All–trans retinoic acid recruits Smad3 to drive TGF–beta ...geb.uni- .All –trans retinoic acid

download All–trans retinoic acid recruits Smad3 to drive TGF–beta ...geb.uni- .All –trans retinoic acid

of 111

  • date post

    16-Aug-2019
  • Category

    Documents

  • view

    212
  • download

    0

Embed Size (px)

Transcript of All–trans retinoic acid recruits Smad3 to drive TGF–beta ...geb.uni- .All –trans retinoic acid

  • All–trans retinoic acid recruits Smad3 to drive TGF–β signaling in lung fibroblasts:

    new insights for the management of bronchopulmonary dysplasia with retinoids

    Inauguraldissertation

    zur Erlangung des Grades eines

    Doktors der Humanmedizin

    des Fachbereichs Medizin der

    Justus-Liebig-Universität Gießen

    vorgelegt von

    Jakob Usemann aus Hamburg

    Gießen, 2016

  • Aus dem Zentrum für Innere Medizin

    Medizinische Klinik II und Poliklinik

    der Justus-Liebig-Universität Gießen

    Supervisor: Prof. Dr. Seeger

    Assessor: PD Dr. Konrad

    Date of Doctoral Defense: 26.01.2016

  • Contents

    1 List of abbrevations 6

    2 Introduction 11 2.1 Normal lung development . . . . . . . . . . . . . . . . . . . . . . . . 11

    2.1.1 Stages of normal lung development . . . . . . . . . . . . . . . 11 2.1.2 Reference time points of lung development in mice and hu-

    mans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.2 Historical background of bronchopulmonary dysplasia . . . . . . . . 13 2.3 Defining bronchopulmonary dysplasia . . . . . . . . . . . . . . . . . 13 2.4 The “old” bronchopulmonary dysplasia . . . . . . . . . . . . . . . . . 15 2.5 The “new” bronchopulmonary dysplasia . . . . . . . . . . . . . . . . 16 2.6 Incidence of bronchopulmonary dysplasia . . . . . . . . . . . . . . . 16 2.7 Bronchopulmonary dysplasia in the context of other preterm infant

    diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.8 Current treatment options for bronchopulmonary dysplasia . . . . . . 18

    2.8.1 Nasal continuous positive airway pressure . . . . . . . . . . . 18 2.8.2 Nasal intermittent positive pressure ventilation . . . . . . . . 19 2.8.3 Surfactant . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.8.4 Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.8.5 Caffeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.8.6 Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.8.7 Inhaled nitric oxide . . . . . . . . . . . . . . . . . . . . . . . 21

    2.9 Pathogenesis of bronchopulmonary dysplasia . . . . . . . . . . . . . 21 2.10 Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.11 Vitamin A and its receptors . . . . . . . . . . . . . . . . . . . . . . . 25 2.12 TGF–β signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

    2.12.1 The TGF–β signaling pathway . . . . . . . . . . . . . . . . . 25 2.12.2 The TGF–β ligands . . . . . . . . . . . . . . . . . . . . . . . 26 2.12.3 The TGF–β receptors . . . . . . . . . . . . . . . . . . . . . . 26 2.12.4 The Smad proteins . . . . . . . . . . . . . . . . . . . . . . . . 27

    2.13 The role of TGF–β and vitamin A in bronchopulmonary dysplasia . . 28

    3 Aims of the study 31

    1

  • 4 Materials and reagents 32 4.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 4.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.3 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

    5 Methods 36 5.1 Cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 5.2 Cultivation of cell lines . . . . . . . . . . . . . . . . . . . . . . . . . 36 5.3 Cell line stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 5.4 Plasmids employed in this study . . . . . . . . . . . . . . . . . . . . 37 5.5 Elution of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 5.6 Preparation of competent E. coli DH5α bacterial cells . . . . . . . . . 38 5.7 Transformation of plasmid DNA into competent E. coli cells . . . . . 38 5.8 Plating and cultivation of E. coli cells . . . . . . . . . . . . . . . . . . 39 5.9 Plasmid maxi preparation . . . . . . . . . . . . . . . . . . . . . . . . 39 5.10 Vector digestion using DNA restriction endonucleases for plasmid ver-

    ification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 5.11 Luciferase assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 5.12 Transient transfection using Lipofectamine™ 2000 . . . . . . . . . . . 40 5.13 NIH/3T3 cell line transfection with small interfering RNA . . . . . . 41 5.14 Transfection of cells with overexpression plasmid constructs . . . . . 42 5.15 Luciferase assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 5.16 Protocol for cell lysis using cell culture lysis 5× reagent . . . . . . . . 43 5.17 Protocol for luciferase measurement . . . . . . . . . . . . . . . . . . . 43 5.18 RNA isolation, cDNA synthesis and PCR . . . . . . . . . . . . . . . 43 5.19 Polymerase chain reaction . . . . . . . . . . . . . . . . . . . . . . . . 44 5.20 Semi-quantitative reverse transcription polymerase chain reaction . . 45 5.21 Real-time reverse transcription polymerase chain reaction . . . . . . 46 5.22 DNA agarose gel electrophoresis . . . . . . . . . . . . . . . . . . . . 47 5.23 Protein isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 5.24 Protein quantification measurement . . . . . . . . . . . . . . . . . . 48 5.25 Separation of proteins by SDS poly-acrylamide gel electrophoresis . . 48 5.26 Western blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 5.27 Statistical analysis of data . . . . . . . . . . . . . . . . . . . . . . . . 50 5.28 Primer table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

    6 Results 53 6.1 ATRA enhances TGF–β signaling assessed by p(CAGA)9 induction . 53

    2

  • 6.2 ATRA effects on TGF–β signaling assessed by Smad phosphorylation using Western blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

    6.3 Identification of TGF–β-responsive genes in NIH/3T3 cells and MLE- 12 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

    6.4 ATRA impacts expression of TGF–β-responsive genes in NIH/3T3 cells 56 6.5 ATRA impacts expression of TGF–β-responsive genes in MLE-12 cells 58 6.6 ATRA effects the expression of components of the TGF–β signaling

    machinery in NIH/3T3 cells . . . . . . . . . . . . . . . . . . . . . . . 59 6.7 ATRA effects the expression of components of the TGF–β signaling

    machinery in MLE-12 cells . . . . . . . . . . . . . . . . . . . . . . . 61 6.8 Smad3 modulates TGF–β signaling in NIH/3T3 cells . . . . . . . . . 62 6.9 Smad3 is crucial for TGF–β signaling in NIH/3T3 cells . . . . . . . . 64

    7 Discussion 66 7.1 TGF–β signaling is critical for lung development . . . . . . . . . . . . 66 7.2 Vitamin A is critical for lung development . . . . . . . . . . . . . . . 67 7.3 ATRA affects Smad3 protein levels in culture . . . . . . . . . . . . . 67 7.4 Retinoids regulate TGF–β signaling at the level of Smad3 in NIH/3T3

    cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 7.5 Perspectives and conclusion . . . . . . . . . . . . . . . . . . . . . . . 71

    8 Abstract 74

    9 Zusammenfassung 75

    10 References 77

    11 Appendix 102

    12 Declaration 105

    13 List of publications 106

    14 Acknowledgements 108

    3

  • List of Figures

    1 ATRA enhances TGF–β signaling in NIH/3T3 cells . . . . . . . . . . 53 2 ATRA effects on phospho-Smad2 and phospho-Smad3 levels in NIH/3T3

    cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 3 TGF–β responsive genes in NIH/3T3 cells and MLE-12 cells . . . . . 56 4 TGF–β target gene expression patterns in NIH/3T3 cells . . . . . . . 56 5 TGF–β target gene expression patterns in NIH/3T3 cells after stim-

    ulation with ATRA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 6 Gene expression patterns in MLE-12 cells after stimulation with ATRA

    and TGF–β . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 7 Gene expression patterns of TGF–β target genes in MLE-12 cells after

    stimulation with ATRA . . . . . . . . . . . . . . . . . . . . . . . . . 60 8 Gene expression patterns of the TGF–β receptor and TGF–β signaling

    Smads in NIH/3T3 cells after stimulation with ATRA . . . . . . . . . 61 9 Gene expression patterns of the TGF–β receptor and signaling Smads

    in MLE-12 cells after stimulation with ATRA . . . . . . . . . . . . . 62 10 Smad3 deficient NIH/3T3 cells lack responsiveness to TGF–β and

    ATRA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 11 Dose dependent effects of Smad 3 expression on TGF–β signaling . . 65 12 The pGEM–T Easy vector map . . . . . . . . . . . . . . . . . . . . . 102 13 The pIRES–hrGFPII vector map . . . . . . . . . . . . . . . . . . . . 102 14 The pRL–SV40 vector map . . . . . . . . . . . . . . . . . . . . . . . 103 15 The pGL3–basic vector map . . . . . . . . . . . . . . . . . . . . . . . 103 16 The pRL–TK vector map . . . . . . . . . . . . . . . . . . . . . . . . 104

    4

  • List of Tables

    1 Lung development stages in human and mice . . . . . . . . . . . . . . 13 2 Diagnostic criteria for bronchopulmonary dysplasia. . . . . . . . . . . 14 3 Physiological criteria for bronchopulmonary dysplasia . . . . . . . . . 15 6 Medium for MLE-12 cells and NIH/3T3 cells . . . . . . . . . . . . . . 37 7 NIH/3T3 cell and MLE-12 cell line stimulation . . . . . . . . . . . . 37 8 E. coli cultivation components . . . . . . . . . . . . . . . . . . . . . . 39 9 Vector digestion components . . . . . . . . . . . . . . . . . . . . . . 40 10 Reverse transcriptase reaction components . . .