Purinergic Signalling in the Reproductive System.

Purinergic effects in vivo are local, specific, transient, and affect target cells in a paracrine or autocrine manner. Purinergic signalling is involved in the regulation of numerous functions in the male and female reproductive tract organs. Analysis of functional expression of purinoceptors suggests that P2Y2 receptors are involved in the regulation of luminal fluid secretion in vivo; P2X1 and P2X2 in the regulation of smooth muscle cell contraction of blood vessels and tubular organs; P2X2 in the regulation of sperm cell maturation; P2X3 in activation of sensory nerve fibers involved in reflex activities and pain; P2X4 in the regulation of tight junctional resistance and epithelial transport in female reproductive tract epithelia, and in the regulation of luminal acidification in the epididymis, vas deferens and probably the vagina and ectocervix; P2Y1 and P2Y2 in the regulation of cell proliferation; P2X5 and P2X7A in the regulation of epithelial cell terminal differentiation and apoptosis; and A1 receptors in the regulation of sperm cell capacitation. Translational studies suggested that cellular levels of the P2X7A receptor could be used as a biomarker for the early detection of breast, ectocervix, endocervix, endometrial and bladder cancers. Data also suggested that the P2X7A mechanism could be used as a pharmaceutical target for the prevention and treatment of epithelial neoplasia through the induction of apoptosis.

P2X7-mediated chemoprevention of epithelial cancers.

BACKGROUND: Anti-apoptotic mechanisms contribute to the development of cancer. The purinergic ligand-gated ion channel receptor P2X(7) system is an important pro-apoptosis modulator in epithelial cells, and augmentation of P2X(7)-mediated apoptosis has been proposed as a novel pharmacological modality for chemoprevention and treatment of epithelial cancers. OBJECTIVE: This review focuses on the progress towards clinical application of therapies that directly modulate P2X(7)-mediated apoptosis. This paper reviews and discusses P2X(7)-mediated apoptosis in the context of prevention and growth-control of epithelial cancer cells, and summarizes recent data in the mouse skin model in vivo. METHODS: Data for this review were identified from the PubMed and MEDLINE databases. SUMMARY/CONCLUSIONS: The growing understanding of mechanisms of P2X(7)-mediated apoptosis has generated a novel strategy for targeting directly and specifically skin neoplasia. The data link directly, for the first time, upregulation of apoptosis with cancer chemoprevention. Striking antitumor efficacy has been achieved in the rodent cancer model, and it is likely that the recently gained wealth of knowledge about the mechanisms of P2X(7)-control of apoptosis will eventually be translated into new clinical therapies for epithelial cancers.

Regulation of P2X(7) gene transcription.

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.

Activation of P2X(7)-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice.

BACKGROUND: The study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X(7) receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X(7) system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X(7) action. METHODS: Skin 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 P2X(7)-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 (P2X(7) pore formation) were determined by confocal laser microscopy. RESULTS: (a) Co-application on the skin of the P2X7 specific 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 P2X(7)-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 P2X(7) receptor, 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 P2X(7) receptor, 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) P2X(7)-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X(7)-dependent apoptosis is mediated by calcium influx via P2X(7) pores, 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 P2X(7) receptor. (d) Activation of P2X(7)-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.

P2X(7) receptor expression is decreased in epithelial cancer cells of ectodermal, uro-genital sinus, and distal paramesonephric duct origin.

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.

MicroRNAs miR-186 and miR-150 down-regulate expression of the pro-apoptotic purinergic P2X7 receptor by activation of instability sites at the 3'-untranslated region of the gene that decrease steady-state levels of the transcript.

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.

Estrogen modulation of epithelial permeability in cervical-vaginal cells of premenopausal and postmenopausal women.

OBJECTIVE: To understand how menopause affects estrogen regulation of epithelial permeability. DESIGN: Experimental study using human normal epithelial vaginal-ectocervical cells obtained from premenopausal and postmenopausal women. Endpoints were paracellular permeability (determined in terms of the resistance of the lateral intercellular space [RLIS] and tight junctions [RTJ]); cellular G-actin; nonmuscle myosin type II-B (NMMII-B) filamentation and magnesium-adenosine triphosphatase activity; and occludin expression (in terms of expression of the functional 65-kd and truncated 50-kd forms). RESULTS: Estrogen induced an early transient decrease in RLIS that correlated in time with increases in cellular G-actin and NMMII-B magnesium-adenosine triphosphatase activity and with decreases in NMMII-B filamentation and a slower decrease in RTJ that correlated with up-regulation of the 50-kd form of occludin. Estrogen modulation of G-actin NMMII-B and occludin could be described in terms of interaction with the estrogen receptor mechanism. The potency of estrogen effects was similar in cells of premenopausal and postmenopausal women, but the effects occurred earlier in cells of premenopausal women. RLIS returned to baseline faster in cells of postmenopausal women, and the effect was associated with faster reversal of estrogen changes in NMMII-B despite the continued presence of estrogen in the culture medium, suggesting that desensitization of the actin-myosin effects to estrogen actions occur distal to the estrogen receptor. CONCLUSIONS: The results suggest that the transient estrogen decrease in RLIS is mediated by modulation of actomyosin, and it is affected by the aging process. In contrast, the late persistent decrease in RTJ is mediated by occludin degradation and is unrelated to aging.

Decreased expression of P2X7 in endometrial epithelial pre-cancerous and cancer cells.

OBJECTIVES: To understand the potential role of P2X(7) as biomarker of endometrial cancer, and the molecular mechanisms by which cancerous epithelial cells maintain low expression of P2X(7). 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 P2X(7) protein was average pixel signal density of tissue immunoreactivity with anti-P2X(7) antibody. Endpoint for P2X(7) 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)-P2X(7) cDNA, using as endpoint steady-state luciferase mRNA levels. RESULTS: Levels of P2X(7) 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<0.0001-0.01). Steady-state levels of luciferase mRNA increased over a 6 h incubation period in hEVEC cells transfected with the 3'UTR-P2X(7)-luciferase vector, but decreased in HeLa cells transfected with the reporter plasmid. CONCLUSIONS: Tissue levels of P2X(7) protein and mRNA can differentiate effectively and accurately between normal and benign hyperplastic endometrial tissues from pre-cancerous and cancer tissues. Cancerous epithelial cells degrade P2X(7) mRNA by activation of instability domains located at the 3'UTR of the P2X(7).

Estrogen decrease in tight junctional resistance involves matrix-metalloproteinase-7-mediated remodeling of occludin.

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.

Estrogen modulation of MgATPase activity of nonmuscle myosin-II-B filaments.

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.

The P2X7 receptor: a novel biomarker of uterine epithelial cancers.

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.

Estrogen regulates epithelial cell deformability by modulation of cortical actomyosin through phosphorylation of nonmuscle myosin heavy-chain II-B filaments.

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 P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization.

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 heterooligomerization 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.

Changes in tight junctional resistance of the cervical epithelium are associated with modulation of content and phosphorylation of occludin 65-kilodalton and 50-kilodalton forms.

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 (R(TJ)). 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 R(TJ) 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 R(TJ) correlated with the 65-kDa form, whereas low levels of R(TJ) 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 R(TJ), 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.

Effects of estrogen on proton secretion via the apical membrane in vaginal-ectocervical epithelial cells of postmenopausal women.

OBJECTIVE: To understand estrogen regulation of proton (H(+)) secretion by human vaginal-ectocervical epithelial cells and the mechanisms involved. DESIGN: Primary-tertiary cultures of human normal vaginal-ectocervical epithelial cells were generated from surgical specimens of premenopausal women (aged 37-46 years) and of postmenopausal women (aged 53-65 years). Cells were grown on filters, and measurements were made of changes in extracellular pH (pHo) in the contraluminal (CL) and luminal (L) solutions 30 minutes after shifting cells to basic salt solution. RESULTS: Upon shifting cells to basic salt solution, CL-pHo decreased from 7.4 to 7.25, and was not affected by removal of intracellular estrogens or treatment with estradiol. L-pHo decreased from 7.4 to 7.05 in cells of premenopausal women, and from 7.4 to 7.20 in cells of postmenopausal women. Removal of intracellular estrogens attenuated the decrease in L-pHo in cells of premenopausal women (only to 7.20). In cells of premenopausal women stripped of estrogens, treatment with 10 nM 17beta-estradiol restored the decrease in L-pHo. In estrogen-stripped cells of postmenopausal women, treatment with estradiol augmented luminal acidification but to a lesser degree than in cells of premenopausal women (L-pHo of 7.15 vs 7.05). In cells of pre- and postmenopausal women, the addition in the L solution of bafilomycin-A(1), a specific inhibitor of the vacuolar-H(+)-ATPase (V-H(+)-ATPase), blocked the decrease in L-pHo. CONCLUSIONS: Human vaginal-ectocervical epithelial cells acidify constitutively their luminal solution, and the effect is mediated by active H(+) secretion by V-H(+)-ATPase expressed predominantly in the apical cell membrane. Estrogen deprivation attenuates, and treatment with 17beta-estradiol augments, active H(+) secretion. Finally, cells of postmenopausal women actively secrete H(+) via apically located V-H(+)-ATPase, but the effect is lesser, and estrogen failed to augment active H(+) secretion, as in cells of premenopausal women. These data suggest that in addition to hypoestrogenism, other factors of the aging process affect the capacity of vaginal-ectocervical cells to secrete acid.

ATP stimulates GRK-3 phosphorylation and beta-arrestin-2-dependent internalization of P2X7 receptor.

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, beta-arrestin-2, and dynamin, and it stimulated beta-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-, beta-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.

Aging and estrogen effects on transcervical-transvaginal epithelial permeability.

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.

Estrogen acidifies vaginal pH by up-regulation of proton secretion via the apical membrane of vaginal-ectocervical epithelial cells.

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 17beta-estradiol restored it. The 17beta-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.

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

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.