RESUMEN
Although the biology the PLUNC (recently renamed BPI fold, BPIF) family of secreted proteins is poorly understood, multiple array based studies have suggested that some are differentially expressed in lung diseases. We have examined the expression of BPIFB1 (LPLUNC1), the prototypic two-domain containing family member, in lungs from CF patients and in mouse models of CF lung disease. BPIFB1 was localized in CF lung samples along with BPIFA1, MUC5AC, CD68 and NE and directly compared to histologically normal lung tissues and that of bacterial pneumonia. We generated novel antibodies to mouse BPIF proteins to conduct similar studies on ENaC transgenic (ENaC-Tg) mice, a model for CF-like lung disease. Small airways in CF demonstrated marked epithelial staining of BPIFB1 in goblet cells but staining was absent from alveolar regions. BPIFA1 and BPIFB1 were not co-localised in the diseased lungs. In ENaC-Tg mice there was strong staining of both proteins in the airways and luminal contents. This was most marked for BPIFB1 and was noted within 2 weeks of birth. The two proteins were present in distinct cells within epithelium. BPIFB1 was readily detected in BAL from ENaC-Tg mice but was absent from wild-type mice. Alterations in the expression of BPIF proteins is associated with CF lung disease in humans and mice. It is unclear if this elevation of protein production, which results from phenotypic alteration of the cells within the diseased epithelium, plays a role in the pathogenesis of the disease.
Asunto(s)
Fibrosis Quística/metabolismo , Proteínas/metabolismo , Regulación hacia Arriba , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Autoantígenos , Líquido del Lavado Bronquioalveolar/química , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Fibrosis Quística/patología , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Proteínas de Unión a Ácidos Grasos , Glicoproteínas/análisis , Células Caliciformes/metabolismo , Células Caliciformes/patología , Humanos , Leucocitos Mononucleares/metabolismo , Pulmón/química , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucina 5AC/análisis , Neutrófilos/metabolismo , Fosfoproteínas/análisis , Neumonía Bacteriana/metabolismo , Neumonía Bacteriana/patología , Proteínas/análisisRESUMEN
Despite being initially identified in mice, little is known about the sites of production of members of the BPI fold (BPIF) containing (PLUNC) family of putative innate defence proteins in this species. These proteins have largely been considered to be specificaly expressed in the respiratory tract, and we have recently shown that they exhibit differential expression in the epithelium of the proximal airways. In this study, we have used species-specific antibodies to systematically localize two members of this protein family; BPIFA1 (PLUNC/SPLUNC1) and BPIFB1 (LPLUNC1) in adult mice. In general, these proteins exhibit distinct and only partially overlapping localization. BPIFA1 is highly expressed in the respiratory epithelium and Bowman's glands of the nasal passages, whereas BPIFB1 is present in small subset of goblet cells in the nasal passage and pharynx. BPIFB1 is also present in the serous glands in the proximal tongue where is co-localised with the salivary gland specific family member, BPIFA2E (parotid secretory protein) and also in glands of the soft palate. Both proteins exhibit limited expression outside of these regions. These results are consistent with the localization of the proteins seen in man. Knowledge of the complex expression patterns of BPIF proteins in these regions will allow the use of tractable mouse models of disease to dissect their function.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Boca/citología , Boca/metabolismo , Cavidad Nasal/citología , Cavidad Nasal/metabolismo , Fosfoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Diferenciación Celular/fisiología , Glicoproteínas/genética , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fosfoproteínas/genéticaRESUMEN
The FOXE1 gene was screened for mutations in a cohort of 34 unrelated patients with congenital hypothyroidism, 14 of whom had thyroid dysgenesis and 18 were normal (the thyroid status for 2 patients was unknown). The entire coding region of the FOXE1 gene was PCR-amplified, then analyzed using single-stranded conformational polymorphism, followed by confirmation by direct DNA sequencing. DNA sequencing analysis revealed a heterozygous A>G transition at nucleotide position 394 in one of the patients. The nucleotide transition changed asparagine to aspartate at codon 132 in the highly conserved region of the forkhead DNA binding domain of the FOXE1 gene. This mutation was not detected in a total of 104 normal healthy individuals screened. The binding ability of the mutant FOXE1 protein to the human thyroperoxidase (TPO) promoter was slightly reduced compared with the wild-type FOXE1. The mutation also caused a 5% loss of TPO transcriptional activity.
Asunto(s)
Factores de Transcripción Forkhead/genética , Hipotiroidismo/genética , Polimorfismo de Nucleótido Simple , Disgenesias Tiroideas/genética , Adenina , Secuencia de Aminoácidos , Animales , Estudios de Cohortes , Anomalías Congénitas/genética , Cartilla de ADN , Amplificación de Genes , Genes Reporteros , Guanina , Células Hep G2 , Humanos , Hipotiroidismo/metabolismo , Malasia , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Regiones Promotoras Genéticas , Valores de Referencia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Disgenesias Tiroideas/metabolismo , TransfecciónRESUMEN
We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response.