Jung n Yoo

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

Aberrant activation of signal transducer and activator of transcription-3 (STAT3) signaling in endometriosis

STUDY QUESTION Are STAT3 signaling molecules differentially expressed in endometriosis? SUMMARY ANSWER Levels of phospho-STAT3 and HIF1A, its downstream signaling molecule, are significantly higher in eutopic endometrium from women with endometriosis when compared with women without the disease. WHAT IS KNOWN ALREADY Endometriosis is an estrogen-dependent inflammatory condition. Interleukin 6 (IL-6) is an inflammatory survival cytokine known to induce prolonged activation of STAT3 via association with the IL-6 receptor. STUDY DESIGN, SIZE, DURATION Cross-sectional measurements of STAT3 and HIF1A protein levels in eutopic endometrium from women with endometriosis versus those without. PARTICIPANTS/MATERIALS, SETTING, METHODS Levels of phospho-STAT3 (pSTAT3) and HIF1A were examined in the endometrium of patients with and without endometriosis as well as in a non-human primate animal model using western blot and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Levels of pSTAT3 were significantly higher in the eutopic endometrium from women with endometriosis when compared with women without the disease in both the proliferative and secretory phases. HIF1A is known to be stabilized by STAT3 and IL-6. Our immunohistochemistry results show abundant HIF1A expression within the eutopic endometrial epithelial cells of women with endometriosis. Furthermore, pSTAT3 and HIF1A proteins are co-localized in endometriosis. This aberrant activation of pSTAT3 and HIF1A is confirmed by sequential analysis of eutopic endometrium using a baboon animal model of induced endometriosis. Lastly, we confirmed this IL-6 induction of both STAT3 phosphorylation and HIF1A mRNA expression in Ishikawa human endometrial adenocarcinoma cell line. LIMITATIONS, REASONS FOR CAUTION Ishikawa cancer cell line was used to study a benign disease. The peritoneal fluid contains various inflammatory cytokines in addition to IL-6 and so it is possible that other cytokines may affect the activity and expression of STAT3 signaling molecules. WIDER IMPLICATIONS OF THE FINDINGS Our results imply that aberrant activation of STAT3 signaling plays an important role in the pathogenesis of endometriosis. Our findings could progress in our understanding of the etiology and pathophysiology of endometriosis and potential therapeutic interventions by targeted pharmacological. STUDY FUNDING/COMPETING INTERESTS This work was supported by NIH R01 HD067721 (to S.L.Y and B.A.L) and NIH R01 HD057873 and American Cancer Society Research Grant RSG-12-084-01-TBG (to J.-W.J.). There are no conflicts of interest.

β-Catenin activation contributes to the pathogenesis of adenomyosis through epithelial–mesenchymal transition

Adenomyosis is defined by the presence of endometrial glands and stroma within the myometrium. Despite its frequent occurrence, the precise aetiology and physiopathology of adenomyosis is still unknown. WNT/β‐catenin signalling molecules are important and should be tightly regulated for uterine function. To investigate the role of β‐catenin signalling in adenomyosis, the expression of β‐catenin was examined. Nuclear and cytoplasmic β‐catenin expression was significantly higher in epithelial cells of human adenomyosis compared to control endometrium. To determine whether constitutive activation of β‐catenin in the murine uterus leads to development of adenomyosis, mice that expressed a dominant stabilized β‐catenin in the uterus were used by crossing PR‐Cre mice with Ctnnb1f(ex3)/+ mice. Uteri of PRcre/+ Ctnnb1f(ex3)/+ mice displayed an abnormal irregular structure and highly active proliferation in the myometrium, and subsequently developed adenomyosis. Interestingly, the expression of E‐cadherin was repressed in epithelial cells of PRcre/+ Ctnnb1f(ex3)/+ mice compared to control mice. Repression of E‐cadherin is one of the hallmarks of epithelial–mesenchymal transition (EMT). The expression of SNAIL and ZEB1 was observed in some epithelial cells of the uterus in PRcre/+ Ctnnb1f(ex3)/+ mice but not in control mice. Vimentin and COUP‐TFII, mesenchymal cell markers, were expressed in some epithelial cells of PRcre/+ Ctnnb1f(ex3)/+ mice. In human adenomyosis, the expression of E‐cadherin was decreased in epithelial cells compared to control endometrium, while CD10, an endometrial stromal marker, was expressed in some epithelial cells of human adenomyosis. These results suggest that abnormal activation of β‐catenin contributes to adenomyosis development through the induction of EMT. Copyright

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 Suppresses Endometrial Cancer Associated with Pten Deficiency and ERK Activation

PTEN mutations are the most common genetic alterations in endometrial cancer. Loss of PTEN and subsequent AKT activation stimulate estrogen receptor α-dependent pathways that play an important role in endometrial tumorigenesis. The major pathologic phenomenon of endometrial cancer is the loss of ovarian steroid hormone control over uterine epithelial cell proliferation and apoptosis. However, the precise mechanism of PTEN/AKT signaling in endometrial cancer remains poorly understood. The progesterone signaling mediator MIG-6 suppresses estrogen signaling and it has been implicated previously as a tumor suppressor in endometrial cancer. In this study, we show that MIG-6 also acts as a tumor suppressor in endometrial cancers associated with PTEN deficiency. Transgenic mice, where Mig-6 was overexpressed in progesterone receptor-expressing cells, exhibited a relative reduction in uterine tumorigenesis caused by Pten deficiency. ERK1/2 was phosphorylated in uterine tumors and administration of an ERK1/2 inhibitor suppressed cancer progression in PR(cre/+)Pten(f/f) mice. In clinical specimens of endometrial cancer, MIG-6 expression correlated inversely with ERK1/2 phosphorylation during progression. Taken together, our findings suggest that Mig-6 regulates ERK1/2 phosphorylation and that it is crucial for progression of PTEN-mutant endometrial cancers, providing a mechanistic rationale for the evaluation of ERK1/2 inhibitors as a therapeutic treatment in human endometrial cancer.

ARID1A Is Essential for Endometrial Function during Early Pregnancy

AT-rich interactive domain 1A gene (ARID1A) loss is a frequent event in endometriosis-associated ovarian carcinomas. Endometriosis is a disease in which tissue that normally grows inside the uterus grows outside the uterus, and 50% of women with endometriosis are infertile. ARID1A protein levels were significantly lower in the eutopic endometrium of women with endometriosis compared to women without endometriosis. However, an understanding of the physiological effects of ARID1A loss remains quite poor, and the function of Arid1a in the female reproductive tract has remained elusive. In order to understand the role of Arid1a in the uterus, we have generated mice with conditional ablation of Arid1a in the PGR positive cells (Pgr cre/+ Arid1a f/f; Arid1a d/d). Ovarian function and uterine development of Arid1a d/d mice were normal. However, Arid1a d/d mice were sterile due to defective embryo implantation and decidualization. The epithelial proliferation was significantly increased in Arid1a d/d mice compared to control mice. Enhanced epithelial estrogen activity and reduced epithelial PGR expression, which impedes maturation of the receptive uterus, was observed in Arid1a d/d mice at the peri-implantation period. The microarray analysis revealed that ARID1A represses the genes related to cell cycle and DNA replication. We showed that ARID1A positively regulates Klf15 expression with PGR to inhibit epithelial proliferation at peri-implantation. Our results suggest that Arid1a has a critical role in modulating epithelial proliferation which is a critical requisite for fertility. This finding provides a new signaling pathway for steroid hormone regulation in female reproductive biology and furthers our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in human reproductive disorders such as endometriosis.

Protein Inhibitor of Activated STAT3 (PIAS3) Is Down-Regulated in Eutopic Endometrium of Women with Endometriosis

ABSTRACT Endometriosis is a major cause of chronic pelvic pain and infertility. Activation of STAT3 appears central to the inflammatory phenotype of eutopic endometrium in women with endometriosis. However, the molecular mechanism by which this occurs remains unknown. Our objective is to determine how STAT3 activity is regulated in endometriosis. Protein inhibitor of activated STAT3 (PIAS3) is a negative regulator of STAT3 activity. We examined the levels of PIAS3 in endometrium from women with and without endometriosis using Western blot analysis and immunohistochemistry. Levels of PIAS3 are significantly lower, in contrast with phosphorylation of STAT3, in women with endometriosis compared to women without endometriosis. Furthermore, induction of endometriosis in the baboon showed a significant reduction of PIAS3 expression during the progression of the disease. Interferon-γ (INFγ) reduces PIAS3 protein levels and increases phospho-STAT3 levels through CXCL10 in endometrial cells, Ishikawa, and 12Z cells. These results suggest that attenuation of PIAS3 causes aberrant activation of STAT3 in endometriosis, leading to inflammatory changes that may impair fertility or cause pain.

KRAS Activation and over-expression of SIRT1/BCL6 Contributes to the Pathogenesis of Endometriosis and Progesterone Resistance

Endometriosis is an inflammatory condition that is associated with progesterone resistance and cell proliferation, resulting in pain, infertility and pregnancy loss. We previously demonstrated phosphorylation of STAT3 in eutopic endometrium of infertile women with this disorder leading to over-expression of the oncogene BCL6 and stabilization of hypoxia-induced factor 1 alpha (HIF-1α). Here we report coordinated activation of KRAS and over-expression of Sirtuin 1 (SIRT1), a histone deacetylase and gene silencer, in the eutopic endometrium from women with endometriosis throughout the menstrual cycle. The mice with conditional activation of KRAS in the PGR positive cells reveal an increase of SIRT1 expression in the endometrium compared to control mice. The expression of progesterone receptor target genes including the Indian Hedgehog pathway genes are significantly down-regulated in the mutant mice. SIRT1 co-localizes with BCL6 in the nuclei of affected individuals and both proteins bind to and suppress the promoter of GLI1, a critical mediator of progesterone action in the Indian Hedgehog pathway, by ChIP analysis. In eutopic endometrium, GLI1 expression is reduced in women with endometriosis. Together, these data suggest that KRAS, SIRT1 and BCL6 are coordinately over-expressed in eutopic endometrium of women with endometriosis and likely participate in the pathogenesis of endometriosis.

DNAJB1 negatively regulates MIG6 to promote epidermal growth factor receptor signaling

Mitogen-inducible gene 6 (MIG6) is a tumor suppressor implicated in the development of human cancers; however, the regulatory mechanisms of MIG6 remain unknown. Here, using a yeast two-hybrid screen, we identified DnaJ homolog subfamily B member I (DNAJB1) as a novel MIG6-interacting protein. We found that DNAJB1 binds to and decreases MIG6 protein, but not mRNA, levels. DNAJB1 overexpression dosage-dependently decreased MIG6 protein levels. Conversely, DNAJB1 knockdown increased MIG6 protein levels. DNAJB1 destabilizes MIG6 by enhancing K48-linked ubiquitination of MIG6. However, knocking-down of DNAJB1 reduced the ubiquitination of MIG6. DNAJB1 positively regulates the epidermal growth factor receptors (EGFR) signaling pathway via destabilization of MIG6; however, DNAJB1 knockdown diminishes activation of EGFR signaling as well as elevation of MIG6. Importantly, the increased levels of MIG6 by DNAJB1 knockdown greatly enhanced the gefitinib sensitivity in A549 cells. Thus, our study provides a new molecular mechanism to regulate EGFR signaling through modulation of MIG6 by DNAJB1 as a negative regulator.

cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.

Endometriosis is a major cause of infertility and pelvic pain, affecting more than 10% of reproductive-aged women. Progesterone resistance has been observed in the endometrium of women with this disease, as evidenced by alterations in progesterone-responsive gene and protein expression. cAMPResponse Element-Binding 3-like protein 1 (Creb3l1) has previously been identified as a progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 function has not been studied in the context of endometriosis and uterine biology. In this study, we validated progesterone (P4) regulation of Creb3l1 in the uteri of wild-type and progesterone receptor knockout (PRKO) mice. Furthermore, we observed that CREB3L1 expression was significantly higher in secretory phase human endometrium compared to proliferative phase and that CREB3L1 expression was significantly decreased in the endometrium of women with endometriosis. Lastly, by transfecting CREB3L1 siRNA into cultured human endometrial stromal cells (hESCs) prior to hormonal induction of in vitro decidualization, we showed that CREB3L1 is required for the decidualization process. Interestingly, phosphorylation of ERK1/2, critical factor for decidualization, was also significantly reduced in CREB3L1-silenced hESCs. It is known that hESCs from patients with endometriosis show impaired decidualization and that dysregulation of the P4-PR signaling axis is linked to a variety of endometrial diseases including infertility and endometriosis. Therefore, these results suggest that CREB3L1 is required for decidualization in mice and humans and may be linked to the pathogenesis of endometriosis in a P4-dependent manner.