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Epigenetic mechanisms silence a disintegrin and metalloprotease 33 expression in bronchial epithelial cells  Youwen Yang, PhD, Hans Michael Haitchi, MD,

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Presentation on theme: "Epigenetic mechanisms silence a disintegrin and metalloprotease 33 expression in bronchial epithelial cells  Youwen Yang, PhD, Hans Michael Haitchi, MD,"— Presentation transcript:

1 Epigenetic mechanisms silence a disintegrin and metalloprotease 33 expression in bronchial epithelial cells  Youwen Yang, PhD, Hans Michael Haitchi, MD, Julie Cakebread, PhD, David Sammut, MD, Anna Harvey, BSc, Robert M. Powell, PhD, John W. Holloway, PhD, Peter Howarth, MD, PhD, Stephen T. Holgate, MD, DSc, Donna E. Davies, PhD  Journal of Allergy and Clinical Immunology  Volume 121, Issue 6, Pages e14 (June 2008) DOI: /j.jaci Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Organization of the 5′ region of the ADAM33 gene and analysis of its expression. A, The predicted promoter region, CpG island, Sp1-binding sites (boxed), RT-PCR primer location, and methylation sequence. B and C, ADAM33 expression in MRC5 and H292 cells, bronchial fibroblasts, and epithelial cells from healthy (Fig 1, B) or asthmatic (Fig 1, C) donors was assessed by means of RT-PCR, with GAPDH as a housekeeping gene. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 A, Promoter activity of ADAM33. The luciferase reporter plasmid was constructed by using a 637-bp 5′ flanking fragment of the ADAM33 gene. Luciferase activity using the ADAM33 promoter in MRC5 fibroblasts was compared with that using the negative control (No insert) plasmid; activities were normalized by using Renilla luciferase activity. Data are from 3 independent experiments. B, Prediction of CpG islands between −1000 and +400 of the ADAM33 gene by using CpGPlot software ( Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 A, Cytosine methylation of the ADAM33 promoter from 3 normal PBECs and PBFs. Each row represents the methylation pattern for individual clones from bisulfite-treated DNA; unmethylated CpG sites (open circles) and methylated CpG sites (filled circles) are shown. B, Methylation-sensitive PCR with HpaII-digested genomic DNA from PBECs and PBFs from patients with asthma. C, Densitometric analysis of ADAM33 promoter methylation; peak intensity, a measure of uncut (ie, methylated) ADAM33 DNA, was normalized to control DNA. ∗P < .001). Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Induction of ADAM33 expression after 5-aza-dC treatment. After 7 days' treatment with 5-aza-dC, expression of ADAM33 in H292 cells (A) or PBECs from asthmatic patients (C) was analyzed by means of RT-PCR (Fig 4, A) or RT-qPCR (Fig 4, C), with GAPDH as a housekeeping gene. Demethylation of the ADAM33 promoter by 5-aza-dC was confirmed in H292 cells by means of bisulphate sequencing (B) and in PBECs from asthmatic patients by means of methylation-sensitive PCR with HpaII-digested genomic DNA (D). Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 TGF-β–induced EMT does not affect ADAM33. A, The morphologic appearance of A549 cells after TGF-β–induced EMT. B, mRNA expression of EMT markers was quantified by using RT-qPCR. Data were normalized by using the ΔΔCt method, taking the lower expression level for each gene as the normalizing value. Data are from 3 experiments. ND, Not detected. C, Methylation-sensitive PCR for ADAM33 by using HpaII-digested genomic DNA from control and TGF-β1–treated A549 cells. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Quantitation of ADAM33 mRNA in bronchial biopsy specimens from healthy subjects and patients with severe asthma. Quantitative RT-PCR was performed by using primers and probes targeting the α (ADAM33-EGF-Alpha) and β (ADAM33-Beta) isoforms of ADAM33, as well as exons G and H in the metalloprotease domain (ADAM33-GH). For comparison, the levels of αSMA in each biopsy specimen are also provided. Data were analyzed by using the Mann-Whitney U test, and no significant differences were detected between the groups. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 A, Validation of probe-based qPCR assays for ADAM33
A, Validation of probe-based qPCR assays for ADAM33. cDNA from HEK293 cells expressing recombinant ADAM33 cells was tested for equivalence by using primers in the EGF domain (ADAM33-EGF-α) and the metalloprotease domain (ADAM33-GH); mock-transfected cells were used as control. B, Comparison of the ADAM33-EGF-α, ADAM33-β, and ADAM33-GH assays by using cDNA from PBFs. Data are presented as means ± SDs. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 A, Analysis of ADAM33 expression in bronchial brushings
A, Analysis of ADAM33 expression in bronchial brushings. cDNA from bronchial epithelial brushings was tested for ADAM33 expression by using the 3 validated probe-based assays, with fibroblast cDNA being used as a positive control. B, The quantity and quality of the RNA from bronchial brushings was assessed by using primers to MUC5AC, with αSMA to control for the signal from fibroblasts. Data are presented as means ± SDs. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Validation of the SYBR Green assays for the ADAM33-α isoform and S9 rRNA. The upper 2 plots in the left panel show amplification curves for ADAM33 expression in HEK293 cells expressing recombinant ADAM33 (red) and mock-transfected cells (orange; upper panel) and fibroblasts (green) and RT− (purple) and water (blue) controls (lower panel). The lower 2 plots show amplification curves for S9 rRNA by using the same samples as used for ADAM33. The right panel shows the corresponding melt curves for each sample, and on the far right, a Western blot using an anti-ADAM33 antibody against cell lysates prepared from ADAM33-transfected or mock-transfected HEK293 cells is shown. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Analysis of mRNA in bronchial epithelial brushings by using the SYBR Green assays for ADAM33-α isoform and S9 rRNA. The left panel shows (in descending order) the amplification curves obtained for ADAM33 expression in bronchial brushings from healthy control volunteers (green), patients with severe asthma (red), and the RT minus and water controls (purple and blue). The bottom plots shows curves for S9 rRNA. The right panel shows the corresponding melt curves for each sample. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Melt curve analysis of PCR products obtained in assays of bronchial epithelial brushings and bronchial biopsy specimens by using the SYBR Green assays for the ADAM33-α isoform. The upper 2 panels show melt curves obtained for PCR products from bronchial brushings (upper panels) and bronchial biopsy specimens (lower panels) from healthy subjects (left) and asthmatic patients (right). The bottom 2 panels are the same on both the left and right and show control data for comparison. These comparative data are melt curves obtained for PCR products from expression fibroblasts (green), the RT minus and water controls (purple and blue), and HEK293 cells expressing ADAM33-transfected (red) or mock-transfected cells (yellow). Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Agarose gel electrophoresis of PCR products from the SYBR Green assays for the ADAM33-α isoform. The plate shows a typical gel for the PCR products from bronchial brushings (B), fibroblasts (F), HEK293 cells expressing ADAM33 (H+), and the RT minus control. The region of the gel that was selected for excision and cloning of the PCR products is indicated. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Quantitation of ADAM33 expression in bronchial biopsy specimens from healthy subjects and patients with severe asthma by using the SYBR Green assays for the ADAM33-α isoform. cDNA from the biopsy specimens used in the probe-based ADAM33 assays was reanalyzed by using the SYBR Green assay. The data were normalized by using S9 rRNA, as described by Foley et al.9 Data were analyzed by using the Mann-Whitney U test; no significant differences were detected between the groups. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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