Studies on the function of CREB3L1 and GPR64 in endometrial changes of the uterus

Abstract

Endometrium consists of epithelial and stromal compartments that undergo dynamic change to allow for embryo implantation and development. These compartments are regulated by the ovarian steroid hormone, estrogen (E2) and progesterone (P4). Imbalanced ovarian steroid hormone in uterine endometrium causes several female reproductive diseases and dysregulation of endometrial environment. In previously study, cAMP-Response Element-Binding 3-like protein 1 (CREB3L1) and G-protein coupled receptor (GPR64) have been identified as a P4 and progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 and GPR64 function have not been studied in the context of human uterine biology. In this study, I investigate P4-PR target molecule CREB3L1 and GPR64 function in uterus endometrium. First study, P4 is regulated endometrial stromal cell decidualization, so I determined CREB3L1 role in endometrium. I validated P4-PR regulation of CREB3L1 in the uteri of wild-type or progesterone receptor knockout (PRKO) mice. CREB3L1 expression level is induced wild-type mice after P4 treatment whereas expression of GPR64 is reduced P4 injected PRKO mice. And I observed that CREB3L1 expression is significantly increase in secretory phase human endometrium compared to proliferative phase. Furthermore, CREB3L1 expression level is significantly lower in women endometrium with endometriosis. I confirmed that CREB3L1 require in vitro decidualization, transfecting CREB3L1 siRNA in human endometrial stromal cell prior to hormonal induction of in vitro decidualization. Interestingly, the decidual markers, IGFBP1 and PRL, are significantly decreased and phosphorylation of ERK1/2, critical factor for decidualization, is also significantly reduced in CREB3L1 knockdown hESCs. Second study, to confirm GPR64 function for endometrial stromal compartment, I examine the expression of GPR64 in ovariectomized wild-type and PRKO mice uteri injected vehicle or P4. The expression of GPR64 is increased after P4 treatment in the wild type mice. However, GPR64 expression is significantly reduced in PRKO mice treated with P4. Furthermore, I observed that GPR64 expression is significantly higher in P4 injected ovariectomized C57BL/6 wild-type mice uterus for 6 hours, and decidua of implantation and post-implantation stage. These result showed that GPR64 is regulated by P4. Finally, to determined GPR64 is require for decidualization, I examine in vitro decidualization with E2, P4, cAMP in hESCs with GPR64 siRNA. The decidual marker, IGFBP1 and PRL, and decidual related gene, COUP-TFII and PR, are significantly decreased in GPR64 knockdown hESCs. Third study is the GPR64 role in endometrial cancer. Mostly endometrial carcinoma is caused by imbalances of steroid hormone unopposed estrogen exposure or progesterone resistance. P4 is inhibited E2 dependent endometrial epithelial cell and GPR64 is regulated by P4 in endometrium. Therefore, I examined GPR64 role in endometrial carcinoma. GPR64 expression is significantly reduced in grade I endometrial carcinoma. Moreover, GPR64 knockdown by GPR64 siRNA treatment in endometrial carcinoma cell, cell colony formation, cell proliferation, cell migration and cell invasion are significantly increased. Moreover, cancer related genes MSX2, SFN, and CD44 are significantly increase and CX43 is significantly decrease in GPR64 knockdown endometrial cancer. CX43 is one of tumor suppressor and reduced gene level, I confirmed expression of CX43 protein level in endometrial carcinoma cell with or without GPR64 siRNA. The CX43 expression level is significantly reduced in GPR64 siRNA treated endometrial carcinoma for day 1 and day 6. These results suggested that GPR64 is tumor suppressor in endometrial carcinoma. The P4-PR target genes, CREB3L1 and GPR64, are required for regulation of endometrial environment involved decidualization. Additionally these genes are linked to the pathogenesis of uterus endometrial diseases such as endometriosis or endometrial carcinoma. Therefore, modulation of P4-PR target gene is an aim of new treatment of uterine endometrial environment dysregulation.