DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Wolfram Lux :
Regulation of tPA genexpression in human neuronal cells
Regulation der tPA Genexpression in neuronalen Zellen des Menschen

 FU Logo

|Abstract| |Table of Contents| |More Information|

Abstract

Tissue-type plasminogen activator (tPA) is a serine protease which catalyzes the conversion of plasminogen to plasmin within the fibrinolytic pathway to aid with blood clot dissolution. As a protease it is also necessary for processes in development (e. g. embryogenesis) and in cellular migration such as tumor outgrowth and metastasis. Recent studies have implicated tPA in neuronal plasticity, synaptic remodelling and neuronal degeneration. TPA is widely expressed in the CNS and neuronal aktivity induces the expression of mRNA of tPA in pyramidal neurons. Transgenic mice overexpressing tPA show an increased and prolonged hippocampal long-term potentiation (LTP) and improved performance in spatial orientation learning tasks while tPA-/- mutants exhibit learning deficits, retarded neuronal migration and resistance towards neuronal destruction by injections of excitotoxins. In MS brain tPA is thought to be responsible for the final enzyme-mediated process of demyelination. A better untestanding of the role of tPA in cognitive processes requires detailed examination of its regulation. No in vivo data and only few in vitro data could be gained regarding the control of neuronal expression of tPA until the beginning of this thesis.. In neuronal cells (SY5Y, SK-N-SH) neurotrophins induced the expression of mRNA of tPA in a dose and time dependent manner as shown by Ribonuclease Protection Assays (RPA) and Transactivation Assays. At 2.4 to 2.5 EC50 NGF (100 ng/ml) stimulated tPA expression 4fold after 48 h whereas BDNF (5 ng/ml) and NT-4 (25 ng/ml) stimulatd tPA expression ~5fold after 4 h. NT-3 (5 ng/ml) showed no effect at all. Neither of these effects was based on enhanced mRNA stability nor dependent of de novo protein synthesis. In vivo genomic analysis of the whole tPA gene in neuronal tissue (Kelly, SNB-19, SK-N-SH) exhibited DNaseI HS sites in the promotor region, 1st and 3rd intron. This promotor region was of further interest for this thesis. DNaseI HS sites spanning a region of ~3.6 kb in the promotor were investigated by in vivo footprinting and revealed 46 protected responsive sites towards 28 known transcription factors and 27 protected DNA elements of unknown protein factors (all based on matrix and core similarity >90%). Many of these factors are known to play roles in processes of development. One NF-?B responsive element at ~3.1 kb upstream of the transcription start site was further investigated. It bears an almost perfect consensus sequence for the NF-?B/Rel family of protein factors (91-100 % similarity) and shows enhancer properties in Transactivation Assays. Cloning into reporter-gene-vectors and site directed mutagenesis identified this element as being responsible for a very strong response to phorbolester (PMA) of 11-13 fold overnight in neuronal cells.

Table of Contents

Download the whole PhDthesis as a zip-tar file or as zip-File

For download in PDF format click the chapter title

0. Titelblatt

2. Inhaltsverzeichnis

3. Abbildungsverzeichnis

4. Tabellenverzeichnis

5. Abkürzungsverzeichnis

6. Einleitung

7. Material und Methoden

7.1 Material und Chemikalien

7.2 Zellbiologische Methoden

7.3 Molekularbiologische Methoden

7.4 Ribonuklease Protektions Assay (RPA)

7.5 DNasel Hypersensitivitätsassay

7.6 In vivo Footprinting

8. Ergebnisse

8.1 Neurotrophine als Mediatoren der tPA Genexpression

8.2 Analyse des tPA Gens auf genomischer Ebene

8.3 Phorbolester induziert tPA über ein NF-kB responsives DNA-Element

9. Diskussion

9.1 Experimentelle Methodik

9.2 Neurotrophine regulieren die tPA Transkription

9.3 Eine funktionelle NF-kB Erkennungssequenz reguliert die Transkription von tPA

9.4 Erkennungssequenzen multipler Transkriptionsfaktoren

Zusammenfassung

Literaturverzeichnis

Danksagung

Veröffentlichungen und Vorträge

Anhang

 

 

More Information:

Online available: http://www.diss.fu-berlin.de/2001/212/indexe.html
Language of PhDThesis: german
Keywords: plasminogen tpa neuronal transcription expression footprint DNase
DNB-Sachgruppe: 32 Biologie
Date of disputation: 12-Oct-2001
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Ferdinand Hucho
Second Referee: Prof. Dr. Peter Donner
Contact (Author): wolfram.lux@gmx.de
Contact (Advisor): Peter.Donner@schering.de
Date created:05-Nov-2001
Date available:07-Nov-2001

 

|| DARWIN|| Digitale Dissertationen || Dissertation|| German Version|| FU Berlin|| Seitenanfang ||

Mail-Icon Fragen und Kommentare an:
darwin@inf.fu-berlin.de

© Freie Universität Berlin 1999