p68 RNA helicase [Elektronische Ressource] : a novel mediator of nitric oxide and mitogen functions in keratinocytes / von Kornelija Kahlina

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• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2004
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

p68 RNA helicase:
a novel mediator of nitric oxide and mitogen
functions in keratinocytes




DISSERTATION
zur Erlangung des Doktorgrades
der Naturwissenschaften




vorgelegt beim Fachbereich
Chemisch und Pharmazeutische Wissenschaften
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main




von
Kornelija Kahlina
aus Gross-Umstadt



Frankfurt am Main 2004
















Vom Fachbereich
Chemische und Pharmazeutische Wissenschaften
der Johann Wolfgang Goethe-Universität
als Dissertation angenommen.




















Dekan: Prof. Dr. Harald Schwalbe
Gutachter: PD Dr. Stefan Frank
Prof. Dr. Bernd Ludwig
Datum der Disputation: 09. November 2004 The work outlined in this thesis is based on experimental studies published in
the following article:


Kahlina K, Goren I, Pfeilschifter J, Frank S
p68 DEAD box RNA helicase expression in keratinocytes: regulation, nuclear
localization, and functional connection to proliferation and VEGF gene expression.
J Biol Chem 279: 44872-44882, 2004
Contents


Contents
___________________________________

1 INTRODUCTION 1
1.1 The skin 1
1.2 Phases of cutaneous wound repair 3
1.2.1 Hemorrhage 3
1.2.2 Inflammation 4
51.2.3 Reepithelialization
1.2.4 Granulation tissue formation 7
1.2.5 Neovascularisation 8
1.2.6 Remodeling 9
1.3 Nitric oxide and skin 10
1.4 RNA helicases 12
1.4.1 p68 RNA helicase 12
1.5 Aim of the thesis 14

2 MATERIALS AND METHODS 15
2.1 Materials 15
2.1.1 Chemicals 15
2.1.2 Other materials and kits 17
2.1.3 Laboratory equipment 17
172.1.4 Enzymes
2.1.4.1 Pretreatment of enzymes 18
2.1.5 Antibodies and antisera 18
2.1.6 Recombinant and purified proteins 18
192.1.7 Plasmids
2.1.7.1 Vectors 19
2.1.7.2 Recombinant plasmids 19
2.1.8 Bacterial strains 19
192.1.9 Eukaryotic cell lines
2.1.10 Buffers 19 Contents

2.1.11 S-nitroso-glutathione 20
2.1.12 Computer Software 20
2.2 Bacterial culture 20
202.2.1 Competent bacteria for transformation
2.3 Nucleic acid techniques 21
2.3.1 Preparation of plasmid DNA 21
222.3.2 RNA isolation
2.3.2.1 Isolation from cultured cells 22
2.3.2.2 Isrom tissue 23
2.3.3 Quantification of nucleic acid concentrations 23
242.3.4 Agarose gel electrophoresis of nucleic acids
2.3.4.1 DNA isolation from agarose gels 24
2.3.5 Reverse transcriptase polymerase chain reaction 25
2.3.5.1 Reverse transcription 25
2.3.5.2 Polymerase chain reaction (PCR) 25
2.3.5.3 Cloning of PCR products 26
2.3.6 Manipulation of DNA 26
2.3.6.1 Restriction 26
2.3.6.2 Ligation 27
2.3.6.3 Dephosphorylation 27
2.3.6.4 Cloning pcDNA-p68 28
2.3.6.5pEGFP-N1-p68 28
2.3.7 DNA sequencing 28
2.3.8 RNase protection assay 29
2.3.8.1 Preparation of a radiolabeled antisense probe 29
2.3.8.2 Hybridization and cleavage 30
2.3.8.3 Analytical gel electrophoresis and signal detection 31
2.3.9 Differential screening of a subtractive library 31
2.3.9.1 PCR-Select cDNA substraction 31
2.3.9.2 Subtracted cDNA Library Construction 32
2.3.9.3 Colony Array 32
32
α 332.3.9.4 Preparation of [ - P]dCTP labeled probe
2.3.9.5 Hybridization 33
2.4 Protein techniques 34
2.4.1 Preparation of lysates 34
2.4.1.1 Cell lysates 34
2.4.1.2 Preparation of fractionated cell lysates 34
352.4.1.3 Tissue lysates
2.4.2 Trichloroacetic acid (TCA) precipitation 35
2.4.3 Determination of protein concentration 36 Contents

2.4.4 Western blot analysis 36
2.4.4.1 SDS gel electrophoresis 37
2.4.4.2 Transfer to PVDF membrane 37
2.4.4.3 Immunodetection 38
2.4.5 Generation of a p68 RNA helicase antibody 38
2.4.6 Enzyme-linked immunosorbent assay (ELISA) 39
2.4.7 Immunohistochemistry 39
2.4.7.1 Preparation of frozen tissue sections 39
2.4.7.2 Immunoperoxidase staining 39
2.4.7.3 Hematoxylin staining 40
2.5 Cell culture 41
412.5.1 Culture and stimulation of HaCaT cells
2.5.2 Culture of HEK 293 cells 41
2.5.3 Transfection 41
2.6 In vitro transcription/translation 42
2.7 Proliferation 43
2.8 Confocal microscopy 43
2.9 Silencing p68 RNA helicase gene expression by siRNA 43
2.10 Wound healing studies 44
2.10.1 Wounding of mice 44

3 RESULTS 46
3.1 Identification of p68 as a novel NO-regulated gene in
keratinocytes 46
3.1.1 Identification of novel NO-regulated genes by a differential
cDNA library from GSNO-stimulated HaCaT keratinocytes 46
3.1.2 NO induces p68 mRNA expression 46
3.1.3 NO regulates p68 expression at the transcriptional level 47
3.1.4 NO r p68 protein expression 48
3.1.4.1 Characterization of the p68 antibody 49
3.1.4.2 NO induces p68 protein expression 50
3.1.5 Potential involvement of cGMP or MAPK pathways in p68 induction 51
3.1.5.1 NO mediated p68 expression is not dependent on activation of
51 soluble guanylate cyclase
3.1.5.2 MAPK cascade is not involved in NO-induced p68 expression 52
3.2 Growth factors, serum and cytokines induce p68 expression 52
3.2.1 growth factors and serum stimulate p68 expression 52 Contents

3.2.2 54 Pro-inflammatory cytokines induce p68 expression
3.3 p68 is localized in the nucleus 55
3.3.1 p68 is localized in nuclei of human and murine keratinocytes in vitro 55
563.3.2 Constuction of a p68/ GFP fusion protein
3.3.3 p68 localizes in nucleoli of keratinocytes 57
3.4 p68 and skin repair 59
3.4.1 p68 protein expression is transiently decreased in wounded skin 59
3.4.2 p68 protein is restrictively localized in nuclei of keratinocytes in vivo 60
3.5 Silencing p68 RNA helicase protein expression by short
interfering RNA (siRNA) 63
3.6 p68 expression is functionally connected to keratinocyte
proliferation and VEGF gene expression 63

4 DISCUSSION 67
4.1 Nitric oxide and skin 67
4.2 Nitric oxide and p68 RNA helicase 67
4.2.1 p68 is a novel NO-induced gene 67
4.2.2 68 How does nitric oxide exert its action on p68?
4.3 p68 expression is triggered by wound-related keratinocyte
mitogens and inflammatory stimuli 69
4.4 p68 and proliferation 70
4.5 Nuclear p68 localizes in nucleoli of keratinocytes 71
4.6 Does p68 alter VEGF gene expression as transcriptional
co-activator? 72
4.7 p68 and wound healing 73
4.8 Clinical relevance 74

5 SUMMARY 76

6 REFERENCES 78
Contents

7 APPENDIX 92
7.1 Abbreviations 92
7.2 List of publications 96
7.2.1 Journal publications 96
7.2.2 Talks 96
967.2.3 Poster presantions
7.3 Acknowledgement 97
7.4 Deutsche Zusammenfassung 98
7.4.1 Haut und Wundheilung 98
7.4.2 Sickstoffmonoxid und Wundheilung 99
7.4.3 p68 RNA Helicase und W100
7.4.4 NO und weitere Keratinozyten-Mitogene induzieren die
Expression der p68 RNA Helicase in vitro 101
7.4.5 p68 lokalisiert in Keratinozyten Nucleoli 102
7.4.6 p68 beeinflußt die Zellteilung in Keratinozyten 102
7.4.7 p68 ist beteiligt an der Regulation der VEGF Expression 103
7.4.8 p68 Expression während der kutanen Wundheilung in vivo 103
7.5 Curriculum vitae 105 Introduction

1
Introduction
___________________________________

1.1 The skin

The skin represents the body´s largest organ system and is the principle site of
interaction with the environment. The skin serves as a barrier which mediates
protection against harmful environmental conditions, such as mechanical damage,
noxious agents, invading pathogens and ultraviolet light. Moreover, the skin limits
water loss and participates in the maintenance of body temperature. Special
receptors enable the skin organ system to sense heat, cold, pain and pressure
(Thews et al., 1999; Freinkel and Woodley, 2001).
The skin consists of the outer epidermal, the underlying dermal and the
subcutaneous layers. Comparable to other epidermal structures in the body, the
epidermis represents a stratified, self-renewing tissue compartment of multiple cell
types. Keratinocytes represent the major epidermal cell type and are responsible for
cutaneous barrier function. To a lesser extent, melanocytes (melanin synthesis),
Langerhans cells (antigen-presenting), and Merkel cells (sensory functions) are
found in the epidermal layer. Keratinocytes are organized into four different layers
that correspond to the progressive stages of differentiation: stratum basale, stratum
spinosum, stratum granulosum, and stratum corneum. While passaging through
these layers, keratinocytes show an increase in size and cell flattening, they loose
organelles, change their keratin (intermediate filament protein) expression and are no
longer able to proliferate (Haake et al., 2001). The