P62 Regulates Estrogen Receptor (Er)-Induced Endometrial Growth through Repressing Autophage

Autophage, alternate with apoptosis, is an important mechanism for regulating cell growth and survival. In the environment with short of food supply, survived cells obtain energy source from surrounding specific cells through the pathway of autophage. In chemotherapy for cancers, drug-induced autophage promotes the effectiveness of cancer therapy. Recent studies have shown that P62 acts an important roles on the pathway of autophage through binding to LC3, a crucial facor for autophage. Specifically, autophage is repressed in the situation when the binding of P62 and LC3 is disturbed. Estrogens induce cell proliferation through both nuclear estrogen receptors (ER) or extra-nuclear GPR30/GPER1 receptor. 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. 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. Recently, we have observed that ER, in the presence of estrogen, binds to P62 and LSD1 to form an ER-P62-LSD1 complex. Thus, estrogen-induced cell growth of the endometrium may be through repressing autophage in the presence of ER-P62-LSD1 complex. In the upcoming three years, we are going to exert the following experiments using immortalized endometrial cells (國科會計劃NSC96-2314-B- 182-016;PI:王馨世) to investigate signal pathways of estrogen-induced cell growth and autophage involving P62. Furthermore, we are going to explore the changes of cell functions on endometrial cells from patients who have endometrial adhesion or poor growth of endometrium, in comparison with normal endometrial cells from uteri with myoma following hysterectomy. Part I：The estrogen-induced cell proliferation activated by increase in nuclear estrogen receptor (ER) in endometrial cells. In this pathway, the mode of P62 regulating ER-induced endometrial growth via inhibiting autophage will be investigated. The aim of the part I is to testify: 1. if estrogen enhances the activity of P62, leading to inhibiting autophage and promoting cell proliferation of the endometrial cells? 2. if P62-repressed autophage may be abolished by addition of ER inhibitor (ICI182,780)? 3. if estrogen-induced cell proliferation may be diminished by knock-down of P62 using siRNA? 4. if estrogen-induced cell proliferation may also be diminished by addition of P62 inhibitor (P62-ZZ)? Part II：The role of P62 in formation of a transcription complex containing ESR1 and LSD1 will be investigated. The aim of the part II is to testify: 1. if estrogen induces the formation of ESR1-P62-LSD1 complex on ERE (estrogen response element)? 2. if estrogen-induced formation of ESR1-P62-LSD1 complex on ERE may be diminished by addition of ER inhibitor (ICI182,780)? 3. if estrogen-induced formation of ESR1-P62-LSD1 complex on ERE may also be diminished by addition of P62 inhibitor (P62-ZZ)? Part III：The role of P62 in the degradation of ESR1 through ubiquitination will be investigated. The aim of the part III is to testify: 1. if P62 involved in the degradation of ESR1 through ubiquitination? Detect the amount of marked ubiquitin on ESR1 by using proteasome inhibitor (MG132). 2. if estrogen-induced formation of ESR1-P62-LSD1 complex on ERE (estrogen response element) may enhance the ubiquitinqtion of ESR1? 3. if the ubiquitinqtion of ESR1 may be diminished by addition of ER inhibitor (ICI182,780)? 4. if the ubiquitinqtion of ESR1 may also be diminished by addition of P62 inhibitor (P62-ZZ)?

Project ID:PC10608-2355
External Project ID:MOST106-2314-B182-056