GATA2 and Progesterone Receptor Interaction in Endometrial Stromal Cells Undergoing Decidualization.

The transcription factor GATA2 is important for endometrial stromal cell decidualization in early pregnancy. Progesterone receptor (PGR) is also critical during decidualization but its interaction with GATA2 in regulating genes and pathways necessary for decidualization in human endometrium are unclear. RNA-sequencing (RNA-seq) was performed to compare gene expression profiles (n = 3), and chromatin immunoprecipitation followed by sequencing (ChIP-seq) using an antibody against GATA2 (n = 2) was performed to examine binding to target genes in human endometrial stromal cells undergoing in vitro decidualization (IVD including estrogen, progestin, and 3',5'-cyclic AMP analogue) or vehicle treatment.

Generation of Progesterone-Responsive Endometrial Stromal Fibroblasts from Human Induced Pluripotent Stem Cells: Role of the WNT/CTNNB1 Pathway.

Defective endometrial stromal fibroblasts (EMSFs) contribute to uterine factor infertility, endometriosis, and endometrial cancer. Induced pluripotent stem cells (iPSCs) derived from skin or bone marrow biopsies provide a patient-specific source that can be differentiated to various cells types. Replacement of abnormal EMSFs is a potential novel therapeutic approach for endometrial disease; however, the methodology or mechanism for differentiating iPSCs to EMSFs is unknown.

The Essential Role of GATA6 in the Activation of Estrogen Synthesis in Endometriosis.

Endometriotic stromal cells synthesize estradiol via the steroidogenic pathway. Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is critical, but alone not sufficient, in activating this cascade that involves at least 5 genes. To evaluate whether another transcription factor is required for the activation of this pathway, we examined whether GATA Binding Protein 6 (GATA6) can transform a normal endometrial stromal cell (NoEM) into an endometriotic-like cell by conferring an estrogen-producing phenotype.

Paracrine Pathways in Uterine Leiomyoma Stem Cells Involve Insulinlike Growth Factor 2 and Insulin Receptor A.

Context: Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown.

Objective: To define differential gene expression and signaling pathways in leiomyoma cell populations.

Estrogen receptor β regulates endometriotic cell survival through serum and glucocorticoid-regulated kinase activation.

Objective: To determine the expression and biological roles of serum and glucocorticoid-regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects.

Design: Case-control.

Setting: University research setting.

Uterine Leiomyoma Stem Cells: Linking Progesterone to Growth.

Uterine leiomyomas (fibroids) represent the most common class of benign tumors in women. Multiple leiomyomas usually arise from the uterus of a symptomatic woman. These tumors cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are responsible for more than 200,000 hysterectomies in the United States annually.

Aberrant expression and localization of deoxyribonucleic acid methyltransferase 3B in endometriotic stromal cells.

Objective: To define the expression and function of DNA methyltransferases (DNMTs) in response to decidualizing stimuli in endometriotic cells compared with healthy endometrial stroma.

Design: Basic science.

Setting: University research center.

Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo.

Context: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo.

Ovarian steroids, stem cells and uterine leiomyoma: therapeutic implications.

Background: Uterine leiomyoma is the most common benign tumor in women and is thought to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a cellular insult. Leiomyomas cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are the most common indication for hysterectomy in the USA. A slow rate of cell proliferation, combined with the production of copious amounts of extracellular matrix, accounts for tumor expansion.

5-Hydroxymethylcytosine promotes proliferation of human uterine leiomyoma: a biological link to a new epigenetic modification in benign tumors.

Context: Uterine leiomyoma, or fibroids, represent the most common benign tumors of the female reproductive tract. A newly discovered epigenetic modification, 5-hydroxymethylation (5-hmC), and its regulators, the TET (Ten Eleven Translocation) enzymes, were implicated in the pathology of malignant tumors; however, their roles in benign tumors, including uterine fibroids, remain unknown.

Objective: To determine the role of 5-hmC and TET proteins in the pathogenesis of leiomyoma using human uterine leiomyoma and normal matched myometrial tissues and primary cells.

Genome-wide DNA methylation analysis predicts an epigenetic switch for GATA factor expression in endometriosis.

Endometriosis is a gynecological disease defined by the extrauterine growth of endometrial-like cells that cause chronic pain and infertility. The disease is limited to primates that exhibit spontaneous decidualization, and diseased cells are characterized by significant defects in the steroid-dependent genetic pathways that typify this process. Altered DNA methylation may underlie these defects, but few regions with differential methylation have been implicated in the disease.

Paracrine activation of WNT/β-catenin pathway in uterine leiomyoma stem cells promotes tumor growth.

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells.

Cutting SRC-1 down to size in endometriosis.

Endometriosis is an estrogen-dependent disease. The biologically active estrogen, estradiol, aggravates the pathological processes (e.g.

Activated glucocorticoid and eicosanoid pathways in endometriosis.

Objective: To define altered gene expression networks in endometriosis.

Design: Experiments using endometriotic tissues and primary cells.

Setting: Division of Reproductive Biology Research, Northwestern University.

Vasoactive intestinal peptide (VIP)-mediated expression and function of steroidogenic acute regulatory protein (StAR) in granulosa cells.

VIP is a peptide hormone capable of activating the cAMP/PKA pathway and modifying gonadal steroidogenic capacity. Less is known about the molecular mechanisms of VIP-mediated steroidogenesis and its role in regulating the steroidogenic acute regulatory protein (STAR). We examined the impact of VIP on STAR expression and function in immortalized (KK1) and primary mouse granulosa cells, where VIP strongly upregulated STAR expression and steroidogenesis.

Involvement of peroxisome proliferator-activated receptor gamma in gonadal steroidogenesis and steroidogenic acute regulatory protein expression.

Peroxisome proliferator-activated receptor (PPAR) gamma belongs to the PPAR family of nuclear transcription factors whose ligands, such as eicosanoids, fatty acids and prostaglandins, are known to affect gonadal function. Although several of these enhance the expression of the steroidogenic acute regulatory protein (STAR) and steroid production, the role of PPARgamma in regulating STAR-mediated steroidogenesis remains unclear. In the present study, we used ciglitazone to selectively activate PPARgamma and examine its role in STAR-mediated steroidogenesis in immortalised KK1 mouse granulosa cells and MA-10 mouse Leydig tumour cells.

Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives.

Steroid hormones are synthesized in the adrenal gland, gonads, placenta and brain and are critical for normal reproductive function and bodily homeostasis. The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroid biosynthesis, i.e.

Role of basic leucine zipper proteins in transcriptional regulation of the steroidogenic acute regulatory protein gene.

The regulation of steroidogenic acute regulatory protein (StAR) gene transcription by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP response element (CRE, TGACGTGA). This regulation is coordinated by multiple transcription factors that bind to sequence-specific elements located approximately 150 bp upstream of the transcription start site. Among the proteins that bind within this region, the basic leucine zipper (bZIP) family of transcription factors, i.

The differential regulation of steroidogenic acute regulatory protein-mediated steroidogenesis by type I and type II PKA in MA-10 cells.

Following tropic hormone challenge, steroidogenic tissues utilize PKA to phosphorylate unique subsets of proteins necessary to facilitate steroidogenesis. This includes the PKA-dependent expression and activation of the steroidogenic acute regulatory protein (STAR), which mediates the rate-limiting step of steroidogenesis by inducing the transfer of cholesterol from the outer to the inner mitochondrial membrane. Since both type I and type II PKA are present in steroidogenic tissues, we have utilized cAMP analog pairs that preferentially activate each PKA subtype in order to examine their impact on STAR synthesis and activity.

Role of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 in protein kinase A- and protein kinase C-mediated regulation of the steroidogenic acute regulatory protein expression in mouse Leydig tumor cells: mechanism of action.

Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) is an orphan nuclear receptor that has been demonstrated to be instrumental to the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. However, its mechanism of action remains obscure. The present investigation was aimed at exploring the molecular involvement of DAX-1 in protein kinase A (PKA)- and protein kinase C (PKC)-mediated regulation of StAR expression and its concomitant impact on steroid synthesis using MA-10 mouse Leydig tumor cells.

Mitochondrial A-kinase anchoring protein 121 binds type II protein kinase A and enhances steroidogenic acute regulatory protein-mediated steroidogenesis in MA-10 mouse leydig tumor cells.

The expression of the steroidogenic acute regulatory protein (STAR) is regulated by PKA in response to trophic hormone stimulation through the second messenger cAMP. However, in steroidogenic cells, the concentrations of hormone necessary to maximally induce cAMP synthesis and PKA activity are often significantly higher than is necessary to achieve maximum steroidogenesis. One general mechanism believed to make PKA signaling more effective is the use of A-kinase anchoring proteins (AKAPs) to recruit PKA to discrete subcellular compartments, which coordinates and focuses PKA action with respect to its substrates.

The involvement of epoxygenase metabolites of arachidonic acid in cAMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression.

The essential role of arachidonic acid (AA) in steroidogenesis has been previously demonstrated. The present study continues the investigation into how AA regulates steroidogenesis by examining the effects of epoxygenase-derived AA metabolites on cAMP-stimulated steroidogenic acute regulatory (StAR) gene expression and steroid hormone production in MA-10 mouse Leydig cells. The HPLC analysis of cell extracts from MA-10 cells treated with the cAMP analog dibutyryl cAMP (dbcAMP) demonstrated an increase in three epoxygenase-generated AA metabolites: 5,6-epoxyeicosatrienoic acid (EET), 8,9-EET, and 11,12-EET.

Cyclooxygenase-2 regulation of the age-related decline in testosterone biosynthesis.

The age-related decline in testosterone biosynthesis in testicular Leydig cells has been well documented, but the mechanisms involved in the decline are not clear. Recent studies have described a cyclooxygenase-2 (COX2)-dependent tonic inhibition of Leydig cell steroidogenesis and expression of the steroidogenic acute regulatory protein (StAR). The present study was conducted to determine whether COX2 protein increases with age in rat Leydig cells and whether COX2 plays a role in the age-related decline in testosterone biosynthesis.

Involvement of 5-lipoxygenase metabolites of arachidonic acid in cyclic AMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression.

To understand the mechanism for the role of arachidonic acid (AA) in steroidogenic acute regulatory (StAR) gene transcription, sections of the -1/-966 StAR promoter were deleted to produce constructs of -1/-426, -1/-211, -1/-151, and -1/-110 and inserted into the PGL3 vector to drive luciferase expression. Results indicated that -1/-151 StAR promoter contains the elements that are most responsive to AA. Electrophoretic mobility shift assays using nuclear extracts from AA-treated MA-10 Leydig tumor cells showed that AA enhanced specific binding of the nuclear extract to a 30bp (-67/-96) sequence of the StAR promoter.

Inhibition of cyclooxygenase-2 activity enhances steroidogenesis and steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells.

To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mM dibutyryl cAMP (Bt(2)cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 microM of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.