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.

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.

Human prolactin gene expression: positive correlation between site-specific methylation and gene activity in a set of human lymphoid cell lines.

From the human B-lymphoblastoid cell line IM-9-P, we derived the IM-9-P series of clonal sublines that differ from each other in the degree of human PRL (hPRL) production. To elucidate the mechanisms underlying the different levels of hPRL gene activity in these cell lines, we investigated the methylation status of the gene, since the methylation pattern of cytosine-residues in CpG dinucleotides has been implicated with the transcriptional activity of eukaryotic genes. Restriction enzyme analysis of the hPRL gene with methylation-sensitive endonucleases disclosed no correlation between the extent of methylation and gene activity for ThaI, AvaI, and HhaI recognition sequences. Hypermethylation of a MspI site (CCGG) in the second protein-coding exon, however, was found to coincide with hPRL gene activity. Exposure of the cells to the nucleoside 1-beta-D-arabinofuranosylcytosine, which has been reported to increase enzymatic DNA-methylation, led to an elevation of hPRL production that persisted after removal of the drug. However, treatment of PRL-positive cells with the demethylating cytidine analog, 5-azacytidine, caused a distinct and heritable reduction of hPRL secretion and hPRL mRNA abundance, concurrent with hypomethylation of the specific MspI site that is hypomethylated in PRL-negative cell lines of the IM-9-P family. Contrasting the generally favored inverse relationship between methylation and transcriptional activity of a gene we describe a system in which site-specific methylation is positively correlated with gene expression.

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.

Nonpituitary human prolactin gene transcription is independent of Pit-1 and differentially controlled in lymphocytes and in endometrial stroma.

Expression of the human PRL (hPRL) gene in extrapituitary sites such as the uterus (decidualized endometrial stroma and myometrium) and cells of the hematopoietic lineage is directed by an alternative promoter which is located approximately 6 kilobases (kb) upstream of the pituitary-specific start site. In order to delineate the tissue-specific mechanisms governing the control of nonpituitary PRL gene expression, we have cloned and sequenced 3 kb 5'-flanking DNA of the upstream decidual/lymphoid (dPRL) promoter. Based on sequence homology we identified two binding motifs for Pit-1 and seven half-sites for glucocorticoid receptor/progesterone receptor (PR) binding. We focused our studies on the role of Pit-1 and of PR as potential transcriptional regulators, since the POU domain protein Pit-1 is essential in the control of pituitary PRL expression, and progesterone induces decidual transformation of the endometrial stroma, a differentiation process during which the decidual PRL gene is activated. We demonstrate in a variety of cell types, including lymphocytes and endometrial stroma, that Pit-1 is not involved in the regulation of dPRL promoter/reporter gene constructs carrying 3 kb 5'-flanking DNA. Our experiments also show that activated PR does not confer direct transcriptional control on the dPRL promoter. When we compared the activity of the transfected dPRL promoter in PRL-secreting and nonsecreting lymphoid cells, we found that the 3 kb 5'-flanking region of the dPRL promoter did not contain elements restricting expression to only those lymphocytes that produce PRL but allowed expression of fusion reporter genes irrespective of the status of the endogenous PRL gene. This was in sharp contrast to endometrial cells where 3 kb 5'-flanking DNA conferred strong transcriptional activation on the dPRL promoter in decidualized endometrial stromal cells actively secreting PRL, but did not allow transcription in undifferentiated non-PRL-secreting endometrial stromal cells. Activation of the dPRL promoter construct in these undifferentiated cells could however be induced by the addition of cAMP, in the absence of progesterone, suggesting that a signal transduced through the cAMP signaling pathway is a primary inducer of decidual PRL gene expression.

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)

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.

A human B-lymphoblastoid cell line produces prolactin.

A variety of cell lines were examined by Northern blot hybridization for the expression of PRL or PRL-related mRNAs. We found that a human B-lymphoblast cell line transcribed a mRNA which hybridized to human PRL cDNA under high stringency conditions. The human lymphoblast cell line of interest is a variant subline of the IM-9 line that we have designated IM-9-P. The lymphoblast-derived PRL mRNA is approximately 150 bases longer than that produced by the human pituitary as determined by Northern blot analysis. IM-9-P PRL was immunoaffinity purified from conditioned medium and found to be identical in mol wt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to human pituitary PRL. Moreover, IM-9-P PRL is biologically active in the rat Nb2 lymphoma mitogenic assay. Ribonuclease-H digestion of mRNA poly(A) tracts indicated that the size difference between pituitary and IM-9-P PRL transcripts was not due to an elongated poly(A) tail on the lymphoid PRL mRNA. Genomic Southern blot analysis showed no major rearrangements of the PRL gene in IM-9-P cells compared to the parent IM-9 line and human placenta DNA. Thus, it is highly likely that an elongation of the 5' and/or 3' untranslated regions of IM-9-P PRL mRNA account for the size difference with pituitary PRL mRNA. The PRL-producing IM-9-P line was cloned by limiting dilution, and a high PRL-producing clone IM-9-P3 and a non-PRL producer IM-9-P6 were isolated for further analysis. IM-9-P3 cells were found to secrete 40-50 ng PRL/10(6) cells.24 h regardless of cell density. The level of PRL mRNA also remained constant during exponential growth of IM-9-P3 cells. The existence of the PRL-producing IM-9-P3 clone and the IM-9-P6 clone which does not produce PRL as well as the IM-9 progenitor line provides a unique system with which to analyze the molecular mechanism of ectopic human PRL expression.

Regulation of prolactin secretion in the human B-lymphoblastoid cell line IM-9-P3 by dexamethasone but not other regulators of pituitary prolactin secretion.

We examined whether the ectopic production of human(h) PRL by the human B-lymphoblastoid cell line IM-9-P3 is under hormonal control. We demonstrate that PRL secretion in this cell line is regulated by dexamethasone but not by other hormones known to modulate PRL secretion in the pituitary or decidua. Dexamethasone caused a reduction of secretion rates to 30% of control values after 3 day paralleled by a decrease in hPRL mRNA levels, without affecting cell viability. Half-life determinations of hPRL mRNA in control and dexamethasone-treated cells revealed a reduction of t1/2 from 16 to 4 h. PRL secreted by IM-9-P3 cells did not control its own secretion in an autocrine loop, nor did it serve as an autocrine growth factor.

Development of a primary culture system for rainbow trout corpuscles of stannius and characterization of secreted teleocalcin.

A primary culture system has been established for rainbow trout corpuscles of Stannius (CS). A RIA has also been developed to monitor and characterize the teleocalcin secreted by these cultures. The primary cultured CS cells actively synthesized and secreted teleocalcin for up to 39 days when maintained in amphibian and mammalian culture media. Numerous teleocalcin cells were also evident after immunocytochemical staining of the CS cultures. When the cultures were labeled with L-[35S]methionine, de novo synthesized and secreted teleocalcin was judged to be a glycosylated protein on the basis of Concanavalin-A-Sepharose binding and was similar in size to the intracellular form of the hormone. This culture system may prove to be ideal for identifying those factors that regulate teleocalcin secretion.

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.

Posttranscriptional regulation of the human prolactin gene in IM-9-P3 cells by retinoic acid.

The IM-9-P3 family of cell lines, which are derived from the B-lymphoblastoid IM-9 cell line, transcribe the human PRL (hPRL) gene by utilization of the decidual-type promoter and provide a model to study factors controlling extrapituitary expression of the hPRL gene. Here we describe regulation of hPRL gene expression in members of the IM-9-P3 family by retinoic acid (RA). When cells were incubated in medium supplemented with fetal calf serum that had been treated with dextran-coated charcoal, the addition of RA caused a 2-fold stimulation of hPRL secretion in the low hPRL-producing clone IM-9-P31 and the moderate producer IM-9-P32 (ED50, 0.53 and 0.13 nM, respectively), but not in the high hPRL-producing IM-9-P33 clone. Secretion from the RA-responsive cell lines increased steadily over the first 24 h of exposure and remained elevated for several days. The concomitant increase in hPRL mRNA steady state levels was not due to enhanced transcription of the hPRL gene, as assessed by nuclear run-on experiments, but, rather, to message stabilization. In RA-treated IM-9-P32 cells, the half-life of hPRL mRNA was significantly increased from 9 to 22 h. The transcripts were found to be preferentially associated with membrane-bound polysomes, thus being available for the secretory pathway. When we studied the expression of potential transducers of the RA signal, namely the RA receptor subtypes hRAR alpha, -beta, and -gamma and cellular RA-binding protein, we did not detect hRAR gamma or cellular RA-binding protein transcripts in the hPRL-negative clone IM-9-P6 or the hPRL-positive clones IM-9-P31, IM-9-P32, and IM-9-P33. hRAR alpha was equally expressed in all cell lines and not regulated by RA, whereas hRAR beta was differentially expressed and controlled by RA. This receptor subtype was absent from hPRL-negative members of the IM-9-P family, strongly induced by RA in the RA-responsive IM-9-P31 and IM-9-P32 cell lines via rapid transcriptional up-regulation, and only slightly induced in the RA-resistant IM-9-P33 cell line, suggesting a function in mediation of the effect of RA on hPRL gene expression.

Decidual-type prolactin expression by the human myometrium.

The human myometrium, in addition to the decidualized endometrium of the late luteal phase and of pregnancy, has been proposed as a second source of uterine PRL, since immunoreactive PRL was found in supernatants from myometrial explant cultures. We demonstrate here that: 1) the human (h) PRL gene is expressed in the myometrium in vivo; 2) myometrial PRL is identical to pituitary hPRL; 3) the encoding transcript differs from pituitary hPRL messenger (m) RNA but is homologous to decidual and IM-9-P3 lymphoid hPRL mRNA; and 4) the expression of myometrial hPRL mRNA is inhibited by progestin. hPRL mRNA was detected in freshly isolated myometrium by Northern blot hybridization and was larger than the pituitary message. Sequence and primer extension analyses revealed that the transcript is identical to pituitary hPRL mRNA downstream of the pituitary cap site and carries an extension of the 5'-untranslated region homologous to that of decidual/IM-9-P3 lymphoid hPRL mRNA. This mRNA species results from alternative transcription initiation and comprises exon 1a of the hPRL gene, which is not transcribed in the pituitary. hPRL mRNA steady state levels and hPRL secretion increased dramatically when myometrial explants were maintained in long term culture. In addition to the 23,000 mol wt form, myometrial explants synthesized a glycosylated hPRL variant (G-hPRL) which was approximately 500 Daltons larger than pituitary G-hPRL but of similar size as lymphoid G-hPRL (26,500). Lactogenic activity of myometrial conditioned medium paralleled that of pituitary hPRL in the Nb2 lymphoma bioassay and was neutralized by the addition of monoclonal antibody to hPRL. hPRL secretion and hPRL mRNA abundance were not affected by estrogen but were markedly reduced by medroxy-progesterone acetate, which was maximally effective at a dose as low as 10(-10) M.

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.

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.

The effect of short- and long-term exercise on aldolase activity in muscles of CW-1 and C57/BL mice of various ages.

The effect of wheel running on the levels of fructose 1,6-bisphosphate aldolase A (EC 4.1.2.13) in striated muscles of young and old mice was compared. Short-term (6-10 weeks) and long-term (over 12 months) regimens were included in the study. The studies were conducted on the CW-1 outbred strain and on the C57/BL inbred strain of mice. It was shown that, in the short-term regimens, old animals of both strains showed either no increase (C57/BL) or a reduction (CW-1) in aldolase activity in hind leg muscles. On the other hand, young animals of both strains showed increases in aldolase activity of 10-20%. In the long-term regimen young and intermediate age animals showed 30-100% increases in aldolase activity in hind leg muscles over control sedentary animals. This adaptive capacity to exercise was not observed in old animals. However, long-term exercise regimen prevented the age-associated decline in aldolase activity found in sedentary animals.

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.