Regulation of the thrombin/protease-activated receptor 1 axis by chemokine (CXC motif) receptor 4.

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.

Endometriosis expresses a molecular pattern consistent with decreased retinoid uptake, metabolism and action.

BACKGROUND: Retinoic acid (RA) regulates key biological processes, including differentiation, apoptosis and cell survival. RA mediates induction of 17 beta-hydroxysteroid dehydrogenase type 2 mRNA, catalyzing the conversion of estradiol to estrone, in endometrium but not endometriosis because of a defect in endometriotic stromal cells. This defect may involve both the uptake and metabolism of RA. In this study, we analyze the expression of genes involved in RA signaling in normal endometrium and endometriosis. METHODS: Tissue and stromal cells from ovarian endometriomas and eutopic endometrium from disease-free women were collected. Real-time reverse transcription-polymerase chain reaction was used to measure mRNA levels. Western blotting was used to evaluate protein expression. RESULTS: We found that endometriotic tissue and stromal cells demonstrated significantly decreased mRNA expression of the major genes involved in RA signaling, including STRA6, CRBP1, ALDH1A2, CRABP2 and FABP5. We found increased levels of CYP26B1, responsible for RA metabolism. Nuclear extracts showed that RARα, RXRα and PPARß/δ were underexpressed in both tissues and stromal cells from endometriotic tissue. Differences in protein levels were confirmed by western blotting. CONCLUSIONS: Endometriosis is characterized by a gene expression pattern suggesting a decrease in uptake and metabolism of RA. Because RA is integral in regulating key biological processes involved in cell survival, this alteration could partially explain the resistance to apoptosis found in endometriosis.

Modulation of Poly ADP Ribose Polymerase (PARP) Levels and Activity by Alcohol Binge-Like Drinking in Male Mice.

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.

Estrogen receptor (ER) beta regulates ERalpha expression in stromal cells derived from ovarian endometriosis.

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.

Aromatase promoter I.f is regulated by estrogen receptor alpha (ESR1) in mouse hypothalamic neuronal cell lines.

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.

Steroidogenic factor-1 and endometriosis.

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.

Upstream stimulatory factor-2 regulates steroidogenic factor-1 expression in endometriosis.

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.

Retinoic acid (RA) regulates 17beta-hydroxysteroid dehydrogenase type 2 expression in endometrium: interaction of RA receptors with specificity protein (SP) 1/SP3 for estradiol metabolism.

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.

CCAAT/enhancer binding protein beta regulates aromatase expression via multiple and novel cis-regulatory sequences in uterine leiomyoma.

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.

Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle.

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.

Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5' CpG island in endometriosis.

CONTEXT: Endometriosis is an estrogen-dependent disease. Steroidogenic factor-1 (SF-1), a transcriptional factor essential for activation of multiple steroidogenic genes for estrogen biosynthesis, is undetectable in normal endometrial stromal cells and aberrantly expressed in endometriotic stromal cells. OBJECTIVE: The objective of the study was to unravel the mechanism for differential SF-1 expression in endometrial and endometriotic stromal cells. DESIGN: We identified a CpG island flanking the SF-1 promoter and exon I region and determined its methylation patterns in endometrial and endometriotic cells. SETTING: The study was conducted at Northwestern University. PATIENTS OR OTHER PARTICIPANTS: Eutopic endometrium from disease-free subjects (n = 8) and the walls of cystic endometriosis lesions of the ovaries (n = 8) were investigated. INTERVENTION(S): Stromal cells were isolated from these two types of tissues. MAIN OUTCOME MEASURE(S): Measures are mentioned in Results. RESULTS: SF-1 mRNA and protein levels in endometriotic stromal cells were significantly higher than those in endometrial stromal cells (P < 0.001). Bisulfite sequencing showed strikingly increased methylation in endometrial cells, compared with endometriotic cells (P < 0.001). Demethylation by 5-aza-2'-deoxycytidine increased SF-1 mRNA levels by up to 55.48-fold in endometrial cell (P < 0.05). Luciferase assays showed that the -85/+239 region bearing the CpG island regulated its activity (P < 0.01). Natural or in vitro methylation of this region strikingly reduced SF-1 promoter activity in both cell types (P < 0.01). Chromatin immunoprecipitation assay showed that methyl-CpG-binding domain protein 2 binds to the SF-1 promoter in endometrial but not endometriotic cells. CONCLUSIONS: This is the first demonstration of methylation-dependent regulation of SF-1 in any mammalian tissue. These findings point to a new mechanism for targeting local estrogen biosynthesis in endometriosis.

Progesterone receptor regulates Bcl-2 gene expression through direct binding to its promoter region in uterine leiomyoma cells.

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.

Stromal cells of endometriosis fail to produce paracrine factors that induce epithelial 17beta-hydroxysteroid dehydrogenase type 2 gene and its transcriptional regulator Sp1: a mechanism for defective estradiol metabolism.

OBJECTIVE: In endometrium, stromal progesterone receptors mediate production of paracrine factors, which enhance binding of the transcription factor specific protein-1 to the promoter of the gene encoding the 17beta-hydroxysteroid dehydrogenase type 2 enzyme responsible for converting biologically active estradiol to estrone in epithelium. The objective of this study is to define the cellular defect responsible for the disruption of this stromal-epithelial interaction in endometriosis. STUDY DESIGN: We determined the effects of conditioned media generated from primary human eutopic endometrial stromal cells vs endometriotic stromal cells on Ishikawa malignant endometrial epithelial cells. RESULTS: Conditioned media from progestin-pretreated eutopic endometrial stromal cells but not endometriotic stromal cells significantly stimulated specific protein-1 protein levels, 17beta-hydroxysteroid dehydrogenase type 2 messenger RNA levels and promoter activity, and binding activity of specific protein-1 to the 17beta-hydroxysteroid dehydrogenase type 2 promoter region in Ishikawa cells. CONCLUSION: A stromal cell defect in endometriosis blocks formation of progesterone-dependent production of factors leading to 17beta-hydroxysteroid dehydrogenase type 2 deficiency and defective conversion of estradiol to estrone in epithelium.

Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function.

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.

Progesterone resistance in endometriosis: link to failure to metabolize estradiol.

Endometriosis is the most common cause of pelvic pain and affects an estimated 5 million women in the US. The biologically active estrogen estradiol (E2) is the best-defined mitogen for the growth and inflammation processes in the ectopic endometriotic tissue that commonly resides on the pelvic organs. Progesterone and progestins may relieve pain by limiting growth and inflammation in endometriosis but a portion of patients with endometriosis and pelvic pain do not respond to treatment with progestins. Moreover, progesterone-induced molecular changes in the eutopic (intrauterine) endometrial tissue of women with endometriosis are either blunted or undetectable. These in vivo 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 receptors (PRs) and the lack of the PR isoform named progesterone receptor B (PR-B). In normal endometrium, progesterone acts on stromal cells to induce secretion of paracrine factor(s). These unknown factor(s) act on neighboring epithelial cells to induce the expression of the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD-2), which metabolizes the biologically active estrogen E2 to estrone (E1). In endometriotic tissue, progesterone does not induce epithelial 17beta-HSD-2 expression due to a defect in stromal cells. The inability of endometriotic stromal cells to produce progesterone-induced paracrine factors that stimulate 17beta-HSD-2 may be due to the lack of PR-B and very low levels of progesterone receptor A (PR-A) observed in vivo in endometriotic tissue. The end result is deficient metabolism of E2 in endometriosis giving rise to high local concentrations of this local mitogen. The cellular and molecular mechanisms underlying progesterone resistance and failure to metabolize E2 in endometriosis are reviewed.

Mitogen-activated protein kinase activation induces corticotrophin-releasing hormone gene expression in human placenta.

Corticotropin-releasing hormone (CRH) gene expression in human placental cells is induced by activation of the cyclic AMP and protein kinase C signal transduction pathways, but the role of the mitogen-activated kinase (MAPK) pathway is unknown. In this study, we showed that the MAPK inhibitor, PD098059, causes a dose-dependent inhibition of placental CRH gene expression. In contrast, overexpression of RAF in human choriocarcinoma JEG cells stimulates CRH promoter activity by 15-fold, and the stimulation is inhibited by 65% by co-transfection of the cells with a plasmid expressing a RAF dominant/negative protein. The stimulation by RAF was completely abolished by mutation of the cyclic AMP response element (CRE) in the proximal region of the CRH promoter. Taken together, these results strongly suggest that the MAPK signal transduction pathway plays a pivotal role in the regulation of CRH gene expression in human placenta, and that the CRE binding site in the proximal CRH promoter acts as a point of convergence for different signal transduction pathways in the regulation of CRH gene expression in placenta cells.

Critical role for transcription factor AP-2alpha in human trophoblast differentiation.

To examine whether AP-2alpha is a critical component of the genetic program that directs human trophoblast differentiation, we used DNA microarray analyses to characterize the effects of a dominant-negative form of the AP-2 protein upon in vitro differentiating cytotrophoblast cells. Human cytotrophoblast cells (>95% pure) were cultured for 3 days in the presence of control medium or medium containing an adenovirus that expresses a dominant-negative mutant of AP-2 (Ad2.AP-2D/N) or an adenovirus lacking the AP-2 mutant gene (Ad.WT). DNA microarray analyses using Affymetrix human U95Av2 GeneChips were performed on RNA extracted from the three groups of cells immediately prior to and after 3 days of cell culture. Cells infected with Ad2.AP-2D/N or Ad2.WT underwent morphological differentiation similar to that of uninfected cells, with greater than 90% of the cells in each group fusing to form multinucleated syncytiotrophoblast cells. However, Ad2.AP-2D/N markedly inhibited the induction or repression of many genes that were regulated in the noninfected and Ad2.WT-infected cells during differentiation. Eighteen of the 25 most induced genes and 17 of the 20 most repressed genes during differentiation were AP-2 dependent, with the majority of these related to extracellular organization, cellular communication, and signal transduction. Taken together, these findings strongly suggest that AP-2 plays a critical role for both the induction and repression of genes that comprise postsyncytialization gene expression programs of trophoblast differentiation and maturation. AP-2, however, is not required for the fusion of cytotrophoblast cells to form a syncytium or the expression of syncytin.

Identification of an enhancer of the human activating protein-2alpha gene that contains a critical Ets1 binding site.

Deletion analysis studies of the 5'-flanking region of the activating protein-2alpha (AP-2alpha) gene indicates that the proximal 152 bp are essential for minimal promoter activity and that a 140-bp fragment from nucleotides -1279 to -1139 acts as an enhancer of basal transcriptional activity. Ligation of the 140-bp fragment to a minimal AP-2alpha promoter or a heterologous simian virus 40 promoter luciferase reporter plasmid conferred enhancer activity in trophoblast cells. In deoxyribonuclease I footprint studies, nuclear extracts from trophoblast cells protected two regions of the 140-bp fragment, E2 and E3. Site-directed mutagenesis of an Ets1-binding site in E2 significantly inhibited AP-2alpha enhancer activity, whereas mutagenesis of two putative polyomavirus enhancer activator-3-binding sites on E2 and an insulin upstream factor 1-binding site in E3 had no effect on enhancer activity. Gel shift and supershift assays indicated that Ets1 binds to the Ets site in E2, and overexpression of Ets1 in transfection studies induced AP-2alpha promoter activity. As the transcription factor Ets1 is abundant in trophoblast cells, these investigations strongly suggest that AP-2alpha gene expression in the placenta is enhanced by a cis-acting element at nucleotides -1279 to -1139 that contains a critical Ets1-binding site.

AP-2alpha modulates human corticotropin-releasing hormone gene expression in the placenta by direct protein-protein interaction.

Since AP-2alpha induces the expression of hPL, hCG and other syncytiotrophoblast-specific marker genes in cytotrophoblast cells during in vitro differentiation, we have examined whether AP-2alpha also induces hCRH gene expression during differentiation of cytotrophoblast cells. Infection of human cytotrophoblast cells in vitro with an adenovirus expressing AP-2alpha resulted in a twofold increase in hCRH mRNA levels, while infection with an adenovirus expressing a dominant/negative mutant of AP-2alpha inhibited basal hCRH mRNA levels by 40% and completely blocked the induction of hCRH mRNA by AP-2alpha. Transient transfection studies in AtT-20 and HepG2 cells indicated that the induction of hCRH mRNA levels was due, at least in part, to transcriptional activation of the hCRH gene. Gel mobility shift and immunoprecipitation assays strongly suggest that AP-2alpha induces hCRH gene expression by interacting with CREB and not by binding directly to the hCRH promoter.

Retinoic acid inhibits endometrial cancer cell growth via multiple genomic mechanisms.

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 6 h 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 12 768 genes out of 47 296 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.