Glycogen Synthase Kinase-3β Activity Is Required for Androgen-Stimulated Gene Expression in Prostate Cancer

Abstract

Despite the specificity inferred by its name, glycogen synthase kinase (GSK)-3beta is an important kinase with a plethora of significant cellular targets, including cytoskeletal proteins and transcription factors, and its activity is regulated by phosphorylation on tyrosine/serine residues. As part of our efforts to dissect the molecular basis responsible for androgen-independent progression of prostate cancer, we investigated the role of GSK-3beta in androgen-stimulated gene expression in human prostate cancer cells. Pretreatment of prostate cancer cells harboring wild-type or mutant androgen receptor with the GSK-3beta inhibitors, lithium chloride (LiCl), RO318220, or GF109203X, inhibited R1881-stimulated androgen-responsive reporter activity in a dose- and time-dependent manner. In addition, the expression of two endogenous androgen-stimulated gene products, prostate-specific antigen and matrix metalloproteinase-2, was suppressed by the GSK-3beta inhibitors in those cells. Most importantly, knocking down GSK-3beta expression via a small interference RNA-mediated gene silencing approach also reduced R1881-stimulated gene expression, demonstrating the specificity of GSK-3beta involvement. Moreover, R1881 treatment of the cells increased phosphorylation status of GSK-3beta on tyrosine residue Y(216) but not on serine residue S(9). Pretreatment of the cells with phosphatidylinositol 3-kinase inhibitor LY294002 or wortmannin, which blocks androgen action in cells, abolished R1881-induced GSK-3beta Y(216) phosphorylation. However, the phosphatidylinositol 3kinase or GSK-3beta inhibitors did not block R1881-induced nuclear translocation of androgen receptor. Finally, knocking down the expression of Akt or beta-catenin, the two GSK-3beta-related signaling molecules, via siRNA-mediated gene silencing did not significant affect R1881-stimulated gene expression. These findings suggest that GSK-3beta activity is required for androgen-stimulated gene expression in prostate cancer cells.

Serum/glucocorticoid-induced protein kinase-1 facilitates androgen receptor-dependent cell survival

“…18 We previously showed that PI3K activity is required for androgen-induced AR transactivation and gene expression, and that Akt is dispensable for AR transactivation. 33 In this study, we found that SGK-1 is required for AR-dependent cellular survival and gene expression, indicating that SGK-1 might be one of the PI3K downstream effectors in androgen signaling. Although both Akt and SGK-1 use a similar phosphorylation preference of RxRxxS/T on their substrates, some distinctions were recently reported in terms of their substrate specificity requirements.…”

“…32 We previously showed that PI3K activity (but not Akt) is required for AR-mediated gene expression. 33 Therefore, we determined whether SGK-1 modulates AR transactivation. First, we tested whether SGK-1 overexpression could enhance androgen-stimulated AR transactivation.…”

“…We used two SEAP reporters driven by different promoters derived from the endogenous AR target genes, human psa (PSA-E2/P-SEAP, termed as PSA-SEAP) and rat probasin (ARR 2 PB-SEAP, termed as PB-SEAP), as described previously. 33,34 LAPC-4 cells were transfected with SGK-1 siRNA for 3 days to eliminate SGK-1 expression. Cells were then transfected again with the reporter constructs.…”

“…The human prostate cancer LNCaP and LAPC-4 cells and their culture conditions were described previously. 12,33,[38][39][40] LAPC-4/ SGK1 and LNCaP/SGK1 sublines were established by stably infecting the parental cells with a retrovirus vector (pMSCV-puro-SGK1) 29 bearing the rat sgk1 gene. The control subline LAPC-4/Puro and LNCaP/Puro were established when an empty vector was used.…”

“…For protein expression analysis, western blot was performed as described previously. 12,33,[38][39][40] Briefly, after treatment, cells were pelleted and lysed in a buffer containing protease inhibitors (Halft Protease Inhibitor Cocktail Kit, PIERCE, Rockford, IL), separated on SDS-PAGE gels and transferred to PVDF membrane (BIO-RAD, Hercules, CA). Membranes were blocked in a Tris-buffered solution plus 0.1% Tween-20 (TBS-T) solution with 5% nonfat dry milk and incubated with primary antibodies overnight at 41C.…”