaginal epithelial cells resemble other H+ secreting cells that regulate acid secretion into the lumen.4-9 FIG. 1 Hypothesis of the present study. Vaginal-ectocervical epithelial cells acidify the lumen by active extrusion of protons via proton transport mechanisms located in the apical cell membrane. Tight junctions (filled horizontal bars) separate the plasma membranes ... After menopause, vaginal-fluid pH increases to 6.5 to 7.0.10 Elevated vaginal-fluid pH has been associated with urinary tract infections,11 vaginal atrophy,12 and dyspareunia.13 Treatment with estrogen can acidify the luminal vaginal pH to 5.5 to 6.5,10 suggesting that alkalinization of the vaginal-fluid pH in postmenopausal women is the result of hypoestrogenism. To test this hypothesis more directly, we used our novel culture system of filter generated vaginal-ectocervical cultured epithelia to determine effects of estrogen on luminal proton secretion in cells of postmenopausal women. The results indicate that treatment with estrogen increases active proton secretion by cells of postmenopausal women. However, luminal pH remained elevated compared with that of cells of premenopausal women, suggesting that in addition to hypoestrogenism, other factors of the aging process affect the capacity of vaginal-ectocervical cells to secrete acid.

Treatment of human cervical epithelial CaSki cells with ATP or with the diacylglyceride sn-1,2-dioctanoyl diglyceride (diC8) induced a staurosporine-sensitive transient increase, followed by a late decrease, in tight-junctional resistance (RTJ). CaSki cells express two immunoreactive forms of occludin, 65 and 50 kDa. Treatments with ATP and diC8 decreased the density of the 65-kDa form and increased the density of the 50-kDa form. ATP also decreased threonine phosphorylation of the 65-kDa form and increased threonine phosphorylation of the 50-kDa form and tyrosine phosphorylation of the 65- and 50-kDa forms. Staurosporine decreased acutely threonine and tyrosine phosphorylation of the two isoforms and in cells pretreated with staurosporine ATP increased acutely the density of the 65-kDa form and threonine phosphorylation of the 65-kDa form. Treatment with N-acetyl-leucinyl-leucinyl-norleucinal increased the densities of the 65- and 50-kDa forms. Pretreatment with N-acetyl-leucinyl-leucinyl-norleucinal attenuated the late decreases in RTJ induced by ATP and diC8 and the decrease in the 65-kDa and increase in the 50-kDa forms induced by ATP. Correlation analyses showed that high levels of RTJ correlated with the 65-kDa form, whereas low levels of RTJ correlated negatively with the 65-kDa form and positively with the 50-kDa form. The results suggest that in CaSki cells 1) occludin determines gating of the tight junctions, 2) changes in occludin phosphorylation status and composition regulate the RTJ, 3) protein kinase-C-mediated, threonine dephosphorylation of the 65-kDa occludin form increases the resistance of assembled tight junctions, 4) the early stage of tight junction disassembly involves calpain-mediated breakdown of occludin 65-kDa form to the 50-kDa form, and 5) increased levels of the 50-kDa form interfere with occludin gating of the tight junctions.

The objective of this study was to assess estrogen-dependent cellular mechanisms that could contribute to the acid pH of the vaginal lumen. Cultures of normal human cervical-vaginal epithelial (hECE) cells and endocervical cells were grown on filters, and acidification of the extracellular solutions on the luminal (L-pHo) and contraluminal (CL-pHo) sides was measured. The hECE cells and endocervical cells decreased CL-pHo from 7.40 to 7.25 within 20–30 min of incubation in basic salt solution. Endocervical cells also produced a similar decrease in L-pHo. In contrast, hECE cells acidified L-pHo down to pH 7.05 when grown as monoculture and down to pH 6.05 when grown in coculture with human cervical fibroblasts. This enhanced acid secretion into the luminal compartment was estrogen dependent because removal of endogenous steroid hormones attenuated the effect, whereas treatment with 17β-estradiol restored it. The 17β-estradiol effect was dose dependent (EC50 0.5 nm) and could be mimicked by diethylstilbestrol and in part by estrone and tamoxifen. Preincubation with ICI-182780, but not with progesterone, blocked the estrogen effect. Preincubation of cells with the V-ATPase blocker bafilomycin A1, when administered to the luminal solution, attenuated the baseline and estrogen-dependent acid secretion into the luminal solution. Treatment with EGTA, to abrogate the tight junctional resistance, blocked the decrease in L-pHo and stimulated a decrease in CL-pHo, indicating that the tight junctions are necessary for maintaining luminal acidification. We conclude that vaginal-ectocervical cells acidify the luminal canal by a mechanism of active proton secretion, driven in part by V-H+-ATPase located in the apical plasma membrane and that the baseline active net proton secretion occurs constitutively throughout life and that this acidification is up-regulated by estrogen.

The present study explored the effects of estrogen on transcervical tight-junctional resistance (R(TJ)) and the mechanisms involved. Treatment of cultured human cervical epithelial cells with 17beta-estradiol decreased in a time- and dose-related manner the R(TJ). Estrogen had no significant effect on the expression of E-Cadherin, zonula-occluden-1, or Claudin-4. In contrast, 17beta-estradiol modulated expression of the transmembrane tight-junctional protein occludin: at low concentrations (1 and 10 nm) estradiol increased the density of occludin 65-kDa form but at the higher concentration of 100 nm estradiol induced only a mild 2-fold increase in the density of this form. Estradiol also increased the expression of occludin 50-kDa form in a dose-related manner. The R(TJ) and occludin effects of estradiol were reversible and could be blocked by tamoxifen but not progesterone. The present results rule out estrogen modulation of occludin transcription. In contrast, the results suggest that the occludin effects of estrogen involve posttranslational up-regulation of occludin turnover, including synthesis and degradation. The effects of estrogen on occludin expression were compared with those of proteinase-K, plasmin, and matrix-metaloproteinase-2 (all added extracellularly). The three proteinases abrogated irreversibly the R(TJ) and induced expression de novo of occludin low-molecular-weight forms. The latter, however, differed from the effect of estrogen, which generated only a single 50-kDa form. Collectively, the present data suggest that the occludin 50-kDa form is an estrogen-specific-induced occludin isoform and that the mechanism of estrogen-abrogation of transcervical R(TJ) involves occludin modulation.

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 P2X7receptor 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 μM in hECE cells and ∼1 μM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-α, 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-α, it had only a mild effect on caspase-8. Both BzATP and TNF-α 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 ∼0.5 μM, which is in the range of values that suffice to activate the P2X7receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X7receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway.

Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures on filters, an acute transient increase ( phase I response; triggered by P2Y2receptor and mediated by calcium mobilization-dependent cell volume decrease) followed by a slower decrease in permeability ( phase II response). Phase II response is mediated by augmented calcium influx and protein kinase C-dependent increase in tight junctional resistance. The objective of the study was to determine the role of P2X4receptor as a mediator of phase II response. Human cervical epithelial cells express P2X4receptor mRNA (1.4-, 2.2-, and 4.4-kb isoforms by Northern blot analysis) and P2X4protein. Depletion of vitamin A reversibly downregulated P2X4receptor mRNA and protein and ATP-induced calcium influx. Depletion of vitamin A abrogated phase II response, and the effect could be partially reversed only with retinoic acid receptor (RAR)-selective retinoids but not retinoid X receptor (RXR) agonists. Depletion of vitamin A also abrogated protein kinase C increase in tight junctional resistance, and the effect could not be reversed with retinoids. Depletion of vitamin A also abrogated phase I increase in permeability and reversibly downregulated P2Y2receptor mRNA and ATP-induced calcium mobilization. However, in contrast to phase II response, both RAR and RXR agonists could fully reverse those effects. These results suggest that phase IIresponse is mediated by a P2X4receptor mechanism.

Objective: To determine the effects of menopause (aging and E) on vaginal-cervical epithelial paracellular permeability. Design: Experimental, basic clinical research. Setting: Academic research environment. Patient(s):Premenopausal, perimenopausal, and postmenopausal women, aged 35–65 years. Intervention(s): Primary to tertiary cultures of normal human ectocervical epithelial cells on filters. Cells were outgrown from surgically discarded ectocervical minces. Main Outcome Measurement(s): Changes in paracellular permeability were determined as changes in transepithelial electrical conductance and pyranine permeability. Result(s): [1] Levels of transepithelial electrical conductance and pyranine permeability decreased as women's age advanced. [2] Removal of E from the culture medium decreased paracellular permeability. Treatment of cells in vitro with 10 nmol/L 17β-E 2 increased transepithelial electrical conductance and pyranine permeability, but the effects were additive to the age-related decrease in permeability. [3] Coadministration of 100 nmol/L tamoxifen blocked the E increase in paracellular permeability in cells of both premenopausal and postmenopausal women. Conclusion(s): [1] Aging and E deficiency decrease independently vaginal-cervical epithelial paracellular permeability. [2] The E increase in vaginal-cervical epithelial paracellular permeability in cells of postmenopausal women is mediated by the E receptor. [3] The E increase in vaginal-cervical epithelial paracellular permeability in cells of postmenopausal women is masked by age-related increase in the tight junctional resistance, leading to overall decrease in paracellular permeability.

Estrogen increases secretion of cervical mucus in women, and the effect depends on fragmentation of the cytoskeleton. The objective of the present study was to understand the molecular mechanism of estrogen action. Treatment of human cervical epithelial cells with 17β-estradiol, sodium nitroprusside (SNP), or 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) increased cellular monomeric G-actin and decreased polymerized F-actin. The effects of estradiol were blocked by tamoxifen, by the guanylate cyclase inhibitor LY-83583, and by the cGMP-dependent protein kinase inhibitor KT-5823. The effects of SNP were blocked by LY-83583 and KT-5823, while the effects of 8-Br-cGMP were blocked only by KT-5823. Treatment with phalloidin decreased paracellular permeability and G-actin. Treatment with 17β-estradiol, SNP, or 8-Br-cGMP attenuated SNP-induced phosphorylation of [32P]adenylate NAD in vitro: tamoxifen blocked the effect of estrogen; LY-83583 blocked the effect of SNP but not that of 8-Br-cGMP, while KT-5823 blocked effects of both SNP and 8-Br-cGMP. These results indicate that estrogen, nitric oxide (NO), and cGMP stimulate actin depolymerization. A possible mechanism is NO-induced, cGMP-dependent protein kinase augmentation of ADP-ribosylation of monomeric actin.

Objective: To determine the effects of human seminal fluid on cervical paracellular resistance. Design: Experimental study. Setting: Healthy volunteers in an academic research environment; cultures of human CaSki cells on filters, with phenotypic characteristics of the endocervix. Patient(s): Healthy men donating sperm to a sperm bank. Intervention(s): Seminal fluid was obtained as the discarded fluid from ejaculates. Main Outcome Measure(s): Changes in transepithelial electrical resistance across CaSki cells on filters were determined in an Ussing chamber from successive measurements of the short-circuit current and the transepithelial potential difference. Changes in the dilution potential (and hence in the ratio of C1− to Na+ mobilities) were determined after lowering the NaCl concentration in the luminal solution. Result(s): Seminal fluid increased transepithelial electrical resistance acutely ( t 1 2 , 2 minutes), reversibly, and in a dose-related manner (ED50, 1%). The effect of seminal fluid was abolished when the extracellular calcium level was lowered, and the increase in transepithelial electrical resistance correlated with a decrease in the ratio of Cl− to Na+ mobilities, indicating an increase in the resistance of the tight junctional complex. The increase in transepithelial electrical resistance in response to seminal fluid was nonadditive to that of sn-1,2-dioctanoyl diglyceride (a stable diacylglyceride and activator of protein kinase C), and it was abolished by prolonged preincubation with the phorbol ester phorbol 12-myristate 13-acetate (to downregulate protein kinase C) or with staurosporin (to inhibit protein kinase C), suggesting that seminal fluid acts through a protein kinase C-dependent mechanism. Slower ( t 1 2 , 3.3 minutes) increases in transepithelial electrical resistance occurred when seminal fluid was added only to the luminal or the subluminal solution. Treatment with pertussis toxin, adenosine triphosphatase, or trypsin had no effect on the changes in transepithelial electrical resistance. Seminal fluid increased cytosolic calcium, but changes in cytosolic calcium are not important for the increases in transepithelial electrical resistance, suggesting that the effect of seminal fluid is not receptor-mediated. Preliminary studies indicate that the factor(s) in seminal fluid that increases transepithelial electrical resistance is a labile, low molecular weight ( Conclusion(s): Seminal fluid may regulate cervical mucus production in vivo by modulating endocervical permeability.

The purpose of the present study was to establish culture conditions for human uterine cervical epithelial cells on permeable support and to determine how it affects cervical cell differentiation. Human ectocervical epithelial cells (hECE), HPV-16 immortalized hECE cells (ECE16-1) and Caski cells were grown on collagen-coated filters. Culture conditions, density of cells in culture and expression of epithelial and cervical-cell phenotypic markers were determined and compared in cells grown on filter and on solid support. Compared with the latter, cultures on filter had a higher cell density, hECE cells stratified to 5–12 cell layers compared to 1–3 on solid support, and cells of all three types expressed intercellular tight junctions. The cytokeratin profiles revealed differences between the three cell types as well as differences within the same cell species when grown on filter, compared to solid support. Of particular importance was the finding of a higher expression of K-13 in hECE grown on filter compared to solid support; K-13 is a marker of ectocervical cell differentiation. The cytokeratin profiles of the cultured hECE, ECE16-1 and Caski cells resembled those of ectocervical, squamous metaplastic and endocervical epithelia, respectively. hECE and ECE16-1 expressed involucrin protein, the level of which in both was higher in cells grown on filter compared to solid support. Polarization of the cultures was determined by morphology (stratification of hECE cells, expression of pseudomicrovilli in the apical cell membrane), selective apical vs. basolateral secretion of [ 35 S]methionine- and [ 35 S]cys-teine-, [ 3 H]fucose- and [ 14 C]glucosamine-labeled mole-cules, and positive short-circuit current (I sc ) under voltage-clamp conditions. Confluency of the cultures was determined by measuring transepithelial unidirectional fluxes of inert molecules with different molecular weights (MWs) through the paracellular pathway, and by measuring transepithelial conductance. The results indicated transepithelial permeability of 7–22 · 10 −6 cm · sec −1 , which was 5–100 fold smaller compared to blank inserts, with a cut-off MW of 40–70 kDa for hECE and Caski cells. Transepithelial conductance ranged 18.5 to 51.5 mS · cm −2 , indicating a leaky but confluent epithelia. Collectively the results indicate the epithelial nature of the cells and their improved differentiation when grown on filter support; hECE is a model for ectocervical epithelium while ECE16-1 and Caski express phenotypic characteristics of squamous metaplastic cervical epithelium and endocervical epithelium, respectively.

Objective To determine the effects of single dose tamoxifen on plasma estrogen (E)-binding equivalents and endometrial estradiol (E 2 ) and progesterone (P) receptors. Design Controlled clinical study. Setting Normal human volunteers were studied in an academic research environment. Patients Premenopausal and postmenopausal women with histologically normal endometrium undergoing curettage or hysterectomy were selected. Interventions Tamoxifen was administered orally; blood and endometrial samples were collected 4 to 96hours after tamoxifen administration. Main Outcome Measures Plasma E-binding equivalents, endometrial cytosolic and nuclear E 2 and P receptors. Results (1) Plasma E-binding equivalents increased eightfold at 4 to 24hours of tamoxifen administration and declined exponentially thereafter, reaching control levels at 73 to 96hours. Plasma E-binding equivalents were not affected by endogenous E 2 levels. (2) Endometrial total E 2 and P receptor levels increased in all women 2.9 to 19.2-fold after tamoxifen. (3) Tamoxifen resulted in an increase in the fraction of the E 2 receptor measured in the nuclear extract 2.1 to 7.5-fold in midcycle, secretory, and menopausal endometria but not in proliferative endometrium. Conclusions (1) Tamoxifen has an E 2 agonistic effect on histologically normal human endometrium. (2) Irrespective of the total level of the endometrial E 2 receptor, the nuclear capacity of that receptor in vivo is limited (approximately 75% to 80% of the total level).

BackgroundThe study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X7receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X7system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X7action.MethodsSkin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas) were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X7-null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA). Changes in cytosolic calcium or in ethidium bromide influx (P2X7pore formation) were determined by confocal laser microscopy.Results(a) Co-application on the skin of the P2X7specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28) the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b) In the normal skin BzATP affected mainly P2X7-receptor – expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c) In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d) Levels of P2X7receptor, protein and mRNA were 4–5 fold lower in cancer tissues than in normal mouse tissues. (e) In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f) The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for BzATP. (g) Pore formation and the augmented calcium influx were depended on the expression of the P2X7receptor, while the BzATP-induced apoptosis depended on calcium influx. (h) The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8.Conclusion(a) P2X7-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X7-dependent apoptosis is mediated by calcium influx via P2X7pores, and involves the caspase-9 (mitochondrial) pathway. (c) The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X7receptor. (d) Activation of P2X7-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.