title: Transcriptional regulation of RKIP expression by androgen in prostate cells.
abstract: OBJECTIVE
  Raf kinase inhibitory protein (RKIP) is a scaffolding molecule in the PEBP family that sequesters certain signaling molecules away from their pathways, thereby abrogating intracellular growth signals. RKIP has been assigned multiple functions and is associated with an increasing number of diseases through its involvement with signal transduction pathways. We previously demonstrated that RKIP is highly expressed in human normal prostate epithelial cells and plays a pivotal role during prostate cancer (PCa) progression. Whether RKIP is subject to endocrine regulation has not been reported.
  
  
  METHODS
  The effect of dihydrotestosterone (DHT) on RKIP expression in normal prostate epithelial cells was determined by real-time RT-PCR and Western blot. Report assay was performed to determine whether the regulation of RKIP by androgens is at the transcriptional level. The binding of androgen receptor (AR) to the RKIP promoter was determined by EMSA and Chromatin Immunoprecipitation (ChIP) assays. To determine whether RKIP was regulated by androgen in vivo, we examined RKIP expression level in response to castration in 6-8 week old C57BL/6 male mice.
  
  
  RESULTS
  Here we report that DHT positively regulates the transcription of RKIP in the normal prostate epithelial cells. The anti-androgen bicalutamide blocked androgen-mediated regulation of RKIP, which indicates that this regulation is mediated through AR. Transfection of the cells with a RKIP promoter-driven luciferase reporter vector showed that DHT increased RKIP promoter activity in parallel with changes in expression. EMSA demonstrates that AR binds to a putative ARE in the RKIP promoter, which was further validated by ChIP assay. Importantly, these data are further supported by our in vivo experiment where castrated mice had less RKIP expression in their prostate glands than sham-operated mice.
  
  
  CONCLUSIONS
  Collectively, the results establish RKIP as a novel androgen target gene. Androgens induce RKIP expression through AR-mediated transcriptional modulation of the RKIP promoter in the prostate. This is the first demonstration of endocrine regulation of the metastasis suppressor gene RKIP.
authors.affiliation: University of Michigan Comprehensive Cancer Center and Department of Urology, University of Michigan Health System, Ann Arbor, MI 48109, USA.
authors.forename: Honglai
authors.initials: H
authors.lastname: Zhang
authors.forename: Jianguo
authors.initials: J
authors.lastname: Wu
authors.forename: Jill M
authors.initials: JM
authors.lastname: Keller
authors.forename: Kam
authors.initials: K
authors.lastname: Yeung
authors.forename: Evan T
authors.initials: ET
authors.lastname: Keller
authors.forename: Zheng
authors.initials: Z
authors.lastname: Fu
mesh_terms: D000728:Androgens / Q000494:pharmacology* / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D016384:Consensus Sequence; D013196:Dihydrotestosterone / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D049488:Phosphatidylethanolamine Binding Protein / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011467:Prostate / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D000728:Androgens; C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein; D012333:RNA, Messenger; D013196:Dihydrotestosterone; D049410:Luciferases, Renilla