Interleukin-4 Induces CpG Site-Specific Demethylation of the Pendrin Promoter in Primary Human Bronchial Epithelial Cells

Pendrin is upregulated in bronchial epithelial cells following IL-4 stimulation via binding of STAT6 to an N4 GAS motif. Basal CpG methylation of the pendrin promoter is cell-specific. We studied if a correlation exists between IL-4 sensitivity and the CpG methylation status of the pendrin promoter in human bronchial epithelial cell models. Methods: Real-time PCR and pyrosequencing were used to respectively quantify pendrin mRNA levels and methylation of pendrin promoter, with and without IL-4 stimulation, in healthy and diseased primary HBE cells, as well as NCI-H292 cells. Results: Increases in pendrin mRNA after IL-4 stimulation was more robust in NCI-H292 cells than in primary cells. The amount of gDNA methylated varied greatly between the cell types. In particular, CpG site 90 located near the N4 GAS motif was highly methylated in the primary cells. An additional CpG site (90bis), created by a SNP, was found only in the primary cells. IL-4 stimulation resulted in dramatic demethylation of CpG sites 90 and 90bis in the primary cells. Conclusions: IL-4 induces demethylation of specific CpG sites within the pendrin promoter. These epigenetic alterations are cell type specific, and may in part dictate pendrin mRNA transcription

A Potassium-Selective Current Affected by Micromolar Concentrations of Anion Transport Inhibitors

Background/Aims: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. Methods: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). Results: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. Conclusion: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated

Allele Drop Out Conferred by a Frequent CYP2D6 Genetic Variation For Commonly Used CYP2D6*3 Genotyping Assays

Background/Aim: Accurate genotyping of CYP2D6 is challenging due to its inherent genetic variation, copy number variation (duplications and deletions) and hybrid formation with highly homologous pseudogenes. Because a relatively high percentage (∼25%) of clinically prescribed drugs are substrates for this enzyme, accurate determination of its genotype for phenotype prediction is essential. Methods: A cohort of 365 patient samples was genotyped for CYP2D6 using Sanger sequencing (as the gold standard), hydrolysis probe assays or pyrosequencing. Results: A discrepant result between the three genotyping methods for the loss of function CYP2D6*3 (g.2549delA, rs35742686) genetic variant was found in one of the samples. This sample also contained the CYP2D6 g.2470T>C (rs17002852) variation, which had an allele frequency of 2.47% in our cohort. Redesign of the CYP2D6*3 pyrosequencing and hydrolysis probe assays to avoid CYP2D6 g.2470 corrected the anomaly. Conclusion: To evidence allele drop out and increase the accuracy of genotyping, intra-patient validation of the same genetic variation with at least two separate methods should be considered

FRET analysis of YFP-tagged 4.1R and CFP/β-actin (C- βactin) interaction.

<p>(A) Example of an acceptor photobleaching FRET experiment in HEK cells over-expressing Y-4.1R¹³⁵ and C-βActin. A FRET signal can be seen under the plasma membrane (asterisks). Scale bar 5 µm. FRETeff was measured in ROIs in the plasma membrane or cytoplasm. (B) Mean FRETeff ± SEM in cells over-expressing Y-4.1R¹³⁵, C-βActin and IRES-DsRED (<i>-ICln</i>) or Y-4.1R¹³⁵, C-βActin and ICln-IRES-DsRED (<i>+ICln</i>). ***p<0.001, -ICln <i>vs</i> +ICln. (C, D) Co-immuprecipitation of over-expressed 4.1R⁸⁰ or 4.1R¹³⁵ and actin. (C) HEK cells were transfected with GFP-IRES-4.1R^80/135, and actin was immunoprecipitated using a goat anti-actin antibody (samples <i>135</i> or <i>80</i>). Goat Ig-G (bovine alkaline peroxidase) was used as a negative control (<i>ctr</i>). (D) C-ICln (<i>+ICln</i>) or C (<i>-ICln</i>) was co-transfected with 4.1R to investigate the effect of ICln on 4.1R/actin interactions. The Western blots are representative of three (4.1R¹³⁵) or two (4.1R⁸⁰) independent experiments, all with comparable results. The 4.1R (<i>anti-4.1R</i>) and actin signals (<i>anti-Act</i>) in the cell lysates (<i>L</i>) and final eluates (<i>E</i>) are shown for all conditions. The additional bands in the 4.1R⁸⁰ Western blot in D are probably residual incompletely denatured (anti-4.1R blot) or denatured (anti-actin blot) antibody chains as they were also recognised by the sole secondary anti-goat antibody.</p

ICln over-expression affects 4.1R membrane localisation.

<p>(A) The images in the first row show the intracellular localisation of the indicated proteins (single confocal planes); the unshown co-transfected protein is indicated in brackets. The images in the second row are enlargements of the insets indicated in the first row images. (B) Exemplificative images of HEK cells co-transfected with GFP-IRES-4.1R⁸⁰ (<i>4.1R⁸⁰</i>) or GFP-IRES-4.1R¹³⁵ (<i>4.1R¹³⁵</i>) and CFP (<i>C</i>) or CFP-ICln (<i>C-ICln</i>) vectors. The samples were immunolabelled with an anti-4.1R antibody to visualise the 4.1R signal. In the panels showing the endogenous 4.1R signal, the asterisks indicate the CFP or C-ICln transfected cells. Scale bar 10 µm. (C) Effect of ICln on endogenous 4.1R membrane localisation: Western blot of total membrane protein extracts (<i>left</i>) and total endogenous 4.1R (<i>right</i>) from HEK cells transfected with C-ICln or C (control). The histograms show the mean OD value of the 4.1R signal normalised for the corresponding cadherin (<i>left</i>) or tubulin (<i>right</i>) signal (n = 4). The values are percentages of the control. **p<0.01; *p<0.05.</p

Downregulation of ICln by siRNA and 4.1R membrane localisation.

<p>(A) Exemplificative images of HEK cells co-transfected with the ptdTOMATO-N1vector and scrambled (<i>ctrl</i>) or ICln siRNA (<i>ICln</i>). The samples were immunolabelled with an anti-4.1R antibody (<i>4.1R</i>) or anti-ICln antibody (<i>ICln</i>). Scale bar 20 µm. (B) Western blot of total protein extracts from HEK cells co-transfected with ICln siRNA (<i>ICln</i>) or scrambled siRNA (<i>ctrl</i>) and the fluorescent tdTomato protein. The histograms show the mean OD value of the ICln signal normalised for the corresponding GAPDH signal (n = 4). The values are percentages of the control. **p<0.01.</p

I_Cl,swell characterisation in cells over-expressing 4.1R^80/135.

<p>(A) Representative whole-cell traces recorded in control cells (over-expressing GFP) or cells over-expressing the 4.1R⁸⁰ or the 4.1R¹³⁵ protein exposed to hypertonic (<i>Hyper</i>) and hypotonic (<i>Hypo</i>) extracellular solutions. (B) Relationship between mean current density, <i>d (pA/pF)</i>, and membrane voltage, <i>V (mV)</i>, in cells over-expressing the indicated proteins and exposed to the hypotonic extracellular solution for 10 min (GFP: n = 22, 4.1R⁸⁰: n = 15, 4.1R¹³⁵: n = 14). (C) Chloride current activation during hypotonic exposure (GFP: n = 24, 4.1R⁸⁰: n = 17, 4.1R¹³⁵: n = 14. (D,E) Relationship between mean current density and membrane voltage in control cells or cells over-expressing the 4.1R⁸⁰ (C) or the 4.1R¹³⁵ protein (D) in the hypertonic extracellular solution (GFP: n = 43, 4.1R⁸⁰: n = 38, 4.1R¹³⁵: n = 27. *p<0.05; ***p<0.001. Two-way ANOVA.</p

4.1R^80/135 and ICln interactions in HEK cells: co-immunoprecipitation and FRET.

<p>(A) Co-immuprecipitation of FLAG-ICln and endogenous 4.1R in HEK cells. Anti-4.1R (<i>4.1R</i>) and anti-FLAG (<i>FLAG</i>) were respectively used to detect 4.1R and FLAGed proteins. Western blot showing immunoprecipitation of 4.1R with Flag-ICln, but not FLAG-BAP (control). (B) Images of an acceptor photobleaching FRET experiment using living cells over-expressing Y-4.1R⁸⁰ and C-ICln. Pre-photobleaching (PRE pb) and post-photobleaching (POST pb) images are shown. Scale bar: 10 µm. (C) Quantification of FRET experiments with CFP-tagged ICln and YFP-tagged 4.1R⁸⁰. The mean FRETeff ± SEM is plotted (*p<0.05 for Y-4.1R⁸⁰+C-ICln <i>vs</i> Y-4.1R⁸⁰+C; one-way ANOVA). The numbers inside the bars represent the number of cells analysed from at least 3 independent experiments. (D) Co-immunoprecipitation of Y-4.1R⁸⁰ or (E) Y-4.1R¹³⁵ with FLAG-ICln (<i>ICln</i>) or FLAG-tagged bovine alkaline peroxidase (<i>BAP</i>, control). The HEK cells were co-transfected with C-terminally FLAGed ICln and Y-4.1R⁸⁰ or Y-4.1R¹³⁵. FLAG-ICln was immunopurified using an anti-FLAG antibody. The 4.1R signal (anti-GFP antibody) and FLAG signal (anti-FLAG antibody) in cell lysates (L), and three sequential 40 µl eluates (E1-E3) are shown for all conditions.</p