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Transcriptional Regulation of MFG‐E8 by Sp1 and AP‐1 in Homeostasis and LPS Stimulation
Author(s) -
Wang Xiao,
Bu HengFu,
Liu Shirley XL,
De plaen Isabelle,
Tan XiaoDi
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.854.10
Subject(s) - luciferase , promoter , microbiology and biotechnology , transcriptional regulation , transcription factor , biology , gene expression , transcription (linguistics) , regulation of gene expression , gene , chemistry , transfection , biochemistry , linguistics , philosophy
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).