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.

Leukemia inhibitory factor (LIF) stimulates the human HLA-G promoter in JEG3 choriocarcinoma cells.

HLA-G is a non-classic class I MHC molecule specifically expressed by human invasive cytotrophoblast cells, which has been suggested to play a role in facilitating the immune tolerance of the conceptus. So far, very little is known about the regulation of the human HLA-G gene. The present study was, thus, designed to investigate the regulation of the human HLA-G promoter. JEG3 choriocarcinoma cells, which express HLA-G endogenously, were used as a model. A 890 bp fragment of the human HLA-G promoter was amplified by nested PCR from genomic DNA, cloned into pCR-Script and, after sequencing, subcloned into pGL3-Luc in front of the luciferase reporter gene. This vector was then used in transient transfection experiments in JEG3 cells. Parallel transfection experiments were performed using an alpha subunit (alphaSU)-Luc reporter plasmid as a control. Using this system, several potential modulating substances were tested in different concentrations and for different periods of time: phorbol ester (TPA), cAMP, IFNgamma, IL-1, and leukemia inhibitory factor (LIF), with only LIF administration resulting in induction of the HLA-G promoter. LIF treatment also resulted in induction of HLA-G mRNA. JEG3 cells are shown to possess LIF receptors. LIF is a pleiotropic cytokine produced at the maternal-fetal interface which has been shown to play an essential role in implantation in mice. LIF is produced in high amounts by the human endometrium and the trophoblast itself, and LIF receptors are present on cytotrophoblast cells. LIF could, thus, play a role in modulating HLA-G production and immune tolerance at the maternal-fetal interface.

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.

Regulation of the human leukaemia inhibitory factor (LIF) promoter in HEC-1B endometrial adenocarcinoma cells.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine which has been found to be expressed in the human endometrium and to play an important role in human reproduction. In the present study we investigated expression and regulation of the human LIF promoter in HEC-1B endometrial adenocarcinoma cells using a luciferase reporter plasmid bearing a 666 bp promoter fragment (h666LIF-Luc) in transient transfection assays. HEC-1B cells were first shown by reverse transcription-polymerase chain reaction (RT-PCR) to be able to produce endogenous LIF mRNA. The LIF promoter was efficiently transcribed in HEC-1B cells, showing much higher levels of basal activity than in the previously studied Jurkat T-lymphoma cells and SKUT-1B uterine mesodermal tumour cells. The activity of the LIF promoter was stimulated in HEC-1B cells by a combination of phorbol ester (TPA) and ionomycin, which we had previously found to strongly induce its activity in Jurkat T-lymphoma cells. We next studied the effect of progestin (medroxyprogesterone acetate; MPA) on the LIF promoter activity in HEC-1B cells. The LIF promoter was not stimulated by MPA treatment in the presence of transfected progesterone receptor B (PR-B) expression vector in HEC-1B cells, while we had previously described its induction by MPA in SKUT-1B cells. This indicates that progestin-dependent regulation of the LIF promoter in uterine tumour cells is different in cells of epithelial and mesodermal origin.

Progestin-dependent stimulation of the human leukemia inhibitory factor promoter in SKUT-1B uterine tumor cells.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which is essential for implantation in rodents and is expressed during the progesterone-dominated secretory phase of the menstrual cycle in the human endometrium. However, the effect of progestin on the transcriptional regulation of the LIF promoter has not been studied so far. In the present study, we used a luciferase reporter plasmid bearing 666 bp of the human LIF promoter (hLIF666-Luc) to investigate the effects of progestin on the transcriptional regulation of LIF in SKUT-1B uterine tumor cells. Jurkat T-lymphoma cells were used for comparison. Since both cell lines are devoid of functional progesterone receptors (PR), we co-transfected the cells with hLIF666-Luc and an expression vector for the human PR form B (PR-B) or A (PR-A). Addition of the progesterone agonist MPA (medroxy-progesterone acetate, 2.5 x 10(-7) M) resulted in induction of LIF transcription only in SKUT-1B cells, while it had no effect in Jurkat cells. Both PR forms were effective in inducing the LIF promoter in SKUT-1B cells when activated by MPA. However, the induction through PR-A was inhibited more efficiently by the progestin antagonist RU 486. We next investigated the stimulatory effect of MPA in SKUT-1B cells on deletion constructs (h274LIF-Luc, h148LIF-Luc and H82LIF-Luc) and found that it is maintained on these fragments. Thus, 82 bp are sufficient to mediate this effect. Our results show that the human LIF promoter is active in uterine tumor cells, and that it is differentially regulated by progestin in cells of uterine and lymphoid origin.

Transcriptional regulation of the human 'leukemia inhibitory factor' gene: modulation by glucocorticoids and estradiol.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite its possible importance as a therapeutic target, very little is known about the regulation of human LIF. In particular, its regulation at the promoter level has not been studied so far, and was, therefore, the focus of the present work. After showing that Jurkat T lymphoma cells can be induced to express endogenous LIF mRNA, we used this cell line as a model to study the regulation of the human LIF promoter in transient transfection assays. For this purpose, a 666 bp fragment of the human LIF 5'-flanking region, amplified from genomic DNA by nested polymerase chain reaction (PCR), was used for the construction of a luciferase reporter plasmid (hLIF666-Luc). In unstimulated Jurkat cells, the human LIF promoter showed low constitutive activity. The promoter was induced upon stimulation with phorbol ester (TPA). Combined stimulation with TPA and the calcium ionophore ionomycin resulted in strong synergistic induction of luciferase activity from the LIF promoter. Transfection experiments with deletion constructs (hLIF274-Luc and hLIF82-Luc) located the region required for this induction to a 192 bp portion of the promoter, which carries two putative c-ets binding sites. We then investigated the effect of glucocorticoids and estradiol by cotransfecting the respective receptors. Both hormones strongly inhibited the stimulation of the LIF promoter by TPA and ionomycin. Since LIF is implicated in the pathogenesis of inflammatory and degenerative conditions, such as rheumatoid arthritis and osteoporosis, the finding that therapeutic agents employed in the treatment of such conditions, i.e. glucocorticoids and estrogens, can modulate the induction of LIF at the transcriptional level, is of particular interest.

Expression of the plasma membrane Ca2+-ATPase in myogenic cells.

To study the physiological function of the plasma membrane calmodulin-dependent calcium ATPase (PMCA) in intact cells, L6 myogenic cell lines stably overexpressing the human PMCA isoform 4CI (= human PMCA isoform 4b) were generated. Several independent L6 clones and controls stably transfected with the empty expression vector were analyzed in detail. The resting cytosolic calcium level in hPMCA4CI-overexpressing muscle cells (measured by the Fura-2 method) was significantly reduced by 20-30% compared with controls. This was shown in a cytosolic window of 1322 single cells (p < 0.01). Furthermore, the differentiation process of these cells was remarkably accelerated compared with control myoblasts and parental nontransfected L6 cells as assessed by multinucleated myotube formation and creatine phosphokinase activity elevation. After 4 and 6 days of differentiation, PMCA-overexpressing L6 cells from four independent clones displayed a 3- and 4-fold higher creatine phosphokinase activity compared with controls (n = 5, p < 0.02). These results may extend the concept of the function of the PMCA from simple prevention of calcium overload to an active involvement in intracellular calcium regulation with potentially important consequences for cellular functions.