Lokalisation und Gen - Expressionsregulation der neutralen Endopeptidase in der humanen Prostata

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.