Differential regulation of Cl- transport proteins by PKC in Calu-3 cells

Cl- transport proteins expressed in a Calu-3 airway epithelial cell line were differentiated by function and regulation by protein kinase C (PKC) isotypes. mRNA expression of Cl- transporters was semiquantitated by RT-PCR after transfection with a sense or antisense oligonucleotide to the PKC isotypes that modulate the activity of the cystic fibrosis transmembrane conductance regulator [CFTR (PKC-epsilon)] or of the Na/K/2Cl (NKCC1) cotransporter (PKC-delta). Expression of NKCC1 and CFTR mRNAs and proteins was independent of antisense oligonucleotide treatment. Transport function was measured in cell monolayers grown on a plastic surface or on filter inserts. With both culture methods, the antisense oligonucleotide to PKC-epsilon decreased the amount of PKC-epsilon and reduced cAMP-dependent activation of CFTR but not alpha(1)-adrenergic activation of NKCC1. The antisense oligonucleotide to PKC-delta did not affect CFTR function but did block alpha(1)-adrenergic activation of NKCC1 and reduce PKC-delta mass. These results provide the first evidence for mRNA and protein expression of NKCC1 in Calu-3 cells and establish the differential regulation of CFTR and NKCC1 function by specific PKC isotypes at a site distal to mRNA expression and translation in airway epithelial cells.     

Cysteine string protein interacts with and modulates the maturation of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel whose phosphorylation regulates both channel gating and its trafficking at the plasma membrane. Cysteine string proteins (Csps) are J-domain-containing, membrane-associated proteins that have been functionally implicated in regulated exocytosis. Therefore, we evaluated the possibility that Csp is involved in regulated CFTR trafficking. We found Csp expressed in mammalian epithelial cell lines, several of which express CFTR. In Calu-3 airway cells, immunofluorescence colocalized Csp with calnexin in the endoplasmic reticulum and with CFTR at the apical membrane domain. CFTR coprecipitated with Csp from Calu-3 cell lysates. Csp associated with both core-glycosylated immature and fully glycosylated mature CFTRs (bands B and C); however, in relation to the endogenous levels of the B and C bands expressed in Calu-3 cells, the Csp interaction with band B predominated. In vitro protein binding assays detected physical interactions of both mammalian Csp isoforms with the CFTR R-domain and the N terminus, having submicromolar affinities. In Xenopus oocytes expressing CFTR, Csp overexpression decreased the chloride current and membrane capacitance increases evoked by cAMP stimulation and decreased the levels of CFTR protein detected by immunoblot. In mammalian cells, the steady-state expression of CFTR band C was eliminated, and pulse-chase studies showed that Csp coexpression blocked the conversion of immature to mature CFTR and stabilized band B. These results demonstrate a primary role for Csp in CFTR protein maturation. The physical interaction of this Hsc70-binding protein with immature CFTR, its localization in the endoplasmic reticulum, and the decrease in production of mature CFTR observed during Csp overexpression reflect a role for Csp in CFTR biogenesis. The documented role of Csp in regulated exocytosis, its interaction with mature CFTR, and its coexpression with CFTR at the apical membrane domain of epithelial cells may reflect also a role for Csp in regulated CFTR trafficking at the plasma membrane.     

Platelet inhibitory effects of the Phase 3 anticancer and normal tissue cytoprotective agent,RRx‐001

The platelet inhibitory effects of the Phase 3 anticancer agent and nitric oxide (NO) donor, RRx‐001, (1‐bromoacetyl‐3,3‐dinitroazetidine) were examined ex vivo and compared with the diazeniumdiolate NO donor, diethylenetriamine NONOate (DETA‐NONOate), which spontaneously releases nitric oxide in aqueous solution. In the absence of red blood cells and in a dose‐dependent manner, DETA‐NONOate strongly inhibited platelet aggregation induced by several stimuli (ADP, epinephrine and collagen) whereas RRx‐001 only slightly inhibited platelet aggregation under the same conditions in a dose‐dependent manner; these antiaggregant effects were blocked when both DETA‐NONOate and RRx‐001 were co‐incubated with carboxy‐PTIO (CPTIO 0.01‐100 micromol), a widely accepted NO scavenger. However, in the presence of red blood cells from healthy human donors, RRx‐001, which binds covalently to haemoglobin (Hb) and catalyses the production of NO from endogenous nitrite, more strongly inhibited the aggregation of platelets than DETA‐NONOate in a dose‐dependent manner likely because haemoglobin avidly scavenges nitric oxide and reduces its half‐life; the RRx‐001‐mediated platelet inhibitory effect was increased in the presence of nitrite. The results of this study suggest that RRx‐001‐bound Hb (within RBCs) plays an important role in the bioconversion of to NO^., which makes RRx‐001 a more physiologically relevant inhibitor of platelet aggregation than other nitric oxide donors, whose effects are attenuated in the presence of red blood cells. Therefore, RRx‐001‐mediated platelet inhibition is a potentially useful therapeutic property, especially in hypercoagulable cancer patients that are at an increased risk of thrombotic complications.     

Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.     

Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to β-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.     

Role of TAB1 in nitric oxide-induced p38 activation in insulin-producing cells

The aim of present study was to elucidate the role of TAB1 in nitric oxide-induced activation of p38 MAPK. For this purpose we over-expressed TAB1 in insulin-producing beta-TC6 cells. We observed in cells transiently over-expressing TAB1 that p38 activation was enhanced in response to DETA/NONOate. A lowering of TAB1 levels, using the siRNA technique, resulted in the opposite effect. The DETA/NONOate-induced cell death rate was increased in cells transiently overexpressing TAB1. In stable beta-TC6 cell clones with very high TAB1 levels p38 phosphorylation was enhanced also at basal conditions. DETA/NONOate increased also the phosphorylation of JNK and ERK in beta-TC6 cells, but these events were not affected by TAB1. Interestingly, the inhibitory effect of SB203580 on p38 phosphorylation was paralleled by a stimulatory effect on JNK phosphorylation and an inhibitory effect on ERK phosphorylation. In summary, we propose that TAB1 promotes nitric oxide-induced p38 autophosphorylation. In addition, nitric oxide-induced p38 activation seems to promote JNK inhibition and ERK activation, but this effect appears to not require TAB1. A better understanding of how the TAB1/p38 pathway promotes beta-cell death in response to nitric oxide might help in the development of novel pharmacological approaches in the treatment of diabetes.     

Expression and roles of Cl- channel ClC-5 in cell cycles of myeloid cells

This study investigated the effect of exogenous nitric oxide (NO) on endothelial glucocorticoid receptor (GR) function. The NO donor diethylenetriamine NONOate (DETA, 50-500microM) caused concentration dependent nuclear localization of transfected chimeric green fluorescent protein GFP-GR and elevated expression of secreted alkaline phosphatase (SEAP) from a glucocorticoid response element (GRE) promoter construct in bovine aortic endothelial cells. Other weaker NO donors (S-nitroso-N-acetylpenicillamine and spermine NONOate) failed to induce GFP-GR nuclear localization, but all the NO donors activated GRE-SEAP expression, a response unaffected by the antioxidant N-acetyl-L-cysteine. Overall, exogenous NO from high concentration donors can directly activate GR, suggesting a potential feedback mechanism for NO to regulate endothelial inducible nitric oxide synthase (iNOS) expression.     

Molecular mechanisms controlling CFTR gene expression in the airway

The low levels of CFTR gene expression and paucity of CFTR protein in human airway epithelial cells are not easily reconciled with the pivotal role of the lung in cystic fibrosis pathology. Previous data suggested that the regulatory mechanisms controlling CFTR gene expression might be different in airway epithelium in comparison to intestinal epithelium where CFTR mRNA and protein is much more abundant. Here we examine chromatin structure and modification across the CFTR locus in primary human tracheal (HTE) and bronchial (NHBE) epithelial cells and airway cell lines including 16HBE14o- and Calu3. We identify regions of open chromatin that appear selective for primary airway epithelial cells and show that several of these are enriched for a histone modification (H3K4me1) that is characteristic of enhancers. Consistent with these observations, three of these sites encompass elements that have cooperative enhancer function in reporter gene assays in 16HBE14o- cells. Finally, we use chromosome conformation capture (3C) to examine the three-dimensional structure of nearly 800 kb of chromosome 7 encompassing CFTR and observe long-range interactions between the CFTR promoter and regions far outside the locus in cell types that express high levels of CFTR.     

A Highlights from MBoC Selection: Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein.     

Characterization of novel airway submucosal gland cell models for cystic fibrosis studies.

Cultured airway epithelial cells are widely used in cystic fibrosis (CF) research as in vitro models that mimic the in vivo manifestations of the disease and help to define a specific cellular phenotype. Recently, a number of in vitro studies have used an airway adenocarcinoma cell line, Calu-3 that expresses submucosal gland cell features and significant levels of the wild-type CFTR mRNA and protein. We further characterized previously described CF tracheobronchial gland cell lines, CFSMEo- and 6CFSMEo- and determined that these cell lines are compound heterozygotes for the F508del and Q2X mutations, produce vestigial amounts of CFTR mRNA, and do not express detectable CFTR protein. Electrophysiologically, both cell lines are characteristically CF in that they lack cAMP-induced Cl- currents. In this study the cell lines are evaluated in the context of their role as the CF correlate to the Calu-3 cells. Together these cell systems provide defined culture systems to study the biology and pathology of CF. These airway epithelial cell lines may also be a useful negative protein control for numerous studies involving gene therapy by cDNA complementation or gene targeting.     

E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells

Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na(+)/H(+) exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl(-) currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actin-binding protein, the formation of a CFTR.E3KARP.ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.     

Human hepatic cell uptake of resveratrol: involvement of both passive diffusion and carrier-mediated process

We evaluated the relationship between apical surface fluid (ASF) and protein secretion in Calu-3 cells grown at an air-liquid interface. Calu-3 monolayers responded to forskolin, a cystic fibrosis transmembrane regulator (CFTR) channel agonist, by secreting a significant amount of ASF. Such a response from Calu-3 monolayers was not observed with CFTR channel blockers glybenclamide and DPC. Other ion channel mediators, PGF-2alpha, PMA, DNDS, and DIDS, had no effect on Calu-3 ASF secretion. Forskolin decreased Calu-3 protein secretion and glybenclamide increased protein secretion. Similarly, forskolin decreased Calu-3 lysozyme secretion, whereas glybenclamide and DPC increased lysozyme secretion. We observed significant changes in Calu-3 fluid and protein secretions with ion channel mediators known to alter CFTR activity. Our results demonstrate a functional link between fluid and protein secretions in Calu-3 apical surface and suggested a possible involvement of CFTR in these processes.     

Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs

Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to beta-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.