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Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein-coupled receptor CD97 shows an expression gradient along the crypt-villus axis in the normal human intestine. We here report that transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine. Intestinal enlargement involves an increase in length and diameter but does not affect microscopic morphology, as typical for cylindrical growth. The megaintestine is acquired after birth and before weaning, independent of the genotype of the mother, excluding altered availability of milk constituents as driving factor. CD97 overexpression does not regulate intestinal growth factors, stem cell markers, and Wnt signaling, which contribute to epithelial differentiation and renewal, nor does it affect suckling-to-weaning transition. Consistent with augmented cylindrical growth, suckling but not adult transgenic mice show enlarged crypts and thus more crypt fissions caused by a transient increase of the crypt transit-amplifying zone. Intestinal enlargement by CD97 requires its seven-span transmembrane/cytoplasmic C-terminal fragment but not the N-terminal fragment binding partner CD55. In summary, ectopic expression of CD97 in intestinal epithelial cells provides a unique model for intestinal cylindrical growth occurring in breast-fed infants.

The members of the epidermal growth factor (EGF)-transmembrane (TM)7 family of adhesion class G-protein coupled receptors are abundantly expressed by cells of the myeloid lineage. A detailed investigation of their expression by functional subsets of activated macrophages is still lacking. Therefore, we determined the expression of CD97, EGF module-containing mucin-like receptor (EMR)2 and EMR3 by monocyte-derived macrophages experimentally polarized in vitro. This was compared to three types of disease-associated lipid-laden macrophages displaying an alternatively activated phenotype in situ. Polarization in vitro towards classically activated M1 versus alternatively activated M2 extremes of macrophage activation did not result in a congruent regulation of EGF-TM7 receptor mRNA and protein except for a down-regulation of CD97 by IL-10. In contrast, macrophages handling lipid overload in vivo displayed differences in the expression of CD97 and EMR2. While foamy macrophages in atherosclerotic vessels expressed both CD97 and EMR2, foam cells in multiple sclerosis brain expressed CD97, but only little EMR2. Foam cell formation in vitro by oxidized LDL and myelin did not affect CD97 or EMR2 expression. Gaucher spleen cells accumulating glucosylceramide expressed very high levels of CD97 and EMR2. These findings indicate that complex cellular expression programmes rather than activation modes regulate the expression of EGF-TM7 receptors in macrophages., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.

The heptahelical receptor CD97 is a defining member of the EGF-TM7 family of adhesion class receptors. In both humans and mice, CD97 isoforms are expressed with variable numbers of tandemly arranged N-terminal epidermal growth factor-like domains that facilitate interactions with distinct cellular ligands. Results from treatment of mice with mAbs in various disease models have suggested a role for CD97 in leukocyte trafficking. Here, we aimed to thoroughly characterize the expression profile of CD97, and delineate its biological function. To this end, we applied a novel polyclonal Ab, which is the first antiserum suitable for immunohistochemistry, and combined this analysis with the study of Cd97-lacZ knock-in mice. We show that similar to the situation in humans, hematopoietic, epithelial, endothelial, muscle, and fat cells expressed CD97. Despite this broad expression pattern, the Cd97(-/-) mouse that we created had no overt phenotype, except for a mild granulocytosis. Furthermore, granulocyte accumulation at sites of inflammation was normal in the absence of CD97. Interestingly, application of CD97 mAbs blocked granulocyte trafficking after thioglycollate-induced peritonitis in wild-type but not in knock-out mice. Hence, we conclude that CD97 mAbs actively induce an inhibitory effect that disturbs normal granulocyte trafficking, which is not perturbed by the absence of the molecule.

The EGF-TM7 receptor CD97 shows different features of expression and function in muscle cells compared to hematopoetic and tumor cells. Since the molecular function and regulation of CD97 are poorly understood, this study aimed at defining its basal transcriptional regulation in smooth muscle cells (SMCs). The computational analysis of the CD97 5'-flanking region revealed that the TATA box-lacking promoter possesses several GC-rich regions as putative Sp1/Sp3 binding sites. Transfection studies with serially deleted promoter constructs demonstrated that the minimal promoter fragment resided in the -218/+45 region containing one out of five identified GC-boxes in the leiomyosarcoma cell line SK-LMS-1 and human bronchial smooth muscle cells (HbSMCs). Mutation of the most proximal GC-site in CD97 reporter gene constructs caused a significant decrease in promoter activity. Gel shift assays and chromatin immunoprecipitation revealed that Sp1 and Sp3 bound specifically to the most proximal GC-site. Furthermore, we showed that Sp1 and Sp3 over-expression activates CD97 promoter activity in HEK293 cells. Our data characterize for the first time the activity of the human CD97 promoter which is controlled by Sp1/Sp3 transcription factors in SMCs.

Invasion of colorectal carcinomas (CC) is characterized by nuclear accumulation of beta-catenin, a key component of the Wnt pathway, in scattered tumor cells. beta-catenin acts in cooperation with T-cell factor (Tcf) HMG-box transcription factors as a transcriptional activator of genes involved in tumor progression. Overexpression of CD97, a molecule that correlates with dedifferentiation and tumor stage in CC, parallels to nuclear translocation of beta-catenin. Here, we investigated whether CD97 is a direct beta-catenin/Tcf target gene. SW480 CC cells were used to mimic the phenotypical switch between central and invasive tumor areas. We demonstrate two-fold higher CD97 expression and nuclear beta-catenin accumulation in cells grown at low density compared to cells cultured at high density, showing membrane-associated beta-catenin. This suggests that CD97 expression correlates with the cellular localization of beta-catenin. However, CD97 mRNA expression levels were not affected by Tcf-1 or Tcf-4 as determined in inducible dominant-negative (dn) Tcf CC cell lines. Furthermore, co-expression of wildtype (wt) or S33A mutated beta-catenin with Tcf-4 did not influence CD97 promoter activity. Inhibition of glycogen synthase kinase (GSK)-3beta, a negative regulator of the canonical Wnt pathway, had no influence on CD97 expression levels. In summary, enhanced CD97 expression in CC cells is regulated independent of beta-catenin/Tcf-4, and is thus not a direct target of the canonical Wnt pathway.

CD97, an epidermal growth factor (EGF)-TM7 receptor, is not restricted to hematopoetic and carcinoma cells but is also found on smooth muscle cells (SMC). We have examined its location and biochemical structure in various normal and tumorigenic SMC-containing tissues. SMC of the urinary bladder, lung bronchi and bronchioles, myometrium, and gastrointestinal tract were immunohistologically stained by using monoclonal antibodies (mabs) to the CD97 stalk region (CD97(stalk)). Mabs directed against an N-glycosylation-dependent epitope within the EGF-domains (CD97(EGF)) did not bind to normal SMC. Vascular SMC, which was also CD97(EGF)-negative, showed further CD97 heterogeneity. Only a few, if any, SMC from the aorta or elastic arteries of the systemic circulation were positive for CD97 mRNA and therefore also for CD97(stalk). CD97(stalk)-positive SMC were slightly more numerous in muscular and peripheral arteries. In contrast, most venous SMC expressed CD97(stalk). A comparison with other SMC molecules revealed a similar but not identical staining pattern for CD97(stalk) and desmin. Further CD97 heterogeneity was observed during SMC transformation. All leiomyomas (n=5) and nine out of 21 leiomyosarcomas were positive for both CD97(stalk) and CD97(EGF). As expected, CD97(EGF)-positive SMC tumors expressed partly N-glycosylated CD97. Seven out of 21 leiomyosarcomas were completely devoid of CD97. Thus, CD97 showed variable expression in vascular and biochemical modification in tumorigenic SMC, suggesting that the function of the molecule is specific for the SMC subtype.

CD97 is an EGF-TM7 receptor found on various carcinomas where expression levels correlate with dedifferentiation and tumor stage, smooth muscle cells and leukocytes. CD97 acts as an adhesion molecule by binding to its cellular ligand, CD55. In this study, we demonstrate that 2 immunodominant CD97 epitopes are not equally present in the various cell types. Differences were apparent in gastrointestinal tumors and smooth muscle cells where monoclonal antibodies (mAbs) to the first epidermal growth factor (EGF) domain (CD97(EGF)) showed a more restricted staining pattern than mAbs to the stalk region (CD97(stalk)). This discrepancy was not detectable in cultured gastrointestinal tumor cell lines. In fact, the selection of the CD97 mAb influences the result of clinical studies. Thus, we clarified the reason(s) for these differences in CD97 mAb staining on various cell types. We provide evidence that epitope accessibility for CD97(EGF) mAbs depends on N-glycosylation. Immunoprecipitation of CD97 from the Colo 205 tumor cell line revealed the established 78 and 83 kDa products, while a 52 and 57 kDa band were obtained from smooth muscle cells. N-glycosidase F reduced the size of CD97 in Colo 205 cells to 52-57 kDa. Culturing these cells with tunicamycin resulted in the same decrease in size and impaired CD97(EGF) mAb binding. As shown by site-directed mutagenesis, deletion of the N-glycosylation sites located within the EGF domains efficiently disturbed CD97(EGF) mAb immunoreactivity and, importantly, binding of CD55. In conclusion, CD97(EGF) epitope accessibility for mAbs and ligand binding is influenced by cell type-specific N-glycosylation., ((c) 2004 Wiley-Liss, Inc.)

CD97, a cell surface molecule on immune cells with potential adhesive function, is a member of the epidermal growth factor seven-span transmembrane (EGF-TM7) family. We have cloned and characterized bovine CD97 and determined its expression in various cells and tissues. Based on sequence alignment with human genomic DNA, as well as human cDNA sequences encoding various CD97 isoforms, we predict that bovine CD97 mRNA occurs in four splice variants. The encoded CD97 isoforms (800, 751, 756, and 707 amino acids in length) contain different numbers of EGF-like modules, a stalk region, and a TM7 domain with a cytoplasmic tail. RT-PCR demonstrated expression of CD97 mRNA in leukocytes and several other tissues. In each cell type, CD97 mRNA encoding the shortest isoform, CD97 (EGF 1,2,5), was detected at the highest level, which was consistent with the cell surface expression of a 90-kDa polypeptide precipitated from lysates of peripheral blood mononuclear cells., (Copyright 2004 Elsevier Ltd.)

The expression of CD97, a member of the EGF-TM7 family with adhesive properties, is proportional to the aggressiveness and lymph node involvement in thyroid tumors. CD97 has never been systematically investigated in other tumors. First, we examined colorectal carcinoma cell lines (n = 18) for CD97 expression and regulation. All cell lines were CD97-positive. The level of CD97 in each line correlated with migration and invasion in vitro. This result was confirmed in CD97-inducible Tet-off HT1080 cells. Transforming growth factor-beta, which inhibits proliferation in transforming growth factor-beta-sensitive LS513 and LS1034 cells, down-regulated CD97 in these cell lines. Examining CD97 during sodium butyrate-induced cell differentiation of Caco-2 cells, we could demonstrate a CD97-decreasing effect. Second, we screened 81 colorectal adenocarcinomas by immunohistology for expression of CD97. Normal colorectal epithelium is CD97-negative. Seventy-five of 81 of the carcinomas expressed CD97. The strongest staining for CD97 occurred in scattered tumor cells at the invasion front compared to cells located within solid tumor formations of the same tumor. Carcinomas with more strongly CD97-stained scattered tumor cells showed a poorer clinical stage as well as increased lymph vessel invasion compared to cases with uniform CD97 staining. In summary, CD97 expression correlates with dedifferentiation, migration, and invasion in colorectal tumor cell lines. Moreover, more strongly CD97-stained tumor cells at the invasion front of colorectal carcinomas indicate the involvement of the molecule in tumor migration and invasion.

CD97 expression is related closely to the dedifferentiation and tumor stage in thyroid carcinomas. We systematically examined the role of CD97 and its closest relative, EMR2, in normal and malignant gastric, esophageal, and pancreatic tissue. The normal tissues were EMR2-, whereas CD97 was expressed slightly in the parietal cells of gastric mucosa and in exocrine pancreatic cells. Interestingly, intralobular and interlobular pancreatic ducts were CD97+. All tumors were EMR2-. CD97 was expressed by 44 of 50 gastric, 14 of 18 pancreatic, and 10 of 13 esophageal carcinomas. Of the 44 gastric cancers, 27 showed disseminated or scattered tumor cells at the invasion front with stronger CD97 expression than tumor cells located in solid tumor formations. There was no correlation between CD97 levels in the tumors or soluble CD97 in the serum samples and the clinicopathologic features of the patients. Taken together, significant numbers of gastric, esophageal, and pancreatic carcinomas are CD97+, whereas its homolog, EMR2, does not have any role in such tumors.

EMR2 and CD97, members of the epidermal growth factor (EGF)-TM7 family, show a very high homology. CD97, whose expression is closely related to clinical tumor stage in colorectal carcinomas, potentially functions as an adhesion molecule. Nothing is known about the presence of EMR2 in these tumors. We systematically examined the expression of EMR2 in colorectal carcinoma cell lines and adenocarcinomas. Of 18 cell lines, 10 were only slightly positive for EMR2 according to flow cytometry. Various EMR2 splice variants, including a new isoform, have been detected at the mRNA level. EMR2 expression did not correlate with in vitro migration or invasion capacity of the cell lines. Normal colorectal epithelial cells were EMR2 negative. In contrast to CD97, which is found in most colorectal adenocarcinomas, only 8 of 81 of these tumors expressed EMR2. No correlation was found between EMR2 expression and clinicopathological parameters of the tumors. In summary, a significant but low number of colorectal carcinomas are positive for EMR2, indicating different roles for this molecule and CD97 in these tumors. [ABSTRACT FROM AUTHOR]