Roflumilast partially reverses smoke-induced mucociliary dysfunction

BACKGROUND: Phosphodiesterases (PDEs) break down cAMP, thereby regulating intracellular cAMP concentrations and diffusion. Since PDE4 predominates in airway epithelial cells, PDE4 inhibitors can stimulate Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by increasing cAMP. Tobacco smoking and COPD are associated with decreased CFTR function and impaired mucociliary clearance (MCC). However, the effects of the PDE4 inhibitor roflumilast on smoke-induced mucociliary dysfunction have not been fully explored. METHODS: Primary normal human bronchial epithelial cells (NHBE) from non-smokers, cultured at the air-liquid interface (ALI) were used for most experiments. Cultures were exposed to cigarette smoke in a Vitrocell VC-10 smoking robot. To evaluate the effect of roflumilast on intracellular cAMP concentrations, fluorescence resonance energy transfer (FRET) between CFP- and YFP-tagged protein kinase A (PKA) subunits was recorded. Airway surface liquid (ASL) was measured using light refraction scanning and ciliary beat frequency (CBF) employing infrared differential interference contrast microscopy. Chloride conductance was measured in Ussing chambers and CFTR expression was quantified with qPCR. RESULTS: While treatment with 100 nM roflumilast had little effect alone, it increased intracellular cAMP upon stimulation with forskolin and albuterol in cultures exposed to cigarette smoke and in control conditions. cAMP baselines were lower in smoke-exposed cells. Roflumilast prolonged cAMP increases in smoke-exposed and control cultures. Smoke-induced reduction in functional, albuterol-mediated chloride conductance through CFTR was improved by roflumilast. ASL volumes also increased in smoke-exposed cultures in the presence of roflumilast while it did not in its absence. Cigarette smoke exposure decreased CBF, an effect rescued with roflumilast, particularly when used together with the long-acting ß-mimetic formoterol. Roflumilast also enhanced forskolin-induced CBF stimulation in ASL volume supplemented smoked and control cells, confirming the direct stimulatory effect of rising cAMP on ciliary function. In active smokers, CFTR mRNA expression was increased compared to non-smokers and ex-smokers. Roflumilast also increased CFTR mRNA levels in cigarette-smoke exposed cell cultures. CONCLUSIONS: Our results show that roflumilast can rescue smoke-induced mucociliary dysfunction by reversing decreased CFTR activity, augmenting ASL volume, and stimulating CBF, the latter particularly in combination with formoterol. As expected, CFTR mRNA expression was not indicative of apical CFTR function

Human LPLUNC1 is a secreted product of goblet cells and minor glands of the respiratory and upper aerodigestive tracts

Long PLUNC1 (LPLUNC1, C20orf114) is a member of a family of poorly described proteins (PLUNCS) expressed in the upper respiratory tract and oral cavity, which may function in host defence. Although it is one of the most highly expressed genes in the upper airways and has been identified in sputum and nasal secretions by proteomic studies, localisation of LPLUNC1 protein has not yet been described. We developed affinity purified antibodies and localised the protein in tissues of the human respiratory tract, oro- and nasopharynx. We have complemented these studies with analysis of LPLUNC1 expression in primary human lung cell cultures and used Western blotting to study the protein in cell culture secretions and in BAL. LPLUNC1 is a product of a population of goblet cells in the airway epithelium and nasal passages and is also present in airway submucosal glands and minor glands of the oral and nasal cavities. The protein is not expressed in peripheral lung epithelial cells. LPLUNC1 is present in bronchoalveolar lavage fluid as two glycosylated isoforms and primary airway epithelial cells produce identical proteins as they undergo mucociliary differentiation. Our results suggest that LPLUNC1 is an abundant, secreted product of goblet cells and minor mucosal glands of the respiratory tract and oral cavity and suggest that the protein functions in the complex milieu that protects the mucosal surfaces in these locations

Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis.

International audienceThyroglobulin (Tg) binds to cell surfaces through various binding sites of high, moderate and low affinity. We have previously shown that binding with low to moderate affinity is pH dependent, selective, but not tissue specific. To identify the regions of Tg involved in this cell surface binding, we studied the binding of (125)I-labeled cyanogen bromide peptides from human Tg to cell surfaces of thyroid cells (inside-out follicles) and of CHO cells. Electrophoretic analysis of cell homogenates after binding of native or of reduced and alkylated (125)I-labeled peptides showed that three peptides, P1, P2 and P3, were always associated with the cells. Sequence analysis allowed the identification of P1 (Ser-2445 to Met-2596 or Met-2610) and P2 (Phe-2156 to Met-2306). P3 proved to be a mixture of several peptides among which two were identified: P3-1 (Cys-1306 to Met-1640) and P3-2 (Cys-2035 to Met-2413) which includes P2. P1, P2 and P3-2 are entirely (P1) or partly (P2 and P3-2) located in the C-terminal domain of Tg homologous with acetylcholinesterase. The smallest peptides, P1 and P2, were purified by preparative electrophoresis. They both displayed strong binding properties towards cell surfaces. Inhibition experiments of (125)I-labeled Tg binding by P1 or P2 indicated that they were involved in Tg binding to cell surfaces. All the other peptides tested for their binding abilities were either not or only poorly involved in Tg binding to cell surfaces, which suggested that P1 and P2 are major Tg sites of binding to cell surfaces. These two peptides are not involved in the binding of Tg to the known Tg 'receptors' described in the literature, to which recycling, transcytosis and regulation functions have been ascribed. Thus they are potential tools to identify cell surface components involved in the process of Tg endocytosis leading to lysosomal degradation

Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis.

International audienceThyroglobulin (Tg) binds to cell surfaces through various binding sites of high, moderate and low affinity. We have previously shown that binding with low to moderate affinity is pH dependent, selective, but not tissue specific. To identify the regions of Tg involved in this cell surface binding, we studied the binding of (125)I-labeled cyanogen bromide peptides from human Tg to cell surfaces of thyroid cells (inside-out follicles) and of CHO cells. Electrophoretic analysis of cell homogenates after binding of native or of reduced and alkylated (125)I-labeled peptides showed that three peptides, P1, P2 and P3, were always associated with the cells. Sequence analysis allowed the identification of P1 (Ser-2445 to Met-2596 or Met-2610) and P2 (Phe-2156 to Met-2306). P3 proved to be a mixture of several peptides among which two were identified: P3-1 (Cys-1306 to Met-1640) and P3-2 (Cys-2035 to Met-2413) which includes P2. P1, P2 and P3-2 are entirely (P1) or partly (P2 and P3-2) located in the C-terminal domain of Tg homologous with acetylcholinesterase. The smallest peptides, P1 and P2, were purified by preparative electrophoresis. They both displayed strong binding properties towards cell surfaces. Inhibition experiments of (125)I-labeled Tg binding by P1 or P2 indicated that they were involved in Tg binding to cell surfaces. All the other peptides tested for their binding abilities were either not or only poorly involved in Tg binding to cell surfaces, which suggested that P1 and P2 are major Tg sites of binding to cell surfaces. These two peptides are not involved in the binding of Tg to the known Tg 'receptors' described in the literature, to which recycling, transcytosis and regulation functions have been ascribed. Thus they are potential tools to identify cell surface components involved in the process of Tg endocytosis leading to lysosomal degradation

Antisense-mediated isoform switching of steroid receptor coactivator-1 in the central nucleus of the amygdala of the mouse brain

Contains fulltext : 125235.pdf (publisher's version ) (Open Access)BACKGROUND: Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function. This nuclear receptor coregulator exists in two splice variants (SRC-1a and SRC-1e) which display differential distribution and opposing activities in the brain, and whose mRNAs differ in a single SRC-1e specific exon. METHODS: For proof of principle of feasibility, we used immunofluorescent stainings to study uptake by different cell types, translocation to the nucleus and potential immunostimulatory effects at different time points after a local injection in the CeA of the mouse brain of a control AON targeting human dystrophin with no targets in the murine brain. To evaluate efficacy we designed an AON targeting the SRC-1e-specific exon and with qPCR analysis we measured the expression ratio of the two splice variants. RESULTS: We found that AONs were taken up by corticotropin releasing hormone expressing neurons and other cells in the CeA, and translocated into the cell nucleus. Immune responses after AON injection were comparable to those after sterile saline injection. A successful shift of the naturally occurring SRC-1a:SRC-1e expression ratio in favor of SRC-1a was observed, without changes in total SRC-1 expression. CONCLUSIONS: We provide a proof of concept for local neuropharmacological use of exon skipping by manipulating the expression ratio of the two splice variants of SRC-1, which may be used to study nuclear receptor function in specific brain circuits. We established that exon skipping after local injection in the brain is a versatile and useful tool for the manipulation of splice variants for numerous genes that are relevant for brain function