title: Identification and characterization of two androgen response regions in the human neutral endopeptidase gene.
abstract: Transcription of the human neutral endopeptidase 24.11 (NEP) gene is androgen regulated in prostate cancer cells. Homology search identified a sequence GTCACAaagAGTTCT similar to the ARE consensus sequence GGTACAnnnTGTTCT within the 3'-untranslated region of the NEP mRNA. A double-stranded radiolabelled oligonucleotide containing this NEP-ARE sequence formed a DNA-protein complex with nuclear proteins from LNCaP cells or COS-7 cells co-transfected with an androgen receptor (AR) expression vector, and with full-length AR synthesized by baculovirus in mobility shift assays. Unlabeled NEP-ARE or consensus ARE but not mutated NEP-ARE replaced radiolabelled NEP-ARE. Steroid-dependent enhancement of transcription was assayed by transfecting ptkCAT reporter constructs containing the NEP-ARE into CV-1/AR cells and prostate cancer cells (PC-3/AR). Enhancement of chloramphenicol acetyltransferase (CAT) activity was increased four-fold by androgen, seven-fold by dexamethasone and three-fold by progesterone in CV-1/AR cells, and the NEP-ARE bound to glucocorticoid and progesterone receptor in mobility shift assays. We next performed DNase-I footprinting analysis of the NEP promoter and identified a 23 bp sequence GGTGCGGGTCGGAGGGATGCCCA (NEP-ARR) which was protected from DNase I cleavage by nuclear extracts from COS-7 cells expressing AR. This sequence was 62.5% homologous to an androgen responsive region (PSA-ARR) identified in the promoter of the prostate specific antigen (PSA) gene. A double-stranded radiolabelled oligonucleotide containing this NEP-ARR sequence formed DNA-protein complex with AR but not GR proteins. Unlabeled NEP-ARR, PSA-ARR and NEP-ARE replaced radiolabelled NEP-ARR. Steroid-dependent enhancement of transcription assays in PC-3/AR cells revealed that the enhancement of CAT activity was increased 2.3-fold by androgen, but not by glucocorticoid or progesterone. In a thymidine kinase promoter, the NEP-ARE and NEP-ARR together stimulated a five-fold increase in promoter activity in PC cells. These data suggest that steroid regulation of the NEP gene involves at least two elements including a typical ARE which binds androgen, progesterone and glucocorticoid receptors, and a unique ARR which only binds androgen receptor.
authors.affiliation: Weill Medical College of Cornell University, 525 E. 68th Street, B-1519, New York, NY 10021, USA.
authors.forename: R
authors.initials: R
authors.lastname: Shen
authors.forename: M
authors.initials: M
authors.lastname: Sumitomo
authors.forename: J
authors.initials: J
authors.lastname: Dai
authors.forename: D O
authors.initials: DO
authors.lastname: Hardy
authors.forename: D
authors.initials: D
authors.lastname: Navarro
authors.forename: B
authors.initials: B
authors.lastname: Usmani
authors.forename: C N
authors.initials: CN
authors.lastname: Papandreou
authors.forename: L B
authors.initials: LB
authors.lastname: Hersh
authors.forename: M A
authors.initials: MA
authors.lastname: Shipp
authors.forename: L P
authors.initials: LP
authors.lastname: Freedman
authors.forename: D M
authors.initials: DM
authors.lastname: Nanus
mesh_terms: D000728:Androgens / Q000378:metabolism* / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D008297:Male; D015260:Neprilysin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011471:Prostatic Neoplasms / Q000473:pathology; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D020218:Response Elements / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
chemical_list: D000728:Androgens; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D015260:Neprilysin