Mechanism Of PGE2-modulated Aromatase Expression And Function Of Estrogen Response Gene EGR-1

Objective Increasing evidences reveal that estrogen is one of the important factors affecting the occurrence and development of benign prostatic hyperplasia (BPH) and prostate cancer (PCa). We previously reported that prostaglandin E2(PGE2) increased the expression of estrogen-related receptor alpha (ERRa) in the prostate stromal cell line WPMY-1, which ultimately promoted17β-estradiol (E2) production by enhancing aromatase gene transcription. Aromatase is a key enzyme that catalyzes the conversion of testosterone to E2. These findings indicate the important role of PGE2in E2production. ERRa was the first identified orphan nuclear receptor; however, its natural endogenous ligand is still unknown and the regulation mechanism of its transcriptional activity has not yet been clearly documented. In the first part of this study, we find for the first time that PGE2can activate ERRa transcriptional activity, and then promote E2production by enhancing aromatase gene transcription. We previously established rat BPH model and the combination of laser-capture microdissection and microarray analysis indicates us the gene expression profiles in different prostate compartments in different phases of BPH. We found early growth response gene-1(EGR-1)was up-regulated during BPH progression and the most obvious increase occurred in the epithelial compartment at the early stage. These findings indicate us that EGR-1plays important roles in the BPH progression, especially during the BPH occurrence stage. EGR-1has been identified as one of the key genes which play important roles in the progression of PCa; but its function in BPH remains unexplored. In the second part, we study the biological function of EGR-1in BPH and the mechanism of estrogen-induced EGR-1and its target gene expression. Our work may provide some experimental evidence to clarify the mechanism of estrogen production and estrogenic effect in BPH progression.Part One PGE2up-regulates aromatase expression via enhancing the transcriptional activity of ERRa in prostate stromal cells Method The effects of PGE2and ERRa expression plasmid on ERRE luciferase activity were determined by dual-luciferase assay. Real-time RT-PCR and Western blot were performed to detect the effects of PGE2, siRNA against ERRa and ERRa expression plasmid on aromatase expression. The effects of PGE2on the distribution of ERRa protein in cells were detected by fluorescence imaging and Western blot. WPMY-1cells and ERRa stablely transfected WPMY-1cells were treated with PGE2, PGE2receptor (EP)2antagonist AH6809, EP3antagonist L798106, EP4antagonist AH23848, EP2agonist Butaprost, EP3agonist Sulprostone, EP4agonist Cay10580, phosphoinositide-3-kinase (PI3K) signaling pathway inhibitor LY294002, extracellular signal-regulated kinase (ERK) signaling pathway inhibitor U0126and cyclic adenosine monophosphate (cAMP) activator Forskolin, and the aromatase expression and ERRE luciferase activity were detected by Real-time RT-PCR and dual-luciferase assay afterwards. The effects of PGE2, Butaprost, Cay10580, Forskolin, LY294002and U0126on p-ERK and ERK expression were determined by Western blot. WPMY-1cells were transfected with ERRa expression plasmid and treated with PGE2, LY294002and U0126, immunopreciptation (1P) and phosphostaining were performed to detect the protein level of p-ERRα.Result When cells were cultured in serum free medium, over-expression of ERRa did not increase ERRE luciferase activity and aromatase expression; however, the up-regulation effect occurred in the presence of PGE2. Knockdown of ERRa expression by specific siRNAs abolished the enhancement of aromatase expression by PGE2. PGE2treatment significantly increased the nuclear localization of ERRa protein. When the WPMY-1cells and ERRa stablely transfected WPMY-1cells were pretreated with AH6809, L798106or AH23848, the effects of PGE2on aromatase expression still occurred; only the simultaneous presence of AH6809and AH23848dramatically reduced the up-regulation of aromatase expression by PGE2; Butaprost, Cay10580and Forskolin enhanced aromatase expression and ERRE luciferase activity; LY294002and U0126could inhibit the induction of aromatase expression and ERRE luciferase activity by PGE2, Butaprost, Cay10580and Forskolin. PGE2, Butaprost, Cay10580and Forskolin stimulated phosphorylation of ERK and these effects could be abolished by LY294002and U0126. PGE2treatment induced ERRa phosphorylation and this effect was blocked by LY294002and U0126.Conclusion PGE2activates the EP2/EP4-cAMP-PI3K-ERK signaling pathway, thus enhancing ERRa transcriptional potentiality by increasing ERRa phosphorylation and nuclear translocation, which promotes the expression of its target gene aromatase.Part Two Estrogen response gene EGR-1promotes proliferation of prostatic hyperplasia epithelial BPH-1cells in cultureMethod The expression of EGR-1in rat BPH tissues was analysed using Real-time RT-PCR. The analysis of EGR-1expression in human BPH tissues was assayed by immunohistochemistry (IHC) method. The expression of EGR-1in RWPE-1and BPH-1cells was analysed using Real-time RT-PCR. The EGR-1expression in the EGR-1stably transfected BPH-1cells was detected by Real-time RT-PCR and Western blot. The cell proliferation of EGR-1stably transfected BPH-1cells was assessed by MTT assay; BPH-1cells were transfected with EGR-1siRNA and cell proliferation was assessed by MTT assay; WPMY-1cells were treated with conditioned medium (CM) harvested from EGR-1stably transfected BPH-1cells and cell proliferation was assessed by MTT assay. The effects of E2on the distribution of EGR-1protein in cells were detected by immunocytochemistry (ICC) analysis and Western blot. The effects of EGR-1expression plasmid, siRNA against EGR-1and E2on the expression of transforming growth factor β1(TGFβ1) and insulin-like growth factor2(IGF2) were detected by Real-time RT-PCR and enzyme linked-immuno-sorbent assay (Elisa). The expression of prostaglandin E synthase (PTGES) and prostaglandin-endoperoxide synthase1(PTGS1) in EGR-1stably transfected BPH-1cells were analysed using Real-time RT-PCR.Result EGR-1expression was enhanced in rat BPH tissues, human BPH tissues and BPH-1cell line. The cell proliferation was promoted by the over-expression of EGR-1; EGR-1siRNA inhibited BPH-1cells proliferation; the CM of EGR-1stablely transfected BPH-1cell line could promote prostate stromal cell line WPMY-1proliferation. E2promoted EGR-1protein nuclear transfer. IGF2and TGFβ1were up-regulated in EGR-1stably transfected BPH-1cells; E2 up-regulated IGF2and TGFβ1expression and this effect could be abolished by knockdown EGR-1expression. PTGES and PTGS1were up-regulated in EGR-1stably transfected BPH-1cells.Conclusion EGR-1expression is enhanced in both rat and human BPH tissues; EGR-1not only promotes prostate epithelial cell proliferation, but also can promote prostate stromal cell proliferation in a paracrine manner; E2enhances EGR-1protein nuclear location which activates EGR-1transcriptional activity, and the E2-induced IGF2and TGFβ1up-regulation is mediated by EGR-1.