Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases

Rationale ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity. Methods The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis. Results We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active. Conclusion We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease

A large kindred of pulmonary fibrosis associated with a novel ABCA3 gene variant

Background: Interstitial lung disease occurring in children is a condition characterized by high frequency of cases due to genetic aberrations of pulmonary surfactant homeostasis, that are also believed to be responsible of a fraction of familial pulmonary fibrosis. To our knowledge, ABCA3 gene was not previously reported as causative agent of fibrosis affecting both children and adults in the same kindred. Methods: We investigated a large kindred in which two members, a girl whose interstitial lung disease was first recognized at age of 13, and an adult, showed a diffuse pulmonary fibrosis with marked differences in terms of morphology and imaging. An additional, asymptomatic family member was detected by genetic analysis. Surfactant abnormalities were investigated at biochemical, and genetic level, as well as by cell transfection experiments. Results: Bronchoalveolar lavage fluid analysis of the patients revealed absence of surfactant protein C, whereas the gene sequence was normal. By contrast, sequence of the ABCA3 gene showed a novel homozygous G > A transition at nucleotide 2891, localized within exon 21, resulting in a glycine to aspartic acid change at codon 964. Interestingly, the lung specimens from the girl displayed a morphologic usual interstitial pneumonitis-like pattern, whereas the specimens from one of the two adult patients showed rather a non specific interstitial pneumonitis-like pattern. Conclusions: We have detected a large kindred with a novel ABCA3 mutation likely causing interstitial lung fibrosis affecting either young and adult family members. We suggest that ABCA3 gene should be considered in genetic testing in the occurrence of familial pulmonary fibrosis

GATA2 deficiency in children and adults with severe pulmonary alveolar proteinosis and hematologic disorders

Background The majority of cases with severe pulmonary alveolar proteinosis (PAP) are caused by auto-antibodies against GM-CSF. A multitude of genetic and exogenous causes are responsible for few other cases. Goal of this study was to determine the prevalence of GATA2 deficiency in children and adults with PAP and hematologic disorders. Methods Of 21 patients with GM-CSF-autoantibody negative PAP, 13 had no other organ involvement and 8 had some form of hematologic disorder. The latter were sequenced for GATA2. Results Age at start of PAP ranged from 0.3 to 64 years, 4 patients were children. In half of the subjects GATA2-sequence variations were found, two of which were considered disease causing. Those two patients had the typical phenotype of GATA2 deficiency, one of whom additionally showed a previously undescribed feature – a cholesterol pneumonia. Hematologic disorders included chronic myeloic leukemia, juvenile myelo-monocytic leukemia, lymphoblastic leukemia, sideroblastic anemia and two cases of myelodysplastic syndrome (MDS). A 4 year old child with MDS and DiGeorge Syndrome Type 2 was rescued with repetitive whole lung lavages and her PAP was cured with heterologous stem cell transplant. Conclusions In children and adults with severe GM-CSF negative PAP a close cooperation between pneumologists and hemato-oncologists is needed to diagnose the underlying diseases, some of which are caused by mutations of transcription factor GATA2. Treatment with whole lung lavages as well as stem cell transplant may be successful

Lung disease caused by ABCA3 mutations

Background Knowledge about the clinical spectrum of lung disease caused by variations in the ATP binding cassette subfamily A member 3 (ABCA3) gene is limited. Here we describe genotype-phenotype correlations in a European cohort. Methods We retrospectively analysed baseline and outcome characteristics of 40 patients with two disease-causing ABCA3 mutations collected between 2001 and 2015. Results Of 22 homozygous (15 male) and 18 compound heterozygous patients (3 male), 37 presented with neonatal respiratory distress syndrome as term babies. At follow-up, two major phenotypes are documented: patients with (1) early lethal mutations subdivided into (1a) dying within the first 6 months or (1b) before the age of 5 years, and (2) patients with prolonged survival into childhood, adolescence or adulthood. Patients with null/null mutations predicting complete ABCA3 deficiency died within the 1st weeks to months of life, while those with null/other or other/other mutations had a more variable presentation and outcome. Treatment with exogenous surfactant, systemic steroids, hydroxychloroquine and whole lung lavages had apparent but many times transient effects in individual subjects. Conclusions Overall long-term (>5 years) survival of subjects with two disease-causing ABCA3 mutations was <20%. Response to therapies needs to be ascertained in randomised controlled trials

The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

<p>Abstract</p> <p>Background</p> <p>Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in <it>SFTPC</it>, 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.</p> <p>Methods</p> <p>SP-C^A116Dwas 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.</p> <p>Results</p> <p>Stable expression of SP-C^A116Din MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-C^A116Dexpression 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-C^A116Dcells 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-C^A116Don neighboring cells in the alveolar space.</p> <p>Conclusions</p> <p>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.</p

Untersuchungen zur Pathobiochemie der extrazellulären Matrix bei Pseudoxanthoma elasticum (PXE)

Zarbock R. Untersuchungen zur Pathobiochemie der extrazellulären Matrix bei Pseudoxanthoma elasticum (PXE). Bielefeld (Germany): Bielefeld University; 2009.Pseudoxanthoma elasticum (PXE) is a rare inheritable disorder that is characterized by extensive remodelling of the extracellular matrix (ECM). PXE is caused by mutations in the ABCC6 gene. By now, no conclusive pathophysiological connection between ABCC6 and the clinical findings of the disease could be established. Particularly, the molecular mechanisms leading to the ECM alterations are unknown. The clinical picture of PXE is highly variable, with age at disease onset and the number and magnitude of its symptoms differing considerably among patients. The individual disease phenotype is believed to be determined by environmental and genetic factors. In order to identify genetic cofactors of PXE, 29 polymorphisms in four candidate genes, namely MMP2, MMP9, MMP14 and VEGFA, have been examined in this study. Comparison of allelic frequencies showed association of the polymorphisms c.-1575G>A, c.-1306C>T and c.-790T>G in the promoter of the MMP2 gene with PXE. Additionally, polymorphisms in the VEGFA gene could be shown to be associated with the retinopathy frequently accompanying PXE. Moreover, logistic regression analysis identified the c.-460T and the c.674C alleles of VEGFA as independent risk factors for the development of severe retinopathy. An extensive analysis of the expression of ECM genes by dermal fibroblasts from PXE patients was performed and the data were compared with gene expression data of fibroblasts originating from healthy donors. The results showed significant alterations of the expression of several ECM genes as well as of genes connected to glycosaminoglycan (GAG) synthesis in PXE patients. Furthermore, a variation of the gene expression profile during prolonged in vitro cultivation could be demonstrated. Using siRNA mediated gene knockdown, ABCC6 deficiency was induced in normal dermal fibroblasts. In this model, gene expression variations that had been found in cells from PXE patients could be reproduced, thereby establishing a direct connection with lack of ABCC6 activity. In another part of this work, [Delta]-disaccharides derived from GAGs produced by PXE fibroblasts were analyzed using a HPLC method and compared with the [Delta]-disaccharide composition of GAGs produced by normal dermal fibroblasts. The results showed an increased GAG synthesis by PXE fibroblasts which was accompanied by increased activity of xylosyltransferase, the initial enzyme in GAG biosynthesis. Both findings also occurred in normal dermal fibroblasts treated with siRNA against ABCC6. These findings point towards a direct interaction between ABCC6 deficiency and modified GAG synthesis. Analysis of serum concentrations of matrix metalloproteinases in PXE patients and healthy control subjects showed significantly increased levels of MMP-2 and MMP-9 in the serum of PXE patients. For the first time, these results show an increased proteolytic potential in the serum of PXE patients, thereby corroborating the involvement of matrix metalloproteinases in PXE pathogenesis. Finally, by transfection with a plasmid coding for the catalytic subunit of human telomerase, fibroblasts from PXE patients were immortalized and a PXE fibroblast cell line was established, which is ready to be used as a cell model for studying consequences of ABCC6 deficiency. In summary, the results of the present work provide new insights into the mechanisms and genes involved in ECM remodelling, which is the hallmark of PXE.Pseudoxanthoma elasticum (PXE) ist eine seltene vererbbare Erkrankung, die durch eine umfangreiche Umstrukturierung der extrazellulären Matrix (ECM) charakterisiert ist. Mutationen im ABCC6-Gen wurden als PXE-verursachend identifiziert. Ein eindeutiger pathophysiologischer Zusammenhang zwischen ABCC6 und den klinischen Symptomen der Krankheit konnte bis heute nicht hergestellt werden. Insbesondere ist unklar, welche molekularen Mechanismen zum beobachteten Umbau der ECM führen. Der Verlauf von PXE sowie die Schwere der Symptomatik sind sehr heterogen. Neben Umweltfaktoren beeinflussen auch genetische Kofaktoren das individuelle Krankheitsbild der Patienten. Zur Identifizierung genetischer Kofaktoren wurden im Rahmen dieser Arbeit insgesamt 29 Polymorphismen in den vier Kandidatengenen MMP2, MMP9, MMP14 und VEGFA untersucht. Der Vergleich der Allelfrequenzen ergab eine signifikante Assoziation der Polymorphismen c.-1575G>A, c.-1306C>T und c.-790T>G im Promotor des MMP2-Gens mit PXE. Außerdem konnte gezeigt werden, dass Polymorphismen im VEGFA-Gen mit dem Schweregrad der mit PXE regelmäßig einhergehenden Retinopathie assoziiert sind und dass die VEGFA-Allele c.-460T und c.674C unabhängige Risikofaktoren für einen schweren Verlauf der Retinopathie darstellen. Um die Veränderung der ECM zu charakterisieren, wurde mittels realtime RT-PCR eine umfangreiche vergleichende Analyse der Genexpression dermaler Fibroblasten von PXE-Patienten und gesunden Spendern durchgeführt. Die Ergebnisse zeigen signifikante Veränderungen der Expression bei PXE-Fibroblasten im Vergleich zu den Zellen gesunder Spender für eine Reihe von Genen, die ECM-Proteine codieren sowie für Gene, die an der Synthese von Glykosaminoglykanen (GAGs) beteiligt sind. Weiterhin konnte eine Veränderung des Genexpressionsmusters der PXE-Fibroblasten im Verlauf der Kultivierung nachgewiesen werden. Anhand einer in normalen Fibroblasten unter Verwendung von siRNA induzierten ABCC6-Defizienz konnten die gefundenen Expressions-Veränderungen im Wesentlichen reproduziert und somit in einen direkten Zusammenhang mit einem ABCC6-Mangel gestellt werden. In einem weiteren Teil dieser Arbeit konnte durch eine Analyse der von GAGs abgeleiteten [Delta]-Disaccharide mittels einer HPLC-Methode eine verstärkte Synthese von GAGs durch PXE-Fibroblasten im Vergleich zu Zellen gesunder Spender demonstriert werden, die auch von einer erhöhten Aktivität der Xylosyltransferase, des initialen Enzyms der GAG-Biosynthese, begleitet wurde. Sowohl die veränderte GAG-Synthese als auch die Erhöhung der Xylosyltransferase-Aktivität bei PXE-Fibroblasten konnte in normalen dermalen Fibroblasten durch eine siRNA-induzierte ABCC6-Defizienz reproduziert werden. Damit gelang es, einen direkten Zusammenhang zwischen einem Mangel an ABCC6 und der bei PXE veränderten Synthese von GAGs herzustellen. Im Rahmen der Suche nach Biomarkern für eine nichtinvasive Erfassung der Remodulierung der ECM bei PXE konnten in dieser Arbeit erstmals signifikant erhöhte Konzentrationen von MMP-2 und MMP-9 im Serum von PXE-Patienten nachgewiesen werden. Damit gelang es, die Hypothese einer Beteiligung von Matrix-Metalloproteinasen an der Degeneration von ECM-Strukturen bei PXE entscheidend zu untermauern. Schließlich konnten durch Transfektion mit einem Telomerase-codierenden Plasmid primäre dermale Fibroblasten von PXE-Patienten immortalisiert und so eine PXE-Dermalfibroblasten-Zelllinie etabliert werden, die nun für Studien der Auswirkungen einer ABCC6-Defizienz zur Verfügung steht. Die Ergebnisse dieser Arbeit gewähren neue Einsichten in die molekularen Mechanismen der ECM-Umstrukturierung, die das hervorstechende Merkmal von PXE ist, sowie in die genetischen Faktoren, die die Ausprägung des klinischen Phänotyps beeinflussen

Untersuchungen zur Pathobiochemie der extrazellulären Matrix bei Pseudoxanthoma elasticum (PXE)

Zarbock R. Untersuchungen zur Pathobiochemie der extrazellulären Matrix bei Pseudoxanthoma elasticum (PXE). Bielefeld (Germany): Bielefeld University; 2009.Pseudoxanthoma elasticum (PXE) is a rare inheritable disorder that is characterized by extensive remodelling of the extracellular matrix (ECM). PXE is caused by mutations in the ABCC6 gene. By now, no conclusive pathophysiological connection between ABCC6 and the clinical findings of the disease could be established. Particularly, the molecular mechanisms leading to the ECM alterations are unknown. The clinical picture of PXE is highly variable, with age at disease onset and the number and magnitude of its symptoms differing considerably among patients. The individual disease phenotype is believed to be determined by environmental and genetic factors. In order to identify genetic cofactors of PXE, 29 polymorphisms in four candidate genes, namely MMP2, MMP9, MMP14 and VEGFA, have been examined in this study. Comparison of allelic frequencies showed association of the polymorphisms c.-1575G>A, c.-1306C>T and c.-790T>G in the promoter of the MMP2 gene with PXE. Additionally, polymorphisms in the VEGFA gene could be shown to be associated with the retinopathy frequently accompanying PXE. Moreover, logistic regression analysis identified the c.-460T and the c.674C alleles of VEGFA as independent risk factors for the development of severe retinopathy. An extensive analysis of the expression of ECM genes by dermal fibroblasts from PXE patients was performed and the data were compared with gene expression data of fibroblasts originating from healthy donors. The results showed significant alterations of the expression of several ECM genes as well as of genes connected to glycosaminoglycan (GAG) synthesis in PXE patients. Furthermore, a variation of the gene expression profile during prolonged in vitro cultivation could be demonstrated. Using siRNA mediated gene knockdown, ABCC6 deficiency was induced in normal dermal fibroblasts. In this model, gene expression variations that had been found in cells from PXE patients could be reproduced, thereby establishing a direct connection with lack of ABCC6 activity. In another part of this work, [Delta]-disaccharides derived from GAGs produced by PXE fibroblasts were analyzed using a HPLC method and compared with the [Delta]-disaccharide composition of GAGs produced by normal dermal fibroblasts. The results showed an increased GAG synthesis by PXE fibroblasts which was accompanied by increased activity of xylosyltransferase, the initial enzyme in GAG biosynthesis. Both findings also occurred in normal dermal fibroblasts treated with siRNA against ABCC6. These findings point towards a direct interaction between ABCC6 deficiency and modified GAG synthesis. Analysis of serum concentrations of matrix metalloproteinases in PXE patients and healthy control subjects showed significantly increased levels of MMP-2 and MMP-9 in the serum of PXE patients. For the first time, these results show an increased proteolytic potential in the serum of PXE patients, thereby corroborating the involvement of matrix metalloproteinases in PXE pathogenesis. Finally, by transfection with a plasmid coding for the catalytic subunit of human telomerase, fibroblasts from PXE patients were immortalized and a PXE fibroblast cell line was established, which is ready to be used as a cell model for studying consequences of ABCC6 deficiency. In summary, the results of the present work provide new insights into the mechanisms and genes involved in ECM remodelling, which is the hallmark of PXE.Pseudoxanthoma elasticum (PXE) ist eine seltene vererbbare Erkrankung, die durch eine umfangreiche Umstrukturierung der extrazellulären Matrix (ECM) charakterisiert ist. Mutationen im ABCC6-Gen wurden als PXE-verursachend identifiziert. Ein eindeutiger pathophysiologischer Zusammenhang zwischen ABCC6 und den klinischen Symptomen der Krankheit konnte bis heute nicht hergestellt werden. Insbesondere ist unklar, welche molekularen Mechanismen zum beobachteten Umbau der ECM führen. Der Verlauf von PXE sowie die Schwere der Symptomatik sind sehr heterogen. Neben Umweltfaktoren beeinflussen auch genetische Kofaktoren das individuelle Krankheitsbild der Patienten. Zur Identifizierung genetischer Kofaktoren wurden im Rahmen dieser Arbeit insgesamt 29 Polymorphismen in den vier Kandidatengenen MMP2, MMP9, MMP14 und VEGFA untersucht. Der Vergleich der Allelfrequenzen ergab eine signifikante Assoziation der Polymorphismen c.-1575G>A, c.-1306C>T und c.-790T>G im Promotor des MMP2-Gens mit PXE. Außerdem konnte gezeigt werden, dass Polymorphismen im VEGFA-Gen mit dem Schweregrad der mit PXE regelmäßig einhergehenden Retinopathie assoziiert sind und dass die VEGFA-Allele c.-460T und c.674C unabhängige Risikofaktoren für einen schweren Verlauf der Retinopathie darstellen. Um die Veränderung der ECM zu charakterisieren, wurde mittels realtime RT-PCR eine umfangreiche vergleichende Analyse der Genexpression dermaler Fibroblasten von PXE-Patienten und gesunden Spendern durchgeführt. Die Ergebnisse zeigen signifikante Veränderungen der Expression bei PXE-Fibroblasten im Vergleich zu den Zellen gesunder Spender für eine Reihe von Genen, die ECM-Proteine codieren sowie für Gene, die an der Synthese von Glykosaminoglykanen (GAGs) beteiligt sind. Weiterhin konnte eine Veränderung des Genexpressionsmusters der PXE-Fibroblasten im Verlauf der Kultivierung nachgewiesen werden. Anhand einer in normalen Fibroblasten unter Verwendung von siRNA induzierten ABCC6-Defizienz konnten die gefundenen Expressions-Veränderungen im Wesentlichen reproduziert und somit in einen direkten Zusammenhang mit einem ABCC6-Mangel gestellt werden. In einem weiteren Teil dieser Arbeit konnte durch eine Analyse der von GAGs abgeleiteten [Delta]-Disaccharide mittels einer HPLC-Methode eine verstärkte Synthese von GAGs durch PXE-Fibroblasten im Vergleich zu Zellen gesunder Spender demonstriert werden, die auch von einer erhöhten Aktivität der Xylosyltransferase, des initialen Enzyms der GAG-Biosynthese, begleitet wurde. Sowohl die veränderte GAG-Synthese als auch die Erhöhung der Xylosyltransferase-Aktivität bei PXE-Fibroblasten konnte in normalen dermalen Fibroblasten durch eine siRNA-induzierte ABCC6-Defizienz reproduziert werden. Damit gelang es, einen direkten Zusammenhang zwischen einem Mangel an ABCC6 und der bei PXE veränderten Synthese von GAGs herzustellen. Im Rahmen der Suche nach Biomarkern für eine nichtinvasive Erfassung der Remodulierung der ECM bei PXE konnten in dieser Arbeit erstmals signifikant erhöhte Konzentrationen von MMP-2 und MMP-9 im Serum von PXE-Patienten nachgewiesen werden. Damit gelang es, die Hypothese einer Beteiligung von Matrix-Metalloproteinasen an der Degeneration von ECM-Strukturen bei PXE entscheidend zu untermauern. Schließlich konnten durch Transfektion mit einem Telomerase-codierenden Plasmid primäre dermale Fibroblasten von PXE-Patienten immortalisiert und so eine PXE-Dermalfibroblasten-Zelllinie etabliert werden, die nun für Studien der Auswirkungen einer ABCC6-Defizienz zur Verfügung steht. Die Ergebnisse dieser Arbeit gewähren neue Einsichten in die molekularen Mechanismen der ECM-Umstrukturierung, die das hervorstechende Merkmal von PXE ist, sowie in die genetischen Faktoren, die die Ausprägung des klinischen Phänotyps beeinflussen

Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases

Rationale ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity. Methods The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis. Results We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active. Conclusion We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease

Respiratory syncytial virus potentiates ABCA3 mutation-induced loss of lung epithelial cell differentiation

International audienceATP-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-beta1, 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

Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule

Cadmium (Cd2+) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, &#946;2-microglobulin, &#945;1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+-metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105-times higher than (Cd2+)-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, &#946;2-microglobulin, &#945;1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+-metallothionein is not toxic, even at &gt;100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+-&#946;2-microglobulin, Cd2+-albumin and Cd2+-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate