Characterizing CEACAM5 interaction with CD8α and CD1d in intestinal homeostasis.

Normal intestinal epithelial cells (IECs) could act as non-professional antigen-presenting cells, selectively activating CD8(+)-suppressor T cells. An epithelial cell surface glycoprotein, gp180, recognized by monoclonal antibodies B9 and L12 was determined to be critical in this process. Purification and sequence analysis of mAb B9 reactive material revealed amino-acid sequence homology with CEACAM5. We demonstrate that CEACAM5 has properties attributed to gp180, such as CD8α binding and activation of CD8-associated Lck. CEACAM5 is the only CEACAM member interacting with CD1d through the B3 domain. Its N domain (recognized by B9) is required for CD8α binding. Removal of the N-domain glycosylated residues reduces B9 recognition, CD8α binding affinity, and activation of LcK. Therefore, conformational changes in CEACAM5 glycosylation site are critical for its interaction with CD8α. CEACAM5-activated CD8(+) T cells acquire the ability to suppress the proliferation of CD4(+) T cells in vitro in the presence of interleukin (IL)-15 or IL-7. We provide new insights into the role of CEACAM5 and define its specific immunoregulatory properties among the CEACAMs expressed on IECs. We suggest that unique set of interactions between CEACAM5, CD1d, and CD8 render CD1d more class I-like molecule, facilitating antigen presentation and activation of CD8(+)-suppressor regulatory T cells.

Host-related carcinoembryonic antigen cell adhesion molecule 1 promotes metastasis of colorectal cancer.

Liver metastasis is the predominant cause of colorectal cancer (CRC)-related mortality in developed countries. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell adhesion molecule with reduced expression in early phases of CRC development and thus functions as a tumor growth inhibitor. However, CEACAM1 is upregulated in metastatic colon cancer, suggesting a bimodal role in CRC progression. To investigate the role of this protein in the host metastatic environment, Ceacam1(-/-) mice were injected intrasplenically with metastatic MC38 mouse CRC cells. A significant reduction in metastatic burden was observed in Ceacam1(-/-) compared with wild-type (WT) livers. Intravital microscopy showed decreased early survival of MC38 cells in Ceacam1(-/-) endothelial environment. Metastatic cell proliferation within the Ceacam1(-/-) livers was also diminished. Bone marrow-derived cell recruitment, attenuation of immune infiltrates and diminished CCL2, CCL3 and CCL5 chemokine production participated in the reduced Ceacam1(-/-) metastatic phenotype. Transplantations of WT bone marrow (BM) into Ceacam1(-/-) mice fully rescued metastatic development, whereas Ceacam1(-/-) BM transfer into WT mice showed reduced metastatic burden. Chimeric immune cell profiling revealed diminished recruitment of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs) to Ceacam1(-/-) metastatic livers and adoptive transfer of MDSCs confirmed the involvement of these immune cells in reduction of liver metastasis. CEACAM1 may represent a novel metastatic CRC target for treatment.

The specificity for the differentiation blocking activity of carcinoembryonic antigen resides in its glycophosphatidyl-inositol anchor.

Ectopic expression of various members of the human carcinoembryonic antigen (CEA) family of intercellular adhesion molecules in murine myoblasts either blocks (CEA, CEACAM6) or allows (CEACAM1) myogenic differentiation. These surface glycoproteins form a subset of the immunoglobulin (Ig) superfamily and are very closely related, but differ in the precise sequence of their external domains and in their mode of anchorage to the cell membrane. CEA and CEACAM6 are glycophosphatidyl-inositol (GPI) anchored, whereas CEACAM1 is transmembrane (TM) anchored. Overexpression of GPI-linked neural cell adhesion molecule (NCAM) p125, also an adhesion molecule of the Ig superfamily, accelerates myogenic differentiation. The molecular requirements for the myogenic differentiation block were investigated using chimeric constructs in which the COOH-terminal hydrophobic domains of CEA, CEACAM1, and NCAM p125 were exchanged. The presence of the GPI signal sequence specifically from CEA in the chimeras was sufficient to convert both CEACAM1 and NCAM into differentiation-blocking proteins. Conversely, CEA could be converted into a neutral protein by exchanging its GPI anchor for the TM anchor of CEACAM1. Since the external domains of CEA, CEACAM1, and NCAM can all undergo homophilic interactions, and mutations in the self-adhesive domains of CEA abrogate its differentiation-blocking activity, the structural requirements for differentiation-inhibition are any self-adhesive domains attached to the specific GPI anchor derived from CEA. We therefore suggest that biologically significant functional information resides in the processed extreme COOH terminus of CEA and in the GPI anchor that it determines.

Radical differences in functions of closely related members of the human carcinoembryonic antigen gene family.

The immunoglobulin superfamily represents an ancient, highly diversified group of cell surface and extracellular molecules responsible for a wide range of molecular and cellular recognition functions. The human carcinoembryonic antigen (CEA) subfamily of the immunoglobulin superfamily presents evidence of continuing diversification of the immunoglobulin family, in that some of its members, including CEA itself and nonspecific cross-reacting antigen (NCA), are expressed only in primates and not in rodents. These "new" members are glycophosphatidylinositol linked to the external cell membrane and are up-regulated in cancer, unlike members present in both rodents and primates, i.e., biliary glycoprotein (BGP), which are transmembrane linked and down-regulated in cancer. CEA, NCA, and BGP have all been shown to function in vitro as intercellular adhesion molecules. We show here that the properties of adhesion are radically different, in that BGP-mediated adhesion is reversibly Ca2+ and Mg2+ dependent, temperature dependent, and ATP inhibitable, whereas CEA- and NCA-mediated adhesion is the opposite in all aspects. Also, the novel double-reciprocal, antiparallel binding observed for CEA-CEA interactions is not seen for BGP. Finally, the myogenic differentiation block demonstrated for the ectopic expression of CEA in myoblasts was also observed for NCA but not for BGP, which is consistent with the changes in expression seen in cancer. The appearance of new CEA family members with such different properties is discussed in the context of evolution and cancer.

Opposite functions for two classes of genes of the human carcinoembryonic antigen family.

The human carcinoembryonic antigen (CEA) family can be divided into two subgroups according to the means of anchorage of member glycoproteins to the cell membrane: glycophosphatidyl inositol (GPI) linkage and transmembrane linkage. The GPI-linked members tend to be up-regulated in human tumours, whereas the transmembrane-linked members tend to be down-regulated. Thus the question as to whether the GPI members could be formally considered to function as oncogenes and the transmembrane members as tumour suppressors deserves consideration. Members of both subgroups function in vitro as intercellular adhesion molecules, but the characteristics of this adhesion, including temperature and divalent-cation dependence, differ markedly between the groups. Even the mechanism of intermolecular adhesion appears to differ fundamentally in that GPI-linked CEA-CEA binding involves a double reciprocal bonding between two domains, whereas transmembrane-linked biliary glycoprotein (BGP)-BGP binding requires only one domain. Finally, the ectopic expression of CEA in myoblasts can block myogenic differentiation leaving the cells with the ability to divide, while expression of BGP does not affect or may even accelerate myogenic differentiation. These differences in phenotypic effects in vitro thus mirror the differences observed in expression in tumours and support the view that the GPI and transmembrane groups have opposite effects on cells in relation to the malignant phenotype.

Human carcinoembryonic antigen, an intercellular adhesion molecule, blocks fusion and differentiation of rat myoblasts.

Human carcinoembryonic antigen (CEA), a widely used tumor marker, is a member of a family of cell surface glycoproteins that are overexpressed in many carcinomas. CEA has been shown to function in vitro as a homotypic intercellular adhesion molecule. This correlation of overproduction of an adhesion molecule with neoplastic transformation provoked a test of the effect of CEA on cell differentiation. Using stable CEA transfectants of the rat L6 myoblast cell line as a model system of differentiation, we show that fusion into myotubes and, in fact, the entire molecular program of differentiation, including creatine phosphokinase upregulation, myogenin upregulation, and beta-actin downregulation are completely abrogated by the ectopic expression of CEA. The blocking of the upregulation of myogenin, a transcriptional regulator responsible for the execution of the entire myogenic differentiation program, indicates that CEA expression intercepts the process at a very early stage. The adhesion function of CEA is essential for this effect since an adhesion-defective N domain deletion mutant of CEA was ineffective in blocking fusion and CEA transfectants treated with adhesion-blocking peptides fused normally. Furthermore, CEA transfectants maintain their high division potential, whereas control transfectants lose division potential with differentiation similarly to the parental cell line. Thus the expression of functional CEA on the surface of cells can block terminal differentiation and maintain proliferative potential.