The Mechanism Of PGE2 In Regulation Of Aromatase And Ca²⁺ Binding Protein S100A8 Expressions In The Prostate

Objective Prostaglandin E2 (PGE2) is an important inflammation factor that is associated with diseases by regulating cell proliferation, migration, and apoptosis. In benign prostatic hyperplasia (BPH) and prostate cancer (PCa), the production of PGE2 is significantly increased; however, the molecular mechanism that PGE2 regulates has not been well studied. Aromatase in prostate stromal cells of BPH and Ca2+binding protein S100A8 in prostate cancer cells are highly expressed. The objective of this article is to investigate the mechanisms by which PGE2 regulates the expressions of aromatase and S100A8, and to provide evidence that illustrates the role of PGE2 in BPH and PCa.Part One PGE2 up-regulates aromatase expression via ERRa in prostate stromal cellsMethod Expressions of PGE2 receptor EPs in prostate stromal cell line WPMY-1 were examined by RT-PCR. WPMY-1 cells were treated with PGE2 and/or EP2 antagonist AH6809, EP2 agonist butaprost, the inhibitor of protein kinase A (PKA) H89, the inhibitor of MEK PD98059 and cAMP activator forskolin. Expressions of estrogen receptor-related receptor a (ERRa) in mRNA and protein levels were determined by real-time RT-PCR and Western blot. The production of cyclic AMP (cAMP) by WPMY-1 was measured by the luciferase assay. The effects of ERRa antagonist chlordane, or siRNA against ERRa or ERRa expression plasmids on PGE2-induced aromatase expression and its promoter activity were determined by real-time RT-PCR, western blot and dual-luciferase assay. Chromatin Immunoprecipitation (ChIP) assay was performed to investigate the effect of PGE2 on the interaction of ERRa with specific aromatase promoter region in vivo. The concentration of estrodial in the medium of WPMY-1 cells which have been transfected with ERRa siRNA or expression plasmid and then treated with or without PGE2 was measured by ELISA. Result Except EP1, all other EPs have been detected in WPMY-1 cells. ERRa expression was up-regulated by PGE2, EP2 agonist butaprost and cAMP activator forskolin. EP2 antagonist AH6809 and PKA inhibitor H89 blocked the up-regulation of ERRa expression by PGE2, while PD98059 had no effect. PGE2 increased the cAMP production in a time-dependent manner in WPMY-1. Suppression of ERRa activity by chlordane or siRNA knockdown of ERRa blocked the increase of expression in mRNA and protein levels and promoter activity of aromatase induced by PGE2. Over-expression of ERRa significantly increased the expression and promoter activity of aromatase which were further augmented by PGE2. ChIP assay demonstrated that ERRa directly bound to the aromatase promoter in vivo and PGE2 enhanced the recruitment of ERRa and promoted transcriptional regulatory effects on aromatase expression in WPMY-1. Estradiol concentration in WPMY-1 medium was up-regulated by ERRa expression and that was further increased by PGE2.Conclusion In prostate stromal cells, PGE2 up-regulated the expression of ERRa via EP2 through PKA, which, as a transcription factor, further mediated the regulatory effects of PGE2 on the expression of aromatase and promoted the estradiol production by WPMY-1, indicating the potential role of PGE2 in estrogen-related benign prostatic hyperplasia.Part Two PGE2 up-regulates S100A8 expression via C/EBPβin prostate cancer cellsMethod Prostate cancer cell line PC-3 cells were treated with PGE2 and/or EP2 antagonist AH6809, EP4 antagonist AH23848, the inhibitor of PKA H89 or cAMP activator forskolin. Expression of S100A8 and its promoter activity were determined by real-time PCR and dual-luciferase assay. The production of cAMP in PC-3 was measured by the luciferase assay. Expressions of C/EBPβin mRNA and protein levels were examined by real-time RT-PCR and Western blot. The transcription activity by phosphorylation was examined by Western blot. The effects of siRNA against C/EBPβor C/EBPβexpression plasmids on PGE2-induced S100A8 expression and its promoter activity were determined by real-time RT-PCR and dual-luciferase assay. The immunohitochemistry (IHC) staining was performed to investigate the distribution and the location of COX-2 and S100A8 in serial sections of prostate cancer tissue.Result PGE2 induced S100A8 expression and the cAMP production in time-and dose-dependent manner in PC-3. EP2 antagonist AH6809, EP4 antagonist AH23848 and the inhibitor of PKA inhibited the up-regulation of S100A8 expression and its promoter activity by PGE2. PGE2 stimulated the expression of C/EBPβin mRNA and protein level in a time-dependent manner. The transcription activity of C/EBPβby phosphorylation was induced by PGE2 in a time-dependent manner. Suppression of C/EBPβexpression by siRNA knockdown blocked the increase of expression and promoter activity of S100A8 induced by PGE2. Over-expression of C/EBPβsignificantly increased the expression and promoter activity of S100A8. COX-2 and S100A8 were positive stained in all the tumor glands of prostate while negative in normal glands in serial sections of prostate cancer tissue.Conclusion In prostate cancer epithelial cell line PC-3, PGE2 up-regulated the expression and the transcription activity of C/EBPβ,which, as a transcription factor, further mediated the regulatory effects of PGE2 on the expression of s100A8 via EP2 and EP4 through PKA. Our results provided envidence that by up-regulating S100A8 expression, PGE2 involved in the prostate cancer progression.