Interaction of human trophoblast cells with gland-like endometrial spheroids: a model system for trophoblast invasion.

STUDY QUESTION: Do maternal endometrial epithelial cell (EEC) differentiation and polarity impact the invasive capacity of extravillous trophoblast (EVT) cells during early human implantation? SUMMARY ANSWER: In a three dimensional (3D) confrontation co-culture the invasiveness of the human trophoblast cell line AC-1M88 was inversely correlated with the degree of differentiation and polarization of human endometrial adenocarcinoma cell spheroids. WHAT IS KNOWN ALREADY: In a previous study desmosomal and adherens junction proteins were shown to spread from a subapically restricted lateral position to the entire lateral membrane in human glandular EECs during the implantation window of the menstrual cycle. Whether this change in EEC junction localization has an impact on the interaction of EVT cells with glandular EECs during early human implantation is not known. STUDY DESIGN, SIZE, DURATION: A new 3D cell culture system was developed in order to mimic early implantation events in humans. As a model for the invasion of endometrial glands by EVT cells, spheroids of three differently differentiated and polarized endometrial adenocarcinoma cell lines were confronted with an EVT cell line in co-culture experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three human adenocarcinoma EEC lines were chosen for this study because of their differences in differentiation and polarization: HEC-1-A, which is well differentiated and highly polarized, Ishikawa, which is well differentiated and moderately polarized, and RL95-2, which is moderately differentiated and poorly polarized. When the cell lines were grown in reconstituted basement membrane, they formed gland-like, multicellular spheroids. The degree of polarization within the different EEC spheroids was assessed by 3D confocal immunofluorescence microscopy detecting the basal membrane protein integrin α6, the apical tight junction-associated protein ZO-1 and the desmosomal plaque protein desmoplakin 1/2 (Dsp). Cells of the human EVT cell line AC-1M88, which is a fusion cell line of primary EVT cells and choriocarcinoma-derived JEG-3 cells, were added to the different EEC spheroids to examine their interaction. For the analyses of trophoblast-endometrial confrontation sites, HLA-G was used as a specific EVT cell marker. MAIN RESULTS AND THE ROLE OF CHANCE: The endometrial HEC-1-A and Ishikawa cells formed gland-like structures in reconstituted basement membrane with apicobasal polarization towards their well-developed internal lumina, while most of the RL95-2 spheroids showed no lumen formation at all. The three EEC lines strongly differed in their apicobasal distribution pattern of Dsp. Ishikawa and HEC-1-A spheroids showed a subapical concentration of Dsp. In contrast, an equal distribution of Dsp was discerned along the entire lateral membranes in RL95-2 spheroids. In 3D confrontation co-cultures the highest invasiveness of AC-1M88 was observed in the poorly polarized RL95-2 spheroids. LIMITATIONS, REASONS FOR CAUTION: Human endometrial and trophoblast cell lines were used for this study because of ethical and legal restrictions for implantation studies with human blastocysts and because of limited access to primary human endometrial cells. WIDER IMPLICATIONS OF THE FINDINGS: The presented 3D cell culture system can be used to investigate the contribution of epithelial junctions to trophoblast-endometrial interactions. The identified impact of endometrial differentiation and polarity on the invasiveness of EVT cells improves our understanding of the relevance of endometrial receptivity for early implantation and may contribute to higher success rates in assisted reproductive technology. STUDY FUNDING/COMPETING INTERESTS: This work was supported by Grant 146/14, 'START-Program', Medical Faculty, RWTH Aachen University, to V.U.B., by Grant Lec_16_12, 'RWTH Lecturer Award', RWTH Aachen University to I.C.-L. and by the German Research Council (Grant LE 566-20-1). The authors declare no conflict of interest.

Invasiveness of human endometrial stromal cells is promoted by decidualization and by trophoblast-derived signals.

BACKGROUND: Extensive invasion of the maternal decidua by extravillous trophoblast is considered of critical importance for implantation and placentation in humans, the decidua being viewed as a passively invaded tissue. In this study, we examined whether decidual cells might contribute to the highly dynamic processes at the fetal-maternal interface by active movement. METHODS: Primary endometrial stromal cells (ESCs) or the telomerase-immortalized ESC line, St-T1b, was induced to decidualize or was left undifferentiated. The AC-1M88 cell line served as a model for extravillous trophoblast cells. Motility of ESCs and trophoblast cells was monitored in transwell invasion and migration assays under co-culture conditions. Secretion of matrix metalloproteinases (MMPs) was assessed by gelatin zymography. RESULTS: AC-1M88 cell invasiveness was unaffected by the presence of ESCs, irrespective of their decidualization status. Surprisingly, decidualized ESCs were significantly more invasive than undifferentiated cells, and this invasive activity was strongly enhanced when cells were cultured in direct contact with AC-1M88 cells. Conditioned medium from AC-1M88 cells also stimulated migration and invasion of ESCs. Secretion of MMP-2 and -9 by ESCs was increased upon decidualization. CONCLUSIONS: Enhanced motility and invasive capacity of decidualized ESCs in the presence of trophoblastic cells lead us to hypothesize a major contribution of the decidua in encapsulating the early conceptus and supporting subsequent trophoblast invasion. Our findings thus suggest a far more active role of the decidua in the implantation process than hitherto recognized.

Non-genomic progesterone actions in female reproduction.

BACKGROUND: The steroid hormone progesterone is indispensable for mammalian procreation by controlling key female reproductive events that range from ovulation to implantation, maintenance of pregnancy and breast development. In addition to activating the progesterone receptors (PRs)-B and -A, members of the superfamily of ligand-dependent transcription factors, progesterone also elicits a variety of rapid signalling events independently of transcriptional or genomic regulation. This review covers our current knowledge on the mechanisms and relevance of non-genomic progesterone signalling in female reproduction. METHODS: PubMed was searched up to August 2008 for papers on progesterone actions in ovary/breast/endometrium/myometrium/brain, focusing primarily on non-genomic signalling mechanisms. RESULTS: Convergence and intertwining of rapid non-genomic events and the slower transcriptional actions critically determine the functional response to progesterone in the female reproductive system in a cell-type- and environment-specific manner. Several putative progesterone-binding moieties have been implicated in rapid signalling events, including the 'classical' PR and its variants, progesterone receptor membrane component 1, and the novel family of membrane progestin receptors. Progesterone and its metabolites have also been implicated in the allosteric regulation of several unrelated receptors, such as gamma-aminobutyric acid type A, oxytocin and sigma(1) receptors. CONCLUSIONS: Identification of the mechanisms and receptors that relay rapid progesterone signalling is an area of research fraught with difficulties and controversy. More in-depth characterization of the putative receptors is required before the non-genomic progesterone pathway in normal and pathological reproductive function can be targeted for pharmacological intervention.

Death or survival--progesterone-dependent cell fate decisions in the human endometrial stroma.

The human endometrium undergoes cyclical waves of proliferation, differentiation and apoptosis in response to the rise and fall in ovarian oestradiol and progesterone levels. These hormonal responses in endometrial cells must be tightly kept in check to safeguard tissue homeostasis throughout reproductive life. The discovery that differentiating endometrium highly expresses the tumour suppressor p53, the forkhead transcription factor FOXO1, and promyelocytic leukaemia zinc finger protein (PLZF) has provided new insights into the molecular basis of life and death decisions in response to sex steroid hormones.

Regulated expression of putative membrane progestin receptor homologues in human endometrium and gestational tissues.

Rapid non-genomic actions of progesterone are implicated in many aspects of female reproduction. Recently, three human homologues of the fish membrane progestin receptor (mPR) have been identified. We combined bioinformatic analysis with expression profiling to define further the role of these mPRs in human reproductive tissues. Sequence analysis confirmed that the mPRs belong to a larger, highly conserved family of proteins, termed 'progestin and adiponectin receptors' (PAQRs). A comparison of the expression of mPR transcripts with that of two related PAQR family members, PAQRIII and PAQRIX, in cycling endometrium and pregnancy tissues revealed markedly divergent expression levels and profiles. For instance, endometrial expression of mPRalpha and gamma and PAQRIX was cycle-dependent whereas the onset of parturition was associated with a marked reduction in myometrial mPRalpha and beta transcripts. Interestingly, mPRalpha and PAQRIX were most highly expressed in the placenta, and the tissue expression levels of both genes correlated inversely with that of the nuclear PR. Phylogenetic analysis demonstrated that PAQRIX belongs to the mPR subgroup of proteins. We also validated a polyclonal antibody raised against the carboxy-terminus of human mPRalpha. Immunohistochemical analysis demonstrated more intense immunoreactivity in placental syncytiotrophoblasts than in endometrial glands or stroma. The data suggest important functional roles for mPRalpha, and possibly PAQRIX, in specific reproductive tissues, particularly those that express low levels of nuclear PR.

Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair.

During the menstrual cycle, the ovarian hormones oestradiol and progesterone control the ordered growth and differentiation of uterine cells. This remodelling process is critical for implantation of the developing embryo, the formation of the placenta, and maintenance of pregnancy. Failure of uterine tIssues to respond appropriately to ovarian hormone signalling results in defective placentation, associated with a spectrum of pregnancy disorders such as recurrent miscarriages and preeclampsia. These obstetrical disorders are a major cause of maternal and perinatal morbidity and mortality. Progesterone exerts its action on target cells, at least in part, through binding to the progesterone receptor (PR), a member of the steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors. The mechanism by which progesterone controls the differentiation of human endometrial stromal cells, a process termed decidualization, in the secretory phase of the menstrual cycle is not well understood. Emerging evidence indicates that locally expressed factors and activation of the cAMP second messenger pathway integrate hormonal inputs and confer cellular specificity to progesterone action through the induction of diverse transcription factors capable of modulating PR function.

Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene.

The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.

The human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene promoter is controlled by Ets and activating protein-1 transcription factors and progesterone.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a key catabolic enzyme in the inactivation of PGF2alpha and PGE2 and therefore serves as an important determinant in regulating their local concentrations. To gain insights into the transcriptional regulation of this enzyme, we have isolated 3.5 kb of the 5'-flanking sequence of the human PGDH promoter and characterized its control in hemopoietic cells and cells of myometrial and placental origin. Several potential binding sites for cAMP-responsive element-binding protein (CREB), Ets, and activating protein-1 (AP-1) transcription factors are present within 2368 bp of the 5'-flanking region. This region and deletions thereof were fused to the luciferase reporter gene and used for transient transfection experiments. In Jurkat leukemic T cells, which express PGDH endogenously, the transfected PGDH promoter was strongly induced by phorbol ester. Induction was reversed by coexpression of A-Fos, a dominant negative to AP-1. In primary cultures of myometrial smooth muscle cells (SMC), the Ets family members Ets-1, Ets-2, and PEA3 potently stimulated transcriptional activity of the PGDH promoter. PEA3-mediated activation was partially repressed by A-Fos, suggesting an involvement of AP-1 proteins, which might be conferred by a distal and a proximal Ets/ AP-1 composite element. The distal Ets/AP-1 element is flanked by two CRE-like sequences. Cotransfection of A-CREB, a dominant negative to CREB, inhibited stimulation of PGDH-2368/luc3 by PEA3 in myometrial SMC, whereas treatment with 8-bromo-cAMP moderately enhanced promoter activity. Progesterone is believed to be an important stimulus for PGDH expression in the utero-placental unit, thus contributing to the maintenance of a quiescent uterus during pregnancy. In myometrial SMC, both isoforms of the progesterone receptor, PR-B and PR-A, caused a ligand-dependent activation of PGDH-2368/luc3. Transcriptional activity of PR-B, but not PR-A, was further enhanced by the addition of 8-bromo-cAMP. We could not confirm a recently proposed transcriptional control of PGDH by mineralocorticoid receptor. No effect of mineralocorticoid receptor, in the absence or presence of aldosterone, with or without 8-bromo-cAMP, was observed on PGDH-2368/luc3. Taken together, these findings demonstrate control of the PGDH promoter by multiple pathways and provide evidence for cross-talk among Ets, AP-1, cAMP, and PR-mediated signaling, suggesting complex regulatory mechanisms for the expression of PGDH.

CCAAT/enhancer-binding proteins are mediators in the protein kinase A-dependent activation of the decidual prolactin promoter.

In the course of decidualization, human endometrial stromal cells (ESC) activate the alternative upstream promoter of the decidual prolactin (dPRL) gene. The dPRL promoter is induced by the protein kinase A pathway in a delayed fashion via the region -332/-270 which contains two overlapping consensus binding sequences, B and D, for CCAAT/enhancer-binding proteins (C/EBP). Here we show that sites B and D both bind C/EBPbeta and -delta from ESC nuclear extracts. When decidualization of cultured ESC was induced by treatment with 8-Br-cAMP, complex formation on sites B and D was enhanced. Western blot analysis revealed an elevation of both C/EBPbeta isoforms, liver-enriched activator protein and liver-enriched inhibitory protein, with a delayed onset between 8 and 24 h of cAMP treatment, while C/EBPdelta expression remained unaffected. Cyclic AMP-mediated activation of dPRL promoter construct dPRL-332/luc3 was abrogated by mutation of sites B and D at -310/-285. An expression vector for liver-enriched activator protein potently induced transcription of dPRL-332/luc3 and further enhanced cAMP-mediated induction, while liver-enriched inhibitory protein expression vector abolished the cAMP response, implying that C/EBPs serve as mediators in the delayed cAMP signal transduction to the dPRL promoter. The ratio between activating and repressing isoforms is likely to dictate the transcriptional output.

DNase I hypersensitivity analysis of non-pituitary human prolactin gene expression.

The expression of non-pituitary human PRL is initiated at a unique 5' untranslated exon located approximately 5.7 kb upstream of the pituitary-specific transcriptional start site. Unlike pituitary PRL expression, transcriptional regulation from the upstream promoter does not rely on the POU-homeodomain protein Pit-1. We have used DNase I mapping of chromatin from PRL-producing and non-producing human lymphoblastoid cell lines to identify hypersensitive sites unique to the PRL expressing phenotype. Analysis of 22 kb of 5' flanking DNA revealed DNase I hypersensitive sites in intron A-1 separating the pituitary from non-pituitary specific transcription start site which were only detected in the PRL-producing cell line. Transient transfection showed strong transcriptional activity directed by this region only in the antisense orientation and in a non cell-type specific manner. Transfection experiments with deletion mutants of 5259 bp of the non-pituitary PRL promoter region also revealed promoter activity not restricted to the PRL expressing phenotype. These data suggest that non-pituitary PRL gene expression may be regulated by elements located in intron A-1 and that recapitulation of cell-specific expression requires a unique cellular context and chromatin assembly.

Biglycan gene expression in the human leiomyosarcoma cell line SK-UT-1. Basal and protein kinase A-induced transcription involves binding of Sp1-like/Sp3 proteins in the proximal promoter region.

In this study we demonstrate that the gene encoding the small leucine-rich proteoglycan biglycan is expressed in human myometrial tissue and in the human leiomyosarcoma cell line SK-UT-1. Treatment of SK-UT-1 cells with forskolin or 8-bromo-cAMP strongly increased biglycan mRNA and this effect was transcriptional as shown by transient transfection experiments with biglycan promoter-luciferase reporter fusion genes. The cAMP-mediated induction of the transfected biglycan promoter in SK-UT-1 cells was abolished by coexpression of a specific protein kinase A inhibitor, and was mimicked by overexpression of the catalytic subunit (Cbeta) of protein kinase A. By 5' deletion analysis, part of the cAMP response was localized to the segment from residues -78 to -46 of the biglycan promoter. This region conferred strong cAMP responsiveness to a heterologous promoter. Electrophoretic mobility shift and antibody supershift assays identified two specific complexes that contained nuclear proteins antigenically related to the ubiquitous transcription factors Sp1 and Sp3, respectively. The binding site of these proteins was mapped to a CT-rich sequence extending from -59 to -49 in the biglycan promoter. Mutating this sequence eliminated complex formation and markedly reduced basal and cAMP-dependent promoter activity of transfected reporter genes. In vitro binding studies using recombinant Sp1 revealed that the nuclear factor binding to the CT element was not Sp1 but a Sp1-like protein(s). Western blot analysis of SK-UT-1 nuclear proteins confirmed expression of Sp3, Sp1 and nuclear proteins that crossreacted with Sp1 antibody but according to their molecular weight were not Sp1. These results indicate that all cAMP-dependent as well as some basal biglycan transcription in SK-UT-1 cells is mediated through activated protein kinase A and that both functions are conferred at the promoter level through the interaction of Sp1-like/Sp3 factors with the CT element at -59 in the biglycan promoter.

Marker genes of decidualization: activation of the decidual prolactin gene.

Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogues and ligands that elevate cellular cAMP levels in a manner resembling the gonadotrophins, prostaglandin E2 and relaxin (RLX). This differentiation process is marked by the onset of decidual prolactin (PRL) production in the late luteal phase of the cycle. Using transfection assays and a primary ES cell culture system, we have demonstrated that decidual PRL gene transcription is driven by an alternative upstream promoter (dPRL), approximately 6 kb upstream of the pituitary transcription start site. In primary cell cultures, RLX not only acutely but also permanently elevated cellular cAMP levels and induced PRL secretion after 6 days. Northern and Western blot analyses revealed all regulatory subunit isoforms (RIalpha, RIbeta, RIIalpha, RIIbeta) and catalytic subunits Calpha and Cbeta of protein kinase A (PKA) in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization, but in decidualized ES cells exposed to elevated cellular cAMP levels by stimulation with RLX for >6 days, RIalpha protein levels were significantly reduced, whereas levels of all other forms remained unchanged. Reducing the availability of R subunits changed the R:C subunit ratio in favour of C and increased kinase activity. In transient transfections of undifferentiated ES cells, the dPRL promoter was activated by 8-Br-cAMP and by C subunit (Cbeta) of PKA. This induction, and the differentiation-dependent activity of the dPRL promoter in transfected decidualized cells, was effectively abolished by the co-expression of protein kinase inhibitor (PKI). A fragment of 332 bp of 5'-flanking region of the dPRL transcription start site was sufficient to mediate full inducibility by cAMP. cAMP activation of the dPRL promoter in ES cells was biphasic as an initial weak induction within 12 hours was followed by a subsequent, much more intense induction after 12 hours. The secondary induction was not seen with a control construct driven by a consensus cAMP response element (CRE) linked to a minimal promoter. The early response of the dPRL promoter depended upon a non-palindromic CRE at position -12 and mutation of this sequence led to omission of the early, but not of the delayed, induction. The major activation of the dPRL promoter depended upon a different region between position -332 and -270 since its deletion significantly reduced inducibility by cAMP. Its action was probably indirect as its kinetics differed from classic CRE-mediated responses, and it was specific to ES cells.

Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial stromal cells.

Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogs and ligands that elevate cellular cAMP levels. A marker of this differentiation process is the activation of the decidual PRL (dPRL) promoter. In a primary ES cell culture system we show that relaxin not only acutely but permanently elevates cellular cAMP levels and leads to induction of PRL secretion after 6 days Northern and Western blot analyses revealed that all regulatory subunit isoforms (RI alpha, RI beta, RII alpha, and RII beta) and catalytic subunits C alpha and C beta of protein kinase A (PKA) are expressed in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization but in decidualized ES cells, exposed to relaxin for more than 6 days a significant reduction of RI alpha protein level occurs, whereas levels of all other forms remain unchanged. Reduction of R subunits might result in a net increase in free C subunit activity. This alteration is not due to a change in the mitotic state of the cells, as proliferating cell nuclear antigen is evenly expressed in undifferentiated and differentiated ES cell cultures. In transient transfections of undifferentiated ES cells, the dPRL promoter is activated by 8-bromo-cAMP and the C subunit (C beta) of PKA. This induction as well as the differentiation-dependent activity of the dPRL promoter in transfected decidualized cells are effectively abolished by the coexpression of protein kinase inhibitor. We demonstrate that 332 bp of the dPRL promoter are sufficient to mediate full inducibility by cAMP. Activation of the dPRL promoter by cAMP in ES cells occurs in two steps: an initial weak induction within 12 h and a subsequent, much more pronounced induction after 12 h. The secondary induction is not seen with a control construct driven by a consensus cAMP response element (CRE) linked to a minimal promoter and is absent from a uterine cell line that does not express the endogenous dPRL gene. The early response of the dPRL promoter depends upon a noncanonical CRE at position -12, as mutation of this sequence leads to abolition of the early, but not the delayed, induction. The major activation depends upon a different region within 332 bp of the dPRL promoter; is probably indirect, as it follows different kinetics compared to a classical CRE-mediated response; and is specific to ES cells.

Cloning of guinea pig cyclooxygenase-2 and 15-hydroxyprostaglandin dehydrogenase complementary deoxyribonucleic acids: steroid-modulated gene expression correlates to prostaglandin F2 alpha secretion in cultured endometrial cells.

Prostaglandin F2 alpha (PGF2 alpha) secretion is lowest at midcycle and highest on day 15 at luteolysis in the cycling guinea pig uterus and is inversely related to serum progesterone levels. An increase in 17-beta estradiol (E2) occurs only towards the end of the cycle. To investigate the effect of steroids on the control of uterine PGF2 alpha metabolism at the level of gene expression we established a primary cell culture model of day 15 cycling guinea pig endometrial cells. We cloned guinea pig cDNAs for cyclooxygenase 2 (COX-2), 15-hydroxyprostaglandin dehydrogenase (PGDH) that converts PGF2 alpha to biologically inactive 13,14-dihydro-15-keto PGF2 alpha (PGFM) and a fragment of cyclooxygenase-1 (COX-1). They were found to bear 87% and 90% homology at the amino acid level to their human counterparts for COX-2 and PGDH, respectively, retaining all functional sites. Purified epithelial and stromal cell subcultures were primed with medium containing either E2 or medroxyprogesterone acetate (MPA) for 24 h. They were then treated for a further 4 or 24 h either withdrawing the steroid, maintaining the priming steroid, or supplementing with both steroids, before harvesting conditioned media and RNA. Epithelial cells secreted 30-fold more PGF2 alpha compared with stromal cells (e.g. 7.8 +/- 0.7 vs. 0.26 +/- 0.09 pg/ng DNA.24 h), and PGF2 alpha secretion levels were approximately 15-fold higher than those of PGFM (e.g. 7.8 +/- 0.7 vs. 0.45 +/- 0.16 pg/ng DNA.24 h, for epithelial cells). COX-1 transcripts were low and unaffected by treatment in both cell types. COX-2 transcripts were more abundant in epithelial than stromal cells. Steroid-modulated, COX-2 dependent changes in PGF2 alpha secretion were observed. The addition of MPA to E2 primed cells caused a decrease in PGF2 alpha secretion and COX-2 messenger RNA levels after 4 h. Conversely, the addition of E2 to MPA primed epithelial cells led to an increase in PGF2 alpha secretion and COX-2 messenger RNA levels after 4 and 24 h. The withdrawal of E2 caused a fall in PGF2 alpha secretion and COX-2 transcripts after 24 h. In contrast, PGDH transcripts were more abundant in stromal than epithelial cells and were up-regulated by the addition of MPA to E2 primed cells. These in vitro observations are in keeping with the secretory profile seen in vivo in the cycling guinea pig uterus suggesting that 1) the fall of E2 and the coinciding rise in progesterone seen in the early cycle lead to a reduction in PGF2 alpha levels; and 2) the rise of E2 in the late cycle on a progesterone primed uterus is the stimulus for an increase in uterine PGF2 alpha production. Our findings suggest a differential role for uterine stroma and epithelium in vivo whereby the former acts to remove (via PGDH), and the latter to produce (via COX-2) biologically active prostaglandin.

Human endometrial stromal cells express novel isoforms of the transcriptional modulator CREM and up-regulate ICER in the course of decidualization.

Decidualization of human endometrial stromal (ES) cells in culture can be triggered by a sustained elevation of intracellular cAMP for several days and is characterized by activation of the cAMP-responsive decidual PRL (dPRL) gene promoter. We investigated the expression of the cAMP response element (CRE) binding protein CREB, and the modulators CREM (cAMP response element modulator) and ICER (inducible cAMP early repressor), in relation to decidualization of ES cells. We isolated all four known ICER isoforms from ES cells, which differ by the presence or absence of the small exon gamma and the presence of either DNA-binding domain (DBD) I or II. Of the various CREM isoforms, we cloned six transcript species, all containing DBD I. These were the known repressor CREM-alpha, the potential activator CREM-tau 2 alpha, and four novel forms whose reading frames were blocked upstream of the DBD. Two of these forms contained a novel exon psi, which is 100 bp in length, resides downstream of the first protein-coding exon of the CREM gene, and introduces an early in-frame stop codon. Surprisingly, in cotransfection assays, all four novel CREM isoforms were potent inhibitors of protein kinase A-stimulated transcription of a reporter gene construct driven by a CRE. By in vitro transcription/translation of all six CREM cDNAs, we demonstrated internal translation initiation at three different methionine residues, giving rise to novel short and very short C-terminal proteins comprising DBD I. These proteins bound to a cAMP response element as homodimers or as heterodimers with each other or with CREB. Immunofluorescence showed nuclear localization of C-terminal CREM proteins expressed from all six CREM cDNAs. Comparison of undifferentiated and decidualized ES cells showed no difference in the level of expression of any of the CREM transcript species. Likewise, CREB was evenly expressed between the two populations. In contrast, ICER transcripts were strongly up-regulated in decidualized ES cells in parallel with the induction of dPRL expression. It appears paradoxical that in vivo, in response to a permanent cAMP stimulus, ICER is up-regulated without displaying negative autoregulation of its own gene or suppression of the dPRL promoter. Elevated ICER levels in decidualized ES cells may be indicative of the presence of overriding amounts of transcriptional activators such as full length CREM-tau or CREB which, in turn, upon cAMP-induced phosphorylation, contribute to the induction of the dPRL gene.

Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells.

The composite transcription factor activating protein 1 (AP-1) integrates various mitogenic signals in a large number of cell types, and is therefore a major regulator of cell proliferation. In the normal human endometrium, proliferation and differentiation alternate in a cyclic fashion, with progesterone being largely implicated in the latter process. However, the effects of progesterone and the progesterone receptor (hPR) on AP-1 activity in the human endometrium are not known. To address this issue, HEC-1-B endometrial adenocarcinoma cells, which are devoid of hPR, were transfected with luciferase reporter constructs driven by two different AP-1-dependent promoters. Unexpectedly, cotransfection of hPR caused a marked induction of luciferase activity in the absence of ligand on both promoters. The magnitude of this induction was similar to that observed in response to the phorbol ester TPA. Addition of ligand reversed the stimulating effect of the unliganded hPR on AM activity in these cells. These effects were specific for hPR, and were not observed with either human estrogen receptor or human glucocorticoid receptor. Furthermore, they strictly depended on the presence of AP-1-responsive sequences within target promoters. Finally, the described effects of hPR on AP-1 activity were shown to be cell-type specific, because they could not be demonstrated in SKUT-1-B, JEG-3, and COS-7 cells. To our knowledge this is the first report of an unliganded steroid receptor stimulating AP-1 activity. This effect and its reversal in the presence of ligand suggest a novel mechanism, through which hPR can act as a key regulator of both proliferation and differentiation in the human endometrium.

Human endometrial fibroblasts immortalized by simian virus 40 large T antigen differentiate in response to a decidualization stimulus.

Human endometrial fibroblasts have been immortalized by infection with simian virus 40 large T antigen and established as a permanent cell line, St-2. Biochemical differentiation of this cell line has been demonstrated by the ability of a decidualizing stimulus, 8-bromo-cAMP plus medroxyprogesterone acetate (MPA), to induce PRL secretion and increase the enzymatic activity of estrone sulfatase. MPA, alone or in combination with estradiol, was unable to elicit this response, but potentiated the effect of 8-bromo-cAMP on PRL production and estrone sulfatase activity. The increase in PRL protein was accompanied by an increase in PRL messenger RNA and increased expression of the insulin-like growth factor-binding protein-1 messenger RNA. The St-2 cell PRL transcript was larger than the pituitary PRL transcript, suggesting its initiation from the distal, nonpituitary, PRL promoter. This was confirmed by reverse transcription-PCR analysis of PRL transcripts using primers specific for the additional sequences present only in the 5'-untranslated region of RNA initiated from the distal promoter. Transient transfection of a reporter construct containing 3000 bp of DNA 5' to the decidual-specific promoter of the human PRL gene demonstrated that cAMP was capable of activating this distal promoter in St-2 cells. In conclusion, this novel cell line provides an interesting new model in which to pursue aspects of biochemical differentiation of human endometrium in vitro.

Prolactin gene expression in human myometrial smooth muscle cells is induced by cyclic adenosine 3',5'-monophosphate.

We have previously characterized PRL production in human myometrial tissue maintained in explant culture. Here we describe PRL gene expression and its regulation in smooth muscle cells isolated from normal human myometrium. Onset of PRL secretion occurred spontaneously after several days in culture and increased over time without exogenous stimulation. PRL secretion could be further simulated by the addition of PGE(2) or relaxin, both of which were also shown to increase cAMP formation in smooth muscle cells. Likewise, treatment with 8-Br-cAMP led to an elevation of PRL secretion. By reverse transcription/polymerase chain reaction, we demonstrate that smooth muscle cells transcribe the PRL gene from the alternative decidual-type dPRL promoter, located upstream of the pituitary promoter. Treatment with PGE(2), relaxin, and 8-Br-cAMP resulted in an increase in dPRL transcript abundancy. The effect of cAMP was transcriptional as shown by the induction of transfected dPRL promoter/reporter gene fusion constructs. A fragment of 332 bp flanking the dPRL transcription start site was sufficient to mediate cAMP inducibility. In parallel with the increase in PRL secretion, we detected an increase in cAMP formation and PGE(2) secretion in cultured smooth muscle cells. We propose the presence of a paracrine positive feedback mechanism that may reflect the physiological situation in vivo where an increase in myometrial adenylate cyclase activity throughout pregnancy has been reported.

Pituitary-type transcription of the human prolactin gene in the absence of Pit-1.

We describe a human (h) PRL-producing cell line, SKUT-1B-20, which we isolated as a subclone of a uterine sarcoma cell line. Although this cell line is of uterine origin, it does not use the decidual-specific upstream promoter of the hPRL gene, but transcribes the hPRL gene from the downstream pituitary-type transcription start site, as determined by Northern blot, reverse transcriptase-polymerase chain reaction and primer extension analyses. This is particularly intriguing because SKUT-1B-20 cells lack the transcription factor Pit-1. No Pit-1 messenger RNA was detectable by reverse transcriptase-polymerase chain reaction, and endogenous Pit-1 target genes (GH, PRL, and Pit-1) were refractory to transfected Pit-1 expression vector, whereas in cotransfection experiments, Pit-1 efficiently activated reporter gene fusion constructs carrying 5'-flanking sequences of the human and rat PRL or the mouse Pit-1 genes. By transfecting reporter genes containing 8.7 kilobases of DNA flanking the hPRL pituitary-specific start site (hPRL-8700/Luc) and deletions thereof, we located a Pit-1-independent cis-active region more than 7 kilobases upstream of the start site. The most distal 1650 or 880 base pairs of the hPRL genomic fragment (which extends to -8784 base pairs), when placed directly upstream of the homologous hPRL or the heterologous thymidine kinase promoters, conferred transcriptional activation to those promoters. SKUT-1B-20 cell-specific activation of hPRL-8700/Luc could not be suppressed by the introduction of an inhibitor of protein kinase A (PKA), PKI. This is the first demonstration of pituitary-type PRL gene transcription independent of Pit-1 and activation of the PKA pathway. The SKUT-1B-20 cell line was then used in reconstitution experiments to delineate the role of Pit-1 in modulating the transcriptional effects of phorbol ester, PKA, and estrogen receptor (ER) on the hPRL gene. The low response of hPRL/luciferase fusion genes to phorbol ester was greatly enhanced by cotransfected Pit-1 and was mediated by the proximal region between -250 and -38. The catalytic subunit of PKA, C beta, was able to elicit a moderate induction of hPRL-8700/Luc even in the absence of Pit-1. A potential estrogen response element has been located in the hPRL gene sequence at a position similar to that of the estrogen response element of the rat PRL gene immediately adjacent to the distal enhancer.(ABSTRACT TRUNCATED AT 400 WORDS)