This is a revised renewal application in which research is proposed to investigate further the cellular and molecular mechanisms whereby ovine interferon-tau (IFN-tau) regulates estrogen receptor (ERalpha) and oxytocin receptor (OTR) gene expression in the endometrial epithelium. IFN-tau is produced by trophectoderm of ruminant concept uses and signals pregnancy recognition by suppressing ERalpha and OTR gene expression in the endometrial epithelium to prevent oxytocin-induced luteolytic pulses of prostaglandin F2alpha. Coincident with suppression of these genes, IFN-tau also induces or up regulates transcription of several Type I IFN-regulated genes. These disparate effects of IFN-tau are mediated by triggering a transcription factor network involving activation of the signal transducers of transcription (STAT) family, primarily STATS 1alpha/beta and 2, and induction of IFN regulatory factors (IRF) that bind to specific response elements and either activate or repress transcription of target genes. Results from the current grant period indicate that IFN-tau acts on the endometrial epithelium in vivo to suppress transcription of both the ERalpha and OTR genes and up regulate IRF-1 and IRF-2 repressor. Cloning and analysis of the 5' flanking promoter region (2.8 kb) of the ovine ERalpha gene revealed five predicted IRF elements (IRF-E) that were functional in binding IRF-1 and IRF-2. The working hypothesis is that IFN-tau induces an IRF repressor, such as IRF-2, which binds to functional IRF-Es in the ERalpha promoter to inhibit transcription. It has been demonstrated that IFN-tau inhibits activity of the 2.8 kb oERalpha promoter in a transient transfection assay using an immortalized ovine lumenal epithelial (LE) cell line. A transfection analysis of the oERalpha promoter indicates that both IRF and STAT binding elements are important for inhibitory effects of IFN-tau. The proposed research will continue to delineate cellular and molecular mechanisms underlying the action of IFN-tau in regulating ERalpha and OTR gene expression in the ovine uterus.
Specific aims are to: 1) clone, sequence and functionally analyze the promoter of the ovine OTR gene, 2) determine which IRF family members are induced by IFN-tau in the endometrial epithelium, 3) analyze effects of IFN-tau regulated IRFs on activity of the ovine ERalpha and OTR promoters, and 4) test the hypothesis that STATs activated by IFN-tau mediate, in part, the inhibitory effects of IFN-tau on ovine ERalpha and OTR promoter activity.
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