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. We mapped genome-wide differences in DNA methylation between healthy human endometrial and endometriotic stromal cells and correlated this with gene expression using an interaction analysis strategy. We identified 42,248 differentially methylated CpGs in endometriosis compared to healthy cells. These extensive differences were not unidirectional, but were focused intragenically and at sites distal to classic CpG islands where methylation status was typically negatively correlated with gene expression. Significant differences in methylation were mapped to 403 genes, which included a disproportionally large number of transcription factors. Furthermore, many of these genes are implicated in the pathology of endometriosis and decidualization. Our results tremendously improve the scope and resolution of differential methylation affecting the HOX gene clusters, nuclear receptor genes, and intriguingly the GATA family of transcription factors. Functional analysis of the GATA family revealed that GATA2 regulates key genes necessary for the hormone-driven differentiation of healthy stromal cells, but is hypermethylated and repressed in endometriotic cells. GATA6, which is hypomethylated and abundant in endometriotic cells, potently blocked hormone sensitivity, repressed GATA2, and induced markers of endometriosis when expressed in healthy endometrial cells. The unique epigenetic fingerprint in endometriosis suggests DNA methylation is an integral component of the disease, and identifies a novel role for the GATA family as key regulators of uterine physiology-aberrant DNA methylation in endometriotic cells correlates with a shift in GATA isoform expression that facilitates progesterone resistance and disease progression.

Paracrine activation of WNT/ß-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. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/ß-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of ß-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of ß-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/ß-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

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. We identified 1232 differentially expressed genes (DEGs) in IVD vs vehicle. GATA2 cistrome in IVD-treated cells was enriched with motifs for GATA, ATF, and JUN, and gene ontology analysis of GATA2 cistrome revealed pathways that regulate cholesterol storage, p38 mitogen-activated protein kinase, and the c-Jun N-terminal kinase cascades. Integration of RNA-seq and ChIP-seq data revealed that the PGR motif is highly enriched at GATA2 binding regions surrounding upregulated genes in IVD-treated cells. The integration of a mined public PGR cistrome in IVD-treated human endometrial cells with our GATA2 cistrome showed that GATA2 binding was significantly enhanced at PGR-binding regions in IVD vs vehicle. Interrogating 2 separate ChIP-seq data sets together with RNA-seq revealed integration of GATA2 and PGR action to coregulate biologic processes during decidualization of human endometrial stromal cells, specifically via WNT activation and stem cell differentiation pathways. These findings reveal the key pathways that are coactivated by GATA2 and PGR that may be therapeutic targets for supporting implantation and early pregnancy.

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. We demonstrate that activation of the PKA and PKC pathways, by a cAMP analog dibutyryl (Bu)2cAMP [(Bu)2cAMP] and phorbol 12-myristate 13-acetate (PMA), respectively, markedly decreased DAX-1 expression, an event that was inversely correlated with StAR protein, StAR mRNA, and progesterone levels. Notably, the suppression of DAX-1 requires de novo transcription and translation, suggesting that the effect of DAX-1 in regulating StAR expression is dynamic. Chromatin immunoprecipitation studies revealed the association of DAX-1 with the proximal but not the distal region of the StAR promoter, and both (Bu)2cAMP and PMA decreased in vivo DAX-1-DNA interactions. EMSA and reporter gene analyses demonstrated the functional integrity of this interaction by showing that DAX-1 binds to a DNA hairpin at position -44/-20 bp of the mouse StAR promoter and that the binding of DAX-1 to this region decreases progesterone synthesis by impairing transcription of the StAR gene. In support of this, targeted silencing of endogenous DAX-1 elevated basal, (Bu)2cAMP-, and PMA-stimulated StAR expression and progesterone synthesis. Transrepression of the StAR gene by DAX-1 was tightly associated with expression of the nuclear receptors Nur77 and steroidogenic factor-1, demonstrating these factors negatively modulate the steroidogenic response. These findings provide insight into the molecular events by which DAX-1 influences the PKA and PKC signaling pathways involved in the regulation of the StAR protein and steroidogenesis in mouse Leydig tumor 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. the delivery of cholesterol from the outer to the inner mitochondrial membrane. The expression of the StAR protein is predominantly regulated by cAMP-dependent mechanisms in the adrenal and gonads. Whereas StAR plays an indispensable role in the regulation of steroid biosynthesis, a complete understanding of the regulation of its expression and function in steroidogenesis is not available. It has become clear that the regulation of StAR gene expression is a complex process that involves the interaction of a diversity of hormones and multiple signaling pathways that coordinate the cooperation and interaction of transcriptional machinery, as well as a number of post-transcriptional mechanisms that govern mRNA and protein expression. However, information is lacking on how the StAR gene is regulated in vivo such that it is expressed at appropriate times during development and is confined to the steroidogenic cells. Thus, it is not surprising that the precise mechanism involved in the regulation of StAR gene has not yet been established, which is the key to understanding the regulation of steroidogenesis in the context of both male and female development and function.

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.e. CRE binding protein (CREB)/CRE modulator (CREM)/activating transcription factor (ATF), activator protein 1 (AP-1; Fos/Jun), and CCAAT enhancer binding protein beta (C/EBPbeta), interact with an overlapping region (-81/-72 bp) in the StAR promoter, mediate stimulus-transcription coupling of cAMP signaling and play integral roles in regulating StAR gene expression. These bZIP proteins are structurally similar and bind to DNA sequences as dimers; however, they exhibit discrete transcriptional activities, interact with several transcription factors and other properties that contribute in their regulatory functions. The 5'-flanking -81/-72 bp region of the StAR gene appears to function as a key element within a complex cAMP response unit by binding to different bZIP members, and the StAR promoter displays variable states of cAMP responsivity contingent upon the occupancy of these cis-elements with these transcription factors. The expression and activities of CREB/CREM/ATF, Fos/Jun and C/EBPbeta have been demonstrated to be mediated by a plethora of extracellular signals, and the phosphorylation of these proteins at several Ser and Thr residues allows recruitment of the transcriptional coactivator CREB binding protein (CBP) or its functional homolog p300 to the StAR promoter. This review will focus on the current level of understanding of the roles of selective bZIP family proteins within the complex series of processes involved in regulating StAR gene transcription.

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. In MA-10 mouse Leydig tumor cells Star gene expression is more dependent upon type I PKA, while the post-transcriptional regulation of STAR appears subject to type II PKA. These experiments delineate the discrete effects that type I and type II PKA exert on STAR-mediated steroidogenesis, and suggest complimentary roles for each subtype in coordinating 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. Cotreatment with both dibutyryl-cAMP and ciglitazone revealed a dose-dependent, significant increase in progesterone synthesis, Star promoter activity, Star mRNA and STAR protein relative to either compound alone. The overexpression of PPARgamma further increased Star-promoter activity. The ciglitazone-induced activity of the Star-promoter appears to be mediated through the cAMP-response element half-sites located within its proximal 151 bp. Combined treatment with ciglitazone and dibutyryl-cAMP significantly increased the expression and activity of transcriptional pathways impacted by the activator protein-1 family member c-JUN. The present study demonstrates that ciglitazone and dibutyryl-cAMP synergistically enhance STAR expression in MA-10 and KK1 cells. Ciglitazone-activated PPARgamma appears to increase the sensitivity of Leydig and granulosa cells to cAMP stimulation, possibly via upregulation of c-JUN expression.

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. We ectopically expressed GATA6 alone or with NR5A1 in NoEM or silenced these transcription factors in endometriotic stromal cells (OSIS) and assessed the messenger RNAs or proteins encoded by the genes in the steroidogenic cascade. Functionally, we assessed the effects of GATA6 expression or silencing on estradiol formation. In OSIS, GATA6 was necessary for catalyzing the conversion of progesterone to androstenedione (CYP17A1; P < .05). In NoEM, ectopic expression of GATA6 was essential for converting pregnenolone to estrogen (HSD3B2, CYP17A1, and CYP19A1; P < .05). However, simultaneous ectopic expression of both GATA6 and NR5A1 was required and sufficient to confer induction of all 5 genes and their encoded proteins that convert cholesterol to estrogen. Functionally, only simultaneous knockdown of GATA6 and NR5A1 blocked estradiol formation in OSIS ( P < .05). The presence of both transcription factors was required and sufficient to transform endometrial stromal cells into endometriotic-like cells that produced estradiol in large quantities ( P < .05). In summary, GATA6 alone is essential but not sufficient for estrogen formation in endometriosis. However, simultaneous addition of GATA6 and NR5A1 to an endometrial stromal cell is sufficient to transform it into an endometriotic-like cell, manifested by the activation of the estradiol biosynthetic cascade.

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 uterus differentiates from the intermediate mesoderm (IM) to form coelomic epithelium (CE) followed by the Müllerian duct (MD). Here, we successfully directed the differentiation of human iPSCs (hiPSCs) through IM, CE, and MD to EMSFs under molecularly defined embryoid body culture conditions using specific hormonal treatments. Activation of CTNNB1 was essential for expression of progesterone receptor that mediated the final differentiation step of EMSFs before implantation. These hiPSC-derived tissues illustrate the potential for iPSC-based endometrial regeneration for future cell-based treatments.

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. Design: Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Setting: Research laboratory. Patients: Eight African American women. Interventions: None. Main Outcomes Measures: Gene expression patterns, cell proliferation, and differentiation. Results: A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. Conclusions: IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.

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. Incubating MA-10 cells with each of the EETs induced a dose-dependent increase in StAR protein expression and steroid hormone production in the presence of dbcAMP. These metabolites also significantly enhanced StAR gene transcription as determined by luciferase assays of StAR promoter activity and reverse transcriptase-PCR analysis of StAR mRNA levels. While the EETs enhanced steroidogenesis, inhibiting the activity of protein kinase A (PKA) abolished the stimulatory effects of these AA metabolites on StAR expression and steroid hormone production. This study suggests that cAMP stimulation of MA-10 cells increases epoxygenase-generated AA metabolites and the co-action of these metabolites with PKA significantly increases StAR gene expression and steroid hormone production.

Estrogen receptor ß 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. PATIENT(S): Premenopausal women. INTERVENTION(S): Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. MAIN OUTCOME MEASURE(S): Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) ß. RESULT(S): Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERß-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERß based on small interfering RNA knockdown and chromatin immunoprecipitation of ERß followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. CONCLUSION(S): ERß leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis.

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. Our results indicate that from 3 months of age to 30 months, COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells, StAR protein decreased to 33%, and blood testosterone concentration and testosterone biosynthesis in Leydig cells decreased to 41 and 33%, respectively. Further experiments demonstrated that overexpressing COX2 in MA-10 mouse Leydig cells inhibited StAR gene expression and steroidogenesis and that the inhibitory effects of COX2 could be reversed by blocking COX2 activity. Notably, incubation of aged Leydig cells with the COX2 inhibitor NS398 enhanced their testosterone biosynthesis. Blood testosterone concentrations in aged rats fed the COX2 inhibitor DFU, at doses of 5, 10, 15, and 20 mg/kg body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in the rats receiving no DFU. The present study suggests a novel mechanism in male aging involving COX2 and a potential application of the mechanism to delay the age-related decline in testosterone biosynthesis.

Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family.

The mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through cAMP-dependent pathways. In many cases transcriptional induction by cAMP is mediated through the interaction of a cAMP response-element binding protein (CREB) family member with a consensus cAMP response element (CRE; 5'-TGACGTCA-3') found in the promoter of target genes. The present investigation was carried out to determine whether a CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM) family] was involved in the regulation of steroidogenesis and StAR protein expression. Transient expression of wild- type CREB in MA-10 mouse Leydig tumor cells further increased the levels of (Bu)2cAMP-induced progesterone synthesis, StAR promoter activity, StAR mRNA, and StAR protein. These responses were significantly inhibited by transfection with a dominant-negative CREB (A-CREB), or with a CREB mutant that cannot be phosphorylated (CREB-M1), the latter observation indicating the importance of phosphorylation of a CREB/CREM family member in steroidogenesis and StAR expression. The CREB/CREM-responsive region in the mouse StAR gene was located between -110 and -67 bp upstream of the transcriptional start site. An oligonucleotide probe (-96/-67 bp) containing three putative half-sites for 5'-canonical CRE sequences (TGAC) demonstrated the formation of protein-DNA complexes in EMSAs with recombinant CREB protein as well as with nuclear extracts from MA-10 or Y-1 mouse adrenal tumor cells. The predominant binding factor observed with EMSA was found to be the CREM protein as demonstrated using specific antibodies and RT-PCR analyses. The CRE elements identified within the -96/-67 bp region were tested for cAMP responsiveness by generating mutations in each of the CRE half-sites either alone or in combination. Although each of the CRE sites contribute in part to the CREM response, the CRE2 appears to be the most important site as determined by EMSA and by reporter gene analyses. Binding specificity was further assessed using specific antibodies to CREB/CREM family members, cold competitors, and mutations in the target sites that resulted in either supershift and/or inhibition of these complexes. We also demonstrate that the inducible cAMP early repressor markedly diminished the endogenous effects of CREM on cAMP-induced StAR promoter activity and on StAR mRNA expression. These are the first observations to provide evidence for the functional involvement of a CREB/CREM family member in the acute regulation of trophic hormone-stimulated steroidogenesis and StAR gene expression.

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. PATIENT(S): Premenopausal women with or without endometriosis. INTERVENTION(S): Primary cultures of stromal cells from healthy endometrium (E-IUM) or endometriomas (E-OSIS) were subjected to in vitro decidualization (IVD) using 1 µM medroxyprogesterone acetate, 35 nM 17ß-estradiol, and 0.05 mM 8-Br-cAMP. MAIN OUTCOME MEASURE(S): Expression of DNMT1, DNMT3A, and DNMT3B in E-IUM and E-OSIS were assessed by quantitative real-time polymerase chain reaction and immunoblotting. Recruitment of DNMT3B to the promoters of steroidogenic factor 1 (SF-1) and estrogen receptor α (ESR1) was examined by chromatin immunoprecipitation. RESULT(S): IVD treatment reduced DNMT3B messenger RNA (74%) and protein levels (81%) only in E-IUM; DNMT1 and DNMT3A were unchanged in both cell types. Significantly more DNMT3B bound to the SF-1 promoter in E-IUM compared with E-OSIS, and IVD treatment reduced binding in E-IUM to levels similar to those in E-OSIS. Enrichment of DNMT3B across 3 ESR1 promoters was reduced in E-IUM after IVD, although the more-distal promoter showed increased DNMT3B enrichment in E-OSIS after IVD. CONCLUSION(S): The inability to downregulate DNMT3B expression in E-OSIS may contribute to an aberrant epigenetic fingerprint that misdirects gene expression in endometriosis and contributes to its altered response to steroid hormones.

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. A common salient feature of leiomyomas is their responsiveness to steroid hormones, thus providing an opportunity for intervention. METHODS: A comprehensive search of PUBMED was conducted to identify peer-reviewed literature published since 1980 pertinent to the roles of steroid hormones and somatic stem cells in leiomyoma, including literature on therapeutics that target steroid hormone action in leiomyoma. Reviewed articles were restricted to English language only. Studies in both animals and humans were reviewed for the manuscript. RESULTS: Estrogen stimulates the growth of leiomyomas, which are exposed to this hormone not only through ovarian steroidogenesis, but also through local conversion of androgens by aromatase within the tumors themselves. The primary action of estrogen, together with its receptor estrogen receptor α (ERα), is likely mediated via induction of progesterone receptor (PR) expression, thereby allowing leiomyoma responsiveness to progesterone. Progesterone has been shown to stimulate the growth of leiomyoma through a set of key genes that regulate both apoptosis and proliferation. Given these findings, aromatase inhibitors and antiprogestins have been developed for the treatment of leiomyoma, but neither treatment results in complete regression of leiomyoma, and tumors recur after treatment is stopped. Recently, distinct cell populations were discovered in leiomyomas; a small population showed stem-progenitor cell properties, and was found to be essential for ovarian steroid-dependent growth of leiomyomas. Interestingly, these stem-progenitor cells were deficient in ERα and PR and instead relied on the strikingly higher levels of these receptors in surrounding differentiated cells to mediate estrogen and progesterone action via paracrine signaling. CONCLUSIONS: It has been well established that estrogen and progesterone are involved in the proliferation and maintenance of uterine leiomyoma, and the majority of medical treatments currently available for leiomyoma work by inhibiting steroid hormone production or action. A pitfall of these therapeutics is that they decrease leiomyoma size, but do not completely eradicate them, and tumors tend to regrow once treatment is stopped. The recent discovery of stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma has the potential to provide the missing link between developing therapeutics that temper leiomyoma growth and those that eradicate them.

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. Each leiomyoma seems to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a mutation. Tumor expansion is sustained by cell proliferation together with the production of large amounts of extracellular matrix. Estrogen and progesterone stimulate the growth of leiomyomas. Estrogen, together with its receptor ERα, enables progesterone action via induction of progesterone receptor (PR) expression. Progesterone induces the growth of leiomyoma by regulation of a set of key genes that control proliferation and apoptosis. A distinct cell population with stem-progenitor properties is indispensable for progesterone-dependent growth of leiomyomas. This stem-progenitor cell population is deficient in ERα and PR and dependent on the much higher levels of these steroid receptors in surrounding mature leiomyoma or myometrial cells. Progesterone sends paracrine signals from these mature cells to stem cells. The WNT/ß-catenin pathway comprises a key component of this paracrine signaling system. The majority of medical treatments currently available for leiomyoma works by inhibiting estrogen or progesterone production or action, but tumors tend to regrow once treatment is stopped. Targeting stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma may lead to the development of more effective therapeutics.

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. OBJECTIVE: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. DESIGN: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. RESULTS: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CONCLUSIONS: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

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 5.7-fold and 34.3-fold, respectively. In the presence of indomethacin, the level of Bt(2)cAMP required for maximal steroidogenesis was reduced from 1.0 mM to 0.25 mM. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression, suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. Although SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt(2)cAMP-stimulated StAR protein expression and steroid production. Overexpression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.