ABSTRACT
Binge drinking is a frequent pattern of ethanol consumption within Alcohol Use Disorders (AUDs). Binge-like ethanol exposure increases Poly(ADP-ribose) polymerase (PARP) expression and activity. PARP enzymes have been implicated in addiction and serve multiple roles in the cell, including gene expression regulation. In this study, we examined the effects of binge-like alcohol consumption in the prefrontal cortex (PFC) of adult C57BL/6J male mice via a 4-day Drinking-in-the-Dark (DID) paradigm. The role of PARP in associated gene expression and behavioral changes was assessed by administering the PARP inhibitor ABT-888 on the last DID day. We then conducted an RNA-seq analysis of the PFC gene expression changes associated with DID-consumed ethanol or ABT-888 treatment. A separate cohort of mice was inoculated with an HSV-PARP1 vector in the PFC and subject to a DID experiment to verify whether overexpressed PARP1 increased ethanol drinking. We confirmed that alcohol increases Parp1 gene expression and PARP activity in the PFC. RNA-seq showed significantly altered expression of 41 genes by DID-consumed ethanol, and of 48 genes by ABT-888. These results were confirmed by qPCR in 7 of the 10 genes validated, 4 of which have been previously associated with addiction. ABT-888 reduced, and overexpression of PFC PARP1 increased DID ethanol consumption. In our model, alcohol binge drinking induced specific alterations in the PFC expression of genes potentially involved in addiction. Pharmacological PARP inhibition proved effective in reversing these changes and preventing further alcohol consumption. Our results suggest an involvement of ethanol-induced PARP1 in reinforcing binge-like addictive behavior.
Subject(s)
Alcoholism , Binge Drinking , Alcohol Drinking , Animals , Ethanol , Male , Mice , Mice, Inbred C57BL , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) PolymerasesABSTRACT
The chemokine receptor CXCR4, a G protein-coupled receptor (GPCR) capable of heteromerizing with other GPCRs, is involved in many processes, including immune responses, hematopoiesis, and organogenesis. Evidence suggests that CXCR4 activation reduces thrombin/protease-activated receptor 1 (PAR1)-induced impairment of endothelial barrier function. However, the mechanisms underlying cross-talk between CXCR4 and PAR1 are not well-understood. Using intermolecular bioluminescence resonance energy transfer and proximity ligation assays, we found that CXCR4 heteromerizes with PAR1 in the HEK293T expression system and in human primary pulmonary endothelial cells (hPPECs). A peptide analog of transmembrane domain 2 (TM2) of CXCR4 interfered with PAR1:CXCR4 heteromerization. In HTLA cells, the presence of CXCR4 reduced the efficacy of thrombin to induce ß-arrestin-2 recruitment to recombinant PAR1 and enhanced thrombin-induced Ca2+ mobilization. Whereas thrombin-induced extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation occurred more transiently in the presence of CXCR4, peak ERK1/2 phosphorylation was increased when compared with HTLA cells expressing PAR1 alone. CXCR4-associated effects on thrombin-induced ß-arrestin-2 recruitment to and signaling of PAR1 could be reversed by TM2. In hPPECs, TM2 inhibited thrombin-induced ERK1/2 phosphorylation and activation of Ras homolog gene family member A. CXCR4 siRNA knockdown inhibited thrombin-induced ERK1/2 phosphorylation. Whereas thrombin stimulation reduced surface expression of PAR1, CXCR4, and PAR1:CXCR4 heteromers, chemokine (CXC motif) ligand 12 stimulation reduced surface expression of CXCR4 and PAR1:CXCR4 heteromers, but not of PAR1. Finally, TM2 dose-dependently inhibited thrombin-induced impairment of hPPEC monolayer permeability. Our findings suggest that CXCR4:PAR1 heteromerization enhances thrombin-induced G protein signaling of PAR1 and PAR1-mediated endothelial barrier disruption.
Subject(s)
Receptor, PAR-1/metabolism , Receptors, CXCR4/metabolism , Thrombin/metabolism , Biopolymers/metabolism , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Energy Transfer , HEK293 Cells , Humans , Lung/cytology , Lung/metabolism , MAP Kinase Signaling System , PhosphorylationABSTRACT
Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in ß-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit ß-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced ß-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.
Subject(s)
Muscle, Smooth, Vascular/metabolism , Protein Multimerization , Receptors, CXCR/metabolism , Receptors, Vasopressin/metabolism , Animals , Arginine Vasopressin/metabolism , Cells, Cultured , HEK293 Cells , Humans , Inositol Phosphates/metabolism , Male , Rats , Rats, Sprague-Dawley , beta-Arrestins/metabolismABSTRACT
Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/ß, CXCL12(3-68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05-0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03-3µM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3-68) was inefficient to activate CXCR4 in Presto-Tango ß-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3-68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/ß, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3-68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.
Subject(s)
Lung/metabolism , Receptors, CXCR4/metabolism , Receptors, CXCR/metabolism , Cell Line , Endothelial Cells/cytology , Endothelial Cells/metabolism , Humans , Ligands , Lung/cytologyABSTRACT
Previous studies have indicated that retinoic acid (RA) may be therapeutic for endometrial cancer. However, the downstream target genes and pathways triggered by ligand-activated RA receptor α (RARα) in endometrial cancer cells are largely unknown. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and immunoblotting assays were used to assess the roles of RA and the RA agonist (AM580) in the growth of endometrial cancer cells. Illumina-based microarray expression profiling of endometrial Ishikawa cells incubated with and without AM580 for 1, 3, and 6âh was performed. We found that both RA and AM580 markedly inhibited endometrial cancer cell proliferation, while knockdown of RARα could block AM580 inhibition. Knockdown of RARα significantly increased proliferating cell nuclear antigen and BCL2 protein levels. Incubation of Ishikawa cells with or without AM580 followed by microarray expression profiling showed that 12â768 genes out of 47â296 gene probes were differentially expressed with significant P values. We found that 90 genes were the most regulated genes with the most significant P value (P<0.0001) using F-test. We selected four highly regulated genes with diverse functions, namely G0S2, TNFAIP2, SMAD3, and NRIP1. Real-time PCR verified that AM580 highly regulated these genes, whereas chromatin immunoprecipitation-PCR assay demonstrated that ligand-activated RARα interacted with the promoter of these genes in intact endometrial cancer cells. AM580 also significantly altered 18 pathways including those related to cell growth, differentiation, and apoptosis. In conclusion, AM580 treatment of Ishikawa cells causes the differential expression of a number of RARα target genes and activation of signaling pathways. These pathways could, therefore, mediate the carcinogenesis of human endometrial cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/genetics , Endometrium/pathology , Tretinoin/therapeutic use , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzoates/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Endometrium/drug effects , Endometrium/physiology , Female , Gene Expression Profiling , Gene Knockdown Techniques , Genome/drug effects , Humans , Microarray Analysis , Molecular Sequence Data , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Retinoic Acid Receptor alpha , Signal Transduction/drug effects , Tetrahydronaphthalenes/pharmacology , Tretinoin/pharmacologyABSTRACT
CONTEXT: Retinoic acid (RA) controls multiple biological processes via exerting opposing effects on cell survival. Retinol uptake into cells is controlled by stimulated by RA 6 (STRA6). RA is then produced from retinol in the cytosol. Partitioning of RA between the nuclear receptors RA receptor α and peroxisome-proliferator-activated receptor ß/δ is regulated by cytosol-to-nuclear shuttling proteins cellular RA binding protein 2 (CRABP2) and fatty acid binding protein 5 (FABP5), which induce apoptosis or enhance survival, respectively. The roles of these mechanisms in endometrium or endometriosis remain unknown. OBJECTIVE: The aim was to determine the regulation of retinoid uptake and RA action in primary stromal cells from endometrium (n = 10) or endometriosis (n = 10). RESULTS: Progesterone receptor was necessary for high STRA6 and CRABP2 expression in endometrial stromal cells. STRA6, which was responsible for labeled retinoid uptake, was strikingly lower in endometriotic cells compared to endometrial cells. CRABP2 knockdown in endometrial cells increased survival, and FABP5 knockdown in endometriotic cells decreased survival without altering the expression of downstream nuclear retinoic acid receptor α and peroxisome-proliferator-activated receptor ß/δ. CONCLUSIONS: In endometrial stromal cells, progesterone receptor up-regulates expression of STRA6 and CRABP2, which control retinol uptake and growth-suppressor actions of RA. In endometriotic stromal cells, decreased expression of these genes leads to decreased retinol uptake and dominant FABP5-mediated prosurvival activity.
Subject(s)
Cell Survival , Endometriosis/metabolism , Endometrium/metabolism , Ovarian Diseases/metabolism , Tretinoin/metabolism , Adult , Analysis of Variance , Blotting, Western , Cells, Cultured , Endometriosis/genetics , Endometrium/drug effects , Female , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Ovarian Diseases/genetics , RNA, Small Interfering , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/drug effects , Stromal Cells/metabolism , Tretinoin/pharmacology , Up-RegulationABSTRACT
Uterine leiomyoma is the most common tumor of the female genital tract and the leading cause of hysterectomy. Although progesterone stimulates the proliferation of uterine leiomyoma cells, the mechanism of progesterone action is not well understood. We used chromatin immunoprecipitation (ChIP)-cloning approach to identify progesterone receptor (PR) target genes in primary uterine leiomyoma smooth muscle cells. We identified 18 novel PR-binding sites, one of which was located 20.5 kb upstream of the transcriptional start site of the Krüppel-like transcription factor 11 (KLF11) gene. KLF11 mRNA levels were minimally downregulated by progesterone but robustly upregulated by the progesterone antagonist RU486. Luciferase reporter assays showed significant baseline and RU486-inducible promoter activity in the KLF11 basal promoter or distal PR-binding region, both of which contained multiple Sp1-binding sequences but lacked classic progesterone response elements. RU486 stimulated recruitment of Sp1, RNA polymerase II, PR, and the coactivators SRC-1 and SRC-2 to the distal region and basal promoter. siRNA knockdown of PR increased KLF11 expression, whereas knockdown of KLF11 increased leiomyoma cell proliferation and abolished the antiproliferative effect of RU486. In vivo, KLF11 expression was significantly lower in leiomyoma tissues compared with adjacent myometrial tissues. Taken together, using a ChIP-cloning approach, we uncovered KLF11 as an integrator of PR signaling and proliferation in uterine leiomyoma cells.
Subject(s)
Cell Cycle Proteins/physiology , Cell Proliferation , Leiomyoma/genetics , Receptors, Progesterone/physiology , Repressor Proteins/physiology , Uterine Neoplasms/genetics , Apoptosis Regulatory Proteins , Cell Cycle Proteins/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Hormone Antagonists/pharmacology , Humans , Leiomyoma/pathology , Mifepristone/pharmacology , Progesterone/pharmacology , Promoter Regions, Genetic/drug effects , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Repressor Proteins/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Trans-Activators/metabolism , Trans-Activators/physiology , Transcription Factors/physiology , Tumor Burden/genetics , Tumor Cells, Cultured , Uterine Neoplasms/pathologyABSTRACT
OBJECTIVE: To evaluate the effects of selective P receptor (PR) modulator CDB4124 on cell proliferation and apoptosis in cultured human uterine leiomyoma smooth muscle (LSM) cells and control myometrial smooth muscle (MSM) cells in matched uteri. DESIGN: Laboratory research. SETTING: Academic medical center. PATIENT(S): Premenopausal women (n = 12) undergoing hysterectomy for leiomyoma-related symptoms. INTERVENTION(S): Treatment of primary LSM and MSM cells with CDB4124 (10(-8)-10(-6) M) or vehicle for 24, 48, or 72 hours. MAIN OUTCOME MEASURE(S): Western blot for protein expression of proliferating cell nuclear antigen, cleaved polyadenosine 5'-diphosphate-ribose polymerase, Bcl-2, and Krüppel-like transcription factor 11; 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate viable cell numbers; and real-time polymerase chain reaction (PCR) to quantify messenger RNA (mRNA) levels. RESULT(S): Treatment with CDB4124 significantly decreased levels of the proliferation marker proliferating cell nuclear antigen, the number of viable LSM cells, and the antiapoptotic protein Bcl-2. On the other hand, treatment with CDB4124 increased levels of the apoptosis marker cleaved polyadenosine 5'-diphosphate-ribose polymerase and the tumor suppressor Krüppel-like transcription factor 11 in a dose- and time-dependent manner in LSM cells. In matched MSM cells, however, CDB4124 did not affect cell proliferation or apoptosis. CONCLUSION(S): CDB4124 selectively inhibits proliferation and induces apoptosis in LSM but not in MSM cells.
Subject(s)
Leiomyoma/pathology , Norpregnadienes/pharmacology , Receptors, Progesterone/drug effects , Adult , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Cell Cycle Proteins/biosynthesis , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Female , Humans , Middle Aged , Myometrium/drug effects , Poly(ADP-ribose) Polymerases/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Proliferating Cell Nuclear Antigen/biosynthesis , Proto-Oncogene Proteins c-bcl-2 , Repressor Proteins/biosynthesis , Uterine Neoplasms/pathologyABSTRACT
Estradiol (E2) stimulates the growth and inflammation in the ectopic endometriotic tissue that commonly resides on the pelvic organs. Several clinical and laboratory-based observations are indicative of resistance to progesterone action in endometriosis. The molecular basis of progesterone resistance in endometriosis may be related to an overall reduction in the levels of progesterone receptor (PR). In normal endometrium, progesterone acts via PR on stromal cells to induce secretion of paracrine factor(s) that in turn stimulate neighboring epithelial cells to express the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (HSD17B2). HSD17B2 is an extremely efficient enzyme and rapidly metabolizes the biologically potent estrogen E2 to weakly estrogenic estrone. In endometriotic tissue, progesterone is incapable of inducing epithelial HSD17B2 expression due to a defect in stromal cells. The inability of endometriotic stromal cells to produce progesterone-induced paracrine factors that stimulate HSD17B2 may be due to the very low levels of PR observed in vivo in endometriotic tissue. The end result is deficient metabolism of E2 in endometriosis giving rise to high local concentrations of this mitogen. The molecular details of this physiological paracrine interaction between the stroma and epithelium in normal endometrium and its lack thereof in endometriosis are discussed.
Subject(s)
17-Hydroxysteroid Dehydrogenases/deficiency , Endometriosis/enzymology , Progesterone/metabolism , Animals , Cell Differentiation/genetics , Endometriosis/metabolism , Female , Gene Expression Regulation , Humans , Mice , Paracrine Communication , Receptors, Progesterone/deficiency , Receptors, Progesterone/metabolism , Stromal Cells/metabolismABSTRACT
Loss of progesterone signaling in the endometrium may be a causal factor in the development of endometriosis, and progesterone resistance is commonly observed in women with this disease. In endometriotic stromal cells, the levels of progesterone receptor (PR), particularly the PR-B isoform, are significantly decreased, leading to a loss of paracrine signaling. PR deficiency likely underlies the development of progesterone resistance in women with endometriosis who no longer respond to progestin therapy. Here we review the complex epigenetic and transcriptional mechanisms leading to PR deficiency. The initial event may involve deficient methylation of the estrogen receptor (ER)beta promoter resulting in pathologic overexpression of ERbeta in endometriotic stromal cells. We speculate that alterations in the relative levels of ERbeta and ERalpha in endometrial tissue dictate E2-regulated PR expression, such that a decreased ERalpha-tauomicron-ERbeta ratio may result in suppression of PR. In this review, we propose a molecular model that may be responsible for changes in ERbeta and ERalpha leading to PR loss and progesterone resistance in endometriosis.
Subject(s)
Endometriosis/metabolism , Epigenesis, Genetic , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/genetics , Gene Expression Regulation, Neoplastic , Progesterone/metabolism , CpG Islands , DNA Methylation , Endometriosis/genetics , Endometrium/metabolism , Female , Humans , Receptors, Progesterone/genetics , Stromal Cells/metabolism , Stromal Cells/pathologyABSTRACT
Aromatase (CYP19A1) catalyzes the conversion of C(19) steroids to estrogens. Aromatase and its product estradiol (E(2)) are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The regulation of aromatase expression in the brain is not well understood. The aromatase (Cyp19a1) gene is selectively expressed in distinct neurons of the hypothalamus through a distal brain-specific promoter I.f located approximately 36 kb upstream of the coding region. Here, we investigated a short feedback effect of E(2) on aromatase mRNA expression and enzyme activity using estrogen receptor alpha (ESR1; also known as ER alpha)-positive or ESR1-negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. Estradiol regulated aromatase mRNA expression and enzyme activity in a time- and dose-dependent manner, whereas an E(2) antagonist reversed these effects. The nucleotide -200/-1 region of promoter I.f conferred E(2) responsiveness. Two activator protein 1 (AP-1) elements in this region were essential for induction of promoter activity by E(2). ESR1 and JUN (c-Jun) bound to these AP-1 motifs in intact cells and under cell-free conditions. The addition of an ESR1 mutant that interacts with JUN but not directly with DNA enhanced E(2)-dependent promoter I.f activity. Independently, we demonstrated an interaction between ESR1 and JUN in hypothalamic cells. Knockdown of ESR1 abolished E(2)-induced aromatase mRNA and enzyme activity. Taken together, E(2) regulates Cyp19a1 expression via promoter I.f by enhanced binding of an ESR1/JUN complex to distinct AP-1 motifs in hypothalamic cells. We speculate that this mechanism may, in part, regulate gonadotropin secretion and sexual activity.
Subject(s)
Aromatase/metabolism , Estrogen Receptor alpha/metabolism , Hypothalamus/metabolism , Neurons/metabolism , Promoter Regions, Genetic/physiology , Analysis of Variance , Animals , Aromatase/genetics , Blotting, Western , Cell Line , Cells, Cultured , Chromatin Immunoprecipitation , Dose-Response Relationship, Drug , Electrophoretic Mobility Shift Assay , Enzyme Assays , Estradiol/analogs & derivatives , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Estrogens/pharmacology , Fulvestrant , Hypothalamus/cytology , Hypothalamus/drug effects , Mice , Neurons/cytology , Neurons/drug effects , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , TransfectionABSTRACT
Endometriosis is a common and chronic disease characterized by persistent pelvic pain and infertility. Estradiol is essential for growth and inflammation in endometriotic tissue. The complete cascade of steroidogenic proteins/enzymes including aromatase is present in endometriosis leading to de novo estradiol synthesis. PGE(2) induces the expression of the genes that encode these enzymes. Upon PGE(2) treatment, coordinate recruitment of the nuclear receptor SF-1 to the promoters of these steroidogenic genes is the key event for estradiol synthesis. SF-1 is the key factor determining that an endometriotic cell will respond to PGE(2) by increased estradiol formation. The presence of SF-1 in endometriosis and its absence in endometrium is determined primarily by the methylation of its promoter. The key steroidogenic enzyme in endometriosis is aromatase encoded by a single gene because its inhibition blocks all estradiol biosynthesis. Aromatase inhibitors diminish endometriotic implants and associated pain refractory to existing treatments in affected women.
Subject(s)
Endometriosis/metabolism , Endometriosis/pathology , Steroidogenic Factor 1/metabolism , Dinoprostone/metabolism , Endometriosis/physiopathology , Endometrium/metabolism , Endometrium/pathology , Estradiol/biosynthesis , Female , Humans , Inflammation/metabolism , Transcription Factors/metabolismABSTRACT
CONTEXT: Estradiol and its nuclear receptors, estrogen receptor (ER) alpha and ERbeta, play critical roles in endometrium and endometriosis. Levels of ERbeta, due to pathological hypomethylation of its promoter, are significantly higher in endometriotic vs. endometrial tissue and stromal cells, whereas ERalpha levels are lower in endometriosis. Estradiol regulates ERalpha gene expression via its alternatively used promoters A, B, and C. OBJECTIVE: The aim of the study was to determine whether high levels of ERbeta in endometriotic stromal cells from ovarian endometriomas regulate ERalpha gene expression. RESULTS: ERbeta knockdown significantly increased ERalpha mRNA and protein levels in endometriotic stromal cells. Conversely, ERbeta overexpression in endometrial stromal cells decreased ERalpha mRNA and protein levels. ERbeta knockdown significantly decreased proliferation of endometriotic stromal cells. Chromatin immunoprecipitation assays demonstrated that estradiol enhanced ERbeta binding to nonclassical activator protein 1 and specificity protein 1 motifs in the ERalpha gene promoters A and C and a classic estrogen response element in promoter B in endometriotic stromal cells. CONCLUSIONS: High levels of ERbeta suppress ERalpha expression and response to estradiol in endometrial and endometriotic stromal cells via binding to classic and nonclassic DNA motifs in alternatively used ERalpha promoters. ERbeta also regulates cell cycle progression and might contribute to proliferation of endometriotic stromal cells. We speculate that a significantly increased ratio of ERbeta:ERalpha in endometriotic tissues may also suppress progesterone receptor expression and contribute to progesterone resistance. Thus, ERbeta may serve as a significant therapeutic target for endometriosis.
Subject(s)
Endometriosis/genetics , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/physiology , Ovarian Diseases/genetics , Stromal Cells/metabolism , Adult , Cell Cycle/drug effects , Cell Cycle/genetics , Cells, Cultured , Endometriosis/metabolism , Endometriosis/pathology , Estradiol/pharmacology , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/antagonists & inhibitors , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Female , Gene Expression Regulation/drug effects , Humans , Middle Aged , Ovarian Diseases/metabolism , Ovarian Diseases/pathology , Promoter Regions, Genetic , Protein Binding , RNA, Small Interfering/pharmacology , Stromal Cells/drug effects , Stromal Cells/pathology , TransfectionABSTRACT
CONTEXT: The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (HSD17B2) exerts a local antiestrogenic effect by metabolizing biologically active estradiol to inactive estrone in endometrial epithelial cells. Retinoic acid (RA) induces HSD17B2 expression, but the underlying mechanism is not known. OBJECTIVE: Our objective was to elucidate the molecular mechanisms responsible for HSD17B2 expression in human endometrial cells. METHOD: Human endometrial Ishikawa and RL95-2 cell lines were cultured in the presence or absence of RA to analyze endogenous HSD17B2 expression, transcription factor complex formation, and promoter activity. RESULTS: RA induced HSD17B2 mRNA levels in a dose- and time-dependent manner in endometrial cells. The RA antagonist ANG11273 abolished RA-induced HSD17B2 expression. Small interfering RNA ablation of RA receptor (RAR)alpha or retinoid X receptor (RXR)alpha completely blocked RA-induced HSD17B2 gene expression. Analysis of serial deletion and site-directed mutants of the HSD17B2 promoter fused to a reporter gene indicated that RA induction requires a cis-regulatory sequence that binds the specificity protein (SP) class of transcription factors. Chromatin-immunoprecipitation-PCR and gel-shift assays showed that RARalpha/RXRalpha and SP1/SP3 interact with this HSD17B2 promoter sequence. Small interfering RNA ablation of SP1 and SP3 expression markedly decreased HSD17B2 basal expression and blocked RA-induced expression. Finally, immunoprecipitationimmunoblotting demonstrated RA-induced interactions between RARalpha/RXRalpha and SP1/SP3 in intact endometrial cells. CONCLUSIONS: In endometrial epithelial cells, RA stimulates formation of a multimeric complex comprised of RARalpha/RXRalpha tethered to transcription factors SP1 and SP3 on the HSD17B2 promoter. Assembly of this transcriptional complex is necessary for RA induction of HSD17B2 expression and may be an important mechanism for local estradiol inactivation in the endometrium.
Subject(s)
17-Hydroxysteroid Dehydrogenases/genetics , Endometrium/enzymology , Estradiol/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Receptors, Retinoic Acid/physiology , Sp1 Transcription Factor/physiology , Sp3 Transcription Factor/physiology , Tretinoin/pharmacology , Cell Line, Tumor , Epithelial Cells/enzymology , Estradiol Dehydrogenases , Female , Humans , Mutagenesis, Site-Directed , Promoter Regions, Genetic , RNA, Small Interfering/pharmacologyABSTRACT
Local estrogen biosynthesis is a major factor in the pathogenesis of endometriosis. Aberrant expression of steroidogenic acute regulatory protein (StAR) and aromatase in endometriotic tissue leads to an up-regulation of estrogen production. The transcription factor steroidogenic factor-1 (SF-1) activates the promoters of both StAR and aromatase in endometriotic tissue. We investigated differences in SF-1 expression in endometriotic tissue and normally located endometrium to elucidate the mechanism underlying increased StAR and aromatase activities in endometriosis. Serial deletion and site-directed mutants of the SF-1 promoter showed that an E-box sequence was critical for its activity in endometriotic stromal cells. EMSAs showed that the upstream stimulatory factor (USF) 1 and 2 in nuclear extracts from endometrial and endometriotic stromal cells bound to the E-box. Chromatin-immunoprecipitation-PCR assay, however, demonstrated in intact cells that binding activity of USF2 to the SF-1 promoter was strikingly higher than that of USF1 in endometriotic stromal cells and that USF1 or USF2 binding activity was hardly detectable in endometrial stromal cells. Moreover, knockdown of USF2 but not USF1 resulted in robust and consistent down-regulation of SF-1 and its target genes StAR and aromatase in endometriotic stromal cells. USF2 but not USF1 mRNA and protein levels were significantly higher in endometriotic vs. endometrial stromal cells. In vivo, USF2 mRNA and immunoreactive USF2 levels in endometriotic tissues were strikingly higher than those in endometrium. Taken together, the elevated levels of USF2 in endometriosis account for, in part, the aberrant expression of SF-1 and its target gene StAR and aromatase.
Subject(s)
Endometriosis/genetics , Gene Expression , Steroidogenic Factor 1/genetics , Upstream Stimulatory Factors/physiology , Aromatase/genetics , Aromatase/metabolism , Cells, Cultured , Chromatin Immunoprecipitation , Endometriosis/metabolism , Endometriosis/pathology , Endometrium/metabolism , Endometrium/pathology , Female , Humans , Immunoblotting , Immunohistochemistry , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Steroidogenic Factor 1/metabolism , Stromal Cells/metabolism , Transcription, Genetic/genetics , Transfection , Upstream Stimulatory Factors/genetics , Upstream Stimulatory Factors/metabolismABSTRACT
CONTEXT: Control of aromatase expression in uterine leiomyoma has significant clinical implications because aromatase inhibitors reduce tumor growth and associated irregular uterine bleeding. The mechanisms that regulate aromatase expression in leiomyoma are unknown. OBJECTIVES: We previously demonstrated that the cAMP-responsive proximal promoters I.3 and II regulate aromatase expression in vivo in uterine leiomyoma tissue. Here, we investigated the cellular and molecular mechanisms responsible for promoter I.3/II usage. RESULTS: In smooth muscle cells isolated from leiomyoma (LSMCs), dibutyryl cAMP significantly induced aromatase mRNA and enzyme activity. Reporter constructs of promoter I.3/II deletion and site-directed mutants with selective disruption of cis-regulatory elements in the -517/-16 bp region revealed that five out of seven elements, including three CCAAT/enhancer binding protein (C/EBP) binding sites and two cAMP response elements, were essential for cAMP-induced promoter activity. EMSAs demonstrated that nuclear extracts from LSMCs contain complexes assembled on four of the five cis-elements, with C/EBP binding sites, including a novel -245/-231 bp sequence, clearly associating with C/EBPbeta. Chromatin immunoprecipitation assays revealed that C/EBPbeta binds specifically to the promoter I.3/II region in intact cells. Dibutyryl cAMP significantly induced nuclear C/EBPbeta protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBPbeta dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity. CONCLUSIONS: C/EBPbeta, which binds to multiple cis-regulatory elements in promoter I.3/II, is a key factor in the transcriptional complex controlling aromatase expression in uterine leiomyoma cells. Definition of this mechanism further may assist in designing inhibitors of aromatase specific for leiomyoma tissue.
Subject(s)
Aromatase/genetics , CCAAT-Enhancer-Binding Protein-beta/physiology , Gene Expression Regulation, Enzymologic , Leiomyoma/enzymology , Uterine Neoplasms/enzymology , CCAAT-Enhancer-Binding Protein-beta/analysis , Cell Line, Tumor , Cyclic AMP/physiology , Cyclic CMP/analogs & derivatives , Cyclic CMP/pharmacology , Female , Humans , Leiomyoma/pathology , Phosphorylation , Promoter Regions, Genetic , Uterine Neoplasms/pathologyABSTRACT
CONTEXT: Uterine leiomyomas are smooth muscle cell tumors that cause irregular uterine bleeding and pregnancy loss in many reproductive-age women. Progesterone stimulates their growth, whereas treatment with progesterone receptor (PR) antagonists or selective progesterone receptor modulators shrinks these tumors. Molecular mechanisms underlying these observations are unknown. OBJECTIVE: Bcl-2 is a key protein that inhibits apoptosis. It was proposed that growth enhancement of leiomyoma cells by progesterone was mediated via bcl-2 induction. Here we test the hypothesis that PR regulates the bcl-2 gene by directly binding to its promoter. RESULTS: The pure progesterone agonist R5020 increased the total number of viable primary human leiomyoma smooth muscle (LSM) cells in culture. Progesterone or R5020 (10(-6) m) significantly increased bcl-2 mRNA levels after 2 and 4 h by 9.2- and 3.4-fold, respectively, in LSM cells. Transient transfection with deletion mutants of bcl-2 promoter showed that the -1281/-258-bp region conferred responsiveness to progesterone induction in the presence of PR-A. We identified a palindromic progesterone response element (PRE) at -553/-539 bp. EMSA showed that PR in nuclear extracts from LSM cells bound specifically to this PRE. Chromatin immunoprecipitation-PCR confirmed in situ recruitment of PR to the -629/-388-bp region bearing the PRE. In vivo, bcl-2 mRNA levels correlated significantly with total PR mRNA levels in leiomyoma tissues. CONCLUSION: Taken together, progesterone via PR interacts with the bcl-2 promoter to induce its expression in leiomyoma tissue. This may explain, in part, the progesterone-dependent enhancement of growth in uterine leiomyoma.
Subject(s)
Gene Expression Regulation/physiology , Genes, bcl-2/genetics , Leiomyoma/genetics , Promoter Regions, Genetic/genetics , Receptors, Progesterone/physiology , Uterine Neoplasms/genetics , Blotting, Western , Cell Line, Tumor , Cell Survival/drug effects , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Female , Gene Expression Regulation/genetics , Humans , Leiomyoma/metabolism , Luciferases/genetics , Progesterone/pharmacology , Promegestone/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Progesterone/agonists , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Uterine Neoplasms/metabolismABSTRACT
Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (-197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2'-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (-197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.
Subject(s)
DNA Methylation , Endometriosis/genetics , Endometrium/metabolism , Estrogen Receptor beta/genetics , Gene Expression Regulation/genetics , Adult , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cells, Cultured , CpG Islands , Decitabine , Endometrium/drug effects , Estrogen Receptor alpha/genetics , Female , Humans , Promoter Regions, Genetic/drug effects , RNA, Messenger/analysis , RNA, Messenger/metabolism , Receptors, Progesterone/genetics , Stromal Cells/drug effects , Stromal Cells/metabolismABSTRACT
Pathogenesis and growth of three common women's cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215 bp from each other and are coordinately stimulated by PGE(2) via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE(2) secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE(2) may play important roles in inducing local production of estrogen that promotes tumor growth.
Subject(s)
Aromatase/metabolism , Breast Neoplasms/enzymology , Endometrial Neoplasms/enzymology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Ovarian Neoplasms/enzymology , Animals , Aromatase/genetics , Breast Neoplasms/genetics , Endometrial Neoplasms/genetics , Female , Humans , Ovarian Neoplasms/geneticsABSTRACT
17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone. HSD17B2 is highly expressed in human placental tissue where it is localized to placental endothelial cells lining the fetal compartment. The aim of this study was to investigate the effects of potential regulatory factors including progesterone, estradiol, and retinoic acid (RA) onHSD17B2 expression in primary human placental endothelial cells in culture.HSD17B2 mRNA expression was not regulated by progesterone, the progesterone agonist R5020, or estradiol treatment. RA significantly induced HSD17B2 mRNA levels and enzyme activity in a dose- and time-dependent manner. Maximal stimulation occurred at Hour 48 at an RA concentration of 10(-6) M. Both retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA) were readily detected by immunoblotting in isolated placental endothelial cells. RNA interference directed against RARA or RXRA led to reduced basal levels of HSD17B2 mRNA levels and significantly abolished RA-stimulated HSD17B2 expression. Together, these data indicate that regulation of HSD17B2 mRNA levels and enzymatic activity by RA in the placenta is mediated by RARA and RXRA.