Transcriptional Regulation of MFG‐E8 by Sp1 and AP‐1 in Homeostasis and LPS Stimulation

Abstract

Intestinal MFG-E8 (Milk fat globule-EGF factor 8) is derived from macrophages in the lamina propria. It maintains the homeostasis and benefits tissue repair intestines by enhancing enterocyte migration and attenuating inflammation. Intestinal MFG-E8 is decreased in inflammation such as sepsis and inflammatory bowel disease. However, the regulation of MFG-E8 gene at transcriptional level is unclear. Here, we characterized the MFG-E8 gene promoter and further clarified its transcriptional regulation both constitutively and following LPS stimulation. C57BL/6J mouse peritoneal macrophages and RAW264.7 cells (macrophage-like cell line) were used. MFG-E8 mRNA and protein were quantified by Real-time PCR and Western Blotting respectively. Its promoter activity was measured by luciferase assay of serial promoter-pGL3-luciferase truncates. ChIP and EMSA were used to verify the interactions of transcription factors with the MFG-E8 promoter. Site-directed mutation or deletion was applied in mechanistic studies. We found that (1) LPS suppressed MFG-E8 expression in macrophages. (2) Sp1 and AP-1 were both necessary for the intact activity of the MFG-E8 promoter, whereas specific binding motifs of Sp1 (-11,-3) and AP-1 (-372) were critical. (3) Sp1 and AP-1 physically interacted with the MFG-E8 promoter. Moreover, gains-in-function study indicated that Sp1 enhanced but AP-1 inhibited the MFG-E8 promoter activity. (4) LPS attenuated MFG-E8 expression through targeting Sp1 and AP-1 binding motifs in the 5'flanking promoter region. Taken together, MFG-E8 gene is transcriptional controlled by Sp1 and AP-1 in both steady-state and inflammatory conditions (Supported by grants from NIH and Department of Veterans Affairs).