RESUMO
Cystic fibrosis (CF) is a genetic disease characterized by the lack of cystic fibrosis transmembrane conductance regulator (CFTR) protein expressed in epithelial cells. The resulting defective chloride and bicarbonate secretion and imbalance of the transepithelial homeostasis lead to abnormal airway surface liquid (ASL) composition and properties. The reduced ASL volume impairs ciliary beating with the consequent accumulation of sticky mucus. This situation prevents the normal mucociliary clearance, favouring the survival and proliferation of bacteria and contributing to the genesis of CF lung disease. Here, we have explored the potential of small molecules capable of facilitating the transmembrane transport of chloride and bicarbonate in order to replace the defective transport activity elicited by CFTR in CF airway epithelia. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of our compounds on some key properties of ASL. The treatment of these functional models with non-toxic doses of the synthetic anionophores improved the periciliary fluid composition, reducing the fluid re-absorption, correcting the ASL pH and reducing the viscosity of the mucus, thus representing promising drug candidates for CF therapy.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Ionóforos , Mucosa Respiratória/metabolismo , Linhagem Celular , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Células Epiteliais/patologia , Humanos , Transporte de Íons/efeitos dos fármacos , Ionóforos/síntese química , Ionóforos/química , Ionóforos/farmacologia , Muco/metabolismo , Mucosa Respiratória/patologiaRESUMO
Pharmacological rescue of mutant cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis (CF) depends on the specific defect caused by different mutation classes. We asked whether a patient with the rare p.Gly970Asp (c.2909G>A) mutation could benefit from CFTR pharmacotherapy since a similar missense mutant p.Gly970Arg (c.2908G>C) was previously found to be sensitive to potentiators in vitro but not in vivo. By complementary DNA transfection, we found that both mutations are associated with defective CFTR function amenable to pharmacological treatment. However, analysis of messenger RNA (mRNA) from patient's cells revealed that c.2908G>C impairs RNA splicing whereas c.2909G>A does not perturb splicing and leads to the expected p.Gly970Asp mutation. In agreement with these results, nasal epithelial cells from the p.Gly970Asp patient showed significant improvement of CFTR function upon pharmacological treatment. Our results underline the importance of controlling the effect of CF mutation at the mRNA level to determine if the pharmacotherapy of CFTR basic defect is appropriate.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Mutação Puntual , Códon , Fibrose Cística/metabolismo , Células HEK293 , Humanos , Fenótipo , Splicing de RNA , TransfecçãoRESUMO
KEY POINTS: Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation. ABSTRACT: TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.
Assuntos
Anoctamina-1/metabolismo , Sinalização do Cálcio , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa Respiratória/metabolismo , Canais de Cátion TRPV/metabolismo , Potenciais de Ação , Animais , Anoctamina-1/genética , Benzamidas/farmacologia , Brônquios/citologia , Células Cultivadas , Células HEK293 , Humanos , Ratos , Ratos Endogâmicos F344 , Receptores Purinérgicos/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/fisiologia , Canais de Cátion TRPV/genética , Tiazóis/farmacologiaRESUMO
In cystic fibrosis, deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. One possible approach to reducing the detrimental health effects of cystic fibrosis could be the identification of proteins whose suppression rescues F508del-CFTR function in bronchial epithelial cells. However, searches for these potential targets have not yet been conducted, particularly in a relevant airway background using a functional readout. To identify proteins associated with F508del-CFTR processing, we used a high-throughput functional assay to screen an siRNA library targeting 6,650 different cellular proteins. We identified 37 proteins whose silencing significantly rescued F508del-CFTR activity, as indicated by enhanced anion transport through the plasma membrane. These proteins included FAU, UBE2I, UBA52, MLLT6, UBA2, CHD4, PLXNA1, and TRIM24, among others. We focused our attention on FAU, a poorly characterized protein with unknown function. FAU knockdown increased the plasma membrane targeting and function of F508del-CFTR, but not of wild-type CFTR. Investigation into the mechanism of action revealed a preferential physical interaction of FAU with mutant CFTR, leading to its degradation. FAU and other proteins identified in our screening may offer a therapeutically relevant panel of drug targets to correct basic defects in F508del-CFTR processing.
Assuntos
Brônquios/metabolismo , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Mutação , Proteínas Ribossômicas/metabolismo , Brônquios/patologia , Membrana Celular/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/patologia , Humanos , Proteólise , Proteínas Ribossômicas/genéticaRESUMO
NEW FINDINGS: What is the central question of this study? What is the precise subcellular localization of the epithelial sodium channel (ENaC) in human airway epithelium? What is the main finding and its importance? ENaC protein has an unexpected localization in the peripheral region of the apical membrane of bronchial epithelial cells, very close to tight junctions. This may be important for the mechanism of Na+ absorption ABSTRACT: The epithelial sodium channel (ENaC) has a key role in absorbing fluid across the human airway epithelium. Altered activity of ENaC may perturb the process of mucociliary clearance, thus impairing the innate defence mechanisms against microbial agents. The proteins forming ENaC are present on the apical membrane of the epithelium. However, their precise localization is unknown. In the present study, we used two antibodies recognizing the α and ß ENaC subunits. Both antibodies revealed a restricted localization of ENaC in the peripheral region of the apical membrane of cultured bronchial epithelial cells, close to but not overlapping with tight junctions. In contrast, the cystic fibrosis transmembrane conductance regulator chloride channel was more diffusely expressed on the whole apical membrane. Modulation of ENaC activity by aprotinin or elastase resulted in a decrease or increase in the peripheral localization, respectively. Our results suggest that sodium absorption is mainly occurring close to tight junctions where this cation may be rapidly expelled by the Na+ /K+ pump present in lateral membranes. This arrangement of channels and pumps may limit Na+ build-up in other regions of the cells.
Assuntos
Brônquios/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Mucosa Respiratória/metabolismo , Animais , Brônquios/citologia , Linhagem Celular , Membrana Celular/metabolismo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/citologia , Humanos , RatosRESUMO
Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl-/- mice, and found that Rankl-/- BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl-/- BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics. Stem Cells 2017;35:1365-1377.
Assuntos
Diferenciação Celular , Vetores Genéticos/metabolismo , Lentivirus/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Ligante RANK/deficiência , Animais , Biomarcadores/metabolismo , Células Clonais , Imunofenotipagem , Camundongos Endogâmicos C57BL , Ligante RANK/metabolismo , Transdução de Sinais , Transdução GenéticaRESUMO
Clâ» channels activated by acidic extracellular pH have been observed in various mammalian cells but their molecular identity and mechanisms of regulation are unknown. The aim of this study was to analyse the acid-activated Cl- current (ICl(H)) by elucidating its functional properties and mechanisms of regulation in three different cell types: primary human bronchial epithelial (HBE) cells, neuroblastoma SK-N-MC cells and HEK-293 cells. We found that outward rectification, sensitivity to acidic pH (50% activation at pH5.15), permeability sequence (SCNâ»>Iâ»>Brâ»>Clâ»>gluconate), voltage dependence and sensitivity to blockers of ICl(H) were identical in all cells. These findings suggest a common molecular basis for ICl(H). We analysed the possible relationship of ICl(H) with members of ClC and TMEM16 protein families. By gene silencing, validated using RT-PCR, we found that ICl(H) is unrelated to ClC-3, ClC-7, TMEM16A, TMEM16D, TMEM16F, TMEM16H and TMEM16K. Analysis of possible mechanisms of regulation indicate that Ca²âº, ATP and phosphorylation by PKA or PKC do not seem to be implicated in channel activation. Instead, the inhibition of ICl(H) by genistein and wortmannin suggest regulation by other kinases, possibly a tyrosine kinase and a phosphatidylinositol-3-kinase. Moreover, by using dynasore, the dynamin inhibitor, we found indications that exo/endocytosis is a mechanism responsible for ICl(H) regulation. Our results provide the first evidence about acid-activated Clâ» channel regulation and, thus, could open the way for a better understanding of the channel function and for the molecular identification of the underlying protein.
Assuntos
Ácidos/metabolismo , Canais de Cloreto/metabolismo , Ativação do Canal Iônico/fisiologia , Androstadienos/farmacologia , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Canais de Cloreto/genética , Cricetinae , Cricetulus , Genisteína/farmacologia , Células HEK293 , Humanos , Hidrazonas/farmacologia , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , WortmaninaRESUMO
TMEM16F is a membrane protein with possible dual function as an ion channel and a phospholipid scramblase. The properties of ion channels associated with TMEM16F and the link between ion channel and scramblase activity are a matter of debate. We studied the properties of four isoforms of TMEM16F generated by alternative splicing. Upregulation of three TMEM16F isoforms or silencing of endogenous TMEM16F increased and decreased, respectively, both scramblase and channel activities. Introduction of an activating mutation in TMEM16F sequence caused a marked increase in phosphatidylserine scrambling and in ion transport indicating direct involvement of the protein in both functions. TMEM16F, also known as ANO6, is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F-dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F variants generated by alternative splicing. Using whole-cell patch-clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular Ca(2+) concentrations and positive membrane potentials. These variants showed different degrees of Ca(2+) sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for Cl(-) over Na(+) . A fourth variant (V3) showing a unique carboxy-terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium-dependent phosphatidylserine externalization. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent Ca(2+) sensitivity of TMEM16F-dependent channels. In parallel, this mutation also enhanced the extent of phosphatidylserine externalization that occurred even under resting conditions. These results support the conclusion that TMEM16F proteins are directly involved in dual activity, as a phospholipid scramblase and as an ion channel.
Assuntos
Canais Iônicos , Proteínas de Transferência de Fosfolipídeos , Anoctaminas , Cálcio/metabolismo , Linhagem Celular , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , RNA Mensageiro/metabolismoRESUMO
TMEM16A is a plasma membrane protein with voltage- and calcium-dependent chloride channel activity. The role of the various TMEM16A domains in expression and function is poorly known. In a previous study, we found that replacing the first ATG of the TMEM16A coding sequence with a nonsense codon (M1X mutation), to force translation from the second ATG localized at position 117, only had minor functional consequences. Therefore, we concluded that this region is dispensable for TMEM16A processing and channel activity. We have now removed the first 116 codons from the TMEM16A coding sequence. Surprisingly, the expression of the resulting mutant, Δ(1-116), resulted in complete loss of activity. We hypothesized that, in the mutant M1X, translation may start at a position before the second ATG, using a non-canonical start codon. Therefore, we placed an HA-epitope at position 89 in the M1X mutant. We found, by western blot analysis, that the HA-epitope can be detected, thus demonstrating that translation starts from an upstream non-ATG codon. We truncated the N-terminus of TMEM16A at different sites while keeping the HA-epitope. We found that stepwise shortening of TMEM16A caused an in parallel stepwise decrease in TMEM16A expression and function. Our results indicate that indeed the N-terminus of TMEM16A is important for its activity. The use of an alternative start codon appears to occur in a naturally-occurring TMEM16A isoform that is particularly expressed in human testis. Future experiments will need to address the role of normal and alternative amino-terminus in TMEM16A structure and function.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/genética , Cloretos/metabolismo , Proteínas de Neoplasias/genética , Iniciação Traducional da Cadeia Peptídica/genética , Testículo/metabolismo , Anoctamina-1 , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Genes Reporter , Células HEK293 , Humanos , Transporte de Íons/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fases de Leitura Aberta , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Testículo/citologia , TransfecçãoRESUMO
TMEM16A and TMEM16B proteins are CaCCs (Ca2+-activated Cl- channels) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a 10-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimaeric proteins in which different domains of the TMEM16A protein were replaced by the equivalent domains of TMEM16B. Replacement of the N-terminus, TMD (transmembrane domain) 1-2, the first intracellular loop and TMD3-4 did not change the channel's properties. Instead, replacement of intracellular loop 3 decreased the apparent Ca2+ affinity by nearly 8-fold with respect to wild-type TMEM16A. In contrast, the membrane currents derived from chimaeras containing TMD7-8 or the C-terminus of TMEM16B showed higher activation and deactivation rates without a change in Ca2+ sensitivity. Significantly accelerated kinetics were also found when the entire C-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B is the site involved in Ca2+-sensitivity, whereas the C-terminal part, including TMD7-8, affect the rate of transition between the open and the closed state.
Assuntos
Canais de Cloreto/química , Canais de Cloreto/genética , Proteínas de Membrana/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusão/química , Anoctamina-1 , Anoctaminas , Canais de Cloreto/fisiologia , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Relação Estrutura-AtividadeRESUMO
In the respiratory system, Na(+) absorption and Cl(-) secretion are balanced to maintain an appropriate airway surface fluid (ASF) volume and ensure efficient mucociliary clearance. In cystic fibrosis (CF), this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the absence of functional CFTR-dependent Cl(-) secretion. The consequences of defective Cl(-) transport are worsened by the persistence of Na(+) absorption, which contributes to airway surface dehydration. We asked whether normal ASF can be restored to an equal extent by recovering Cl(-) secretion from mutated CFTR or by reducing Na(+) absorption. This is highly relevant in the selection of the best strategy for the treatment of patients with CF. We analyzed the ASF thickness of primary cultured bronchial CF and non-CF epithelia after silencing the epithelial Na(+) channel (ENaC) with specific short, interfering RNAs (siRNAs) and after the pharmacological stimulation of CFTR. Our results indicate that (1) single siRNAs complementary to ENaC subunits are sufficient to reduce ENaC transcripts, Na(+) channel activity, and fluid transport, but only silencing both the α and ß ENaC subunits at the same time leads to an increase of ASF (from nearly 7 µm to more than 9 µm); (2) the ASF thickness obtained in this way is about half that measured after maximal CFTR stimulation in non-CF epithelia (10-14 µm); and (3) the pharmacological rescue of mutant CFTR increases the ASF to the same extent as ENaC silencing. Our results indicate that CFTR rescue and ENaC silencing both produce a significant and long-lasting increase of airway hydration in vitro.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Bloqueadores do Canal de Sódio Epitelial/metabolismo , Canais Epiteliais de Sódio/genética , RNA Interferente Pequeno/metabolismo , Líquidos Corporais , Brônquios/metabolismo , Brônquios/patologia , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/patologia , Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Transporte de Íons , Mutação , Cultura Primária de Células , RNA Interferente Pequeno/genética , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologiaRESUMO
The TMEM16A protein has a potential role as a Ca(2+)-activated Cl(-) channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o-). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with short-interfering RNAs caused a marked inhibition of CaCC activity, thus demonstrating that a low TMEM16A expression is sufficient to support Ca(2+)-dependent Cl(-) transport. Following treatment for 24-72 h with interleukin-4 (IL-4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL-4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15 and 60% of TMEM16A-positive cells when epithelia were treated with IL-4 for 24 or 72 h, respectively. In contrast, ciliated cells showed expression of the cystic fibrosis transmembrane conductance regulator Cl(-) channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL-4, and that TMEM16A upregulation by IL-4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.
Assuntos
Canais de Cloreto/fisiologia , Células Caliciformes/fisiologia , Metaplasia/fisiopatologia , Proteínas de Neoplasias/fisiologia , Anoctamina-1 , Brônquios/citologia , Linhagem Celular , Células Cultivadas , Células Epiteliais , Células HEK293 , Humanos , Interleucina-4/farmacologia , RNA Interferente Pequeno/administração & dosagemRESUMO
A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Fibrose Cística/genética , Ativação do Canal Iônico , Mutação , Tiazóis/farmacologia , Transporte Biológico/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Ativação do Canal Iônico/genética , Técnicas de Patch-Clamp , Relação Estrutura-Atividade , Tiazóis/uso terapêuticoRESUMO
TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca(2+)-activated Cl(-) channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca(2+)-activated Cl(-) channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca(2+)-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.
Assuntos
Canais de Cloreto/química , Proteínas de Membrana/química , Proteínas de Neoplasias/química , Processamento Alternativo , Anoctamina-1 , Proteínas de Bactérias/metabolismo , Cálcio/química , Cloretos/farmacologia , Células HEK293 , Humanos , Íons , Proteínas Luminescentes/metabolismo , Potenciais da Membrana , Microscopia de Fluorescência/métodos , Técnicas de Patch-Clamp , Conformação Proteica , Isoformas de Proteínas , TransfecçãoRESUMO
Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-a, IL-4 and IFN-g to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as G(T) values; (d) epidermal growth factor receptor (EGFR)-dependent mitogenactivated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-a (47±3%), IL-4 (43±1%) and INF-g (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. G(T) values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm²)1000 in control cultures and in those exposed to TNF-a, IFN-g and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 µM) or a MAP kinase inhibitor (U0126, 25 µM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.
Assuntos
Citocinas/metabolismo , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Animais , Linhagem Celular Tumoral , Dano ao DNA , Impedância Elétrica , Células Epiteliais/citologia , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Microscopia de Fluorescência , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Ocludina , Fosfoproteínas/metabolismo , Coelhos , Mucosa Respiratória/citologia , Mucosa Respiratória/enzimologia , Mucosa Respiratória/metabolismo , Junções Íntimas/enzimologia , Proteína da Zônula de Oclusão-1RESUMO
Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fosfatidilinositol 3-Quinase , Animais , Classe Ib de Fosfatidilinositol 3-Quinase , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Inflamação , Camundongos , Peptídeos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
The F508del mutation, the most frequent in cystic fibrosis (CF), impairs the maturation of the CFTR chloride channel. The F508del defect can be partially overcome at low temperature (27°C) or with pharmacological correctors. However, the efficacy of correctors on the mutant protein appears to be dependent on the cell expression system. We have used a bronchial epithelial cell line, CFBE41o-, to determine the efficacy of various known treatments and to discover new correctors. Compared with other cell types, CFBE41o- shows the largest response to low temperature and the lowest one to correctors such as corr-4a and VRT-325. A screening of a small-molecule library identified 9-aminoacridine and ciclopirox, which were significantly more effective than corr-4a and VRT-325. Analysis with microarrays revealed that 9-aminoacridine, ciclopirox, and low temperature, in contrast to corr-4a, cause a profound change in cell transcriptome. These data suggest that 9-aminoacridine and ciclopirox act on F508del-CFTR maturation as proteostasis regulators, a mechanism already proposed for the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). However, we found that 9-aminoacridine, ciclopirox, and SAHA, in contrast to corr-4a, VRT-325, and low temperature, do not increase chloride secretion in primary bronchial epithelial cells from CF patients. These conflicting data appeared to be correlated with different gene expression signatures generated by these treatments in the cell line and in primary bronchial epithelial cells. Our results suggest that F508del-CFTR correctors acting by altering the cell transcriptome may be particularly active in heterologous expression systems but markedly less effective in native epithelial cells.
Assuntos
Temperatura Baixa , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Perfilação da Expressão Gênica , Aminacrina , Proteínas de Bactérias , Benzamidas , Linhagem Celular , Membrana Celular , Cloretos/metabolismo , Ciclopirox , Fibrose Cística/genética , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos , Proteínas Luminescentes , Mutação , Piperazinas , Transporte Proteico/genética , Transporte Proteico/fisiologia , Piridonas , Quinazolinas , Tiazóis , VorinostatRESUMO
A novel class of transmembrane anion carriers, the click-tambjamines, display remarkable anionophoric activities in model liposomes and living cells. The versatility of this building block for the generation of molecular diversity offers promise to develop future drugs based on this design.
Assuntos
Antineoplásicos/farmacologia , Pirróis/farmacologia , Células A549 , Ânions/síntese química , Ânions/química , Ânions/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Transporte de Íons , Lipossomos/química , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Pirróis/síntese química , Pirróis/química , Relação Estrutura-AtividadeRESUMO
The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal-distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.
Assuntos
Brônquios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Mucosa Nasal/metabolismo , Estudos de Casos e Controles , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Linhagem Celular , Meios de Cultura , Fibrose Cística/patologia , Fatores de Transcrição Forkhead/genética , Humanos , Transcriptoma , TransfecçãoRESUMO
F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.