Treatment Effects of B23 Inhibitor on the Endometrium with Poor Growth or Adhesion

Estrogens induce cell proliferation through both nuclear estrogen receptors (ER) or extra-nuclear GPR30/GPER1 receptor. In general, classic ER takes a longer time to induce cell proliferation (24 hours or longer) whereas intracellular signals through GPR30/GPER1 takes only a few minutes (5-60 min) and frequently co-regulated by other growth factors. The endometrium is a target tissue of estrogens. Through estrogen receptor (ER), estrogens stimulate proliferation of the endometrial cell. Our previous studies have shown that nucleophosmin (NPM/B23), a nucleolar protein acting as a stimulator to synthesize ribosome proteins, is found to be responded to estrogens and induce cell proliferation in endometrial cells. By contrast, knock-down of NPM/B23 gene expression using siRNA for NPM/B23 increases ERα of the uterine endometrial cells which enhances the response to estrogens and then in turn stimulates cell proliferation substantially. Similarly, treatment of endometrial cells with NSC348884 (an inhibitor for NPM/B23) also increases the production of ERα.Recently, we have successfully established immortalized human endometrial cells (IEM17-1) by infection of endometrial cells with retrovirus containing human telomerase reverse transcriptase (hTERT) (國科會計劃 NSC96-2314-B- 182-016;PI:王馨世). In both IEM17-1 cells and uterine endometrial cells from primary culture (in uterus with myoma undergoing hysterectomy), increases in ERα and improvement of cell proliferation were found following treatment with NSC348884. Clinically, poor growth of endometrium due to adhesion or impaired function of growth leads to failure of embryo implantation. Using this model, a practicable treatment may offer to improve the embryo implantation. In the upcoming three years, we are going to exert the following experiments using immortalized endometrial cells, including the investigation of signal pathways involved in estrogen receptors. Furthermore, we are going to explore the changes of cell functions on endometrial cells from patients who have endometrial lesions, in comparison with normal endometrial cells from uteri with myoma following hysterectomy. Part I：The estrogen-induced cell proliferation and migration induced by increase in nuclear ERα following treatment with NSC348884 (B23 inhibitor) in endometrial cells. The aim of the part I is to testify: 1. if estrogen enhances the gene expression of Src, ERK1/2, cFos and cJun of endometrial cells after treatment with NSC348884 (B23 inhibitor)? 2. if estrogen-induced cell proliferation and migration after treatment with NSC348884 (B23 inhibitor) may be abolished by addition of ER inhibitor (ICI182,780)? 3. if estrogen-induced production of other signal proteins may be diminished by addition of one of inhibitors for Src, ERK1/2, cFos and cJun? 4. if estrogen-induced cell proliferation and migration may also be stimulated by knock-down of NPM/B23 using siRNA? Part II：The study of increased gene expression of membrane ER (GPR30) on estrogen-induced cell proliferation and migration following treatment with NSC348884 (B23 inhibitor) in endometrial cells. The aim of the part II is to testify: 1. if estrogen enhances the phosphrylation of PI3K, ERK and Akt in EGFR signal pathway? 2. if estrogen-induced phosphrylation of FAK and activity of MMP-2 may be diminished by addition of inhibitors for PI3K, ERK and Akt…? 3. if estrogen-induced cell proliferation and migration are through activation of ER by E2-ER-ETS1 complex? Part III：Estrogens activate Src kinase through GPR30 which in turn phosphorylates ETS1. Phosphorylated ETS1 may avoid to be degraded by the ubiquitin ligase (COP I). The aim of the part III is to testify: 1. explore the interaction between GPR30 and ERα. if GPR30 regulate ERα through Src kinase, leading to degradation of ERα? 2. if ERα, Src and ETS1 form a complex of Src-ERα-ETS1 or an interaction domain in endometrial cells under the regulation of E2 and GPR30? 3. how many genes involving in cell proliferation and migration affected by the Src-ERα-ETS1 complex? We are going to treat ETS1-knockdown cells with E2, then analyze the change of gene expression using human transcriptome array (HTA, Affymetrix).

Project ID:PC10507-0629
External Project ID:MOST105-2314-B182-059