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G‐protein alpha‐s and ‐12 subunits are involved in androgen‐stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells
Author(s) -
Liu Jianjun,
Youn Hyewon,
Yang Jun,
Du Ningchao,
Liu Jihong,
Liu Hongwei,
Li Benyi
Publication year - 2011
Publication title -
the prostate
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.295
H-Index - 123
eISSN - 1097-0045
pISSN - 0270-4137
DOI - 10.1002/pros.21345
Subject(s) - transactivation , androgen receptor , biology , small interfering rna , microbiology and biotechnology , androgen , chromatin immunoprecipitation , cancer research , prostate cancer , chemistry , gene expression , transfection , cell culture , endocrinology , gene , cancer , biochemistry , promoter , genetics , hormone
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. CONCLUSION These data suggest that caveolae‐associated G‐protein alpha subunits are involved in AR transactivation by modulating the activities of different PI3K isoforms. Prostate 71:1276–1286, 2011. © 2011 Wiley‐Liss, Inc.