Milk fat globule-EGF factor 8 (MFG-E8) is expressed by macrophages and plays an important role in attenuating inflammation and maintaining tissue homeostasis. Previously, we and others found that LPS inhibits MFG-E8 gene expression in macrophages. Here, we characterized the 5′-flanking region of the mouse MFG-E8 gene. To functionally analyze the upstream regulatory region of the MFG-E8 gene, a series of luciferase reporter gene constructs containing deleted or mutated regulatory elements were prepared. Using the luciferase assay, we revealed that Sp1 binding motifs within the proximal promoter region were necessary for full activity of the MFG-E8 promoter, whereas AP-1 like binding sequence at −372 played a role in governing the promoter activity at a homeostatic level. With chromatin immunoprecipitation assay, we showed that Sp1 and c-Jun physically interact with the MFG-E8 promoter region in vivo. In addition, Sp1 was found to regulate the MFG-E8 promoter activity positively and c-Jun negatively. Furthermore, we demonstrated that LPS inhibited MFG-E8 promoter activity via targeting Sp1 and AP-1-like motifs in the 5′-flanking region. Collectively, our data indicate that Sp1 and AP-1-related factors are involved in the regulation of MFG-E8 gene transcription by targeting their binding sites in the 5′-flanking region under physiological and inflammatory states.
Necrotizing enterocolitis (NEC) is a severe intestinal disease affecting premature infants. Its pathogenesis remains poorly understood. Vascular endothelial growth factor (VEGF) is an angiogenic protein that plays a role in several pathologic processes. While VEGF polymorphisms have been associated with NEC, the role of VEGF in the pathogenesis of NEC remains unknown. Here, we examined the developmental expression of VEGF in the mouse intestine and in a neonatal mouse NEC model. To determine VEGF developmental expression, pups were sacrificed at embryonic day (E) 16, postnatal day (D) 0 or 3. To examine intestinal VEGF in our NEC model, animals were submitted to the NEC protocol for 48 hours or allowed to be dam fed. Intestinal VEGF protein was assessed by western blot followed by band densitometry (values were normalized to β‐actin). Compared to D0, only a low amount of VEGF was detected in the mouse intestine at E16 (2.6±0.42 % of D0 value; p<0.05). At D3, there was a marginal increase in VEGF compared to D0 (194 ±20 %), although this was not statistically significant. In pups exposed to the NEC protocol, intestinal VEGF was not detectable while it was strongly present in dam fed controls. In conclusion, we found that intestinal VEGF is increased after birth in dam fed pups, but undetectable at 48 hours in our neonatal mouse NEC model. Therefore, we speculate that VEGF plays a role in the pathogenesis of this disease.
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