Signaling Transduction Pathway Involves in Mifepristone (RU486) Regulating hCG-induced Cyclooxygenase-2 Expression in Human Granulosa-Luteal Cells

• Main Authors: Chiao-Ya Chuang, 莊喬雅
• Other Authors: Eing-Mei Tsai
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/60207813978015044447

碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 92 === In the past decade, a critical role for the luteinizing hormone (LH) surge in initiating periovulatory events in mammals is well established. There are also several evidence indicate that gonadotropins, i.e., LH and human chorionic gonadotropin (hCG), treatments were able to increase cyclooxygenase-2 (COX-2) protein expression through the cAMP/type I protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) family activation signaling pathway. COX-2, a rate-limiting enzyme of prostaglandins (PGs) synthesis, and PGs-deficient mice will lead to infertility. RU486, a synthetic steroid, can provide a significant degree of pregnancy protection, preventing implantation or ovulation by inhibiting progesterone (P), COX-2 and prostaglandin E2 (PGE2) productions in primates. However, most of researches about RU486 in human reproduction realm still focus on progesterone synthesis and its effect on implantation. In the present study, we examine the effects of RU486 on COX-2, prostaglandin E synthase (PGEsyn) mRNA and PGE2 secretion levels in human granulosa-luteal cells (hGLC), and whether RU486 can affect COX-2 expression by altering phosphorylation level of mitogen-activated protein kinase (MAPK) family proteins. Thus, we have used primary cultures of hGLC collected from follicular fluid of women participated in in vitro fertilization (IVF) program and hGLC cell line, HO23, as our target, by using reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunocytochemistry, we found hCG stimulation could increase PGEsyn mRNA expression in time- and does-dependent manner; COX-2 protein and ERK1/2 phosphorylation level were also increased in a does-dependent character. However, in time-course experiment, both COX-2 protein and phosphorylated ERK1/2 protein expressions decreased after 12 hours hCG administration. By using MEK1/2 inhibitor, PD98059, hCG increased COX-2 mRNA and protein expression via activating ERK1/2 protein. Meanwhile, in HO23 cells co-cultured with hCG and RU486, we found that hCG-induced ERK1/2 phosphorylation level and COX-2 expressions were profoundly suppressed by RU486 in a similar pattern. However, RU486 has no significant influence on PGEsyn mRNA expression level. However, an increasing level of prostaglandin E2 secretion in the HO23 cell culture medium after giving hCG have been discovered, and RU486 administration will inhibit hCG from increasing PGE2 secretion level in a does-dependent manner. In summery, we conclude that RU486 might inhibit hCG-induced COX-2 expression by direct or indirect suppression of ERK1/2 protein phosphorylation, leading to a decrease in PGE2 secretion, and as a result inhibiting ovulation and luteinization in women.