Interferon regulatory factor 6 (IRF6) is expressed in the ovine uterus and functions as a transcriptional activator

Abstract

Interferon tau (IFNT), the maternal recognition of pregnancy signal in sheep and other ruminants, is secreted by the conceptus and regulates the expression of a number of genes in a cell-specific manner within the uterus. The response of different endometrial cell types to IFNT appears to be specified by IFN regulatory factors (IRFs). IRF2, a potent repressor of gene transcription, is expressed only by luminal (LE) and superficial glandular epithelia (sGE), whereas IRF1 and IRF9, activators of gene transcription, are expressed only in GE and stromal cells of the uterus during early pregnancy. In the present study, IRF6 was found to be expressed in LE/sGE and middle GE of the ovine uterine endometrium as well as conceptus trophectoderm. IRF family members can regulate transcription via IFN-stimulated response elements (ISREs). Transient transfection analyses found that IRF6 enhanced basal activity of ISRE-containing promoters, but did not enhance IFNT stimulation of ISRE-containing promoters in variety of different cell types. Further, IRF6 did not cooperate with IRF1 or reduce IRF2 repression of ISRE-containing promoter activity. These results establish that IRF6 is a transcriptional activator that is preferentially expressed in the endometrial epithelia and conceptus trophectoderm. IRF6 is hypothesized to play critical roles in endometrial gene expression as well as in conceptus trophectoderm growth and differentiation.

Fig. 1. In situ hybridization analysis of IRF6 mRNA in the ovine uterus

Cross-sections of the uterine wall from cyclic (C) and pregnant (P) ewes were hybridized with radiolabeled antisense or sense ovine IRF6 cRNAs. IRF6 mRNA was detected in the uterine LE/sGE and GE as well as trophectoderm of the conceptus on Day 17 of pregnancy. Note the apparent increase in IRF6 mRNA in LE/sGE during pregnancy. Legend: GE, glandular epithelium; LE, luminal epithelium; S, stroma; Tr, trophectoderm. Scale bar represents 10 μm.

Fig. 2. Immunohistochemical localization of IRF6 protein in uteri from cyclic and pregnant ewes

In the IgG control, normal rabbit IgG was substituted for rabbit IgG specific for human IRF6. Sections were not counterstained. Note the immunoreactive IRF6 protein in uterine LE/sGE and GE, as well as conceptus trophectoderm. IRF6 protein increased in uterine LE/sGE and GE during pregnancy. Legend: LE, luminal epithelium; GE, glandular epithelium; S, stroma; Tr, trophectoderm; En, endoderm. Scale bar represents 10 μm in [A] and 100 μm in [B].

Fig. 3. Effect of IRFs and IFNT on 5xISRE-TATA-LUC reporter activity in 2fTGH cells

Cells were co-transfected with indicated constructs (amount indicated in ng/well) and treated with nothing or IFNT (10⁴ antiviral units) for 24 h. Transfection assays and luciferase activities were determined as described in Materials and Methods. The data are presented as mean relative light units (RLU) with standard deviation (SD). Four replicate determinations for each treatment group were conducted in each of three independent experiments. The asterisk (*) denotes a significant effect of treatment with IFNT on reporter activity (P<0.05).

Fig. 4. Effect of IRFs and IFNT on 5xISRE-TATA-LUC reporter activity in U3A STAT1 null cells

Cells were co-transfected with indicated constructs (amount indicated in ng/well) and treated with nothing or IFNT (10⁴ antiviral units) for 24 h. Transfection assays and luciferase activities were determined as described in Materials and Methods. The data are presented as mean relative light units (RLU) with SD. Four replicate determinations for each treatment group were conducted in each of three independent experiments. Note the lack of IFNT stimulation of reporter activity (P>0.10) in the transfected U3A cells.

Fig. 5. Effect of IRFs and IFNT on 5xISRE-TATA-LUC reporter activity in 2fTGH cells

Cells were co-transfected with indicated constructs (amount indicated in ng/well) and treated with nothing or IFNT (10⁴ antiviral units) for 24 h. Transfection assays and luciferase activities were determined as described in Materials and Methods. The data are presented as mean relative light units (RLU) with SD. Four replicate determinations for each treatment group were conducted in each of three independent experiments. The asterisk (*) denotes a significant effect of treatment with IFNT on reporter activity (P<0.05).

Fig. 6. Effect of IRFs and IFNT on 5xISRE-TATA-LUC reporter activity in immortalized ovine endometrial luminal epithelia (LE) cells

Cells were co-transfected with indicated constructs (amount indicated in ng/well) and treated with nothing or IFNT (10⁴ antiviral units) for 24 h. Transfection assays and luciferase activities were determined as described in Materials and Methods. The data are presented as mean relative light units (RLU) with SD. Four replicate determinations for each treatment group were conducted in each of three independent experiments. The asterisk (*) denotes a significant effect of treatment with IFNT on reporter activity (P<0.05).

Fig. 7. Effect of IRFs and IFNT on 5xISRE-TATA-LUC reporter activity in a bovine endometrial cell line (BEND)

Cells were co-transfected with indicated constructs (amount indicated in ng/well) and treated with nothing or IFNT (10⁴ antiviral units) for 24 h. Transfection assays and luciferase activities were determined as described in Materials and Methods. The data are presented as mean relative light units (RLU) with SD. Four replicate determinations for each treatment group were conducted in each of three independent experiments. The asterisk (*) denotes a significant effect of treatment with IFNT on reporter activity (P<0.05).

Fig. 8. Effect of IRFs and IFNT on 5xISRE-TATA-LUC reporter activity in a bovine endometrial cell line (BEND)

Cells were co-transfected with indicated constructs (amount indicated in ng/well) and treated with nothing or IFNT (10⁴ antiviral units) for 24 h. Transfection assays and luciferase activities were determined as described in Materials and Methods. The data are presented as mean relative light units (RLU) with SD. Four replicate determinations for each treatment group were conducted in each of three independent experiments. The asterisk (*) denotes a significant effect of treatment with IFNT on reporter activity (P<0.05).

Fig. 9. Effect of IRFs and IFNT on 5xISRE-TATA-LUC and TAT-LUC reporter activity in ovine trophectoderm (oTr1) cells

Cells were co-transfected with indicated constructs (amount indicated in ng/well) and treated with nothing or IFNT (10⁴ antiviral units) for 24 h. Transfection assays and luciferase activities were determined as described in Materials and Methods. The data are presented as mean relative light units (RLU) with SD. Four replicate determinations for each treatment group were conducted in each of three independent experiments. The asterisk (*) denotes a significant effect of treatment with IFNT on reporter activity (P<0.05).