Dual androgen-response elements mediate androgen regulation of MMP-2 expression in prostate cancer cells.
- Author:
Ben-Yi LI
1
;
Xin-Bo LIAO
;
Atsuya FUJITO
;
J Brantley THRASHER
;
Fang-Yun SHEN
;
Ping-Yi XU
Author Information
- Publication Type:Journal Article
- MeSH: Androgens; pharmacology; Cell Line, Tumor; Chromatin; genetics; DNA Primers; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; genetics; Male; Matrix Metalloproteinase 2; genetics; metabolism; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Prostatic Neoplasms; enzymology; RNA, Messenger; genetics; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion
- From: Asian Journal of Andrology 2007;9(1):41-50
- CountryChina
- Language:English
-
Abstract:
AIMTo characterize the matrix metalloproteinases (MMP)-2 promoter and to identify androgen response elements (AREs) involved in androgen-induced MMP-2 expression.
METHODSMMP-2 mRNA levels was determined by reverse transcription-polymerase chain reaction (RT-PCR). MMP-2 promoter-driven luciferase assays were used to determine the fragments responsible for androgen-induced activity. Chromatin-immunoprecipitation assay and electrophoretic mobility shift assays (EMSA) were used to verify the identified AREs in the MMP-2 promoter.
RESULTSAndrogen significantly induced MMP-2 expression at the mRNA level, which was blocked by the androgen antagonist bicalutamide. Deletion of a region encompassing base pairs -1591 to -1259 (relative to the start codon) of the MMP-2 promoter led to a significant loss of androgen-induced reporter activity. Additional deletion of the 5'-region up to -562 bp further reduced the androgen-induced MMP-2 promoter activity. Sequence analysis of these two regions revealed two putative ARE motifs. Introducing mutations in the putative ARE motifs by site-directed mutagenesis approach resulted in a dramatic loss of androgen-induced MMP-2 promoter activity, indicating that the putative ARE motifs are required for androgen-stimulated MMP-2 expression. Most importantly, the androgen receptor (AR) interacted with both motif-containing promoter regions in vivo in a chromatin immunoprecipitation assay after androgen treatment. Furthermore, the AR specifically bound to the wild-type but not mutated ARE motifs-containing probes in an in vitro EMSA assay.
CONCLUSIONTwo ARE motifs were identified to be responsible for androgen-induced MMP-2 expression in prostate cancer cells.