Endometrial factors similarly induced by IFNT2 and IFNTc1 through transcription factor FOXS1

Abstract

In ruminants, Interferon tau (IFNT) is the pregnancy recognition protein produced by the mononuclear trophectoderm of the conceptus, and is secreted into the uterine lumen during the peri-attachment period. In our previous study, the high-throughput RNA sequencing (RNA-seq) data obtained from bovine conceptuses during the peri-attachment period identified two IFNT mRNAs, IFNT2 and IFNTc1. However, how each of these IFNT variants regulates endometrial gene expression has not been characterized. Using RNA-seq analysis, we evaluated how IFNT2 and IFNTc1 affected transcript expression in primary bovine endometrial epithelial cells (EECs). IFNT treatment induced 348 differentially expressed genes (DEGs); however, there are few DEGs in IFNT2 or IFNTc1 treated EECs, indicating that IFNT2-induced DEGs were similar to those induced by IFNTc1 treatment. In in silico analysis, we identified four IFNT2- and IFNTc1-induced pathways: 1) type II interferon signaling, 2) proteasome degradation, 3) type III interferon signaling, and 4) DNA damage response. We further demonstrated that IFNT2 and IFNTc1 up-regulated several transcription factors, among which forkhead box S1 (FOXS1) was identified as the most highly expressed gene. Furthermore, the knockdown of FOXS1 in IFNT2- or IFNTc1-treated EECs similarly down-regulated 9 genes including IRF3 and IRF9, and up-regulated 9 genes including STAT1, STAT2, and IRF8. These represent the first demonstration that effects of each IFNT on EECs were studied, and suggest that endometrial response as well as signaling mechanisms were similar between two IFNT variants existed in utero.

Fig 1. Differential gene expression in bovine endometrial epithelial cells treated with IFNT2 or IFNTc1.

(A) Venn diagram shows the number of gene with 1.5-fold changes among Control (Ctrl), IFNT2, and IFNTc1 treatment groups, and right table lists increased or decreased genes in IFNT2 vs. IFNTc1 group, which overlap with Ctrl vs. IFNT2 or Ctrl vs. IFNTc1 group. (B) EECs were incubated without (Ctrl) or with IFNT2 or IFNTc1 (2 x 10⁵ cells/5000 IU/well) for 24 h. RNA was extracted from the EECs and subjected to real-time PCR analysis on mRNA expression with overlapping IFNT2 vs. IFNTc1 group with other groups. GAPDH mRNA was used as an internal control for RNA integrity. Value represent the mean ± SEM from three independent experiments in each treatment. (C) these diagrams show pair plots comparison among Ctrl, IFNT2, and IFNTc1, and density plots in each groups. Figures show correlation coefficient among Ctrl, IFNT2, and IFNTc1.

Fig 2. Identification of gene expression induced by IFNTs in EECs.

EECs were incubated without (Ctrl) or with IFNT2 or IFNTc1 (2 x 10⁵ cells/5000 IU/well) for 24 h. RNA was extracted from the EECs and subjected to real-time PCR analysis to determine gene expression related to type II interferon, proteasome degradation, type III interferon, and DNA damage response signaling in Ctrl, IFNT2-, or IFNTc1-treated EECs (n = 3 each group). GAPDH mRNA was used as an internal control for RNA integrity. ^aP < 0.01, ^bP<0.05 vs. Ctrl. Value represent the mean ± SEM from three independent experiments in each treatment.

Fig 3. Determination of IFNTs’ downstream transcription factors.

EECs were incubated without (Ctrl) or with IFNT2 or IFNTc1 (2 x 10⁵ cells/5000 IU/well) for 24 h. RNA was extracted from the EECs and subjected to real-time PCR analysis to determine the expression of transcription factors in Ctrl, IFNT2-, or IFNTc1-treated EECs (n = 3 each group). GAPDH mRNA was used as an internal control for RNA integrity. ^aP < 0.01, ^bP<0.05 vs. Ctrl. Value represent the mean ± SEM from three independent experiments in each treatment.

Fig 4. Effects of FOXS1 knockdown on the expression of IFNTs downstream factors.

(A-C) EECs were transfected with non-targeting control (Ctrl: 200 nM) or FOXS1 siRNA (#1 or #2: 200 nM) for 48h, and then incubated with IFNT2 (B) or IFNTc1 (C) (2 x 10⁵ cells/5000 IU/well) for 24 h. RNA was extracted from the EECs and subjected to real-time PCR analysis (n = 3 each group). GAPDH mRNA was used as an internal control for RNA integrity. Values represent the mean ± SEM from three independent experiments in each treatment.

Fig 5. Diagram illustrating the potential role of IFNT through FOXS1 in EECs.

IFNT2 and IFNTc1 bind to their receptor and then activate STAT1 or STAT2. Activated STATs up-regulate FOXS1 expression, which down-regulates STATs expression, resulting in a negative feedback loop between STATs and FOXS1.