Escherichia coli-induced epithelial hyporesponsiveness to secretagogues is associated with altered CFTR localization

Both pathogenic and commensal strains of Escherichia coli colonize the human intestinal tract. Pathogenic strains differ only in the expression of virulence factors, many of which comprise a type III secretion system (TTSS). Little is known regarding the effect of E. coli on the intestinal epithelial response to the secretagogues that drive ion secretion, despite its importance in causing clinically significant diarrhoea. Using Ussing chambers to measure electrogenic ion transport of T84 intestinal epithelial cell monolayers, we found that all strains of E. coli tested (pathogenic, commensal, probiotic and lab strain) significantly reduced cAMP-dependent ion secretion after 4-8 h exposure. Enteropathogenic E. coli mutants lacking a functional TTSS caused similar hyposecretion while not causing significant apoptosis (as shown by caspase-3 cleavage) or necrosis (lactate dehydrogenase release), as did the commensal strain F18, indicating that epithelial cell death was not the cause of hyposecretion. Enteropathogenic E. coli and the TTSS mutant significantly reduced cell surface expression of the apical anion channel, cystic fibrosis transmembrane conductance regulator, which is likely the mechanism behind the pathogen-induced hyposecretion. However, F18 did not cause cystic fibrosis transmembrane conductance regulator mislocalization and the commensal-induced mechanism remains unclear.     

CFTR targeting in epithelial cells

We used polarized and nonpolarized colonic cell lines (HT-29) to correlate CFTR function and expression with epithelial cell morphogenesis. Unpolarized cells express levels of CFTR mRNA and protein that are equivalent to those observed in polarized cells, and the extent of CFTR glycosylation is also similar. Despite these similarities in CFTR expression, the polarized cells secreted Cl in response tocAMP, but there was nocAMP-stimulated Cl conductance response in the unpolarized cells. In the polarized cells, CFTR is localized in the apical membrane domain, but in unpolarized cells the protein is retained at a perinuclear location. These findings indicate that a peripheral targeting mechanism, distal to the Golgi cisternae, controls the progression of N-linked glycoproteins like CFTR to the apical membrane. This targeting process does not become active until epithelial cells polarize. It may determine whether mutant forms of CFTR are targeted to the apical membrane.     

Differential effects of Matrigel and its components on functional activity of CFTR and ENaC in mouse endometrial epithelial cells

Our previous studies have observed an effect of Matrigel, a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, on the expression of ion channels in mouse endometrial epithelia; namely the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-)channel, and the epithelial sodium channel (ENaC). The present study further investigated the effects of Matrigel and its individual components on the functional expression of CFTR and ENaC using the short-circuit current (Isc) technique. The results showed that different components of Matrigel, namely growth factors, laminin and collagen, had differential effects on the functional activity of the two ion channels in murine endometrial epithelium. The information obtained may be useful for designing future in vitro culture models to investigate the functional roles of these ion channels in the endometrium.     

cAMP-activated anion conductance is associated with expression of CFTR in neonatal mouse cardiac myocytes

In this study,patch-clamp techniques were applied to cultured neonatal mouse cardiacmyocytes (NMCM) to assess the contribution of cAMP stimulation to theanion permeability in this cell model. Addition of either isoproterenolor a cocktail to raise intracellular cAMP increased the whole cellcurrents of NMCM. The cAMP-dependent conductance was largely anionic,as determined under asymmetrical (low intracellular)Cl conditions and symmetrical Clin the presence of various counterions, including Na⁺,Mg²⁺, Cs⁺, andN-methyl-D-glucamine. Furthermore, thecAMP-stimulated conductance was also permeable to ATP. ThecAMP-activated currents were inhibited by diphenylamine-2-carboxylate,glibenclamide, and an anti-cystic fibrosis transmembrane conductanceregulator (CFTR) monoclonal antibody. The anti-CFTR monoclonal antibodyfailed, however, to inhibit an osmotically activated anion conductance,indicating that CFTR is not linked to osmotically stimulated currentsin this cell model. Immunodetection studies of both neonatal mouse heart tissue and cultured NMCM revealed that CFTR is expressed in thesepreparations. The implication of CFTR in the cAMP-stimulated Cl- and ATP-permeable conductance was furtherverified with NMCM of CFTR knockout mice[cftr(/)] in which cAMP stimulationwas without effect on the whole cell currents. In addition, stimulation with protein kinase A and ATP induced Cl-permeablesingle-channel activity in excised, inside-out patches from control,but not cftr(/) NMCM. The data in this report indicate that cAMP stimulation of NMCM activates an anion-permeable conductance with functional properties similar to those expected forCFTR, thus suggesting that CFTR may be responsible for the cAMP-activated conductance. CFTR may thus contribute to the permeation and/or regulation of Cl- and ATP-permeable pathwaysin the developing heart.

     

Hepatocyte growth factor is a pleiotropic factor protecting epithelial cells from apoptosis

Hepatocyte Growth Factor (HGF) is a pleiotropic factor, capable of evoking complex biological responses such as mitogenesis, motogenesis and morphogenesis in a variety of epithelial and endothelial cells. Nonetheless, the meaning of the acronym is consistent with the key role of the factor in liver regeneration, in vivo and in liver development during embryogenesis. The receptor for HGF is the tyrosine kinase encoded by the c-MET proto-oncogene. Upon ligand binding, the receptor kinase is activated by tyrosine autophosphorylation and recruits cytoplasmic transducers involved in HGF-triggered signal transduction. We investigated the role of HGF as a survival factor in protecting cells from apoptosis and we show that HGF is able to counteract staurosporin-induced apoptosis of epithelial cells.     

Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function

Polymorphonuclear leukocyte-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease and may be associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant DeltaF508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause "cell stress" and activation of nuclear factor (NF)-kappaB. G551D mutants also lack Cl- channel function, but CFTR is trafficked normally. We compared the effects of CFTR mutations on the endogenous activation of an NF-kappaB reporter construct. In transfected Chinese hamster ovary cells, the mistrafficked DeltaF508 allele caused a sevenfold activation of NF-kappaB compared with wild-type CFTR or the G551D mutant (P < 0.001). NF-kappaB was also activated in 9/HTEo-/pCep-R cells and in 16HBE/pcftr antisense cell lines, which lack CFTR Cl- channel function but do not accumulate mutant protein in the ER. This endogenous activation of NF-kappaB was associated with elevated interleukin-8 expression. Impaired CFTR Cl- channel activity as well as cell stress due to accumulation of mistrafficked CFTR in the ER contributes to the endogenous activation of NF-kappaB in cells with the CFTR mutation.     

Growth-promoting effects of gastrin on mouse colon cancer cells in vitro: Absence of autocrine effects

Summary We have previously demonstrated trophic effects of gastrin on mouse colon cancer (MC-26) cells, in vivo, and demonstrated the presence of gastrin receptors (GR) on these cells. The cellular and intracellular mechanism by which gastrin expresses trophic effects on colon cancer cells is, however, as yet unknown. For us to start investigating the possible mechanisms involved, it was important that we first develop an in vitro model, in which gastrin expresses its trophic effects directly on the MC-26 cells. The growth-promoting effects of gastrin on the MC-26 cells were examined in various in vitro culture models, in terms of [³H]thymidine incorporation and cell number. A significant trophic effect of gastrin could be demonstrated on quiescent cells in culture, in the absence of serum. The optimal cell-culture conditions for observing trophic effects of gastrin were defined and included a 24-h period of rapid growth of MC-26 cells in serum-supplemented normal growth medium, followed by a 24-h period of culture in serum-free medium containing an optimal dose (1.0 mM) of thymidine, to achieve growth-arrest of the cells. Addition of gastrin (0.5 to 25 nM) to the quiescent, growth-arrested cells resulted in significant dose-dependent increases in both the incorporation of [³H]thymidine uptake by the cells, and a significant increase in cell number. The concentration of GR on the growth-arrested quiescent MC-26 cells in culture was significantly increased compared to the GR concentration on the control, asynchronized cells. The increased presence of GR on the growth-arrested, synchronized MC-26 cells may have allowed us to observe a significant trophic effect of gastrin on the MC-26 cells, in vitro itself. To determine if gastrin was functioning as an autocrine growth factor for MC-26 cells, we examined the effect of gastrin antibodies on the growth of MC-26 cells; no significant effect of the antigastrin IgG on the growth of MC-26 cells was observed. Supported by grants from the National Institutes of Health, Bethesda, MD (PO1 DK 35608 and CA 38651, and a grant from the American Cancer Society, Washington, DC (PDT-220).     

L-MBP is expressed in epithelial cells of mouse small intestine

The mannan-binding proteins (L-MBP and S-MBP, also denoted MBL-C and MBL-A), mainly produced in liver and existing in liver and serum, play important roles in the innate immunity against a variety of pathogens. Total RNA from mouse tissues were screened for MBP mRNA by RT-PCR. In addition to liver, S-MBP mRNA was detected in lung, kidney, and testis, and L-MBP mRNA was detected in kidney, thymus, and small intestine. Quantitative RT-PCR revealed that the small intestine is a predominant site of extrahepatic expression of L-MBP. Western blotting with polyclonal Abs against rat L-MBP demonstrated this protein in Triton X-100 extracts of the small intestine obtained from mice that had undergone systemic perfusion. Immunohistochemical staining with an mAb against mouse L-MBP and in situ hybridization revealed that L-MBP is selectively expressed in some villous epithelial cells of the small intestine. These findings suggest that L-MBP plays a role in mucosal innate immunity.     

Calcitonin receptor-mediated CFTR activation in human intestinal epithelial cells

High levels of calcitonin (CT) observed in medullary thyroid carcinoma and other CT‐secreting tumours cause severe diarrhoea. Previous studies have suggested that CT induces active chloride secretion. However, the involvement of CT receptor (CTR) and the molecular mechanisms underlying the modulation of intestinal electrolyte secreting intestinal epithelial cells have not been investigated. Therefore, current studies were undertaken to investigate the direct effects of CT on ion transport in intestinal epithelial cells. Real time quantitative RT‐PCR and Western blot analysis demonstrated the expression of CTR in intestinal epithelial T84 cells. Exposure of T84 cells to CT from the basolateral but not from apical side significantly increased short circuit current (I_SC) in a dose‐dependent manner that was blocked by 1 μM of CTR antagonist, CT8–32. CT‐induced I_SC was blocked by replacing chloride in the bath solutions with equimolar gluconate and was significantly inhibited by the specific cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, CFTR_127inh. Further, biotinylation studies showed that CT increased CFTR levels on the apical membrane. The presence of either the Ca²⁺ chelator, bis(2‐aminophenoxy)ethane tetraacetic acid‐acetoxymethyl (BAPTA‐AM) ester or the protein kinase A (PKA) inhibitor, H89, significantly inhibited I_SC induced by CT (～32–58% reduction). Response to CT was retained after permeabilization of the basolateral or the apical membranes of T84 cells with nystatin. In conclusion, the activation of CTR by CT induced chloride secretion across T84 monolayers via CFTR channel and the involvement of PKA‐ and Ca²⁺‐dependent signalling pathways. These data elucidate the molecular mechanisms underlying CT‐induced diarrhoea.     

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.     

Characterization of mucins from cultured normal human tracheobronchial epithelial cells

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.     

WDNM1 is associated with differentiation and apoptosis of mammary epithelial cells

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFalpha treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFalpha induced WDNM1 expression showed the NFkappaB-dependent mechanism since it's expression was abrogated in IkappaBalphaM (super-repressor of NFkappaB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFalpha-NFkappaB signal pathway, is associated with HC11 cell apoptosis.     

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells

Cystic fibrosis iscaused by mutations in the cystic fibrosis transmembrane conductanceregulator (CFTR) Clchannel, which mediates transepithelialCl transport in a varietyof epithelia, including airway, intestine, pancreas, and sweat duct. Insome but not all epithelial cells, cAMP stimulatesCl secretion in part byincreasing the number of CFTRCl channels in the apicalplasma membrane. Because the mechanism whereby cAMP stimulates CFTRCl secretion is cell-typespecific, our goal was to determine whether cAMP elevates CFTR-mediatedCl secretion across serousairway epithelial cells by stimulating the insertion of CFTRCl channels from anintracellular pool into the apical plasma membrane. To this end westudied Calu-3 cells, a human airway cell line with a serous cellphenotype. Serous cells in human airways, such as Calu-3 cells, expresshigh levels of CFTR, secrete antibiotic-rich fluid, and play a criticalrole in airway function. Moreover, dysregulation of CFTR-mediatedCl secretion in serouscells is thought to contribute to the pathophysiology of cysticfibrosis lung disease. We report that cAMP activation of CFTR-mediatedCl secretion across humanserous cells involves stimulation of CFTR channels present in theapical plasma membrane and does not involve the recruitment of CFTRfrom an intracellular pool to the apical plasma membrane.

     

Nitric oxide inhibits cAMP-dependent CFTR trafficking in intestinal epithelial cells

Nitric oxide is produced during intestinal inflammation and inhibits the epithelial responsiveness to cAMP-dependent secretagogues. The effect is presumably due to inhibition of activation of the CFTR. However, because insertion of CFTR into the epithelial apical membrane is also a cAMP-dependent process, we tested the hypothesis that NO could inhibit cAMP-dependent CFTR trafficking. SCBN intestinal epithelial cells were treated with forskolin to activate adenylate cyclase activity. The cells were fixed at various times and immunostained for CFTR. Some cells were pretreated with the nitric oxide donor PAPA-NONOate, the protein kinase A inhibitor H89, or the microtubule blocker nocodazole. Cross sections of epithelial monolayers were then studied under fluorescence, and the ratio of apical to basolateral CFTR immunoreactivity was determined. Stimulation of adenylate cyclase activity caused an increase in the apical-to-basolateral ratio of CFTR within 30 s. This effect was transient and preceded changes in short-circuit current in SCBN monolayers mounted in Ussing chambers. PAPA-NONOate, H89, and nocodazole all reduced forskolin-stimulated CFTR trafficking. The inhibitory effect of the NO donor was not affected by pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. PAPA-NONOate reduced forskolin-stimulated increases in intracellular cAMP. The data suggest that a portion of the inhibitory effect of nitric oxide donors on cAMP-dependent chloride secretion is through the inhibition of cAMP-dependent insertion of CFTR into the apical plasma membrane. These data provide insight into the mechanism of secretory dysfunction in inflammatory diseases of the gut where mucosal nitric oxide is elevated.     

Brush cells of the mouse gallbladder

Summary The brush cells (BC) of the mouse gallbladder were studied using light and electron microscopy (transmission and scanning) to determine their shape and distribution. Specimens were fixed in glutaraldehyde and postfixed in ferrocyanide-reduced osmium tetroxide. BC selectively stained with toluidine blue could be identified by means of light microscopy and subsequently studied in serial semithin and ultrathin sections. The results revealed that the shape of the BC is flask-like. A slender, occasionally branched cytoplasmic process emerges from the bulk cell body and extends through the basal region of neighboring epithelial elements to the basement membrane. Examination of the entire gallbladder epithelial surface by scanning electron microscopy revealed that the BC are numerous in the neck region of the organ but only scanty or even absent in wide areas of the corpus region. Their number increases again in the fundic region. These results demonstrate a preferential regional distribution of BC in the gallbladder, which is discussed in relation to a possible functional significance of the BC.