Overexpression of CD97 in intestinal epithelial cells of transgenic mice attenuates colitis by strengthening adherens junctions

Abstract

The adhesion G-protein-coupled receptor CD97 is present in normal colonic enterocytes but overexpressed in colorectal carcinoma. To investigate the function of CD97 in colorectal carcinogenesis, transgenic Tg(villin-CD97) mice overexpressing CD97 in enterocytes were generated and subjected to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated tumorigenesis. Unexpectedly, we found a CD97 cDNA copy number-dependent reduction of DSS-induced colitis in Tg compared to wild-type (WT) mice that was confirmed by applying a simple DSS protocol. Ultrastructural analysis revealed that overexpression of CD97 strengthened lateral cell-cell contacts between enterocytes, which, in contrast, were weakened in CD97 knockout (Ko) mice. Transepithelial resistance was not altered in Tg and Ko mice, indicating that tight junctions were not affected. In Tg murine and normal human colonic enterocytes as well as in colorectal cell lines CD97 was localized preferentially in E-cadherin-based adherens junctions. CD97 overexpression upregulated membrane-bound but not cytoplasmic or nuclear beta-catenin and reduced phospho-beta-catenin, labeled for degradation. This was associated with inactivation of glycogen synthase kinase-3beta (GSK-3beta) and activation of Akt. In summary, CD97 increases the structural integrity of enterocytic adherens junctions by increasing and stabilizing junctional beta-catenin, thereby regulating intestinal epithelial strength and attenuating experimental colitis.

Figure 1. Generation of CD97 Tg mice that overexpress CD97 in intestinal cells.

A Schematic representation of the pBS-KS-Villin-CD97 construct showing the villin promoter region (hatched box), the mouse CD97 cDNA containing EGF-like domains 1 to 4, and the SV40 polyadenylation sequence (dotted box). B Three CD97 Tg lines with different CD97 cDNA copy numbers were established (n = 3 mice/line, mean ± SEM). C CD97 Tg mice showed impaired reproductive performance. The number of births per mating pair correlated inversely with the CD97 cDNA copy number integrated (n = 6 mating pairs over 4 months). The percentage of stillbirths dramatically increased in the Tg3 line. The number of pups per birth was comparable between Tg and WT mice. (***, p<0.001). D Quantitative RT-PCR of tissues from WT and Tg mice. Tissues with a high percentage of villin-positive cells as the small and large intestine expressed high levels of CD97 mRNA in Tg mice. E Flow-cytometric analysis revealed comparable levels of CD97 expression in splenic lymphocytes of WT and Tg mice. In contrast, enterocytes were CD97 negative in WT but strongly CD97 positive in Tg2 mice. F CD97 immunostaining (c = negative control) of tissues of WT (a, c, e) and Tg (b, d, f) mice. In WT colon (a), cells of the lamina propria mucosae (arrow) were stained whereas in the colon of Tg1 (b) and Tg2 (d) mice, epithelial cells were CD97 positive. The muscular coat (asterisk) slightly stained for CD97. In the spleen (e, f), leukocytes equally expressed CD97 in WT and Tg mice. Scale bar = 50 µm. G Western blot analysis of CD97 with α- and β-chain-specific antibodies revealed strong signals in the small intestine of Tg1 but not WT mice. In the spleen, CD97 was detected in WT and Tg mice. Two bands at 80 and 85 kDa, derived from the α-chain-specific antibody, probably present alternatively spliced CD97 isoforms.

Figure 2. CD97 Tg mice are protected from colitis in the AOM/DSS model.

A Treatment protocol: Mice underwent three cycles of AOM/DSS treatment (n = 16/group). B Intestinal CD97 overexpression changed the number of tumors in the colon of Tg mice (*, p<0.05). C–F Disease activity index (DAI) and separate scores of this index during the first treatment cycle causing acute colitis in WT animals. Disease activity (C), fecal blood (D), diarrhea (E), and weight loss (F) were strongly reduced in Tg mice. Lowest disease activity was found in Tg2 mice.

Figure 3. CD97 Tg mice show reduced clinical and microscopic signs of DSS-induced colitis.

A Treatment protocol: Mice were killed at days 4, 7, or 27 after initiation of the experiment (n = 8/group/time point of termination). Shown here is the disease activity index (DAI) of mice that stayed in the experiment for 27 days. B Crypt damage and inflammation of the distal colon was strongly reduced in Tg2 (b, d) compared to WT mice (a, c) at day 7. Only few neutrophils (arrow) were detected in the distal colon of Tg mice. HE- (a, b) and myeloperoxidase- (c, d) stained sections; scale bar = 20 µm. C Histological examination at days 4 and 7 revealed gradually lowered colitis in Tg mice depending on the CD97 cDNA copy number integrated (*, p<0.05; **, p<0.01). D Decreased secretion of CXCL1 in whole colon cultures of Tg mice at day 7 (*, p<0.05). E Colon shortening as a hallmark of DSS-induced damage was stronger in WT compared to Tg mice at days 4, 7, and 27 (*, p<0.05; **, p<0.01). F Increase in spleen and mesenteric lymph node weight as signs of local and systemic activation of the immune system were reduced in Tg mice at days 4, 7, and 27 (*, p<0.05).

Figure 4. Overexpression of CD97 strengthens apicolateral cell-cell contacts between enterocytes.

A Examination by electron microscopy revealed a correlation between CD97 expression and the formation of cell-cell junctions (arrows). In CD97 Tg mice especially the cytoplasmic sites of the contacts were condensed, and microfilaments were pronounced. Upper images, scale bar = 1 µm; lower images, scale bar = 0.25 µm. B Transepithelial resistance in the distal colon was comparable between untreated WT, Tg, and Ko mice (n = 4). C Transepithelial resistance remained comparable between WT and Tg mice after 4, 7, and 27 days of DSS-treatment (n = 4).

Figure 5. CD97 is localized in adherens junctions.

A CD97 (green) was concentrated at the apicolateral region of colonic enterocytes of Tg mice as shown by doublestaining with the epithelial cell marker EpCAM (red). CD97 co-localized with the adherens junction receptor E-cadherin. In contrast, ZO-1 and desmoplakin as marker for tight junctions and desmosomes, respectively, showed only partial co-localization with CD97. Scale bar = 10 µm. B In polarized HT-29/B6 human colorectal carcinoma cells, CD97 co-localized with β-catenin but not with ZO-1 or desmoplakin. Photographs for ZO-1 co-staining were taken from the most apical part of the cells. Scale bar = 10 µm. C (a) Immunostaining of HT29/B6 cells for CD97 (green) showed partial co-localization in the lateral cell-cell contacts with cortical F-actin (red). (b) F-actin disappeared after destruction by cytochalasin while CD97 remained partly in the cortical belt (arrow, cell-cell contacts). Scale bar = 10 µm. D In human colonic enterocytes, CD97 had a similar distribution as β-catenin in lateral cell contacts. In biopsies of ulcerative colitis, CD97 expression at cell-cell contacts was partly lost. Infiltrating leukocytes strongly expressed CD97. Scale bar = 20 µm (a, b, e, f) or 10 µm (c, d, g, h).

Figure 6. CD97 stabilizes membrane-associated β-catenin.

A Western blot analysis of colonic lysates revealed that α-catenin, β-catenin, and p120 catenin were increased in Tg compared to WT mice. Lowest levels of these catenins were seen in Ko mice. Expression of E-cadherin was not affected. α-tubulin was used as a loading control. B Western blot analysis of the membrane, cytoplasmic and nuclear fraction of colonic lysates showed an increase only in membrane-associated β-catenin in Tg mice. C Phosphorylation of β-catenin, leading to its degradation, inversely correlated with the amount of CD97 in colonic lysates. Phosphorylation of the kinases GSK-3β and Akt that are involved in the regulation of β-catenin degradation was regulated by CD97 (see Results for details). The same lysates as in (A) were analyzed. A–C All experiments were performed in triplicate, typical runs are shown. Colonic lysates of the various mouse lines were always analyzed in one run in parallel.