Combinatorial roles of protein kinase A, Ets2, and 3',5'-cyclic-adenosine monophosphate response element-binding protein-binding protein/p300 in the transcriptional control of interferon-tau expression in a trophoblast cell line

In ruminants, conceptus interferon-tau (IFNT) production is necessary for maintenance of pregnancy. We examined the role of protein kinase A (PKA) in regulating IFNT expression through the activation of Ets2 in JAr choriocarcinoma cells. Although overexpression of the catalytic subunit of PKA or the addition of 8-bromo-cAMP had little ability to up-regulate boIFNT1 reporter constructs on their own, coexpression with Ets2 led to a large increase in gene expression. Progressive truncation of reporter constructs indicated that the site of PKA/Ets2 responsiveness lay in a region of the promoter between -126 and -67, which lacks a cAMP response element but contains the functional Ets2-binding site and an activator protein 1 (AP1) site. Specific mutation of the former reduced the PKA/Ets2 effects by more than 98%, whereas mutation of an AP1-binding site adjacent to the Ets2 site or pharmacological inhibition of MAPK kinase 2 led to a doubling of the combined Ets2/PKA effects, suggesting there is antagonism between the Ras/MAPK pathway and the PKA signal transduction pathway. Although Ets2 is not a substrate for PKA, lowering the effective concentrations of the coactivators, cAMP response element-binding protein-binding protein (CBP)/p300, known PKA targets, reduced the ability of PKA to synergize with Ets2, suggesting that PKA effects on IFNT regulation might be mediated through CBP/p300 coactivation, particularly as CBP and Ets2 occupy the proximal promoter region of IFNT in bovine trophoblast CT-1 cells. The up-regulation of IFNT in the elongating bovine conceptus is likely due to the combinatorial effects of PKA, Ets2, and CBP/p300 and triggered via growth factors released from maternal endometrium.     

Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.     

Coculture system that mimics in vivo attachment processes in bovine trophoblast cells

The establishment of pregnancy requires bidirectional communication between the developing conceptus and the uterine endometrium. The aim of this study was to establish an in vitro coculture system with bovine trophoblast cells and uterine epithelial cells (EECs) that mimics the in vivo attachment process. We previously reported that expression of interferon tau (IFNT), a major secretory product from the trophectoderm, decreases with changes in chromatin structure when the conceptus successfully attaches to the uterine epithelium. Thus, IFNT is a good marker to assess whether attachment has successfully occurred. In this study, bovine trophoblast CT-1 cells were cultured to generate spheroids, which were then placed on type I collagen-coated plates (monoculture) or bovine EECs (coculture) with or without uterine flushings collected from Day 15 cyclic or Days 15, 17, or 19 pregnant animals. In the coculture but not the monoculture, addition of uterine flushings from Day 15 or 17 pregnant animals resulted in decreased IFNT and CDX2 mRNA expression in CT-1 spheroids, accompanied with changes in histone modifications. In monocultured CT-1 spheroids, integrin subunit ITGA8 and ITGB3 mRNAs were minimally expressed but were induced in cocultured CT-1 spheroids with or without uterine flushings. Expression of CDH2, another marker for bovine conceptus attachment to the uterine epithelium, was also induced in the cocultured CT-1 spheroids. These results suggest that this in vitro coculture system could be used to isolate processes essential for conceptus attachment to uterine EECs.     

Comparison of the effects of early pregnancy with human interferon, alpha 2 (IFNA2), on gene expression in bovine endometrium

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.     

Protein kinase C delta mediates fibroblast growth factor-2-induced interferon-tau expression in bovine trophoblast

Interferon-tau (IFNT) is the trophoblast-secreted factor responsible for establishing and maintaining pregnancy in ruminants. Several uterine- and embryo-derived factors, including fibroblast growth factor-2 (FGF2), regulate IFNT production. The objective of the present study was to decipher the intracellular signaling mechanisms employed by FGF2 to regulate IFNT production. In bovine trophoblast cells (CT1), mitogen-activated protein kinase kinase-dependent pathways mediated constitutive IFNT mRNA concentrations. However, FGF2-mediated increases in IFNT mRNA levels occurs independent of mitogen-activated protein kinase. Exposure to the pan-protein kinase C (PKC) inhibitor, calphostin C, did not affect basal IFNT mRNA levels but limited the ability of FGF2 to increase IFNT mRNA abundance. Also, supplementation with phorbol 12-myristate 13-acetate (PMA) stimulated IFNT mRNA levels to the same extent as with FGF2. PMA and FGF2 cosupplementation did not elicit an additive effect on IFNT mRNA abundance. Pharmacological antagonists for classic PKCs (G?6976) or novel PKCs, including PKC delta (rottlerin), were used to identify the specific PKC isoform utilized by FGF2. Supplementation of CT1 cells with G?6976 did not affect FGF2 or PMA activities, whereas rottlerin prevented FGF2- and PMA-dependent increases in IFNT mRNA abundance in CT1 cells. Rottlerin also prevented FGF2 from increasing IFNT mRNA levels in Vivot trophoblast cells and primary trophoblast outgrowths. Modifications in PRKCD phosphorylation status were evident following FGF2 and PMA treatment. Also, reducing PRKCD expression by RNA interference attenuated FGF2-dependent increases in IFNT mRNA abundance. In conclusion, these results provide evidence that FGF2 regulates IFNT production in bovine trophectoderm by acting through PRKCD.     

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS.

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.