Defect in CEACAM family member expression in Crohn's disease IECs is regulated by the transcription factor SOX9

Abstract

Background: CEACAM1, CEACAM5, and CEACAM6 represent 3 of the CEACAM (carcinoembryonic antigen-related cell adhesion molecule) subfamily members expressed on intestinal epithelial cells (IECs). Deficiency in their expression, as seen in inflammatory bowel disease (IBD), results in the lack of activation of CD8+ regulatory T cells in the mucosa. Since CEACAM expression was shown to be regulated by the transcription factor SOX9, we sought to determine whether the defect in CEACAM expression in IBD was related to aberrant SOX9 expression.

Methods: IECs and lamina propria lymphocytes (LPLs) were freshly isolated from colonic tissues. T84 and HT29 16E cells were cocultured with LPLs. SOX9 and CEACAM subfamily member expression was assessed by real-time polymerase chain reaction (PCR), Western blot, immunohistochemistry, and immunofluorescence.

Results: In Crohn's disease (CD) but not in ulcerative colitis (UC), a significant reduction in mRNA and protein expression for CEACAM1 and 5 was noted; in contrast, no difference in SOX9 mRNA expression was seen. However, nuclear SOX9 immunostaining was increased in CD IECs. Furthermore, SOX9 protein was reduced in the cytoplasm of LPL-stimulated T84 and HT29 16E cells, while CEACAM5 expression was increased.

Conclusions: The defect in CEACAM family members in CD IECs appears to be related to the aberrant nuclear localization of SOX9. Changes in SOX9 expression in the CD mucosa relate to the local microenvironment and altered IEC:LPL crosstalk.

Figure 1

CEACAM1, 5 and 6 mRNA expression in surface epithelial cells of Nor, CD, and UC colonic tissues. The IECs were isolated and the mRNA was purified and analyzed by Real-Time PCR. The fold increase of mRNA expression was calculated as described in the Materials and Methods section.

Figure 2

Immunofluorescence staining for CEACAM1 and 5 antibodies (red) in Nor, CD and UC colonic tissues. The nuclei were staining with Hoechst 33342 (blue). All the samples were analyzed with a Leica SP5-DM Confocal microscope at 40x magnification. These data are representative of 5 experiments.

Figure 3

A. SOX9 mRNA expression in surface epithelial cells of Nor, CD, and UC colonic tissues. The IECs were isolated and the mRNA was purified and analyzed by Real-Time PCR. The fold increase in mRNA expression was calculated as described in the Materials and Methods section. B. Nor, CD and UC colonic tissue sections were immunostained using an anti-SOX9 antibody. The slides were counterstained with Mayer’s Hematoxylin solution and examined with a Zeiss Axioskop Light Microscope at 20x and 40x magnifications. These data are representative of 5 experiments.

Figure 4

A. SOX9, CEACAM1, 5 and 6 mRNA expression in T84 cells co-cultured with Nor or CD LPL for 4 days. After removing the LPL, mRNA from T84 cells was purified and analyzed by Real Time-PCR. The fold increase in mRNA expression was calculated as described in the Materials and Methods section. B. Immunoblotting for SOX9, CEACAM5 and ERK2 in lysates obtained from T84 cells co-cultured with freshly isolated Nor or CD LPL for 4 days. These data are representative of 5 experiments. C. Immunoflurorescence staining for SOX9 (green) in HT29 16E cells co-cultured with Nor or CD LPL for 4 days. The nuclei were staining with Hoechst 33342 (blue). All the samples were analyzed with a Leica SP5-DM Confocal microscope at 100x magnification. These data are representative of 5 experiments.