Developmental Expression of Claudins in the Mammary Gland.

Claudins are a large family of membrane proteins whose classic function is to regulate the permeability of tight junctions in epithelia. They are tetraspanins, with four alpha-helices crossing the membrane, two extracellular loops, a short cytoplasmic N-terminus and a longer and more variable C-terminus. The extracellular ends of the helices are known to undergo side-to-side (cis) interactions that allow the formation of claudin polymers in the plane of the membrane. The extracellular loops also engage in head-to-head (trans) interactions thought to mediate the formation of tight junctions. However, claudins are also present in intracellular structures, thought to be vesicles, with less well-characterized functions. Here, we briefly review our current understanding of claudin structure and function followed by an examination of changes in claudin mRNA and protein expression and localization through mammary gland development. Claudins-1, 3, 4, 7, and 8 are the five most prominent members of the claudin family in the mouse mammary gland, with varied abundance and intracellular localization during the different stages of post-pubertal development. Claudin-1 is clearly localized to tight junctions in mammary ducts in non-pregnant non-lactating animals. Cytoplasmic puncta that stain for claudin-7 are present throughout development. During pregnancy claudin-3 is localized both to the tight junction and basolaterally while claudin-4 is found only in sparse puncta. In the lactating mouse both claudin-3 and claudin-8 are localized at the tight junction where they may be important in forming the paracellular barrier. At involution and under challenge by lipopolysaccharide claudins -1, -3, and -4 are significantly upregulated. Claudin-3 is still colocalized with tight junction molecules but is also distributed through the cytoplasm as is claudin-4. These largely descriptive data provide the essential framework for future mechanistic studies of the function and regulation of mammary epithelial cell claudins.

Claudin-4 activity in ovarian tumor cell apoptosis resistance and migration.

BACKGROUND: Claudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile. The objective of this study was to determine the functional role that claudin-4 plays in apoptosis resistance and migration as well as the therapeutic utility of targeting claudin-4 activity with a small mimic peptide. METHODS: We examined claudin-4 activity in human ovarian tumor cell lines (SKOV3, OVCAR3, PEO4) using in vitro caspase and scratch assays as well as an in vivo mouse model of ovarian cancer. Claudin-4 activity was disrupted by treating cells with a small peptide that mimics the DFYNP sequence in the second extracellular loop of claudin-4. Claudin-4 expression was also altered using shRNA-mediated gene silencing. RESULTS: Both the disruption of claudin-4 activity and the loss of claudin-4 expression significantly increased tumor cell caspase-3 activation (4 to 10-fold, respectively) in response to the apoptotic inducer staurosporine and reduced tumor cell migration by 50 %. The mimic peptide had no effect on cells that lacked claudin-4 expression. Female athymic nude mice bearing ZsGreen-PEO4 ovarian tumors showed a significant decrease in ovarian tumor burden, due to increased apoptosis, after treatment with intraperitoneal injections of 4 mg/kg mimic peptide every 48 h for three weeks, compared to control peptide treated mice. CONCLUSION: Claudin-4 functionally contributes to both ovarian tumor cell apoptosis resistance and migration and targeting extracellular loop interactions of claudin-4 may have therapeutic implications for reducing ovarian tumor burden.

Claudins play a role in normal and tumor cell motility.

BACKGROUND: Claudins are key integral proteins of the tight junction. Although they play an essential role in controlling paracellular diffusion in epithelia, increasing evidence supports a role for these proteins in non-barrier forming activities. To elucidate a potential function for claudins outside of their traditional role in tight junctions, subcellular localization of claudin-4 was determined in normal mammary epithelial cells as well as breast and ovarian cancer cell lines and the effects of a claudin mimic peptide on cell motility were determined. RESULTS: Immunofluorescence revealed that claudin-4 was localized along cellular projections. Using a fluorescent peptide that mimics a conserved sequence in the second extracellular loop of a set of claudin subtypes, that includes claudin-4, exposure of this loop to the extracellular environment was confirmed in non-polarized cells. This peptide inhibited cell motility when normal mammary epithelial cells as well as breast and ovarian tumor cells were subjected to a wound healing assay. Knockdown of claudin-4 also inhibited cell motility and the mimic peptide had no effect on motility in the claudin-4 knockdown cells. This effect on motility was seen when cells were grown on collagen, but not when cells were grown on non-physiological cell adhesive or fibronectin. CONCLUSION: The second extracellular loop of claudins is able to interact with the extracellular environment to promote normal and tumor cell motility when it is not associated with tight junction structures.

Claudin-4 localization in epithelial ovarian cancer.

Claudin-4, a protein with the structure of classic claudins most often found in cell-cell junctions, is frequently overexpressed in epithelial cancers where its localization has not been studied. In this study we aimed to find out where this membrane protein is localized in an ovarian tumor model, OVCAR3 cells, that express high levels of the protein. Immunohistochemical studies showed claudin-4 staining in a perinuclear region, at most plasma membranes and in cytoplasmic puncta. Native claudin-4 did not overlap with phosphorylated claudin-4, which was partially located in focal adhesions. Using claudin-4 BioID technology we confirmed that large amounts of claudin-4 are localized to the Golgi compartment, including in dispersed Golgi in cells where claudin-4 is partially knocked down and in dividing cells. Claudin-4 appears to be present in the vicinity of several types of cell-cell junctions, but there is no evidence that it forms tight junctions in these tumor cells. Both claudin-4, the Golgi marker GM130, and the plasma membrane receptor Notch2 were found in dispersed Golgi in dividing cells. This definition of the cellular architecture of claudin-4 should provide a framework for better understanding of the function of claudin-4 in tumor cells and its molecular interactions.

Loss of Claudin-4 Reduces DNA Damage Repair and Increases Sensitivity to PARP Inhibitors.

High-grade serous ovarian cancer is the deadliest gynecologic malignancy due to progression to resistant disease. Claudin-4 is classically defined as a tight junction protein and is often associated with epithelial cancers. Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors and conveys a worse overall prognosis. Elevated claudin-4 expression correlates to increased DNA repair activity and resistance to DNA damaging agents. PARP inhibitors are emerging as an effective therapeutic option for patients with ovarian cancer and function by promoting DNA damage. The study examines the relationship between claudin-4 expression and the response to PARP inhibitors using both genetic and pharmacologic inhibition of claudin-4 in in vitro and ex vivo models of ovarian cancer to examine DNA repair markers and functional activity. Genetic inhibition of claudin-4 results in the downregulation of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4 knockdown did not change homology-directed repair but inhibited nonhomologous end-joining and reduced 53BP1 foci formation. In 15 primary ovarian cancer tumors, higher claudin-4 expression significantly correlated to a dampened PARP inhibitor-mediated antiproliferation response. Further, claudin-4 inhibition in high claudin-4 tumors sensitized tumor sections to PARP inhibition. These data highlight that claudin-4 expression in ovarian cancer tumors could serve as both a marker of PARP inhibitor response and a therapeutic target to improve PARP inhibitor response.

Calcium elevation in mitochondria is the main Ca2+ requirement for mitochondrial permeability transition pore (mPTP) opening.

We have investigated in detail the role of intra-organelle Ca2+ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca2+ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca2+ responses because of the release of Ca2+ from both ER and acidic stores. Ca2+ responses to menadione were accompanied by elevation of Ca2+ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca2+ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca2+ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca2+ with 10 mum RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam. We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.

Disruption of occludin function in polarized epithelial cells activates the extrinsic pathway of apoptosis leading to cell extrusion without loss of transepithelial resistance.

BACKGROUND: Occludin is a tetraspanin protein normally localized to tight junctions. The protein interacts with a variety of pathogens including viruses and bacteria, an interaction that sometimes leads to its extrajunctional localization. RESULTS: Here we report that treatment of mammary epithelial monolayers with a circularized peptide containing a four amino acid sequence found in the second extracellular loop of occludin, LHYH, leads to the appearance of extrajunctional occludin and activation of the extrinsic apoptotic pathway. At early times after peptide treatment endogenous occludin and the LYHY peptide were co-localized in extrajunctional patches, which were also shown to contain components of the death inducing signaling complex (DISC), caspases 8 and 3, the death receptor FAS and the adaptor molecule FADD. After this treatment occludin could be immunoprecipitated with FADD, confirming its interaction with the DISC. Extrusion after LYHY treatment was accomplished with no loss of epithelial resistance. CONCLUSION: These observations provide strong evidence that, following disruption, occludin forms a complex with the extrinsic death receptor leading to extrusion of apoptotic cells from the epithelial monolayer. They suggest that occludin has a protective as well as a barrier forming role in epithelia; pathogenic agents which utilize this protein as an entry point into the cell might set off an apoptotic reaction allowing extrusion of the infected cell before the pathogen can gain entry to the interstitial space.

Ovarian Tumor Cell Expression of Claudin-4 Reduces Apoptotic Response to Paclitaxel.

A significant factor contributing to poor survival rates for patients with ovarian cancer is the insensitivity of tumors to standard-of-care chemotherapy. In this study, we investigated the effect of claudin-4 expression on ovarian tumor cell apoptotic response to cisplatin and paclitaxel. We manipulated claudin-4 gene expression by silencing expression [short hairpin RNA (shRNA)] in cells with endogenously expressed claudin-4 or overexpressing claudin-4 in cells that natively do not express claudin-4. In addition, we inhibited claudin-4 activity with a claudin mimic peptide (CMP). We monitored apoptotic response by caspase-3 and Annexin V binding. We examined proliferation rate by counting the cell number over time as well as measuring the number of mitotic cells. Proximity ligation assays, immunoprecipitation (IP), and immunofluorescence were performed to examine interactions of claudin-4. Western blot analysis of tubulin in cell fractions was used to determine the changes in tubulin polymerization with changes in claudin-4 expression. Results show that claudin-4 expression reduced epithelial ovarian cancer (EOC) cell apoptotic response to paclitaxel. EOCs without claudin-4 proliferated more slowly with enhanced mitotic arrest compared with the cells expressing claudin-4. Furthermore, our results indicate that claudin-4 interacts with tubulin, having a profound effect on the structure and polymerization of the microtubule network. In conclusion, we demonstrate that claudin-4 reduces the ovarian tumor cell response to microtubule-targeting paclitaxel and disrupting claudin-4 with CMP can restore apoptotic response. IMPLICATIONS: These results suggest that claudin-4 expression may provide a biomarker for paclitaxel response and can be a target for new therapeutic strategies to improve response.

Characterization of choline transporters in the human placenta over gestation.

INTRODUCTION: The developing fetus relies on the maternal blood supply to provide the choline it requires for making membrane lipids, synthesizing acetylcholine, and performing important methylation reactions. It is vital, therefore, that the placenta is efficient at transporting choline from the maternal to the fetal circulation. Although choline transporters have been found in term placenta samples, little is known about what cell types express specific choline transporters and how expression of the transporters may change over gestation. The objective of this study was to characterize choline transporter expression levels and localization in the human placenta throughout placental development. METHODS: We analyzed CTL1 and -2 expression over gestation in human placental biopsies from 6 to 40 weeks gestation (n = 6-10 per gestational window) by immunoblot analysis. To determine the cellular expression pattern of the choline transporters throughout gestation, immunofluorescence analysis was then performed. RESULTS: Both CTL1 and CTL2 were expressed in the chorionic villi from 6 weeks gestation to term. Labor did not alter expression levels of either transporter. CTL1 localized to the syncytial trophoblasts and the endothelium of the fetal vasculature within the chorionic villous structure. CTL2 localized mainly to the stroma early in gestation and by the second trimester co-localized with CTL1 at the fetal vasculature. DISCUSSION: The differential expression pattern of CTL1 and CTL2 suggests that CTL1 is the key transporter involved in choline transport from maternal circulation and both transporters are likely involved in stromal and endothelial cell choline transport.

A D-peptide analog of the second extracellular loop of claudin-3 and -4 leads to mislocalized claudin and cellular apoptosis in mammary epithelial cells.

Claudins are cell adhesion proteins thought to mediate cell-cell contacts at the tight junction. Although a major role of claudins is to control paracellular diffusion, increasing evidence suggests that they may also function in tumor progression. To examine the role of the second extracellular loop in cell adhesion, a small peptide was designed, which mimics a conserved sequence, DFYNP, within specific 'classic' claudin subtypes. Using fluorescent indicators with mammary epithelial cells, treatment with both the L- and D-forms of this peptide showed mislocalization of claudin-4 and claudin-3 and activation of caspase-8 and caspase-3, indicating apoptosis. To test specificity, peptides were made both with various end-groups and with glycine substitutions at each of the five residues. Changing end-groups did not influence the activity of the peptide. Amino acid substitutions at F147, Y148, N149, or P150, however, prevented peptide activity. A fluorescent-labeled peptide was shown to associate with the tight junction at 4 °C and cause apoptosis when the cultures were warmed to 37 °C. In conclusion, both the D- and L-forms of a small peptide that mimics a sequence in the second extracellular loop of claudins can target and disrupt claudin proteins in an epithelial monolayer and initiate apoptosis.

Disruption of the Cox-1 gene slows repair of microscopic lesions in the mouse gastric epithelium.

Cyclooxygenase-1 (Cox-1) contributes to gastric defense of healthy tissue, but the role in the protection of the gastric epithelium after minor, acute damage has been difficult to study in vivo. Using 710-nm two-photon light absorption to create microscopic gastric damage in anesthetized mice with the gastric mucosal surface surgically exposed and perfused on the microscope stage, the acute response of surface cells to injury could be monitored using in vivo microscopy within seconds after injury. Using exogenous (Cl-NERF) and endogenous fluorophores, extracellular pH and cell death were monitored in real time during the entire damage and repair cycle. Two-photon damage was initiated by scanning approximately 200 microm(2) of gastric surface cells with high laser intensity, causing rapid bleaching of NAD(P)H fluorescence in optically targeted cells. In both Cox-1(+/-) and Cox-1(-/-) mice, a similar initial damage area expanded to include bystander epithelial cells over the next 2-5 min, with larger maximal damage noted in Cox-1(-/-) mice. The maximal damage size seen in Cox-1(-/-) mice could be reduced by exogenous dimethyl-PGE(2). All damaged cells exfoliated, and the underlying epithelium was coincidently repaired over a time interval that was briefer in Cox-1(+/-) (12 +/- 2 min, n = 12) than in Cox-1(-/-) (24 +/- 4 min, n = 14) mice. Directly after damage, pH increased transiently in the juxtamucosal layer (maximal at 3-6 min). A smaller peak pH change was noted in Cox-1(-/-) mice (DeltapH = 0.3 +/- 0.04) than in Cox-1(+/-) mice (DeltapH = 0.6 +/- 0.2). Recovery to normal surface pH took longer in Cox-1(-/-) mice (27 +/- 5 min) than in Cox-1(+/-) mice (12 +/- 1 min). In conclusion, constitutive loss of Cox-1 leaves the gastric mucosa more prone to damage and slowed repair of microlesions.

Caspase-8-mediated apoptosis induced by oxidative stress is independent of the intrinsic pathway and dependent on cathepsins.

Cell-death programs executed in the pancreas under pathological conditions remain largely undetermined, although the severity of experimental pancreatitis has been found to depend on the ratio of apoptosis to necrosis. We have defined mechanisms by which apoptosis is induced in pancreatic acinar cells by the oxidant stressor menadione. Real-time monitoring of initiator caspase activity showed that caspase-9 (66% of cells) and caspase-8 (15% of cells) were activated within 30 min of menadione administration, but no activation of caspase-2, -10, or -12 was detected. Interestingly, when caspase-9 activation was inhibited, activation of caspase-8 was increased. Half-maximum activation (t(0.5)) of caspase-9 occurred within approximately 2 min and was identified at or in close proximity to mitochondria, whereas t(0.5) for caspase-8 occurred within approximately 26 min of menadione application and was distributed homogeneously throughout cells. Caspase-9 but not caspase-8 activation was blocked completely by the calcium chelator BAPTA or bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore. In contrast, caspase-8 but not caspase-9 activation was blocked by the destruction of lysosomes (preincubation with Gly-Phe beta-naphthylamide, a cathepsin C substrate), loss of lysosomal acidity (bafilomycin A1), or inhibition of cathepsin L or D. Using pepstatin A-BODIPY FL conjugate, we confirmed translocation of cathepsin D out of lysosomes in response to menadione. We conclude that the oxidative stressor menadione induces two independent apoptotic pathways within pancreatic acinar cells: the classical mitochondrial calcium-dependent pathway that is initiated rapidly in the majority of cells, and a slower, caspase-8-mediated pathway that depends on the lysosomal activities of cathepsins and is used when the caspase-9 pathway is disabled.

Regulated alkali secretion acts in tandem with unstirred layers to regulate mouse gastric surface pH.

BACKGROUND & AIMS: The physical basis for the protective pH gradient at the gastric surface is unconfirmed. This study examined the role of mucus, the unstirred layer, and acid/alkali secretion in controlling gastric surface pH in vivo. METHODS: Stomachs of anesthetized mice were exteriorized, and exposed gastric mucosa was imaged by confocal microscopy. RESULTS: Accessibility of molecules at the gastric surface was determined by monitoring the decrease in probe fluorescence over time after dyes were removed from perfusate. On dye removal, Cl-NERF (400 molecular weight) fluorescence decreased more slowly at the gastric surface in the presence of mucus (rate constant [k] = 0.08 +/- 0.02 per second) than in the absence of mucus (k = 0.15 +/- 0.02 per second) or 90 microm distant from the surface (k = 0.22 +/- 0.03 per second). In contrast, 70-kilodalton Cl-NERF/dextran washed from the gastric surface more slowly in the presence (k = 0.05 +/- 0.01 per second) or absence (k = 0.09 +/- 0.01 per second) of mucus compared with 90 microm from the tissue surface (k = 0.36 +/- 0.08 per second). Two-photon uncaging of fluorescein near nonsuperfused gastric surface showed that diffusion was not slowed at the gastric surface compared with diffusion in free solution. Surface pH was measured by Cl-NERF ratio imaging. Increasing the superfusion rate decreased the thickness of the surface pH gradient without significantly changing surface pH values, suggesting a pH set point of approximately 4 for control of surface pH. Increasing perfusate pH buffers decreased surface pH toward perfusate values. CONCLUSIONS: Proton concentration at the gastric surface is the result of regulating acid/alkali secretion to a set point in combination with an unstirred layer and not by trapping of proton or small-molecular-weight buffers in the unstirred layer.

Coordinated regulation of gastric chloride secretion with both acid and alkali secretion.

Gastric secretion of hydrochloric acid requires protons and chloride, yet the mechanisms and regulation of gastric chloride secretion remain unclear. We developed an in vivo technique to simultaneously measure acid/base and chloride secretion into the gastric lumen of anesthetized rats. The cannulated stomach lumen was perfused with weakly pH-buffered chloride-free solution containing a chloride-sensitive fluorophore [5 microM N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)]. Gastric acid and chloride secretion was detected in gastric effluents by 1) flow-through pH electrode and 2) MQAE fluorescence. Gastric effluent was also collected at 1-min intervals for independent determination of chloride amount by chloridometer. In all conditions, both optical and chemical determinations of chloride report similar amounts of secreted chloride. During luminal perfusion with pH 5 solution, net acid and chloride secretion into the lumen was observed. Pentagastrin stimulated both secretions. In contrast, proton pump inhibition (omeprazole) caused alkalinization of the gastric effluent, but chloride secretion was not diminished. During luminal pH 3 perfusion, net alkali secretion was observed, and chloride secretion at luminal pH 3 was greater than pH 5. When tissue is pretreated with omeprazole at luminal pH 3, the addition of prostaglandin E2 synchronously stimulates both alkali and chloride secretion. Results suggest that both acid and alkali secretions are separately coupled with chloride secretion.

Endogenous cyclo-oxygenase activity regulates mouse gastric surface pH.

In the stomach, production of prostaglandins by cyclo-oxygenase (COX) is believed to be important in mucosal defence. We tested the hypothesis that endogenous COX activity is required for protective gastric surface pH control. Intact stomachs of anaesthetized mice were perfused with a weakly buffered solution (150 mM NaCl + 4 mM Homopipes) at pH values from 2.5 to 7.0. Gastric effluents were collected to measure pH and estimate amounts of acid or alkali secretion in nanomoles secreted per minute. A switch from net acid to net alkali secretion was seen in response to acidifying luminal pH with an apparent 'set point' between pH 4 and 5. At luminal pH 3, the net alkali secretion (12.7 +/- 2.8 nmol OH(-) equivalents min(-1)) was abolished (2.2 +/- 1.7 nmol OH(-) min(-1)) by the non-specific COX inhibitor indomethacin (5 mg kg(-1) I.P.). Similar inhibition was observed using a COX-1 inhibitor (SC-560; 10 mg kg(-1) I.P.), but not a COX-2 inhibitor (NS-398; 10 mg kg(-1) I.P.). Subsequent treatment with 16,16-dimethyl prostaglandin E(2) (dm-PGE(2); 1 mg kg(-1) I.P.) rescued the alkali secretion (21.8 +/- 2.7 nmol OH(-) min(-1)). In either the absence or presence of the H(+),K(+)-ATPase inhibitor omeprazole (60 mg kg(-1) I.P.), indomethacin blocked similar amounts of net alkali secretion (10.5 +/- 2.7 and 16.4 +/- 3.4 nmol OH(-) min(-1), respectively). We also used in vivo confocal microscopy to examine pH near the mucosal surface. The gastric mucosal surface of anaesthetized mice was exposed and mucosal surface pH was imaged using the fluorescence intensity ratio of Cl-NERF as a pH indicator. Results showed a switch from a continuous net acid to net alkali secretion by the stomach in response to changing superfusate pH from 5 to 3. At luminal pH 3, the relatively alkaline surface pH (4.3 +/- 0.1) was acidified (3.6 +/- 0.2) by indomethacin, and subsequent dm-PGE(2) restored surface pH (4.2 +/- 0.2). We conclude that the pre-epithelial alkaline layer is regulated by endogenous COX activity.