Jae Hee Lee

Signal transducer and activator of transcription-3 (Stat3) plays a critical role in implantation via progesterone receptor in uterus.

Recent studies have shown that activation of the signal transducer and activator of transcription‐3 (Stat3) is required for decidualization, interacting with progesterone receptor (PR) in uterus. Based on previous reports, we hypothesized that crosstalk between STAT3 and PR signaling is required for successful implantation. To identify the interaction between STAT3 and PR isoforms, we performed immunoprecipitation following transient cotransfection and found that STAT3 physically interacted with PR‐A, which is known to be important for uterine development and function, but not with PR‐B. To further investigate the role of Stat3 in uterine function, Stat3 was conditionally ablated only in the PR‐positive cells (PRcre/+ Stat3f/f; Stat3d/d). Our studies revealed that ovarian function and uterine development of Stat3d/d mice were normal. However, Stat3d/d female mice were infertile due to defective embryo implantation. Unlike Stat3f/f mice, Stat3d/d mice exhibited an unclosed uterine lumen. Furthermore, uteri of Stat3d/d mice were unable to undergo a well‐characterized hormonally induced decidual reaction. The expression of stromal PR was decreased during decidualization and preimplantation period in Stat3d/d mice, and PR target genes were significantly down‐regulated after progesterone induction. Our results suggest that STAT3 and PR crosstalk is required for successful implantation in the mouse uterus.—Lee, J. H., Kim, T. H., Oh, S. J., Yoo, J.‐Y., Akira, S., Ku, B. J., Lydon, J. P., Jeong, J.‐W. Signal transducer and activator of transcription‐3 (Stat3) plays a critical role in implantation via progesterone receptor in uterus. FASEB J. 27, 2553–2563 (2013). www.fasebj.org

Extracellular Signal-Regulated Kinase 1/2 Signaling Pathway Is Required for Endometrial Decidualization in Mice and Human

Decidualization is a crucial change required for successful embryo implantation and the maintenance of pregnancy. During this process, endometrial stromal cells differentiate into decidual cells in response to the ovarian steroid hormones of early pregnancy. Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are known to regulate cell proliferation and apoptosis in multiple cell types, including uterine endometrial cells. Aberrant activation of ERK1/2 has recently been implicated in the pathological processes of endometriosis and endometrial cancer. However, the function of ERK1/2 signaling during implantation and decidualization is still unknown. To determine the role and regulation of ERK1/2 signaling during implantation and decidualization, we examine ERK1/2 signaling in the mouse uterus during early pregnancy using immunostaining and qPCR. Interestingly, levels of phospho-ERK1/2 were highest within decidual cells located at the implantation sites. Expression levels of ERK1/2 target genes were also significantly higher at implantation sites, when compared to either inter-implantation sites. To determine if ERK1/2 signaling is also important during human endometrial decidualization, we examined levels of phospho-ERK1/2 in cultured human endometrial stromal cells during in vitro decidualization. Following treatment with a well-established decidualization-inducing steroidogenic cocktail, levels of phospho-ERK1/2 were markedly increased. Treatment with the ERK1/2 inhibitor, U0126, significantly decreased the expression of the known decidualization marker genes, IGFBP1 and PRL as well as inhibited the induction of known ERK1/2 target genes; FOS, MSK1, STAT1, and STAT3. Interestingly, the phosphorylation level of CCAAT/ enhancer binding protein β (C/EBPβ), a protein previously shown to be critical for decidualization, was significantly reduced in this model. These results suggest that ERK1/2 signaling is required for successful decidualization in mice as well as human endometrial stromal cells and implicates C/EBPβ as a downstream target of ERK1/2.

Mig-6 Plays a Critical Role in the Regulation of Cholesterol Homeostasis and Bile Acid Synthesis

The disruption of cholesterol homeostasis leads to an increase in cholesterol levels which results in the development of cardiovascular disease. Mitogen Inducible Gene 6 (Mig-6) is an immediate early response gene that can be induced by various mitogens, stresses, and hormones. To identify the metabolic role of Mig-6 in the liver, we conditionally ablated Mig-6 in the liver using the Albumin-Cre mouse model (Albcre/+Mig-6f/f; Mig-6d/d). Mig-6d/d mice exhibit hepatomegaly and fatty liver. Serum levels of total, LDL, and HDL cholesterol and hepatic lipid were significantly increased in the Mig-6d/d mice. The daily excretion of fecal bile acids was significantly decreased in the Mig-6d/d mice. DNA microarray analysis of mRNA isolated from the livers of these mice showed alterations in genes that regulate lipid metabolism, bile acid, and cholesterol synthesis, while the expression of genes that regulate biliary excretion of bile acid and triglyceride synthesis showed no difference in the Mig-6d/d mice compared to Mig-6f/f controls. These results indicate that Mig-6 plays an important role in cholesterol homeostasis and bile acid synthesis. Mice with liver specific conditional ablation of Mig-6 develop hepatomegaly and increased intrahepatic lipid and provide a novel model system to investigate the genetic and molecular events involved in the regulation of cholesterol homeostasis and bile acid synthesis. Defining the molecular mechanisms by which Mig-6 regulates cholesterol homeostasis will provide new insights into the development of more effective ways for the treatment and prevention of cardiovascular disease.

Requirement of leukemia inhibitory factor for establishing and maintaining embryonic stem cells in mice

OBJECTIVE To evaluate the necessity of leukemia inhibitory factor (LIF) in establishing and self-renewing embryonic stem cells (ESCs). DESIGN Prospective animal model study. SETTING Gamete and Stem Cell Biotechnology Laboratory, Seoul National University, Korea. ANIMAL(S) F1 hybrid B6D2F1 mice. INTERVENTION(S) Inner cell mass (ICM) cells of blastocysts were cultured or commercially available ESCs were maintained in LIF-free or LIF-containing medium on mouse embryonic fibroblast (MEF) feeder. MAIN OUTCOME MEASURE(S) Cell morphology, LIF concentration, and mRNA expression. RESULT(S) The MEFs themselves secreted 146.5-175.3 pg/mL LIF in LIF-free medium. The ICM cells formed ESC-like colonies on MEF feeder, and E14 and R1 ESCs were successfully maintained in LIF-free medium. Expression of the genes either mediating LIF function or regulating stemness was not altered significantly, and change in the growth of ESCs was not prominent in LIF-free medium. Neither mRNA expression of differentiation-related genes nor differentiation into embryoid body was changed in the ESCs. CONCLUSION(S) Addition of LIF to culture medium is not necessary for establishing ICM-derived ESC-like colonies in the presence of fibroblast monolayer, and established ESCs can be maintained in an LIF-free medium.

Replacement of mouse embryonic fibroblasts with bone marrow stromal cells for use in establishing and maintaining embryonic stem cells in mice

We have investigated the use of BMSC (bone marrow stromal cell) as a feeder cell for improving culture efficiency of ESC (embryonic stem cell). B6CBAF1 blastocysts or ESC stored after their establishment were seeded on to a feeder layer of either SCA‐1+/CD45−/CD11b− BMSC or MEF (mouse embryonic fibroblast). Feeder cell activity in promoting ESC establishment from the blastocysts and in supporting ESC maintenance did not differ significantly between BMSC and MEF feeders. However, the highest efficiency of colony formation after culturing of inner cell mass cells of blastocysts was observed with the BMSC line that secreted the largest amount of LIF (leukaemia inhibitory factor). Exogenous LIF was essential for the ESC establishment on BMSC feeder, but not for ESC maintenance. Neither change in stem cell‐specific gene expression nor increase in stem cell aneuploidy was detected after the use of BMSC feeder. We conclude that BMSC can be utilized as the feeder of ESC, which improves culture efficiency.

MIG-6 negatively regulates STAT3 phosphorylation in uterine epithelial cells

Endometrial cancer is the most common malignancy of the female genital tract. Progesterone (P4) has been used for several decades in endometrial cancer treatment, especially in women who wish to retain fertility. However, it is unpredictable which patients will respond to P4 treatment and which may have a P4-resistant cancer. Therefore, identifying the mechanism of P4 resistance is essential to improve the therapies for endometrial cancer. Mitogen-inducible gene 6 (Mig-6) is a critical mediator of progesterone receptor (PGR) action in the uterus. In order to study the function of Mig-6 in P4 resistance, we generated a mouse model in which we specifically ablated Mig-6 in uterine epithelial cells using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f). Female mutant mice develop endometrial hyperplasia due to aberrant phosphorylation of signal transducers and activators of transcription 3 (STAT3) and proliferation of the endometrial epithelial cells. The results from our immunoprecipitation and cell culture experiments showed that MIG-6 inhibited phosphorylation of STAT3 via protein interactions. Our previous study showed P4 resistance in mice with Mig-6 ablation in Pgr-positive cells (Pgrcre/+Mig-6f/f). However, Sprr2fcre+Mig-6f/f mice were P4-responsive. P4 treatment significantly decreased STAT3 phosphorylation and epithelial proliferation in the uterus of mutant mice. We showed that Mig-6 has an important function of tumor suppressor via inhibition of STAT3 phosphorylation in uterine epithelial cells, and the antitumor effects of P4 are mediated by the endometrial stroma. These data help to develop a new signaling pathway in the regulation of steroid hormones in the uterus, and to overcome P4 resistance in human reproductive diseases, such as endometrial cancer.

Abstract 3674: MIG-6 suppresses epithelial cell proliferation via inhibiting AKT phosphorylation during endometrial tumorigenesis

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Endometrial cancer is the most frequently diagnosed malignancy of the female genital tract. Hysterectomy is typically the first line therapeutic strategy for endometrial cancer. However, there is an increasing demand for nonsurgical approaches for endometrial cancer, especially for women of reproductive age with endometrial cancer who wish to preserve their fertility. Most endometrial cancers are characterized by actively proliferating epithelial cells, increased AKT signaling and association with prolonged, unopposed E2 exposure. Mitogen-inducible gene 6 (Mig-6) is an important mediator of progesterone receptor action in the murine uterus. As P4 achieves the inhibition of proliferation by coordinating stromal-epithelial cross-talk, we generated a mouse model in which we specifically ablate epithelial endometrial Mig-6 using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f) to understand the role of epithelial Mig-6 in the uterus. Sprr2fcre+Mig-6f/f mice displayed endometrial hyperplasia upon 10 weeks of age and develop endometrial cancer by E2 treatment for 3 months. The levels of epithelial proliferation by Ki67 staining were significantly increased in epithelial cells of Sprr2fcre+Mig-6f/f mice at 10 weeks of age. The levels of phospho-AKT and phospho-S6, downstream of AKT, were remarkably higher in Sprr2fcre+Mig-6f/f mice. Interestingly, the hyperplasia exhibited by Sprr2fcre+Mig-6f/f mice was prevented by P4 treatment for 1 week in morphological and histological analysis. Aberrant activation of proliferation as well as AKT signaling was decreased in the epithelium of Sprr2fcre+Mig-6f/f mice by P4 treatment. Furthermore, we identified that MIG-6 inhibits AKT phosphorylation in human endometrial cancer cells. These data suggest that stromal P4 signaling including Mig-6 is critical in regulation of epithelial cell proliferation via regulating AKT phosphorylation during endometrial cancer development and progression. (This work was supported by American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.) Citation Format: Tae Hoon Kim, Jung-Yoon Yoo, Jae Hee Lee, Byung Gak Kim, Russell R. Broaddus, Jae-Wook Jeong. MIG-6 suppresses epithelial cell proliferation via inhibiting AKT phosphorylation during endometrial tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3674.

Abstract 2069: Stromal-epithelial crosstalk ofMig-6has an important role for tumor suppression via progesterone in endometrial cancer

Endometrial cancer is the most common malignancy of the female genital tract. This disease is commonly caused by an imbalance of progesterone (P4) and estrogen (E2) action that E2 stimulates proliferation of uterine epithelial cells while P4 is inhibitory to estrogen-mediated proliferation of the epithelium. Endometrial hyperplasia is also associated with endometrial cancer, which is a proliferative process in epithelium by E2. Responsiveness of progesterone is required to treat endometrial cancer and preserve the fertility of patients. Furthermore, PR expression in the uteri of patients with endometrial cancer has been used as an indicator for a first-rate prognosis. However, more than 30% of patients do not respond to progesterone treatment as a result of progesterone resistance and the mechanism of progesterone resistance is still unknown. Thus, identifying the mechanism of progesterone in endometrial cancer is essential to improve the therapy of this disease. Mitogen-inducible gene 6 (Mig-6) is an important mediator of progesterone receptor (PR) action in the murine uterus. As P4 achieves the inhibition of proliferation by coordinating stromal-epithelial cross-talk, we generated a mouse model in which we specifically ablate epithelial endometrial Mig-6 using Sprr2f-cre mice (Sprr2fcre+ Mig-6f/f) to understand the role of epithelial Mig-6 in the uterus. Sprr2fcre+ Mig-6f/f mice displayed endometrial hyperplasia upon 10 weeks of age and develop endometrial cancer by E2 treatment for 3 months. Interestingly, the hyperplasia exhibited by Sprr2fcre+ Mig-6f/f mice was prevented by P4 treatment for 1 week in morphological and histological analysis. Proliferation decreased in the glandular epithelium of Sprr2fcre+ Mig-6f/f mice by P4, and apoptotic signal was also reduced. The expressions of stromal PR and its target genes were induced in Sprr2fcre+ Mig-6f/f mice after P4 treatment, indicating that P4-induced stromal Mig-6 can prevent the hyperplasia via regulating steroid hormonal signaling. This study suggested that stromal-epithelial communication of Mig-6 is critical in tumor suppressor function via regulating steroid hormonal signaling by progesterone. (This work was supported by American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.) Citation Format: Tae Hoon Kim, Byung Gak Kim, Jung-Yoon Yoo, Jae Hee Lee, Diego H. Castrillon, Jae-Wook Jeong. Stromal-epithelial crosstalk of Mig-6 has an important role for tumor suppression via progesterone in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2069. doi:10.1158/1538-7445.AM2015-2069