Selective dysfunction of p53 for mitochondrial biogenesis induces cellular proliferation in bronchial smooth muscle from asthmatic patients Thomas Trian,

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Presentation transcript:

Selective dysfunction of p53 for mitochondrial biogenesis induces cellular proliferation in bronchial smooth muscle from asthmatic patients Thomas Trian, PhD, Benoit Allard, PhD, Annaig Ozier, MD, PhD, Elise Maurat, Isabelle Dupin, PhD, Matthieu Thumerel, MD, Olga Ousova, PhD, Jennifer Gillibert-Duplantier, PhD, Valérie Le Morvan, PhD, Hugues Begueret, MD, PhD, Pierre-Olivier Girodet, MD, PhD, Roger Marthan, MD, PhD, Patrick Berger, MD, PhD Journal of Allergy and Clinical Immunology Volume 137, Issue 6, Pages 1717-1726.e13 (June 2016) DOI: 10.1016/j.jaci.2015.10.031 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Bronchial specimens and BSM were obtained from control subjects (open circles) and asthmatic patients (solid circles). A, Normalized BSM area was assessed on biopsy specimens and is presented as a percentage of the whole biopsy area (control subjects, n = 6; asthmatic patients, n = 7). B, BSM cell proliferation was assessed by using BrdU incorporation in DMEM supplemented with 10% FBS (control subjects, n = 4; asthmatic patients, n = 8). C, Number of mitochondria per BSM cell was assessed from electronic microscopic images (control subjects, n = 6; asthmatic patients, n = 9). D, Quantification of porin content was assessed from Western blots and normalized by β-actin content (control subjects, n = 4; asthmatic patients, n = 4). E and F, Expression of p53 in BSM was assessed in vivo by using RT-PCR after laser microdissection (control subjects, n = 4; asthmatic patients, n = 5; Fig 1, E) and in vitro by means of Western blotting of BSM cell lysates (control subjects, n = 10; asthmatic patients, n = 10; Fig 1, F). Individual data are shown, and lines represent means. *P < .05, Student t tests. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 BSM cells were obtained from control subjects (open circles) and asthmatic patients (solid circles). A, Quantification of whole Mdm2 content was assessed from Western blots and normalized by β-actin content (control subjects, n = 4; asthmatic patients, n = 4). B, Mean fluorescence intensity of Mdm2 antibody was assessed by using flow cytometry and normalized by irrelevant antibody (control subjects, n = 4; asthmatic patients, n = 4). C, After coimmunoprecipitation of p53, quantification of Mdm2 was assessed from Western blots and normalized by p53 (control subjects, n = 3; asthmatic patients, n = 4). D, Quantification of phospho-ser20-p53 content was assessed from Western blots and normalized by p53 content (control subjects, n = 6; asthmatic patients, n = 5). Individual data are shown, and lines represent means. *P < .05, Student t tests. NS, Nonsignificant. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 BSM cells were obtained from control subjects (white bars) and asthmatic patients (black bars). A and B, Expression of p53 in BSM cells was assessed by using quantitative RT-PCR (control subjects, n = 7; asthmatic patients, n = 10; Fig 3, A) and Western blotting (control subjects, n = 5; asthmatic patients, n = 5; Fig 3, B) and expressed as a percentage from nontransduced BSM cells (baseline). C and D, BSM cell proliferation was assessed by using cell counting (control subjects, n = 5; asthmatic patients, n = 7; Fig 3, C) or BrdU incorporation (control subjects, n = 4; asthmatic patients, n = 5; Fig 3, D) in DMEM supplemented with 10% FBS or bronchial epithelial cell supernatant (Epi Sup). BSM cells were not transduced (empty bars), transduced with an empty lentivirus (vertical hatched bars), or transduced with a lentivirus producing p53 shRNA (horizontal hatched bars). Data are means ± SEMs. *P < .05, ANOVA with the Turkey test. NS, Nonsignificant. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Expression of PGC-1α (A), TFAM (B), p21 (C), and Bax (D) was assessed by using quantitative RT-PCR. BSM cells were obtained from control subjects (white bars, n = 7) and asthmatic patients (black bars, n = 10). BSM cells were either not transduced (empty bars) or transduced with a lentivirus producing p53 shRNA (horizontal hatched bars). Data are means ± SEMs. *P < .05, Student paired t tests. NS, Nonsignificant. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 BSM cells were obtained from control subjects (white bars, n = 7) and asthmatic patients (black bars, n = 7). BSM cell proliferation was assessed by using cell counting in DMEM supplemented with 10% FBS. BSM cells were not transduced (empty bars), transduced with an empty lentivirus (vertical hatched bars), or transduced with a lentivirus producing TFAM shRNA (horizontal hatched bars). Data are means ± SEMs. *P < .05, ANOVA with the Turkey test. NS, Nonsignificant. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 BSM cells were obtained from control subjects (white bars, n = 5) and asthmatic patients (black bars, n = 6). BSM cell proliferation was assessed by using cell counting in DMEM supplemented with 10% FBS. Data are means ± SEMs. *P < .05, ANOVA with Turkey tests. NS, Nonsignificant. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Representative electronic microscopic images from bronchial sections were obtained from a control subject (A) and an asthmatic patient (B) and observed at ×6000 magnification. Some smooth muscle mitochondria were visualized (arrows). Bars = 1 μm. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 BSM cells were obtained from control subjects (white bars, n = 5) and asthmatic patients (black bars, n = 10). Mean fluorescence intensity of TOM20 antibody was assessed by using flow cytometry and normalized by that of irrelevant antibody. Data are means ± SEMs. *P < .05, Student t tests. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 BSM cells were obtained from control subjects (white bars, n = 4) and asthmatic patients (black bars, n = 4). Mean fluorescence intensity of p53 antibody was assessed by using flow cytometry and normalized by that of irrelevant antibody. Data are means ± SEMs. *P < .05, Student t tests. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Lentivirus transduction was assessed by means of flow cytometry with lentiviral production of GFP. BSM cells were obtained from control subjects (white bars, n = 7) and asthmatic patients (black bars, n = 10). BSM cells were either transduced with an empty lentivirus (vertical hatched bars) or transduced with a lentivirus producing p53 shRNA (horizontal hatched bars). Data are means ± SEMs. NS, Nonsignificant. ANOVA with Turkey tests. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Expression of PGC-1α (A), TFAM (B), p21 (C), and Bax (D) was assessed by using quantitative RT-PCR. BSM cells were obtained from control subjects (white bars, n = 7) and asthmatic patients (black bars, n = 10). BSM cells were either not transduced (empty bars) or transduced with a lentivirus producing p53 shRNA (horizontal hatched bars). Data are expressed as fold changes compared with control values and presented as means ± SEMs. *P < .05, Student paired t tests. NS, Nonsignificant. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 The p53 binding assay was performed on nuclear protein extracted from BSM cells by using the p53 transcription factor binding assay. BSM cells were obtained from control subjects (white bars, n = 5) and asthmatic patients (black bars, n = 10). Data are expressed as a ratio between the OD value measured at 450 nm and the quantity of nuclear protein assessed by using the Lowry method in nanograms. Data are presented as means ± SEMs. NS, Nonsignificant by using Student t tests. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E8 Proposed schematic diagram of the cellular pathway involved in BSM proliferation in asthmatic patients compared with that in control subjects. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E9 A and B, Expression of p53 in BSM was assessed in vitro by means of Western blotting (patients with moderate asthma, n = 5; patients with severe asthma, n = 5; Fig E9, A) and flow cytometry (patients with moderate asthma, n = 8; patients with severe asthma, n = 6; Fig E9, B). C and D, Expression of PGC-1α (Fig E9, C) and TFAM (Fig E9, D) was assessed by using quantitative RT-PCR. BSM cells were transduced with a lentivirus producing p53 shRNA and obtained from patients with moderate asthma (n = 5) and those with severe asthma (n = 5). Individual data are shown, and lines represent means. NS, Nonsignificant by using Student t tests. Journal of Allergy and Clinical Immunology 2016 137, 1717-1726.e13DOI: (10.1016/j.jaci.2015.10.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions