Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 May 9;18(1):497-506.
doi: 10.2119/molmed.2011.00369.

Milk fat globule-epidermal growth factor 8 is decreased in intestinal epithelium of ulcerative colitis patients and thereby causes increased apoptosis and impaired wound healing

Qiu-jie Zhao  1 , Yan-bo Yu, Xiu-li Zuo, Yan-yan Dong, Yan-qing Li
Affiliations

Milk fat globule-epidermal growth factor 8 is decreased in intestinal epithelium of ulcerative colitis patients and thereby causes increased apoptosis and impaired wound healing

Qiu-jie Zhao et al. Mol Med. .

Abstract

Milk fat globule-epidermal growth factor 8 (MFG-E8) plays an important role in maintaining intestinal barrier homeostasis and accelerating intestinal restitution. However, studies of MFG-E8 expression in humans with ulcerative colitis are lacking. We examined MFG-E8 expression in colonic mucosal biopsies from ulcerative colitis patients and healthy controls (n = 26 each) by real-time quantitative polymerase chain reaction (PCR), Western blot analysis and immunohistochemistry. MFG-E8 mRNA and protein expression was lower in ulcerative colitis patients than in controls. MFG-E8 expression was inversely correlated with mucosal inflammatory activity and clinical disease activity in patients. MFG-E8 was present in human intestinal epithelial cells both in vivo and in vitro. Apoptosis induction was also detected in the intestinal epithelium of ulcerative colitis patients by terminal-deoxynucleoitidyl transferase mediated nick-end labeling assay. We used lentiviral vectors encoding human MFG-E8 targeting short hairpin RNA to obtain MFG-E8 knockdown intestinal epithelia cell clones. MFG-E8 knockdown could promote apoptosis in intestinal epithelial cell lines, accompanied by a decrease in level of the antiapoptotic protein B-cell lymphoma 2 (BCL-2) and induction of the proapoptotic protein BCL2-associated protein X (BAX). The addition of recombinant human MFG-E8 led to decreased BAX and cleaved caspase-3 levels and induction of BCL-2 level in intestinal epithelia cells. MFG-E8 knockdown also attenuated wound healing on scratch assay of intestinal epithelial cells. The mRNA level of intestinal trefoid factor 3, a pivotal factor in intestinal epithelial cell migration and restitution, was downregulated with MFG-E8 knockdown. In conclusion, we demonstrated that decreased colonic MFG-E8 expression in patients with ulcerative colitis may be associated with mucosal inflammatory activity and clinical disease activity through basal cell apoptosis and preventing tissue healing in the pathogenesis of ulcerative colitis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
MFG-E8 mRNA and protein level in colonic biopsies from control subjects and UC patients. (A) Semiquantitative PCR of the mRNA level of MFG-E8 in colons of controls (HC) and UC patients and in human colon cancer cell lines HT-29 and Caco-2. β-Actin was an internal control. Real-time quantitative PCR analysis of mRNA level (B) and Western blot analysis of protein level of MFG-E8 (C) in colons from UC patients and healthy controls. *Not representative outliers. Human β-actin was a loading control. Quantification is median (line within the box) and interquartile range (top and bottom of the box). Upper and lower whiskers indicate the 95th and 5th percentile, respectively.
Figure 2
Figure 2
Association of colonic MFG-E8 protein expression with inflammation severity and clinical disease activity. (A) MFG-E8 protein expression in UC biopsies with colitis classified as mild (n = 5) and moderate or severe (n = 9) according to Matts grade (19). (B) Spearman rank correlation of MFG-E8 protein level with Mayo clinical disease activity in UC patients (r = −0.7713; P < 0.01).
Figure 3
Figure 3
Immunohistochemical staining for MFG-E8 expression in human colonic biopsies. (A, B) Normal colons from control subjects. (C, D) Inflamed colons from UC patients. (E) Negative control. Original magnification: 400×.
Figure 4
Figure 4
TUNEL staining of inflamed colons of patient with UC (A, B) and normal colons from healthy controls (C, D). Arrows indicate TUNEL-positive IECs. Original magnification: 400×.
Figure 5
Figure 5
Validation of MFG-E8 expression in IEC lines and MFG-E8 knockdown. (A) Western blot analysis of MFG-E8 protein level in HT-29 and Caco-2 cells. (B) Fluorescence microscopy of HT-29 and Caco-2 cells 72 h after transduction with copGFP. Scale bar, 100 μm. (C) Real-time quantitative PCR analysis of MFG-E8 mRNA expression in LV-M– and LV-C–transduced HT-29 and Caco-2 cells. LV-M, lentiviral particles encoding short hairpin RNA sequences targeted to human MFG-E8 mRNA. LV-C, control lentiviral particles encoding a scrambled shRNA sequence. (D) Western blot analysis of MFG-E8 protein level in LV-M– and LV-C–transduced HT-29 and Caco-2 cells.
Figure 6
Figure 6
Basal and TNF-α– and flagellin-induced IL-8 mRNA expression in LV-C– and LV-M–transduced HT-29 (A) and Caco-2 cells (B). To detect TNF-α– and flagellin-induced IL-8 mRNA expression, total RNA was isolated after stimulation with 100 ng/mL TNF-α or 100 ng/mL flagellin for 12 h. (C) IL-8 mRNA levels in flagellin-stimulated Caco-2 cells pre-treated with rhMFG-E8 at different concentrations for 12 h. A, B: □, LV-C; formula image, LV-M.
Figure 7
Figure 7
Apoptosis assay in MFG-E8 knockdown IEC lines. (A) Flow cytometry of annexin V staining with LV-M or LV-C in HT-29 and Caco-2 cells. (B) Hoechst 33342 staining results. Arrows indicate typical apoptotic nuclei with chromatin condensation or fragmentation. Scale bar, 50 μm. □ LV-C; formula image, LV-M.
Figure 8
Figure 8
Assessment of apoptotic-related proteins in MFG-E8 knockdown IEC lines. The mRNA level of the antiapoptotic factor BCL-2 in Caco-2 cells (A) and proapoptotic factor BAX in Caco-2 (B) and HT-29 cells (C) with LV-M or LV-C transduction is shown. (D) Western blot analysis of cleaved caspase-3, BCL-2 and BAX protein level with LV-M or LV-C transduction in HT-29 and Caco-2 cells. (E) Western blot analysis of cleaved caspase-3, BCL-2 and BAX protein levels in IECs incubated with rhMFG-E8 at different concentrations for 24 h.
Figure 9
Figure 9
MFG-E8 knockdown attenuated IEC restitution in vitro. (A) Wound healing assay of MFG-E8 knockdown HT-29 cells and cells transduced with control lentivirus. The mRNA expression of TGF-β1 (B) and intestinal TFF3 (C) in MFG-E8 knockdown HT-29 cells is shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427–34. - PubMed
    1. Strober W, Fuss I, Mannon P. The fundamental basis of inflammatory bowel disease. J Clin Invest. 2007;117:514–21. - PMC - PubMed
    1. Podolsky DK. Inflammatory bowel disease. N Engl J Med. 2002;347:417–29. - PubMed
    1. Yamamoto-Furusho JK, Podolsky DK. Innate immunity in inflammatory bowel disease. World J Gastroenterol. 2007;13:5577–80. - PMC - PubMed
    1. Matsuda R, et al. Quantitive cytokine mRNA expression profiles in the colonic mucosa of patients with steroid naive ulcerative colitis during active and quiescent disease. Inflamm Bowel Dis. 2009;15:328–34. - PubMed

Publication types

MeSH terms