G-protein alpha-s and -12 subunits are involved in androgen-stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells.
G-protein alpha-s and -12 subunits are involved in androgen-stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells.
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DOI：
10.1002/pros.21345
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发表时间：
2011-09
期刊：
影响因子：
2.8
通讯作者：
Li, Benyi
中科院分区：
文献类型：
--
作者：
Liu, Jianjun;Youn, Hyewon;Yang, Jun;Du, Ningchao;Liu, Jihong;Liu, Hongwei;Li, Benyi
雄激素受体（AR）是一种配体依赖性转录因子，介导细胞中的雄激素作用。我们最近证实了磷脂酰肌醇3-OH激酶（PI 3 K）p110 β参与AR反式激活和基因表达。在这项研究中，我们确定了雄激素刺激后导致PI 3 K/p110 β激活和AR反式激活的上游信号。人前列腺癌LAPC-4和22 Rv 1细胞系用于实验。使用雄激素反应元件驱动的荧光素酶（ARE-LUC）测定评估AR反式激活。用BrdU掺入法和MTT法检测细胞增殖。使用小干扰RNA（siRNA）方法沉默靶基因。在mRNA水平（实时RT-PCR）和蛋白质水平（Western印迹）评估基因表达。使用基于免疫沉淀的体外激酶测定法测量PI 3 K激酶活性。用染色质免疫沉淀法（ChIP）测定AR-DNA结合活性。首先，在细胞质膜上，用甲基-β-环糊精（M-β-CD）破坏小窝微区的完整性，消除了雄激素诱导的AR反式激活和基因表达。然后，敲低小窝结构蛋白小窝蛋白-1或-2与基因特异性siRNA显着降低雄激素诱导的AR反式激活。接下来，沉默Gαs和Gα12基因而不是其他G蛋白阻断雄激素诱导的AR反式激活和细胞增殖。Gαs或Gα12活性突变体的过表达增强雄激素诱导的AR反式激活，其中Gαs活性突变体使AR对去势水平的雄激素敏感（R1881）。最有趣的是，敲低Gαs而非Gα12亚基显著抑制雄激素刺激的PI 3 K p110 β激活。然而，染色质免疫沉淀（ChIP）分析显示，Gαs或Gα12亚基均参与雄激素诱导的AR与AR靶基因PSA启动子区的相互作用。这些数据表明，小窝相关的G蛋白α亚基参与AR的反式激活，通过调节不同的PI 3 K亚型的活动。
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. 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. 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 Gαs and Gα12 genes but not other G-proteins blocked androgen-induced AR transactivation and cell proliferation. Consistently, overexpression of Gαs or Gα12 active mutants enhanced androgen-induced AR transactivation, of which Gαs active mutant sensitized the AR to castration-level of androgen (R1881). Most interestingly, knocking down Gαs but not Gα12 subunit significantly suppressed androgen-stimulated PI3K p110beta activation. However, chromatin-immunoprecipitation (ChIP) analysis revealed that both Gαs or Gα12 subunits are involved in androgen-induced AR interaction with the AR target gene PSA promoter region. These data suggest that caveolae-associated G-protein alpha subunits are involved in AR transactivation by modulating the activities of different PI3K isoforms.
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影响因子：
4.8
作者：
Pi, M;Spurney, RF;Quarles, LD
通讯作者：
Quarles, LD
影响因子：
82.9
作者：
Nasu, Y;Timme, TL;Thompson, TC
通讯作者：
Thompson, TC
DOI：
10.1016/s0960-0760(99)00070-9
发表时间：
1999-04-01
影响因子：
4.1
作者：
Rosner, W;Hryb, DJ;Romas, NA
通讯作者：
Romas, NA
影响因子：
13.6
作者：
Franch, HA;Wang, XN;Du, J
通讯作者：
Du, J
影响因子：
2.7
作者：
Rosner, W;Hryb, DJ;Romas, NA
通讯作者：
Romas, NA