Dual androgen-response elements mediate androgen regulation of mmp-2 gene expression in prostate cancer cells

Ben-Yi Li^1,2,3,4, Xin-Bo Liao¹, Atsuya Fujito¹, J. Brantley Thrasher^1,4, Fangyun Shen⁵, Ping-Yi Xu⁶
Departments of ¹Urology, ²Molecular and Integrative Physiology, ³Pathology and Laboratory Medicine, and ⁴Kansas Masonic Cancer Research Institute, the University of Kansas Medical Center; Kansas City, KS 66160, USA; ⁵Department of Urology, The Affiliated Shenzhen Sun Hospital, Dalian Medical University, Shenzhen 518001, ⁶Department of Neurology, Sun Yat-sen University, Guangzhou 510080, China.
DOI: 10.1111/j.1745-7262.2006.00226.x

Abstract
Aim: To analyze the MMP-2 promoter and to identify androgen response elements involved in androgen-induced MMP-2 expression. Methods: MMP-2 mRNA 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 gel retardation assay were used to verify the identified androgen response elements 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 enhanced the reduction of androgen-induced MMP-2 promoter activity. Sequence analysis of these two regions revealed two putative androgen response element (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 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 gel retardation assay. Conclusion: Two ARE motifs were identified to be responsible for androgen-induced MMP-2 gene expression in prostate cancer cells.
Keywords: androgen, androgen receptor, androgen response element, MMP-2, promoter, prostate cancer

Correspondence to: Dr Ben-Yi Li, MD, PhD, Department of Urology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160, USA.
Tel: 913-588-4773 Fax: 913-588-4756
E-mail: bli@kumc.edu
Received 2006-05-12 Accepted 2006-07-16