Nuclear receptor interaction protein, a coactivator of androgen receptors (AR), is regulated by AR and Sp1 to feed forward activate its own gene expression through AR protein stability

• Main Authors: Pei-Hong Chen, 陳沛鴻
• Other Authors: Show-Li Chen
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/56767418531660865306

博士 === 國立臺灣大學 === 微生物學研究所 === 96 === Prostate cancer is the second-leading male-specific death in the world. The androgen-AR signaling has been considered as the primary cause in the prostate cancer progression. AR coactivators play a pivotal role in the AR signaling and are expressed abnormally in some prostate cancer tissues. Androgens act through the AR to target androgen response elements (AREs) in the promoter of AR target genes (e.g., p21, FGF8, etc.) which stimulate cell proliferation and ultimately tumor formation. Nuclear receptor interaction protein (NRIP) is recently found in our lab in the yeast-two hybrid system using C-terminus of AR as a bait. NRIP is an AR coactivator which can activate PSA transcription in an androgen-dependent manner. NRIP directly interacts with AR and is involved in cell proliferation and survival in AR-sensitive LNCaP cells, a prostate cancer cell line. Small interfering RNA (siRNA)-mediated knockdown of NRIP expression reduces the transcription of AR target genes and results in cell apoptosis. NRIP expression is increased in AR-regulated LNCaP cells in the presence of androgens. In this thesis, we further investigated the regulation mechanism of NRIP and the role for AR protein stability. We found that NRIP is a novel AR target gene and NRIP plays a role in the AR signaling and the prostate cancer cell line. The core promoter region of NRIP ranges from -413 to +94 upstream the transcription initiation site. The promoter DNA sequences are composed of hormone response elements (ARE, GRE) and three Sp1 binding sites (Sp1-1, Sp1-2, Sp1-3). NRIP is a TATA-less promoter with GC-rich elements which is regulated by Sp1. Transcription of NRIP is increased in the presence of androgens in LNCaP cells in a dose-dependent manner and induced in the AR-deficient 293T cells as AR expression plasmids are transduced. In chromatin immunoprecipitation (ChIP) assays, AR not only associates on the ARE within the NRIP promoter region but also indirectly on Sp1 binding site through interaction with Sp1. In addition, the activity of NRIP is induced by androgens in the ARE-containing promoter as well as the ARE-losing, Sp1 binding site-containing promoter, but not in the ARE and Sp1 double mutated promoter. This result indicates that androgen-AR activates NRIP promoter not only through ARE but Sp1 binding site. Knockdown of NRIP by siRNAs against NRIP gene (siNRIP) reduces the stability of the AR protein regardless of the presence of androgens. However, knockdown of NRIP has no effect on AR transcription and nuclear translocation. The expression of AR protein was restored in the NRIP-knockdowned LNCaP cells which are treated with proteasome inhibitor MG132, demonstrating that knockdown of NRIP contributes to AR degradation through proteasmoe activity. Knockdown of NRIP decreases the AR protein level and consequently results in cell growth arrest and apoptosis. NRIP forms a complex with AR and Sp1 which are recruited to the promoter of AR target genes of PSA and NRIP. The promoter activity and transcription of NRIP gene are increased with the transiently introduced NRIP expression plasmids in a dose-dependent and AR-dependent manner, suggesting NRIP regulates its own promoter through AR activity. Taken together, NRIP acts as an AR coactivator that stabilizes AR protein and enhances AR transcriptional activity in which NRIP autoregulates its own promoter through androgen-mediated AR transactivation.