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Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Lesions and neurologic disability in inflammatory CNS diseases such as multiple sclerosis (MS) result from the translocation of leukocytes and humoral factors from the vasculature, first across the endothelial blood-brain barrier (BBB) and then across the astrocytic glia limitans (GL). Factors secreted by reactive astrocytes open the BBB by disrupting endothelial tight junctions (TJs), but the mechanisms that control access across the GL are unknown. Here, we report that in inflammatory lesions, a second barrier composed of reactive astrocyte TJs of claudin 1 (CLDN1), CLDN4, and junctional adhesion molecule A (JAM-A) subunits is induced at the GL. In a human coculture model, CLDN4-deficient astrocytes were unable to control lymphocyte segregation. In models of CNS inflammation and MS, mice with astrocyte-specific Cldn4 deletion displayed exacerbated leukocyte and humoral infiltration, neuropathology, motor disability, and mortality. These findings identify a second inducible barrier to CNS entry at the GL. This barrier may be therapeutically targetable in inflammatory CNS disease.

BackgroundDifferences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity.ObjectivesGrantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability.MethodsConsortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells.ResultsThe overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β.ConclusionsThe results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

We investigated the influence of extracellular matrix on transport properties of mouse alveolar epithelial cell (AEC) monolayers (MAECM) and transdifferentiation of isolated mouse alveolar epithelial type II (AT2) cells into an alveolar epithelial type I (AT1) cell-like phenotype. Primary mouse AT2 cells plated on laminin 5-coated polycarbonate filters formed monolayers with transepithelial resistance ([R.sub.T]) and equivalent short-circuit current ([I.sub.EQ]) of 1.8 k[OMEGA] x [cm.sup.2] and 5.3 [micro]A/[cm.sup.2], respectively, after 8 days in culture. Amiloride (10 [micro]M), ouabain (0.1 mM), and pimozide (10 [micro]M) decreased MAECM [I.sub.EQ] to 40%, 10%, and 65% of its initial value, respectively. Sequential addition of pimozide and amiloride, in either order, revealed that their inhibitory effects are additive, suggesting that cyclic nucleotide-gated channels contribute to amiloride-insensitive active ion transport across MAECM. Ussing chamber measurements of unidirectional ion fluxes across MAECM under short-circuit conditions indicated that net absorption of [Na.sup.+] in the apical-to-basolateral direction is comparable to net ion flux calculated from the observed short-circuit current: 0.38 and 0.33 [micro]eq x [cm.sup.-2] x [h.sup.-1], respectively. Between days 1 and 9 in culture, AEC demonstrated increased expression of aquaporin-5 protein, an AT1 cell marker, and decreased expression of pro-surfactant protein-C protein, an AT2 cell marker, consistent with transition to an AT1 cell-like phenotype. These results demonstrate that AT1 cell-like MAECM grown on laminin 5-coated polycarbonate filters exhibit active and passive transport properties that likely reflect the properties of intact mouse alveolar epithelium. This mouse in vitro model will enhance the study of AEC derived from mutant strains of mice and help define important structure-function correlations. alveolar epithelium; ion transport; short-circuit current; transepithelial resistance; phenotypic transition

We investigated mechanisms underlying GATA-6-mediated transcriptional activation of the alveolar epithelial type I cell-enriched gene aquaporin-5 (AQP5). GATA-6 expression increases in alveolar epithelial cells in primary culture, concurrent with upregulation of AQP5 and transition to a type I cell-like phenotype. Cotransfections in MLE-15 and NIH 3T3 cells demonstrated trans-activation by GATA-6 of a rat 1,716-bp-AQP5-luciferase (-1716-AQP5-Luc) reporter. Electrophoretic mobility shift assay and chromatin immunoprecipitation identified an interaction between GATA-6 and putative binding sites in the AQP5 promoter. However, mutation of these sites did not reduce GATA-6-mediated activation, implicating mechanisms in addition to direct binding of GATA-6 to DNA. A 5'-deletion construct, -358-AQP5-Luc, that does not encompass GATA motifs was still activated by GATA-6 by as much as 50% relative to -1716-AQP5-Luc. Internal deletion of the -358/--173 G-Crich domain, which includes several putative Spl consensus sites, reduced trans-activation by ~60%, suggesting importance of this region for GATA-mediated activity. -358-AQP5-Luc was similarly activated by both GATA-6 and a GATA DNA-binding defective mutant, whereas cotransfections in Schneider $2 cells demonstrated dose-dependent trans-activation of -358-AQP5-Luc by Spl. Activation of -358-AQP5-Luc by GATA-6 was dramatically reduced by Spl small-interfering RNA, and -358-AQP5-Luc was activated synergistically by GATA-6 and Spl in NIH 3T3 cells. Furthermore, association between endogenous GATA-6 and Spl was demonstrated by coimmunoprecipitation. These results suggest that transcriptional activation of AQP5 by GATA-6 is mediated at least in part through cooperative interactions with Spl occurring at the proximal promoter. transcription; alveolar epithelium; gene regulation; transfection; protein-protein interaction

We studied the capacity of adult human bone marrow-derived cells (BMDC) to incorporate into distal lung of immunodeficient mice following lung injury. Immunodeficient NOD/SCID and NOD/SCID/[[beta].sub.2] microglobulin [([[beta].sub.2]M).sup.null] mice were administered bleomycin (bleo) or saline intranasally. One, 2, 3 and 4 days after bleo or saline, human BMDC labeled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) were infused intravenously. Retention of [CMFDA.sup.+] cells was maximal when delivered 4 days after bleo treatment. Seven days after bleo, stromal-derived factor-1; CXCR4; CCXL12; homing; engraftment

We previously cloned a 4.3-kb genomic fragment encompassing 5'-flanking regulatory elements of rat aquaporin-5 (Aqp5) that demonstrated preferential transcriptional activity in lung and salivary cells in vitro. To investigate the ability of Aqp5 regulatory elements to direct transgene expression in vivo, transgenic (TG) mice and rats were generated in which the 4.3-kb Aqp5 fragment directed the expression of enhanced green fluorescent protein (EGFP). RT-PCR revealed relative promoter specificity for the lung and salivary glands in TG mice. Immunofluorescence microscopy showed strong EGFP expression in salivary acinar cells but not in lung type I (AT1) cells, both known sites of endogenous AQP5 expression. Similar results were obtained in TG rats generated by lentiviral transgenesis. EGFP mRNA was detected in both salivary glands and lung. Robust EGFP fluorescence was observed in frozen sections of the rat salivary gland but not in the lung or other tested tissues. The percentage of EGFP-positive acinar cells was increased in parotid and submandibular glands of TG rats receiving a chronic injection of the [beta]-adrenergic receptor agonist isoproterenol. EGFPpositive cells in the lung that were also reactive with the ATl-cell specific monoclonal antibody VIIIB2 were identified by flow cytometry. These findings demonstrate that the 4.3-kb Aqp5 promoter/enhancer directs strong cell-specific transgene expression in salivary gland and low-level AT1 cell-specific expression in the lung. While these Aqp5 regulatory elements should be useful for functional studies in salivary glands, additional upstream or intronic cis-active elements are likely required for robust expression in the lung. alveolar epithelium; isoproterenol; transcriptional regulation

We characterized immunoglobulin G (IgG) transport across rat alveolar epithelial cell monolayers cultured on permeable supports. Unidirectional fluxes of biotin-labeled rat IgG (biot-rIgG) were measured in the apical-to-basolateral (ab) and opposite (ba) directions as functions of [rIgG] in upstream fluids at 37 and 4[degrees]C. We explored specificity of IgG transport by measuring fluxes in the presence of excess Fc, Fab, F[(ab').sub.2], or chicken Ig (IgY). Expression of the IgG receptor FcRn and the effects of dexamethasone on FcRn expression and biot-rIgG fluxes were determined. Results show that ab flux of biot-rIgG is about fivefold greater than ba flux at an upstream concentration of 25 nM biot-rIgG at 37[degrees]C. Both ab and ba fluxes of rIgG saturate, resulting in net absorption with half-maximal concentration and maximal flow of 7.1 nM and 1.3 fmol x [cm.sup.-2] x [h.sup.-1]. At 4[degrees]C, both ab and ba fluxes significantly decrease, nearly collapsing net absorption. The presence of excess unlabeled Fc [but not Fab, F[(ab').sub.2], or IgY] significantly reduces biot-rIgG fluxes. RT-PCR demonstrates expression of [alpha]- and [beta]-subunits of rat FcRn. Northern analysis further confirms the presence of [alpha]-subunit of rat FcRn mRNA of ~1.6 kb. Dexamethasone exposure for 72 h decreases the steady-state level of mRNA for rat FcRn [alpha]-subunit and the ab (but not ba) flux of biot-rIgG. These data indicate that IgG transport across alveolar epithelium takes place via regulable FcRn-mediated transcytosis, which may play an important role in alveolar homeostasis in health and disease. receptor mediated; saturable transcytosis; net IgG absorption; lung defense; pulmonary immune system

Transforming growth factor-[beta]1 (TGF-[beta]1) may be a critical mediator of lung injury and subsequent remodeling during recovery. We evaluated the effects of TGF-[beta]1 on the permeability and active ion transport properties of alveolar epithelial cell monolayers. Rat alveolar type II cells plated on polycarbonate filters in defined serum-free medium form confluent monolayers and acquire the phenotypic characteristics of alveolar type I cells. Exposure to TGF-[beta]1 (0.1-100 pM) from day 0 resulted in a concentration- and time-dependent decrease in transepithelial resistance ([R.sub.t]) and increase in short-circuit current ([I.sub.sc]). Apical amiloride of basolateral ouabain on day 6 inhibited [I.sub.sc] by 80 and 100%, respectively. Concurrent increases in expression of [Na.sup.+]-[K.sup.+]-ATPase [[alpha.sub.1]- and [[beta].sub.1]-subunits were observed in TGF-[beta]1-treated monolayers. No change in the [alpha]-subunit of the rat epithelial sodium channel ([alpha]-rENaC) was seen. Exposure of confluent monolayers to TGF-[beta]1 from day 4 resulted in an initial decrease in [R.sub.t] within 6 h, followed by an increase in [I.sub.sc] over 72-96 h. These results demonstrate that TGF-[beta]1 modulates ion conductance and active transport characteristics of the alveolar epithelium, associated with increased [Na.sup.+]-[K.sup.+]-ATPase, but without a change in [alpha]-rENaC. transforming growth factor; alveolar epithelium; alveolar epithelial cells; epithelial sodium channel; sodium pump; acute lung injury

Transport characteristics of intact albumin were investigated using primary cultured rat alveolar epithelial cell monolayers. The apical-to-basolateral (ab) flux of intact fluorescein isothiocyanate (FITC)-labeled albumin (F-Alb) is greater than basolateral-to-apical (ba) flux at the same upstream [F-Alb]. Net absorption of intact F-Alb occurs with half-maximal concentration of ~1.6 [micro]M and maximal transport rate of ~0.15 fmol*[cm.sup.-2]*[s.sup.-1]. At 15 and 4[degrees]C, both ab and ba F-Alb fluxes are not different from zero, collapsing net absorption. The presence of excess unlabeled albumin (but not other macromolecule species) in either the apical or basolateral fluid significantly reduces both ab and ba unidirectional F-Alb fluxes. Photoaffinity labeling of apical cell membranes revealed an ~60-kDa protein that exhibits specificity for albumin. These data indicate that net absorption of intact albumin takes place via saturable receptor-mediated transcellular endocytotic processes recognizing albumin, but not other macromolecules, that may play an important role in alveolar homeostasis in the mammalian lung. protein transport; air-blood barrier; saturable transcytosis; albumin-binding sites; alveolar fluid balance

Hypoxia has been reported to inhibit activity and expression of ion transporters of alveolar epithelial cells. This study extended those observations by investigating the mechanisms underlying inhibition of active Na transport across primary cultured adult rat alveolar epithelial cell monolayers grown on polycarbonate filters. Cell monolayers were exposed to normoxia and hypoxia (1.5% and 5% [O.sub.2], 5% C[O.sub.2]), and resultant changes in bioelectric properties [i.e., short-circuit current ([I.sub.sc]) and transepithelial resistance ([R.sub.t])] were measured in Ussing chambers. Results showed that [I.sub.sc] decreased with duration of exposure to hypoxia, while relatively little change was observed for [R.sub.t]. In normoxia, amiloride inhibited ~70% of [I.sub.sc]. The amiloride-sensitive portion of [I.sub.sc] decreased over time of exposure to hypoxia, whereas the magnitude of the amiloride-insensitive portion of [I.sub.sc] was not affected. Na pump capacity measured aider permeabilization of the apical plasma membrane with amphotericin B decreased in monolayers exposed to 1.5% [O.sub.2] for 24 h, as did the capacity of amiloride-sensitive Na uptake measured aider imposing an apical to basolateral Na gradient and permeabilization of the basolateral membrane. These results demonstrate that exposure to hypoxia inhibits alveolar epithelial Na reabsorption by reducing the rates of both apical amiloride-sensitive Na entry and basolateral Na extrusion. alveolar type II cells; Ussing chambers; sodium channels; sodium pump; amphotericin B

We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined. Monolayers in HC[O.sup.-.sub.3]-free [Na.sup.+] buffer (140 mM [Na.sup.+], 6 mM HEPES, pH 7.4) maintained a transepithelial pH gradient between the two fluid compartments over 30 min. Replacement of apical fluid by acidic (6.4) or basic (8.0) buffer resulted in minimal changes in intracellular pH. Replacement of basolateral fluid by acidic or basic buffer resulted in transmembrane proton fluxes and intracellular acidification or alkalinization. Intracellular alkalinization was blocked [greater than or equal to] 80% by 100 [micro]M dimethylamiloride, an inhibitor of [Na.sup.+]/[H.sup.+] exchange, whereas acidification was not affected by a series of acid/base transport inhibitors. Additional experiments in which AEC monolayers were grown in the presence of acidic (6.4) or basic (8.0) medium revealed differential effects on bioelectric properties depending on whether extracellular pH was altered in apical or basolateral fluid compartments bathing the cells. Acid exposure reduced (and base exposure increased) short-circuit current from the basolateral side; apical exposure did not affect short-circuit current in either case. We conclude that AEC monolayers are relatively impermeable to transepithelial acid/base fluxes, primarily because of impermeability of intercellular junctions and of the apical, rather than basolateral, cell membrane. The principal basolateral acid exit pathway observed under these experimental conditions is [Na.sup.+]/[H.sup.+] exchange, whereas proton uptake into cells occurs across the basolateral cell membrane by a different, undetermined mechanism. These results are consistent with the ability of the alveolar epithelium to maintain an apical-to-basolateral (air space-to-blood) pH gradient in situ. alveolar epithelium; 2',T-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; sodium/hydrogen exchange; acidosis

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the [alpha]-subunit of the rat (r) epithelial Na channel ([alpha]-ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump [[alpha].sub.1]-subunit in AEC was assessed in situ. Na pump subunit isoform and [alpha]-rENaC expression was also evaluated by RT-PCR in highly purified (~95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-[[alpha].sub.1] and anti-[[beta].sub.1] Na pump sub-unit and anti-[alpha]-rENaC antibodies was detected, while reactivity with anti-[[alpha].sub.2] Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-[[alpha].sub.1] Na pump subunit antibody. Na pump [[alpha].sub.1]- and [[beta].sub.1] (but not [[alpha].sub.2]-) subunits and [alpha]-rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport. alveolar epithelium; sodium pumps; ion transport; immunofluorescence; sodium channel

A study was conducted to examine the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on TI1-alpha expression. AT2 cells were determined from Sprague-Dawley rats by elastase disaggregation while total RNA was obtained using the acid phenol-guanidinium-chloroform method. Results indicated that soluble factors and changes in cell shape influence T1-alpha expression in association with AEC phenotypes.

A study was conducted to investigate the influence of epidermal growth factor (EGF) on active Na2+ absorption by alveolar epithelium. Disaggregation was carried out to obtain cells from male Sprague-Dawley rats while total RNA was determined from monolayers using the acid phenol-guanidinium-chloroform technique. Results indicated that EGF supports active Na+ resorption across alveolar epithelial cell monolayers.

Epidermal growth factor (EGF) is involved in the regulation of cell permeability to water and solutes in the alveolar epithelium. EGF receptors in the basolateral side of these epithelial cells respond to EGF treatment by reducing tight junctional permeability while stimulating active transport of sodium ions. Sodium enters the cell through apical sodium channels and leaves through basolateral sodium pumps. The EGF receptor inhibitor tyrphostin RG-50864 prevented these effects.