RESUMO
INTRODUCTION: Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The most common mutation, F508del, induces protein misprocessing and loss of CFTR function. The discovery through in vitro studies of the CFTR correctors (i.e. lumacaftor, tezacaftor) that partially rescue the misprocessing of F508del-CFTR with the potentiator ivacaftor is promising in giving an unprecedented clinical benefit in affected patients. AREAS COVERED: Online databases were searched using key phrases for CF and CFTR modulators. Tezacaftor-ivacaftor treatment has proved to be safer than lumacaftor-ivacaftor, although clinical efficacy is similar. Further clinical efficacy has ensued with the introduction of triple therapy, i.e. applying second-generation correctors, such as VX-569 and VX-445 (elexacaftor) to tezacaftor-ivacaftor. The triple combinations will herald the availability of etiologic therapies for patients for whom no CFTR modulators are currently applied (i.e. F508del/minimal function mutations) and enhance CFTR modulator therapy for patients homozygous for F508del. EXPERT OPINION: CF patient-derived tissue models are being explored to determine donor-specific response to current approved and future novel CFTR modulators for F508del and other rare mutations. The discovery and validation of biomarkers of CFTR modulation will complement these studies in the long term and in real-life world.
Assuntos
Aminofenóis/administração & dosagem , Benzodioxóis/administração & dosagem , Fibrose Cística/tratamento farmacológico , Indóis/administração & dosagem , Quinolonas/administração & dosagem , Aminofenóis/efeitos adversos , Aminofenóis/farmacologia , Benzodioxóis/efeitos adversos , Benzodioxóis/farmacologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Desenvolvimento de Medicamentos , Descoberta de Drogas , Quimioterapia Combinada , Humanos , Indóis/efeitos adversos , Indóis/farmacologia , Mutação , Quinolonas/efeitos adversos , Quinolonas/farmacologiaRESUMO
The treatment of cystic fibrosis (CF) patients homozygous for the F508del mutation with Orkambi®, a combination of a corrector (lumacaftor) and a potentiator (ivacaftor) of the mutated CFTR protein, resulted in some amelioration of the respiratory function. However, a great variability in the clinical response was also observed. The aim of this study was to evaluate the response to Orkambi® in a small cohort of F508del/F508del patients (n = 14) in terms of clinical and laboratory parameters, including ex vivo CFTR activity in mononuclear cells (MNCs), during a 12-month treatment. Patients responded with an increase in percent predicted forced expiratory volume in 1 s (FEV1%) and body mass index (BMI) as well as with a decrease in white blood cell (WBC) total counts and serum C-reactive protein (CRP) levels, although not significantly. Sweat chloride and CFTR-dependent chloride efflux were found to decrease and increase, respectively, as compared with pre-therapy values. CFTR and BMI showed a statistically significant correlation during Orkambi® treatment. Clustering analysis showed that CFTR, BMI, sweat chloride, FEV1%, and WBC were strongly associated. These data support the notion that CFTR-dependent chloride efflux in MNCs should be investigated as a sensitive outcome measure of Orkambi® treatment in CF patients.
Assuntos
Aminofenóis/uso terapêutico , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Fibrose Cística/genética , Fibrose Cística/terapia , Leucócitos/metabolismo , Quinolonas/uso terapêutico , Adolescente , Adulto , Índice de Massa Corporal , Criança , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Combinação de Medicamentos , Feminino , Volume Expiratório Forçado , Homozigoto , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Mutação , Pacientes , Testes de Função Respiratória , Adulto JovemRESUMO
The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2:F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR.
Assuntos
Fibrose Cística/genética , Fibrose Cística/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Aminopiridinas/farmacologia , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células HEK293 , Humanos , Mutação/genética , Fenilalanina/genética , Estrutura Terciária de Proteína/genética , Tiazóis/farmacologiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Its aggressiveness is driven by an intense fibrotic desmoplastic reaction in which the increasingly collagen I-rich extracellular matrix (ECM) and several cell types, including cancer stem cells (CSCs), create a tumor-supportive environment. However, how ECM composition regulates CSC dynamics and their relationship with the principle parenchymal tumor population to promote early invasive growth is not yet characterized. For this, we utilized a platform of 3D organotypic cultures composed of laminin-rich Matrigel, representative of an early tumor, plus increasing concentrations of collagen I to simulate malignant stroma progression. As ECM collagen I increases, CSCs progress from a rapidly growing, vascular phenotype to a slower growing, avascular phase, while maintaining their endothelial-like gene signatures. This transition is supported autocrinically by the CSCs and paracrinically by the parenchymal cells via their ECM-dependent secretomes. Indeed, when growing on an early tumor ECM, the CSCs are dedicated toward the preparation of a vascular niche by (a) activating their growth program, (b) secreting high levels of proangiogenic factors which stimulate both angiogenesis and vasculogenic mimicry, and (c) overexpressing VEGFR-2, which is activated by VEGF secreted by both the CSC and parenchymal cells. On Matrigel, the more differentiated parenchymal tumor cell population had reduced growth but a high invasive capacity. This concerted high local invasion of parenchymal cells into the CSC-derived vascular network suggests that a symbiotic relationship between the parenchymal cells and the CSCs underlies the initiation and maintenance of early PDAC infiltration and metastasis.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Plasticidade Celular/genética , Invasividade Neoplásica/genética , Neovascularização Patológica/genética , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica/patologia , Metástase Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/patologia , Tecido Parenquimatoso/efeitos dos fármacos , Tecido Parenquimatoso/patologia , Microambiente Tumoral/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium-hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent Km for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.
Assuntos
Guanidinas/farmacologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Sulfonas/farmacologia , Animais , Concentração de Íons de Hidrogênio , Masculino , Ovinos , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismoRESUMO
We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.
RESUMO
AIM: The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiator ivacaftor (Kalydeco®) improves clinical outcome in G551D cystic fibrosis (CF) patients. Here, we have investigated whether ivacaftor has a clinical impact on non-G551D gating mutations and function of circulating leukocytes as well. METHODS: Seven patients were treated with ivacaftor and evaluated at baseline, and at 1-3 and 6 months. Besides clinical and systemic inflammatory parameters, circulating mononuclear cells (MNC) were evaluated for CFTR-dependent chloride efflux by spectrofluorimetry, neutrophils for oxidative burst by cytofluorimetry and HVCN1 mRNA expression by real time PCR. RESULTS: Ivacaftor determined a significant decrease in sweat chloride concentrations at all time points during treatment. Body mass index (BMI), FEV1 , and FVC showed an increasing trend. While C-reactive protein decreased significantly at 2 months, the opposite behavior was noticed for circulating monocytes. CFTR activity in MNC was found to increase significantly at 3 and 6 months. Neutrophil oxidative burst peaked at 2 months and then decreased to baseline. HVCN1 mRNA expression was significantly higher than baseline at 1-3 months and decreased after 6 months of treatment. The chloride efflux in MNC correlated positively with both FEV1 and FVC. On the other hand, sweat chloride correlated positively with CRP and WBC, and negatively with both respiratory function tests. A cluster analysis confirmed that sweat chloride, FEV1 , FVC, BMI, and MNC chloride efflux behaved as a single entity over time. DISCUSSION: In patients with non-G551D mutations, ivacaftor improved both chloride transport in sweat ducts and chloride efflux in MNC, that is, functions directly imputed to CFTR.
Assuntos
Aminofenóis/farmacologia , Agonistas dos Canais de Cloreto/farmacologia , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Quinolonas/farmacologia , Adolescente , Adulto , Aminofenóis/uso terapêutico , Proteína C-Reativa/metabolismo , Criança , Agonistas dos Canais de Cloreto/uso terapêutico , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Mutação , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Quinolonas/uso terapêutico , Testes de Função Respiratória , Suor/metabolismo , Adulto JovemRESUMO
Cystic fibrosis (CF), one of the most common genetic disorders affecting primarily Caucasians, is due to mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, encoding for a chloride channel also acting as regulator of other transmembrane proteins. In healthy subjects, CFTR is maintained in its correct apical plasma membrane location via the formation of a multiprotein complex in which scaffold proteins (such as NHERF1) and signaling molecules (such as cAMP and protein kinases) guarantee its correct functioning. In CF, a disorganized and dysfunctional airway epithelium brings an altered flux of ions and water into the lumen of bronchioles, consequent bacterial infections and an enormous influx of inflammatory cells (mainly polymorphonuclear neutrophils) into the airway lumen. Recent evidence in healthy airway cells supports the notion that CFTR protein/function is strictly correlated with the actin cytoskeleton and tight junctions status. In CF cells, the most frequent CFTR gene mutation, F508del, has been shown to be associated with a disorganized actin cytoskeleton and altered tight junction permeability. Thus, the correct localization of CFTR on the apical plasma membrane domain through the formation of the scaffolding and signaling complex is likely fundamental to determine a physiological airway epithelium. The correction of CFTR mutations by either gene or drug therapies, as well as by stem cell-based interventions, can determine the resumption of a physiological organization of actin stress fibers and TJ structure and barrier function, further indicating the close interrelationship among these processes.
Assuntos
Actinas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Mucosa Respiratória/patologia , Junções Íntimas/patologia , Humanos , Mucosa Respiratória/metabolismo , Junções Íntimas/metabolismoRESUMO
Goblet cell hyperplasia, a feature of asthma and other respiratory diseases, is driven by the Th-2 cytokines IL-4 and IL-13. In human bronchial epithelial cells, we find that IL-4 induces the expression of many genes coding for ion channels and transporters, including TMEM16A, SLC26A4, SLC12A2, and ATP12A. At the functional level, we find that IL-4 enhances calcium- and cAMP-activated chloride/bicarbonate secretion, resulting in high bicarbonate concentration and alkaline pH in the fluid covering the apical surface of epithelia. Importantly, mucin release, elicited by purinergic stimulation, requires the presence of bicarbonate in the basolateral solution and is defective in cells derived from cystic fibrosis patients. In conclusion, our results suggest that Th-2 cytokines induce a profound change in expression and function in multiple ion channels and transporters that results in enhanced bicarbonate transport ability. This change is required as an important mechanism to favor release and clearance of mucus.
Assuntos
Bicarbonatos/metabolismo , Células Caliciformes/metabolismo , Hiperplasia/patologia , Mucinas/metabolismo , Células Cultivadas , Cloretos/metabolismo , Citocinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Concentração de Íons de HidrogênioRESUMO
Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The most common mutation, deletion of phenylalanine 508 (F508del), disrupts tertiary assembly, causing protein misprocessing and loss of CFTR function in epithelial tissues. Lumacaftor (VX-809) is a Class 1 corrector molecule shown to partially rescue misprocessing of F508del and together with the potentiator of channel activity: ivacaftor (VX-770) has been approved for treatment of CF patients homozygous for the F508del mutation. The specificity of these modulators for CFTR is thought to be conferred through direct binding. Trimethylangelicin (TMA) is a distinct small molecule modulator, previously shown to exhibit both corrector and potentiator activities. We were prompted to determine if TMA also mediates these activities by direct binding. Interestingly, we found that like VX-770, TMA was effective in enhancing anion efflux mediated by purified WT-CFTR reconstituted in phospholipid liposomes. Furthermore, like VX-809, TMA was effective in stabilizing the functional expression of CFTR lacking the regulatory "R" domain or second nucleotide-binding domain (NBD2). The smallest domain that was stabilized by TMA binding was the first membrane-spanning domain (MSD1) as previously observed for VX-809. Together, our findings support the claim that TMA binds directly to CFTR, and despite its distinct chemical structure, shares similar mechanisms as VX-770 and VX-809 to potentiate and stabilize CFTR, respectively.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Furocumarinas/farmacologia , Aminopiridinas/química , Aminopiridinas/farmacologia , Benzodioxóis/química , Benzodioxóis/farmacologia , Fibrose Cística , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Furocumarinas/química , Células HEK293 , Humanos , Estrutura Molecular , Domínios ProteicosRESUMO
Evidence supporting the occurrence of oxidative stress in Cystic Fibrosis (CF) is well established and the literature suggests that oxidative stress is inseparably linked to mitochondrial dysfunction. Here, we have characterized mitochondrial function, in particular as it regards the steps of oxidative phosphorylation and ROS production, in airway cells either homozygous for the F508del-CFTR allele or stably expressing wt-CFTR. We find that oxygen consumption, ΔΨ generation, adenine nucleotide translocator-dependent ADP/ATP exchange and both mitochondrial Complex I and IV activities are impaired in CF cells, while both mitochondrial ROS production and membrane lipid peroxidation increase. Importantly, treatment of CF cells with the small molecules VX-809 and 4,6,4'-trimethylangelicin, which act as "correctors" for F508del CFTR by rescuing the F508del CFTR-dependent chloride secretion, while having no effect per sè on mitochondrial function in wt-CFTR cells, significantly improved all the above mitochondrial parameters towards values found in the airway cells expressing wt-CFTR. This novel study on mitochondrial bioenergetics provides a springboard for future research to further understand the molecular mechanisms responsible for the involvement of mitochondria in CF and identify the proteins primarily responsible for the F508del-CFTR-dependent mitochondrial impairment and thus reveal potential novel targets for CF therapy.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Doenças Mitocondriais/fisiopatologia , Mutação , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Células Cultivadas , Cloretos/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Furocumarinas/farmacologia , Furocumarinas/uso terapêutico , Humanos , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/metabolismo , Sistema Respiratório/citologiaRESUMO
The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTR-dependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Actinas/metabolismo , Animais , Cloretos/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Fibrose Cística/enzimologia , Fibrose Cística/genética , Proteínas do Citoesqueleto/genética , Citoesqueleto/genética , Humanos , Fosforilação , Ratos , Deleção de Sequência , Transdução de SinaisAssuntos
Antibacterianos/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Leucócitos Mononucleares/metabolismo , Adolescente , Adulto , Antibacterianos/classificação , Criança , Humanos , Contagem de Leucócitos , Leucócitos Mononucleares/efeitos dos fármacos , Infecções Respiratórias/tratamento farmacológico , Adulto JovemRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers principally because of early invasion and metastasis. The epidermal growth factor receptor (EGFR) is essential for PDAC development even in the presence of Kras, but its inhibition with erlotinib gives only a modest clinical response, making the discovery of novel EGFR targets of critical interest. Here, we revealed by mining a human pancreatic gene expression database that the metastasis promoter Na(+)/H(+) exchanger (NHE1) associates with the EGFR in PDAC. In human PDAC cell lines, we confirmed that NHE1 drives both basal and EGF-stimulated three-dimensional growth and early invasion via invadopodial extracellular matrix digestion. EGF promoted the complexing of EGFR with NHE1 via the scaffolding protein Na+/H+ exchanger regulatory factor 1, engaging EGFR in a negative transregulatory loop that controls the extent and duration of EGFR oncogenic signaling and stimulates NHE1. The specificity of NHE1 for growth or invasion depends on the segregation of the transient EGFR/Na+/H+ exchanger regulatory factor 1/NHE1 signaling complex into dimeric subcomplexes in different lipid raftlike membrane domains. This signaling complex was also found in tumors developed in orthotopic mice. Importantly, the specific NHE1 inhibitor cariporide reduced both three-dimensional growth and invasion independently of PDAC subtype and synergistically sensitized these behaviors to low doses of erlotinib.
Assuntos
Carcinoma Ductal Pancreático/secundário , Proteínas de Transporte de Cátions/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pancreáticas/patologia , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Antiarrítmicos/uso terapêutico , Western Blotting , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Quimioterapia Combinada , Cloridrato de Erlotinib , Guanidinas/uso terapêutico , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Transdução de Sinais , Trocador 1 de Sódio-Hidrogênio , Sulfonas/uso terapêuticoRESUMO
BACKGROUND: The chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) is expressed by many cell types, including hematopoietic stem/progenitor cells (HSPCs). In this study, we sought to better comprehend the regulation of CFTR activity in HSPCs, namely by beta-adrenergic stimuli. METHODS: The expression of ß2-adrenergic receptor (ß2-AR) in murine Sca-1(+) HSPCs was investigated by immunofluorescence/confocal microscopy and flow-cytometric analysis. Association with CFTR was assessed by immunoprecipitation. HSPCs were evaluated for ATP content and CFTR activity by means of luminometric and spectrofluorometric methods, respectively, upon stimulation with salbutamol. RESULTS: HSPCs express ß2-AR over the whole plasma membrane and are associated in cellula with both the immature and mature forms of CFTR. ß2-AR was predominantly expressed by HSPCs with bigger size. CFTR channel activity was increased by salbutamol treatment and this activation was inhibited by either a specific CFTR inhibitor (CFTRinh172) or a ß2-AR receptor inhibitor (ICI 118,551). Intracellular ATP levels were reduced by salbutamol stimulation and this effect was reversed when ICI 118,551 or CFTR inhibitors were present. A trend in the increase of extracellular ATP upon salbutamol stimulation was observed. CONCLUSIONS: In HSPCs, CFTR is regulated by ß2-adrenergic receptor stimulation determining intracellular ATP depletion.
Assuntos
Trifosfato de Adenosina/metabolismo , Albuterol/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Análise de Variância , Animais , Biomarcadores/metabolismo , Células Cultivadas , Canais de Cloreto/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Modelos Animais de Doenças , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Distribuição Aleatória , Sensibilidade e Especificidade , Células-TroncoRESUMO
In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to "healthy" condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and ß-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.
Assuntos
Antibacterianos/uso terapêutico , Fibrose Cística/sangue , Fibrose Cística/genética , Perfilação da Expressão Gênica , Genoma Humano , Neutrófilos/metabolismo , Escarro/citologia , Adolescente , Adulto , Antibacterianos/farmacologia , Estudos de Casos e Controles , Criança , Fibrose Cística/tratamento farmacológico , Fibrose Cística/fisiopatologia , Demografia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Humanos , Inflamação/sangue , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Neutrófilos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Testes de Função Respiratória , Escarro/microbiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Adulto JovemRESUMO
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.
Assuntos
Âmnio/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mucosa Respiratória/metabolismo , Actinas/metabolismo , Âmnio/citologia , Western Blotting , Diferenciação Celular , Células Cultivadas , Cloretos/metabolismo , Técnicas de Cocultura , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/patologia , Canais Epiteliais de Sódio/genética , Imunofluorescência , Humanos , Células-Tronco Mesenquimais/citologia , Mucosa Respiratória/patologia , Junções Íntimas/fisiologia , Engenharia TecidualRESUMO
Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Furocumarinas/farmacologia , Animais , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibrose Cística/genética , Fibrose Cística/patologia , Retículo Endoplasmático/metabolismo , Furocumarinas/uso terapêutico , Células HEK293 , Humanos , Pulmão/patologia , Transporte Proteico/genética , RatosRESUMO
Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease-dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na+/H+ exchanger 1 (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs preferentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.