Dual androgen-response elements mediate androgen regulation of MMP-2 expression in prostate cancer cells.
OBJECTIVE
To characterize the matrix metalloproteinases (MMP)-2 promoter and to identify androgen response elements (AREs) involved in androgen-induced MMP-2 expression.


METHODS
MMP-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.


RESULTS
Androgen 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.


CONCLUSIONS
Two ARE motifs were identified to be responsible for androgen-induced MMP-2 expression in prostate cancer cells.
Li
Ben-Yi
BY
Department of Urology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160, USA. bli@kumc.edu
Liao
Xin-Bo
XB
Fujito
Atsuya
A
Thrasher
J Brantley
JB
Shen
Fang-Yun
FY
Xu
Ping-Yi
PY
D000728:Androgens / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion
D000728:Androgens; D002843:Chromatin; D017931:DNA Primers; D012333:RNA, Messenger; D008156:Luciferases; D020778:Matrix Metalloproteinase 2