Lokalisation und Gen - Expressionsregulation der neutralen Endopeptidase in der humanen Prostata
Loading...
Files
Date
Authors
Publisher
Philipps-Universität Marburg
Supervisors
Abstract
Neutral endopeptidase (NEP / CD10) is a cell
surface zinc metalloproteinase that functions as part of a
regulatory loop controlling local concentrations of peptide
substrates and associated peptide-mediated signal transduction
processes. In contrast to the encouraging data dealing with NEP
activity and regulation in prostate epithelial cells, only a
few studies are available on the cellular expression and
localization of neutral endopeptidase in the prostatic stromal
and cancer cells. Immunofluorescence and western blot
experiments were performed to control the expression and
distribution of the NEP in normal and malignant human prostatic
tissues and cell lines. NEP gene expression was monitored by
RT-PCR, NEP mRNA was detected in human prostatic tissue and in
cultured cells by means of in situ RT-PCR. Prostatic tissue
showed strong signals in the glandular epithelium and weak
signals in the stroma, cultured cells displayed strong signals
in prostate cancer cells (LNCaP) and weak signals in stromal
cells (hPCPs). NEP-immunofluorescence was strong in normal
prostatic epithelium and confined to the apical plasma
membrane. In dedifferentiated prostate cancer specimens,
immunofluorescence of apical plasma membranes was lost, and
both the cytoplasm and portions of the plasma membrane were
immunoreactive for NEP. Prostate cancer cells (LNCaP) showed a
strong immunoreaction of the plasma membrane and the cytoplasm.
In comparison with LNCaP cells, only a weak cytoplasmic
immunofluorescence was found in some stromal cells (hPCPs).
Western blot experiments were performed using whole cells
extracts to proof the presence of NEP protein in LNCaP and
hPCPs. The experiments confirm the expression of NEP by both
cell types, however, the experiment with hPCPs cells showed two
bands. To study the regulation of NEP expression, Northern blot
analysis of total RNA from LNCaP cells showed that NEP-mRNA was
4-fold increased by DHT and bombesin. Expression of NEP in
hPCPs cells was very low and was not induced by DHT or
bombesin. Sequence analysis reveals two androgen response
elements including a typical ARE which binds androgen,
progesterone and glucocorticoid receptors, and a unique ARR
which only binds androgen receptor. Analysis of promoter
deletions using luciferase reporter constructs showed that
-379~-154 fragment of the promoter still containing the Sp-1
binding site are sufficient to confer androgen responsiveness
to the reporter gene. DNase I footprinting identified two
factors binding to the proximal promoter region that were found
to be the ubiquitous Sp family transcription factors and
CCAAT-box transcription factor NF-Y. Analyses of promoter
constructs with mutations in the Sp and NF-Y binding sites
demonstrated that NF-Y factor significantly contributes to the
basal activity, and that Sp helps to mediate the androgen
response.
Review
Metadata
Contributors
Supervisor:
Dates
Created: 2004Issued: 2004-03-18Updated: 2011-08-10
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
In Situ RT-PCRNEPin situ RT-PCRNeprilysin , gene expressionProstate
DFG-subjects
Androgene , NeuropeptideNeprilysinLokalisationProstataTranskription
DDC-Numbers
610
show more
Song, Jian (111997186): Lokalisation und Gen - Expressionsregulation der neutralen Endopeptidase in der humanen Prostata. : Philipps-Universität Marburg 2004-03-18. DOI: https://doi.org/10.17192/z2004.0167.
License
This item has been published with the following license: In Copyright