The Regulation Of Prostaglandin Pathway In Mouse Endometrial Stromal Cells

The implantation of the blastocyst into the maternal uterus is a crucial step in mammalian reproduction. The interaction between embryo and uterus must be executed within an optimal time frame for successful pregnancy. Implantation beyond the normal window of uterine receptivity will lead to increased risk of pregnancy losses in women. Prostaglandins play important roles in female reproductive processes that include ovulation, fertilization, implantation, decidualization and placentation. Pre-implantation administration of prostaglandins can rescue delayed implantation deficiencies in Cox-2-, cPLA_2α- and Lpar3-deficient mice, respectively, but the aberrant uterine spacing of embryos in cPLA_2α- and Lpar3-deficient mice is not rescued by prostaglandins administration. Although Cox-2 and cPLA_2α play important roles during reproduction, the regulation of prostaglandins pathway in pregnant uterus is still largely unknown. The expression patterns of COX-2, cPLA_2α, EP₁, EP₄, PPARδ, C/EBPβ, STAT3 andβ-catenin in the implantation site are very similar. Recent studies show that interactions between PPARδ, C/EBPβ, STAT3,β-catenin and prostaglandin pathway exist. This study was to investigate the regulation of prostaglandin pathway in mouse endometrial stromal cells employing Western Blot, Real-time PCR and luciferase assay.In this study, PGE₂ increased the phosphorylation of cPLA_2α at Ser505 in a concentration-dependent manner after 15 minutes treatment in mouse endometrial stromal cells by Western Blot. The phosphorylation status was still higher in treatment group after 24 hours, at the same time, cPLA_2α mRNA and protein was also up-regulated by PGE₂ treatment. PGE₂ activated p38, ERK1/2 and JNK in mouse endometrial stromal cells, and the phosphorylation of cPLA_2α induced by PGE₂ was inhibited by MEK inhibitor U0126 and partially inihibited by p38 inhibitor SB203580. The phosphorylation of cPLA_2α, p38 and ERK1/2 induced by PGE₂ was inhibited by EP₁ antagonist, so PGE₂ stimulated cPLA_2α activation mainly through EP₁/ERK pathway. PGE₂ stimulated the phosphorylation of STAT3 Tyr705 and the expression of COX-2 significantly in mouse endometrial stromal cells. A STAT3 binding site at -586^-579 position of the Cox-2 promoter was found. When STAT3 overexpression vector was cotransfected with Cox-2 promoter reporter, the promoter activity was stimulated significantly. When STAT3 with mutant Tyr705 was overexpressed with Cox-2 promoter reporter, no up-regulation of promoter activity was observed suggesting that the phosphorylation of STAT3 Tyr705 was important for Cox-2 promoter activity. The phosphorylation of STAT3 and the expression of COX-2 induced by PGE₂ was inhibited by EP₁ antagonist, MEK inhibitor and STAT3 inhibitor, so PGE₂ stimulated COX-2 expression through EP₁/ERK/STAT3 pathway.Arachidonic acid has many bioactivities in mouse endometrial stromal cells. The phosphorylation of p38, ERK1/2 and JNK was induced by arachidonic acid peaking after 5 minutes treatment. The phosphorylation of cPLA_2α induced by arachidonic acid was inhibited by MEK inhibitor U0126 and partially inhibited by p38 inhibitor SB203580, so arachidonic acid stimulated cPLA_2α phosphorylation mainly through ERK1/2. Cox-2 mRNA was up-regulated by arachidonic acid after 1 hour treatment, the COX-2 protein up-regulation was observed after 3 hours treatment. The phosphorylation and the total protein of C/EBPβwas also induced by arachidonic acid treatment. In the Cox-2 promoter from -1053 to +77 four C/EBPβbinding sites were found, but only -872^-864 and -117^-109 sites contributed to Cox-2 promoter activity. The induction of COX-2 by arachidonic acid was inhibited by p38 inhibitor, MEK inhibitor, siRNA against C/EBPβand inhibitory C/EBPβ(LIP). The expression of C/EBPβprotein induced by arachidonic acid was inhibited by p38 inhibitor, and the phosphorylation of C/EBPβinduced by arachidonic acid was inhibited by p38 inhibitor and ERK1/2 inhibitor. Collectively, Arachidonic acid induced COX-2 expression through C/EBPβ, which was activated by p38 and ERK1/2. ETYA (5, 8, 11, 14-eicosatetraynoic acid) is a non-metabolized analog of arachidonic acid, which blocks arachidonic acid oxidization by acting as a false substrate. ETYA can be used to verify the function of AA itself. Stimulation of mouse endometrial stromal cells with ETYA induced the expression of COX-2 and C/EBPβ, as well as the phosphorylation of cPLA_2α, p38, ERK1/2, JNK and C/EBPβindicating that arachidonic acid exerted its bioactivity independent of prostaglandin and leukotriene biosynthesis.Based on our data, AA and PGE₂ activate cPLA_2α/COX-2 pathway in mouse endometrial stromal cells, forming positive regulation within the prostaglandin pathway. Because prostaglandins are unstable, the internal regulation of prostaglandin pathway may be important for the consistent production of prostaglandins in uterus.