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C-Jun Contributes to Transcriptional Control of GNA12 Expression in Prostate Cancer Cells.

ABSTRACT: Abstract: GNA12 is the α subunit of a heterotrimeric G protein that possesses oncogenic potential. Activated GNA12 also promotes prostate and breast cancer cell invasion in vitro and in vivo, and its expression is up-regulated in many tumors, particularly metastatic tissues. In this study, we explored the control of expression of GNA12 in prostate cancer cells. Initial studies on LnCAP (low metastatic potential, containing low levels of GNA12) and PC3 (high metastatic potential, containing high GNA12 levels) cells revealed that GNA12 mRNA levels correlated with protein levels, suggesting control at the transcriptional level. To identify potential factors controlling GNA12 transcription, we cloned the upstream 5' regulatory region of the human GNA12 gene and examined its activity using reporter assays. Deletion analysis revealed the highest level of promoter activity in a 784 bp region, and subsequent in silico analysis indicated the presence of transcription factor binding sites for C/EBP (CCAAT/enhancer binding protein), CREB1 (cAMP-response-element-binding protein 1), and c-Jun in this minimal element for transcriptional control. A small interfering RNA (siRNA) knockdown approach revealed that silencing of c-Jun expression significantly reduced GNA12 5' regulatory region reporter activity. In addition, chromatin immunoprecipitation assays confirmed that c-Jun binds to the GNA12 5' regulatory region in PC3 cells. Silencing of c-Jun expression reduced mRNA and protein levels of GNA12, but not the closely-related GNA13, in prostate cancer cells. Understanding the mechanisms by which GNA12 expression is controlled may aid in the development of therapies that target key elements responsible for GNA12-mediated tumor progression.

SUBMITTER: Udayappan UK

PROVIDER: S-EPMC6153990 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. c-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P<0.05) compared to noninfected controls. Increased NOS3 expression was associated with increased total NOS activity. Transient transfections showed that c-Jun overexpression augmented the transactivation of the sheep NOS3 promoter-driven luciferase/reporter constructs with the AP-1 site but not of deletion constructs without the AP-1 site. When the AP-1 site was mutated, c-Jun failed to trans-activate the sheep NOS3 promoter. AP-1 DNA binding activity also increased in c-Jun overexpressed UAEC. Lastly, the pharmacological AP-1 activator phorbol myristate acetate increased AP-1 binding, trans-activated the wild-type but not the AP-1 mutant NOS3 promoter and dose-dependently stimulated UAEC NOS3 and c-Jun protein expression. Hence, our data show that c-Jun/AP-1 regulates NOS3 transcription involving the proximal AP-1 site in the 5'-regulatory region of the sheep NOS3 gene.

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C-Jun Contributes to Transcriptional Control of GNA12 Expression in Prostate Cancer Cells.

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