Regulation of ectodomain shedding of ADAM33 in vitro and in vivo

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Regulation of ectodomain shedding of ADAM33 in vitro and in vivo Elizabeth R. Davies, PhD, Laura Denney, PhD, Marieke Wandel, MSc, Clare M. Lloyd, PhD, Donna E. Davies, PhD, Hans Michael Haitchi, MD, PhD Journal of Allergy and Clinical Immunology DOI: 10.1016/j.jaci.2019.01.026 Copyright © 2019 The Authors Terms and Conditions

Fig 1 Regulation of ADAM33 shedding. Cos-7 cells expressing full-length catalytically active murine ADAM33 (A-F) or inactive ADAM33 E347A (Fig 1, A and B) were treated without or with TGF-β (5 ng/mL) for 8 hours and SMAD (SB431542) (Fig 1, C and D) or MAPK (PD98059) (Fig 1, E and F) inhibitors, as indicated. Soluble ADAM33 immunoreactivity and enzymatic activity in cell-free supernatants were assessed by Western blotting (inset) with densitometry (arbitrary units, AU) (Fig 1, A, C, and E) or in a fluorescence resonance energy transfer (FRET) peptide cleavage assay, respectively; in panel Fig 1, B, enzymatic activity in the supernatants is expressed as a percent of that measured in supernatants from untreated cells expressing full-length active ADAM33, whereas in panels D and F, activity is expressed as a percent of that measured in supernatants from TGF-β–treated cells. Data are expressed as mean + SD (n = 4 independent experiments). #P < .05, ##P < .01 vs control, *P < .05, **P < .01, ***P < .001. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2019.01.026) Copyright © 2019 The Authors Terms and Conditions

Fig 2 Epithelial TGF-β enhances shedding of ADAM33 in vivo. Epithelial (Epi)Tgfb−/− or littermate Tgfb+/+ control mice were challenged with intranasal house dust mite (HDM) extract (A and B) or recombinant IL-33 (C and D) and, where indicated, recombinant TGF-β (Fig 2, C and D). Soluble ADAM33 immunoreactivity and enzyme activity in BALF were assessed by Western blotting (Fig 2, A and C) or FRET peptide cleavage assay, respectively; enzyme activity is expressed as relative fluorescent units per minute (RFU/min) (Fig 2, B and D). FRET, Fluorescence Resonance Energy Transfer. Data are mean + SD (n = 4-6 mice per group). Data are representative of 2 independent experiments. ##P < .01, ###P < .001 vs control, *P < .05, ***P < .001. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2019.01.026) Copyright © 2019 The Authors Terms and Conditions

Fig E1 TGF-β enhances shedding of murine ADAM33. Cos-7 cells transiently transfected with full-length murine ADAM33 or green fluorescent protein (GFP) as control were treated with TGF-β as indicated. Soluble ADAM33 immunoreactivity and enzyme activity in cell-free supernatants were assessed by Western blotting (A) with densitometry (arbitrary units, AU) (B) or fluorescence resonance energy transfer peptide cleavage assay, respectively; enzyme activity is expressed as percent of activity found in supernatants from control untreated cells (C). Data are mean + SD and are representative of 4 independent experiments. MW, Molecular weight. **P < .01. ***P < .001. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2019.01.026) Copyright © 2019 The Authors Terms and Conditions

Fig E2 The broad-spectrum MMP inhibitor GM6001 does not inhibit ADAM33 shedding. Cos-7 cells transiently transfected with full-length murine ADAM33 were preincubated with GM6001 (0-10 μM) for 1 hour before addition of 5 ng/mL TGF-β for 8 hours. Cell-free supernatants were assessed for soluble ADAM33 by Western blotting (A) with densitometry (arbitrary units, AU) (B) and by fluorescence resonance energy transfer peptide cleavage assay (C). Purified recombinant ADAM33 protein was incubated with GM6001 and tested for activity in a fluorescence resonance energy transfer peptide cleavage assay (D). Activity is presented as a percent of that measured in the absence of GM6001. MW, Molecular weight. Data are expressed as mean + SD and are representative of 4 independent experiments. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2019.01.026) Copyright © 2019 The Authors Terms and Conditions