RESUMO
ATP-binding cassette transporter A3 (ABCA3) is a lipid transporter active in lung alveolar epithelial type II cells (ATII) and is essential for their function as surfactant-producing cells. ABCA3 mutational defects cause respiratory distress in newborns and interstitial lung disease (ILD) in children. The molecular pathomechanisms are largely unknown; however, viral infections may initiate or aggravate ILDs. Here, we investigated the impact of the clinically relevant ABCA3 mutations, p.Q215K and p.E292V, by stable transfection of A549 lung epithelial cells. ABCA3 mutations strongly impaired expression of the ATII differentiation marker SP-C and the key epithelial cell adhesion proteins E-cadherin and zonula occludens-1. Concurrently, cells expressing ABCA3 mutation acquired mesenchymal features as observed by increased expression of SNAI1, MMP-2 and TGF-ß1, and elevated phosphorylation of Src. Infection with respiratory syncytial virus (RSV), the most common viral respiratory pathogen in small children, potentiated the observed mutational effects on loss of epithelial and acquisition of mesenchymal characteristics. In addition, RSV infection of cells harboring ABCA3 mutations resulted in a morphologic shift to a mesenchymal phenotype. We conclude that ABCA3 mutations, potentiated by RSV infection, induce loss of epithelial cell differentiation in ATII. Loss of key epithelial features may disturb the integrity of the alveolar epithelium, thereby comprising its functionality. We suggest the impairment of epithelial function as a mechanism by which ABCA3 mutations cause ILD.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Mutação , Vírus Sinciciais Respiratórios/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Criança , Células Epiteliais/patologia , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Recém-Nascido , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Doenças Pulmonares Intersticiais/genética , Doenças Pulmonares Intersticiais/virologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mesoderma/metabolismo , Mesoderma/patologia , Microscopia de Fluorescência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/virologia , Proteína C Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/metabolismo , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
BACKGROUND: Mutations in the gene encoding surfactant protein C (SP-C) cause familial and sporadic interstitial lung disease (ILD), which is associated with considerable morbidity and mortality. Unfortunately, effective therapeutic options are still lacking due to a very limited understanding of pathomechanisms. Knowledge of mutant SP-C proprotein (proSP-C) trafficking, processing, intracellular degradation and aggregation is a crucial prerequisite for the development of specific therapies to correct aberrant trafficking and processing of proSP-C and to hinder accumulation of cytotoxic aggregates. MATERIALS AND METHODS: To identify possible starting points for therapeutic intervention, we stably transfected A549 alveolar epithelial cells with several proSP-C mutations previously found in patients suffering from ILD. Effects of mutant proSP-C were assessed by Western blotting, immunofluorescence and Congo red staining. RESULTS: A group of mutations (p.I73T, p.L110R, p.A116D and p.L188Q) resulted in aberrant proSP-C products, which were at least partially trafficked to lamellar bodies. Another group of mutations (p.P30L and p.P115L) was arrested in the endoplasmic reticulum (ER). Except for p.I73T, all mutations led to accumulation of intracellular Congo red-positive aggregates. Enhanced ER stress was detectable in none of these stably transfected cells. CONCLUSIONS: Different SP-C mutations have unique consequences for alveolar epithelial cell biology. As these cannot be predicted based upon the localization of the mutation, our data emphasize the importance of studying individual mutations in detail in order to develop mutation-specific therapies.
Assuntos
Doenças Pulmonares Intersticiais/genética , Mutação/genética , Proteína C Associada a Surfactante Pulmonar/genética , Estresse Fisiológico/genética , Células Epiteliais Alveolares , Autofagia/genética , Sobrevivência Celular/genética , Células Cultivadas , Humanos , Proteína C Associada a Surfactante Pulmonar/metabolismo , Ubiquitinação/genéticaRESUMO
BACKGROUND: Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. METHODS: SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. RESULTS: Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. CONCLUSIONS: We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.
Assuntos
Células Epiteliais/efeitos dos fármacos , Doenças Pulmonares Intersticiais/genética , Chaperonas Moleculares/genética , Alvéolos Pulmonares/efeitos dos fármacos , Proteína C Associada a Surfactante Pulmonar/genética , Surfactantes Pulmonares/metabolismo , Animais , Azatioprina/farmacologia , Linhagem Celular , Ciclofosfamida/farmacologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Hidroxicloroquina/farmacologia , Doenças Pulmonares Intersticiais/imunologia , Metilprednisolona/farmacologia , Camundongos , Chaperonas Moleculares/metabolismo , Mutação , Fosfolipídeos/análise , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Proteína C Associada a Surfactante Pulmonar/imunologiaRESUMO
BACKGROUND: ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER). METHODS: Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level. RESULTS: We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. CONCLUSION: Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Apoptose , Retículo Endoplasmático/patologia , Células Epiteliais/patologia , Pulmão/patologia , Mutação , Estresse Fisiológico , Transportadores de Cassetes de Ligação de ATP/metabolismo , Processamento Alternativo , Anexina A5/metabolismo , Apoptose/genética , Caspase 3/metabolismo , Caspases Iniciadoras/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Separação Celular/métodos , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Células Epiteliais/metabolismo , Citometria de Fluxo , Imunofluorescência , Glutationa/metabolismo , Glicosilação , Proteínas de Choque Térmico/metabolismo , Humanos , Pulmão/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfolipídeos/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Transfecção , Proteína 1 de Ligação a X-BoxRESUMO
BACKGROUND: Heterozygous mutations of SFTPC, the gene encoding surfactant protein C (SP-C), cause sporadic and familial interstitial lung disease (ILD) in children and adults. The most frequent SFTPC mutation in ILD patients leads to a threonine for isoleucine substitution at position 73 (I73T) of the SP-C preprotein (proSP-C), however little is known about the cellular consequences of SP-CI73T expression. RESULTS: To address this, we stably expressed SP-CI73T in cultured MLE-12 alveolar epithelial cells. This resulted in increased intracellular accumulation of proSP-C processing intermediates, which matched proSP-C species recovered in bronchial lavage fluid from patients with this mutation. Exposure of SP-CI73T cells to drugs currently used empirically in ILD therapy, cyclophosphamide, azathioprine, hydroxychloroquine or methylprednisolone, enhanced expression of the chaperones HSP90, HSP70, calreticulin and calnexin. SP-CI73T mutants had decreased intracellular phosphatidylcholine level (PC) and increased lyso-PC level without appreciable changes of other phospholipids. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CI73T cells secreted into the medium soluble factors that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine influence of SP-CI73T on neighboring cells in the alveolar space. CONCLUSION: We show that I73T mutation leads to impaired processing of proSP-C in alveolar type II cells, alters their stress tolerance and surfactant lipid composition, and activates cells of the immune system. In addition, we show that some of the mentioned cellular aspects behind the disease can be modulated by application of pharmaceutical drugs commonly applied in the ILD therapy.
Assuntos
Células Epiteliais/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismo , Substituição de Aminoácidos , Animais , Azatioprina/uso terapêutico , Líquido da Lavagem Broncoalveolar , Linfócitos T CD4-Positivos/imunologia , Calnexina/metabolismo , Calreticulina/metabolismo , Linhagem Celular , Ciclofosfamida/uso terapêutico , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Hidroxicloroquina/uso terapêutico , Doenças Pulmonares Intersticiais/tratamento farmacológico , Metilprednisolona/uso terapêutico , Camundongos , Mutação , Fosfatidilcolinas/metabolismo , Proteína C Associada a Surfactante Pulmonar/análise , Proteína C Associada a Surfactante Pulmonar/genética , Receptores CCR2/metabolismo , Receptores de Interleucina-8A/metabolismo , Transdução de SinaisRESUMO
ABCA3 is a surfactant lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ATP-binding cassette, sub-family A (ABC1), member 3 (ABCA3) gene cause respiratory distress syndrome in newborns, and chronic interstitial lung disease in children and adults. ABCA3 belongs to the class of full ABC transporters, which are supposed to be functional in their monomeric forms. Although other family members e.g., ABCA1 and ABCC7 have been shown to function as oligomers, the oligomerization state of ABCA3 is unknown. In the present study, the oligomerization of ABCA3 was investigated in cell lysates and crude membrane preparations from transiently and stably transfected 293 cells using blue native PAGE (BN-PAGE), gel filtration and co-immunoprecipitation. Additionally, homooligomerization was examined in vivo in cells using bioluminescence resonance energy transfer (BRET). Using BN-PAGE and gel filtration, we demonstrate that non-denatured ABCA3 exists in different oligomeric forms, with monomers (45%) and tetramers (30%) being the most abundant forms. Furthermore, we also show the existence of 20% dimers and 5% trimers. BRET analyses verified intermolecular interactions in vivo. Our results also demonstrated that the arrest of ABCA3 in the endoplasmic reticulum (ER), either through drug treatment or induced by mutations in ABCA3, inhibited the propensity of the protein to form dimers. Based on our results, we suggest that transporter oligomerization is crucial for ABCA3 function and that a disruption of oligomerization due to mutations represents a novel pathomechanism in ABCA3-associated lung disease.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Multimerização Proteica , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Gel , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacosRESUMO
ABCA3 mutations cause fatal surfactant deficiency and interstitial lung disease. ABCA3 protein is a lipid transporter indispensible for surfactant biogenesis and storage in lamellar bodies (LB). The protein folds in endoplasmic reticulum and is glycosylated in Golgi en route to the membrane of mature LB and their precursor multivesicular bodies (MVB). In immunoblots, C-terminally labeled ABCA3 appears as two protein bands of 150 and 190 kDa. Using N- and C-terminal protein tags and hindering ABCA3 processing we show that the 150 kDa protein represents the mature ABCA3 whose N-terminus is cleaved by a cysteine protease inside MVB/LB.