title: G-protein alpha-s and -12 subunits are involved in androgen-stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells.
abstract: BACKGROUND
  The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgenic hormone action in cells. We recently demonstrated the involvement of phosphoinositide 3-OH kinase (PI3K) p110beta in AR transactivation and gene expression. In this study, we determined the upstream signals that lead to PI3K/p110beta activation and AR transactivation after androgen stimulation.
  
  
  METHODS
  Human prostate cancer LAPC-4 and 22Rv1 cell lines were used for the experiments. AR transactivation was assessed using an androgen responsive element-driven luciferase (ARE-LUC) assay. Cell proliferation was examined using BrdU incorporation and MTT assays. Target genes were silenced using small interfering RNA (siRNA) approach. Gene expression was evaluated at the mRNA level (real-time RT-PCR) and protein level (Western blot). PI3K kinase activities were measured using immunoprecipitation-based in vitro kinase assay. The AR-DNA-binding activity was determined using chromatin-immunoprecipitation (ChIP) assay.
  
  
  RESULTS
  First, at the cellular plasma membrane, disrupting the integrity of caveolae microdomain with methyl-β-cyclodextrin (M-β-CD) abolished androgen-induced AR transactivation and gene expression. Then, knocking down caveolae structural proteins caveolin-1 or -2 with the gene-specific siRNAs significantly reduced androgen-induced AR transactivation. Next, silencing Gα(s) and Gα(12) genes but not other G-proteins blocked androgen-induced AR transactivation and cell proliferation. Consistently, overexpression of Gα(s) or Gα(12) active mutants enhanced androgen-induced AR transactivation, of which Gα(s) active mutant sensitized the AR to castration-level of androgen (R1881). Most interestingly, knocking down Gα(s) but not Gα(12) subunit significantly suppressed androgen-stimulated PI3K p110beta activation. However, ChIP analysis revealed that both Gα(s) or Gα(12) subunits are involved in androgen-induced AR interaction with the AR target gene PSA promoter region.
  
  
  CONCLUSIONS
  These data suggest that caveolae-associated G-protein alpha subunits are involved in AR transactivation by modulating the activities of different PI3K isoforms.
authors.affiliation: Department of Urology, the Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Liu
authors.forename: Hyewon
authors.initials: H
authors.lastname: Youn
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.forename: Ningchao
authors.initials: N
authors.lastname: Du
authors.forename: Jihong
authors.initials: J
authors.lastname: Liu
authors.forename: Hongwei
authors.initials: H
authors.lastname: Liu
authors.forename: Benyi
authors.initials: B
authors.lastname: Li
mesh_terms: D021941:Caveolae / Q000378:metabolism; D045744:Cell Line, Tumor; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D008297:Male; D015741:Metribolone / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017430:Prostate-Specific Antigen / Q000235:genetics; D011471:Prostatic Neoplasms / Q000378:metabolism*; D011944:Receptors, Androgen / Q000235:genetics*; D015398:Signal Transduction; D015533:Transcriptional Activation; D015854:Up-Regulation
chemical_list: D011944:Receptors, Androgen; D015741:Metribolone; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D058543:Class Ia Phosphatidylinositol 3-Kinase; D017430:Prostate-Specific Antigen; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019205:GTP-Binding Protein alpha Subunits, Gs