RESUMEN
Cystic fibrosis (CF) is an inherited disorder caused by a deleterious mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Given that the CFTR protein is a chloride channel expressed on a variety of cells throughout the human body, mutations in this gene impact several organs, particularly the lungs. For this very reason, research regarding CF disease and CFTR function has historically focused on the lung airway epithelium. Nevertheless, it was discovered more than two decades ago that CFTR is also expressed and functional on endothelial cells. Despite the great strides that have been made in understanding the role of CFTR in the airway epithelium, the role of CFTR in the endothelium remains unclear. Considering that the airway epithelium and endothelium work in tandem to allow gas exchange, it becomes very crucial to understand how a defective CFTR protein can impact the pulmonary vasculature and overall lung function. Fortunately, more recent research has been dedicated to elucidating the role of CFTR in the endothelium. As a result, several vascular dysfunctions associated with CF disease have come to light. Here, we summarize the current knowledge on pulmonary vascular dysfunctions in CF and discuss applicable therapies.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Pulmón , Humanos , Fibrosis Quística/fisiopatología , Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Animales , Pulmón/metabolismo , Pulmón/fisiopatología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Endotelio Vascular/patología , Mutación , Circulación Pulmonar/fisiologíaRESUMEN
The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.
Asunto(s)
Plaquetas , Megacariocitos , Animales , Humanos , Ratones , Transportadoras de Casetes de Unión a ATP/metabolismo , Plaquetas/metabolismo , Diferenciación Celular , Megacariocitos/metabolismo , Mercaptopurina/farmacología , Mercaptopurina/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
Cystic fibrosis (CF) is a fatal autosomal recessive disorder caused by the loss of function mutations within a single gene for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CFTR is a chloride channel that regulates ion and fluid transport across various epithelia. The discovery of CFTR as the CF gene and its cloning in 1989, coupled with extensive research that went into the understanding of the underlying biological mechanisms of CF, have led to the development of revolutionary therapies in CF that we see today. The highly effective modulator therapies have increased the survival rates of CF patients and shifted the epidemiological landscape and disease prognosis. However, the differential effect of modulators among CF patients and the presence of non-responders and ineligible patients underscore the need to develop specialized and customized therapies for a significant number of patients. Recent advances in the understanding of the CFTR structure, its expression, and defined cellular compositions will aid in developing more precise therapies. As the lifespan of CF patients continues to increase, it is becoming critical to clinically address the extra-pulmonary manifestations of CF disease to improve the quality of life of the patients. In-depth analysis of the molecular signature of different CF organs at the transcriptional and post-transcriptional levels is rapidly advancing and will help address the etiological causes and variability of CF among patients and develop precision medicine in CF. In this review, we will provide an overview of CF disease, leading to the discovery and characterization of CFTR and the development of CFTR modulators. The later sections of the review will delve into the key findings derived from single-molecule and single-cell-level analyses of CFTR, followed by an exploration of disease-relevant protein complexes of CFTR that may ultimately define the etiological course of CF disease.
Asunto(s)
Fibrosis Quística , Humanos , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Calidad de Vida , Medicina de Precisión , Transducción de Señal , MutaciónRESUMEN
Autophagy, a cellular stress response to starvation and bacterial infection, is executed by double-membrane-bound organelles called autophagosomes. Autophagosomes transfer cytosolic material to acidified lysosomes for degradation following soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-dependent fusion processes. Many of the autophagy-related disorders stem from defective end-step proteolysis inside lysosomes. The role of epithelial cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel has been argued to be critical for efficient lysosomal clearance; however, its context to autophagic clearance and the underlying mechanism is poorly defined. Here, we report that syntaxin17 (Stx17), an autophagic SNARE protein interacts with CFTR under nutritional stress and bacterial infection and incorporates it into mature autophagosomes to mediate an efficient lysosomal clearance. Lack of CFTR function and Stx17 and loss of CFTR-Stx17 interaction impairs bacterial clearance. We discover a specialized role of the Stx17-CFTR protein complex that is critical to prevent defective autophagy as has been the reported scenario in CF airway epithelial cells, infectious diseases, and lysosomal clearance disorders.
Asunto(s)
Autofagosomas/metabolismo , Autofagia/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas Qa-SNARE/metabolismo , Transducción de Señal/genética , Estrés Fisiológico , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Lisosomas/metabolismo , Nutrientes/deficiencia , Unión Proteica , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Proteínas Qa-SNARE/genética , TransfecciónRESUMEN
Motile cilia are hairlike structures that line the respiratory and reproductive tracts and the middle ear and generate fluid flow in these organs via synchronized beating. Cilium growth is a highly regulated process that is assumed to be important for flow generation. Recently, Kif19a, a kinesin residing at the cilia tip, was identified to be essential for ciliary length control through its microtubule depolymerization function. However, there is a lack of information on the nature of proteins and the integrated signaling mechanism regulating growth of motile cilia. Here, we report that adenylate cyclase 6 (AC6), a highly abundant AC isoform in airway epithelial cells, inhibits degradation of Kif19a by inhibiting autophagy, a cellular recycling mechanism for damaged proteins and organelles. Using epithelium-specific knockout mice of AC6, we demonstrated that AC6 knockout airway epithelial cells have longer cilia compared with the WT cells because of decreased Kif19a protein levels in the cilia. We demonstrated in vitro that AC6 inhibits AMP-activated kinase (AMPK), an important modulator of cellular energy-conserving mechanisms, and uncouples its binding with ciliary kinesin Kif19a. In the absence of AC6, activation of AMPK mobilizes Kif19a into autophagosomes for degradation in airway epithelial cells. Lower Kif19a levels upon pharmacological activation of AMPK in airway epithelial cells correlated with elongated cilia and vice versa. In all, the AC6-AMPK pathway, which is tunable to cellular cues, could potentially serve as one of the crucial ciliary growth checkpoints and could be channeled to develop therapeutic interventions for cilia-associated disorders.
Asunto(s)
Adenilil Ciclasas/metabolismo , Cilios/fisiología , Cinesinas/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/metabolismo , Adenilil Ciclasas/química , Adenilil Ciclasas/deficiencia , Adenilil Ciclasas/genética , Animales , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia/antagonistas & inhibidores , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Cloroquina/farmacología , Cilios/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Masculino , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Tráquea/citología , Tráquea/metabolismoRESUMEN
Cystic fibrosis is a deadly multiorgan disorder caused by loss of function mutations in the gene that encodes for the cystic fibrosis transmembrane conductance regulator (CFTR) chloride/bicarbonate ion channel. More than 1,700 CFTR genetic variants exist that can cause CF, and majority of these are extremely rare. Because of genetic and environmental influences, CF patients exhibit large phenotypic variation. These factors make clinical trials difficult and largely impractical due to limited and heterogeneous patient pools. Also, the benefit of approved small-molecule CF modulators in a large number of rare mutation patients remains unknown. The goal of this study is to perform a comprehensive bench-side study using in vitro patient enteroids and in vivo mice implanted human intestinal organoids (HIOs) to test CF modulator-Ivacaftor response for a rare CF mutation patient. Based on the positive Ivacaftor response in the enteroids, the patient was enrolled in a (N = 1) clinical trial and showed improved clinical outcomes upon Ivacaftor treatment. HIO implantation model allowed in vivo modulator dosing and provided an elegant human organ-based demonstration of bench-to-bedside testing of modulator effects. Additionally, using the CF HIO model the role of CFTR function in the maturation of human intestine was reported for the first time. In all, we demonstrate that these models effectively serve to translate data from the lab to the clinic and back so that patient-specific therapies could be easily identified and disease-relevant developmental abnormalities in CF organs could be studied and addressed.NEW & NOTEWORTHY In this study, we report an example of laboratory models informing clinical care for rare CF mutation patient, with subsequent recapitulation of clinical benefit in a unique and disease relevant, human-derived in vivo model, effectively translating data from the lab to the clinic and back. This extensive work outlines a potential platform to identify patient-specific therapies and to understand relevant developmental abnormalities associated with CF disease.
Asunto(s)
Aminofenoles/uso terapéutico , Agonistas de los Canales de Cloruro/uso terapéutico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Mutación , Quinolonas/uso terapéutico , Aminofenoles/farmacología , Animales , Niño , Agonistas de los Canales de Cloruro/farmacología , Fibrosis Quística/genética , Humanos , Ratones , Organoides/efectos de los fármacos , Medicina de Precisión , Quinolonas/farmacologíaRESUMEN
BACKGROUND: The pathology of eosinophilic esophagitis (EoE) is characterized by eosinophil-rich inflammation, basal zone hyperplasia (BZH), and dilated intercellular spaces, and the underlying processes that drive the pathologic manifestations of the disease remain largely unexplored. OBJECTIVE: We sought to investigate the involvement of the calcium-activated chloride channel anoctamin 1 (ANO1) in esophageal proliferation and the histopathologic features of EoE. METHODS: We examined mRNA and protein expression of ANO1 in esophageal biopsy samples from patients with EoE and in mice with EoE. We performed molecular and cellular analyses and ion transport assays on an in vitro esophageal epithelial 3-dimensional model system (EPC2-ALI) and murine models of EoE to define the relationship between expression and function of ANO1 and esophageal epithelial proliferation in patients with EoE. RESULTS: We observed increased ANO1 expression in esophageal biopsy samples from patients with EoE and in mice with EoE. ANO1 was expressed within the esophageal basal zone, and expression correlated positively with disease severity (eosinophils/high-power field) and BZH. Using an in vitro esophageal epithelial 3-dimensional model system revealed that ANO1 undergoes chromatin modification and rapid upregulation of expression after IL-13 stimulation, that ANO1 is the primary apical IL-13-induced Cl- transport mechanism within the esophageal epithelium, and that loss of ANO1-dependent Cl- transport abrogated esophageal epithelial proliferation. Mechanistically, ANO1-dependent regulation of basal cell proliferation was associated with modulation of TP63 expression and phosphorylated cyclin-dependent kinase 2 levels. CONCLUSIONS: These data identify a functional role for ANO1 in esophageal cell proliferation and BZH in patients with EoE and provide a rationale for pharmacologic intervention of ANO1 function in patients with EoE.
Asunto(s)
Anoctamina-1/inmunología , Esofagitis Eosinofílica/inmunología , Células Epiteliales/inmunología , Esófago/inmunología , Regulación de la Expresión Génica , Proteínas de Neoplasias/inmunología , Animales , Línea Celular , Modelos Animales de Enfermedad , Esofagitis Eosinofílica/patología , Células Epiteliales/patología , Esófago/patología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Food-induced anaphylaxis (FIA) is an IgE-dependent immune response that can affect multiple organs and lead to life-threatening complications. The processes by which food allergens cross the mucosal surface and are delivered to the subepithelial immune compartment to promote the clinical manifestations associated with food-triggered anaphylaxis are largely unexplored. OBJECTIVE: We sought to define the processes involved in the translocation of food allergens across the mucosal epithelial surface to the subepithelial immune compartment in FIA. METHODS: Two-photon confocal and immunofluorescence microscopy was used to visualize and trace food allergen passage in a murine model of FIA. A human colon cancer cell line, RNA silencing, and pharmacologic approaches were used to identify the molecular regulation of intestinal epithelial allergen uptake and translocation. Human intestinal organoid transplants were used to demonstrate the conservation of these molecular processes in human tissues. RESULTS: Food allergens are sampled by using small intestine (SI) epithelial secretory cells (termed secretory antigen passages [SAPs]) that are localized to the SI villous and crypt region. SAPs channel food allergens to lamina propria mucosal mast cells through an IL-13-CD38-cyclic adenosine diphosphate ribose (cADPR)-dependent process. Blockade of IL-13-induced CD38/cADPR-dependent SAP antigen passaging in mice inhibited induction of clinical manifestations of FIA. IL-13-CD38-cADPR-dependent SAP sampling of food allergens was conserved in human intestinal organoids. CONCLUSION: We identify that SAPs are a mechanism by which food allergens are channeled across the SI epithelium mediated by the IL-13/CD38/cADPR pathway, regulate the onset of FIA reactions, and are conserved in human intestine.
Asunto(s)
Alérgenos/inmunología , Anafilaxia/inmunología , Hipersensibilidad a los Alimentos/inmunología , Interleucina-13/inmunología , Mucosa Intestinal/inmunología , Alérgenos/metabolismo , Anafilaxia/metabolismo , Animales , Hipersensibilidad a los Alimentos/metabolismo , Humanos , Inmunoglobulina E/inmunología , Interleucina-13/metabolismo , Mucosa Intestinal/metabolismo , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
The World Health Organization(WHO) has reported a worldwide surge in cases of cholera caused by the intestinal pathogen Vibrio cholerae, and, combined, such surges have claimed several million lives, mostly in early childhood. Elevated cAMP production in intestinal epithelial cells challenged with cholera toxin (CTX) results in diarrhea due to chloride transport by a cAMP-activated channel, the cystic fibrosis transmembrane conductance regulator (CFTR). However, the identity of the main cAMP-producing proteins that regulate CFTR in the intestine and may be relevant for secretory diarrhea is unclear. Here, using RNA-Seq to identify the predominant AC isoform in mouse and human cells and extensive biochemical analyses for further characterization, we found that the cAMP-generating enzyme adenylate cyclase 6 (AC6) physically and functionally associates with CFTR at the apical surface of intestinal epithelial cells. We generated epithelium-specific AC6 knockout mice and demonstrated that CFTR-dependent fluid secretion is nearly abolished in AC6 knockout mice upon CTX challenge in ligated ileal loops. Furthermore, loss of AC6 function dramatically impaired CTX-induced CFTR activation in human and mouse intestinal spheroids. Our finding that the CFTR-AC6 protein complex is the key mediator of CTX-associated diarrhea may facilitate development of antidiarrheal agents to manage cholera symptoms and improve outcomes.
Asunto(s)
Adenilil Ciclasas/metabolismo , Cólera/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Complejos Multiproteicos/metabolismo , Vibrio cholerae/metabolismo , Adenilil Ciclasas/genética , Animales , Línea Celular , Cólera/genética , Cólera/patología , Toxina del Cólera/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Diarrea/genética , Diarrea/metabolismo , Diarrea/patología , Células Epiteliales/microbiología , Células Epiteliales/patología , Humanos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Vibrio cholerae/patogenicidadRESUMEN
BACKGROUND: Eosinophilic esophagitis (EoE) is characterized by histopathologic modifications of esophageal tissue, including eosinophil-rich inflammation, basal zone hyperplasia, and dilated intercellular spaces (DIS). The underlying molecular processes that drive the histopathologic features of EoE remain largely unexplored. OBJECTIVE: We sought to investigate the involvement of solute carrier family 9, subfamily A, member 3 (SLC9A3) in esophageal epithelial intracellular pH (pHi) and DIS formation and the histopathologic features of EoE. METHODS: We examined expression of esophageal epithelial gene networks associated with regulation of pHi in the EoE transcriptome of primary esophageal epithelial cells and an in vitro esophageal epithelial 3-dimensional model system (EPC2-ALI). Molecular and cellular analyses and ion transport assays were used to evaluate the expression and function of SLC9A3. RESULTS: We identified altered expression of gene networks associated with regulation of pHi and acid-protective mechanisms in esophageal biopsy specimens from pediatric patients with EoE (healthy subjects, n = 6; patients with EoE, n = 10). The most dysregulated gene central to regulating pHi was SLC9A3. SLC9A3 expression was increased within the basal layer of esophageal biopsy specimens from patients with EoE, and expression positively correlated with disease severity (eosinophils/high-power field) and DIS (healthy subjects, n = 10; patients with EoE, n = 10). Analyses of esophageal epithelial cells revealed IL-13-induced, signal transducer and activator of transcription 6-dependent SLC9A3 expression and Na+-dependent proton secretion and that SLC9A3 activity correlated positively with DIS formation. Finally, we showed that IL-13-mediated, Na+-dependent proton secretion was the primary intracellular acid-protective mechanism within the esophageal epithelium and that blockade of SLC9A3 transport abrogated IL-13-induced DIS formation. CONCLUSIONS: SLC9A3 plays a functional role in DIS formation, and pharmacologic interventions targeting SLC9A3 function may suppress the histopathologic manifestations in patients with EoE.
Asunto(s)
Esofagitis Eosinofílica/metabolismo , Células Epiteliales/química , Espacio Extracelular , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Línea Celular , Esofagitis Eosinofílica/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Esófago/patología , Guanidinas/farmacología , Humanos , Concentración de Iones de Hidrógeno , Interleucina-13/farmacología , Metacrilatos/farmacología , Intercambiador 3 de Sodio-Hidrógeno/antagonistas & inhibidoresAsunto(s)
Carcinoma Ductal Pancreático , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Mutación de Línea Germinal , Neoplasias Pancreáticas , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Neoplasias Pancreáticas/genética , Pruebas Genéticas/métodos , Carcinoma Ductal Pancreático/genética , Masculino , Femenino , Persona de Mediana Edad , Anciano , Fenotipo , Valor Predictivo de las Pruebas , Biomarcadores de Tumor/genética , Adulto , Análisis Mutacional de ADNRESUMEN
Cystic fibrosis (CF) is the most common life-shortening genetic disease affecting ~1 in 3,500 of the Caucasian population. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. To date, more than 2,000 CFTR mutations have been identified, which produce a wide range of phenotypes. The CFTR protein, a chloride channel, is normally expressed on epithelial cells lining the lung, gut, and exocrine glands. Mutations in CFTR have led to pleiotropic effects in CF patients and have resulted in early morbidity and mortality. Research has focused on identifying small molecules, or modulators, that can restore CFTR function. In recent years, two modulators, ivacaftor (Kalydeco) and lumacaftor/ivacaftor (Orkambi), have been approved by the U.S. Food and Drug Administration to treat CF patients with certain CFTR mutations. The development of these modulators has served as proof-of-concept that targeting CFTR by modulators is a viable therapeutic option. Efforts to discover new modulators that could deliver a wider and greater clinical benefit are still ongoing. However, traditional randomized controlled trials (RCTs) require large numbers of patients and become impracticable to test the modulators' efficacy in CF patients with CFTR mutations at frequencies much lower than 1%, suggesting the need for personalized medicine in these CF patients.
Asunto(s)
Agonistas de los Canales de Cloruro/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/historia , Fibrosis Quística/terapia , Terapia Genética , Mutación , Medicina de Precisión , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Historia del Siglo XXI , Humanos , Transporte Iónico , Fenotipo , Transducción de SeñalRESUMEN
KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene-deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.
Asunto(s)
Alérgenos/inmunología , Aspergillus fumigatus/inmunología , Asma/inmunología , Células Epiteliales/inmunología , Cinesinas/inmunología , Mucosa Respiratoria/inmunología , Células Th2/inmunología , Animales , Asma/inducido químicamente , Asma/genética , Asma/patología , Citocinas/genética , Citocinas/inmunología , Células Epiteliales/patología , Cinesinas/genética , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Transgénicos , Mucosa Respiratoria/patología , Células Th2/patologíaRESUMEN
Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.
Asunto(s)
Membranas Intracelulares/metabolismo , Fagosomas/metabolismo , Canales Catiónicos TRPC/metabolismo , Ácidos/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Diglicéridos/metabolismo , Exocitosis/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Humanos , Membranas Intracelulares/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Ratones , Viabilidad Microbiana/efectos de los fármacos , Modelos Biológicos , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , Fagosomas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Purinas/química , Purinas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Roscovitina , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Canal Catiónico TRPC6Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Combinación de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Mutación/efectos de los fármacos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Aminofenoles/uso terapéutico , Benzodioxoles/uso terapéutico , Niño , Femenino , Humanos , Indoles/uso terapéutico , Masculino , Persona de Mediana Edad , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Pirrolidinas/uso terapéutico , Quinolonas/uso terapéutico , Adulto JovenRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and cGMP-regulated chloride (Cl-) and bicarbonate (HCO3-) channel localized primarily at the apical plasma membrane of epithelial cells lining the airway, gut and exocrine glands, where it is responsible for transepithelial salt and water transport. Several human diseases are associated with altered CFTR channel function. Cystic fibrosis (CF) is caused by the absence or dysfunction of CFTR channel activity, resulting from mutations in the gene. Secretory diarrhea is caused by the hyperactivation of CFTR channel activity in the gastrointestinal tract. CFTR is a validated target for drug development to treat CF, and extensive research has been conducted to develop CFTR inhibitors for therapeutic interventions of secretory diarrhea. The intracellular processing, trafficking, apical membrane localization, and channel function of CFTR are regulated by dynamic protein-protein interactions in a complex network. In this paper, we review the current knowledge of a macromolecular complex of CFTR, Naâº/H⺠exchanger regulatory factor 2 (NHERF2), and lysophosphatidic acids (LPA) receptor 2 (LPA2) at the apical plasma membrane of airway and gut epithelial cells, and discuss its relevance in human physiology and diseases. We also explore the possibilities of targeting this complex to fine tune CFTR channel activity, with a hope to open up new avenues to develop novel therapies for CF and secretory diarrhea.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mucosa Intestinal/metabolismo , Complejos Multiproteicos/metabolismo , Fosfoproteínas/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Mucosa Respiratoria/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Membrana Celular/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Diarrea/tratamiento farmacológico , Diarrea/genética , Diarrea/metabolismo , Descubrimiento de Drogas , Células Epiteliales/metabolismo , Humanos , Terapia Molecular Dirigida , Complejos Multiproteicos/antagonistas & inhibidores , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores del Ácido Lisofosfatídico/química , Receptores del Ácido Lisofosfatídico/genética , Transducción de Señal/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Pulmonary surfactant, a mixture of proteins and phospholipids, plays an important role in facilitating gas exchange by maintaining alveolar stability. Saturated phosphatidylcholine (SatPC), the major component of surfactant, is synthesized both de novo and by the remodeling of unsaturated phosphatidylcholine (PC) by lyso-PC acyltransferase 1 (LPCAT1). After synthesis in the endoplasmic reticulum, SatPC is routed to lamellar bodies (LBs) for storage prior to secretion. The mechanism by which SatPC is transported to LB is not understood. The specificity of LPCAT1 for lyso-PC as an acyl acceptor suggests that formation of SatPC via LPCAT1 reacylation is a final step in SatPC synthesis prior to transport. We hypothesized that LPCAT1 forms a transient complex with SatPC and specific phospholipid transport protein(s) to initiate trafficking of SatPC from the endoplasmic reticulum to the LB. Herein we have assessed the ability of different StarD proteins to interact with LPCAT1. We found that LPCAT1 interacts with StarD10, that this interaction is direct, and that amino acids 79-271 of LPCAT1 and the steroidogenic acute regulatory protein-related lipid transfer (START) domain of START domain-containing protein 10 (StarD10) are sufficient for this interaction. The role of StarD10 in trafficking of phospholipid to LB was confirmed by the observation that knockdown of StarD10 significantly reduced transport of phospholipid to LB. LPCAT1 also interacted with one isoform of StarD7 but showed no interaction with StarD2/PC transfer protein.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Metabolismo de los Lípidos , Fosfolípidos/biosíntesis , Fosfoproteínas/metabolismo , Alveolos Pulmonares/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Ratones , Fosfatidilcolinas/biosíntesis , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Fosfoproteínas/genética , Mapas de Interacción de Proteínas/genética , Alveolos Pulmonares/citología , Surfactantes Pulmonares/metabolismoRESUMEN
Diarrhea is one of the most common adverse side effects observed in â¼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3'-azido-3'-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.
Asunto(s)
AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Diarrea/inducido químicamente , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Animales , Camptotecina/efectos adversos , Camptotecina/análogos & derivados , Aprobación de Drogas , Células HT29 , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Irinotecán , Ratones , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Conformación Proteica , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Cystic fibrosis (CF) is a life-shortening disease caused by the mutations that generate nonfunctional CF transmembrane conductance regulator (CFTR) protein. A rare serine-to-tyrosine (S1045Y) CFTR mutation was earlier reported to result in CF-associated fatality. We identified an African-American patient with the S1045Y mutation in CFTR, as well as a stop-codon mutation, who has a mild CF phenotype. The underlying mechanism of CF caused by S1045Y-CFTR has not been elucidated. In this study, we determined that S1045Y-CFTR exhibits twofold attenuated function compared with wild-type (WT)-CFTR. We report that serine-to-tyrosine mutation leads to increased tyrosine phosphorylation of S1045Y-CFTR, followed by recruitment and binding of E3-ubiquitin ligase c-cbl, resulting in enhanced ubiquitination and passage of S1045Y-CFTR in the endosome/lysosome degradative compartments. We demonstrate that inhibition of tyrosine phosphorylation partially rescues S1045Y-CFTR surface expression and function. Based on our findings, it could be suggested that consuming genistein (a tyrosine phosphorylation inhibitor) would likely ameliorate CF symptoms in individuals with S1045Y-CFTR, providing a unique personalized therapy for this rare CF mutation.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/diagnóstico por imagen , Genisteína/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Preescolar , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Análisis Mutacional de ADN , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Masculino , Mutación Missense , Fosforilación , Medicina de Precisión , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-cbl/metabolismoRESUMEN
Cyclic nucleotides [e.g., cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)] are ubiquitous second messengers that affect multiple cell functions from maturation of the egg to cell division, growth, differentiation, and death. The concentration of cAMP can be regulated by processes within membrane domains (local regulation) as well as throughout a cell (global regulation). The phosphodiesterases (PDEs) that degrade cAMP have well-known roles in both these processes. It has recently been discovered that ATP-binding cassette (ABC) transporters contribute to both local and global regulation of cAMP. This regulation may require the formation of macromolecular complexes. Some of these transporters are ubiquitously expressed, whereas others are more tissue restricted. Because some PDE inhibitors are also ABC transporter inhibitors, it is conceivable that the therapeutic benefits of their use result from the combined inhibition of both PDEs and ABC transporters. Deciphering the individual contributions of PDEs and ABC transporters to such drug effects may lead to improved therapeutic benefits.