Mutational analysis of the cytoplasmic domain of CEACAM1-4L in humanized mammary glands reveals key residues involved in lumen formation: Stimulation by Thr-457 and inhibition by Ser-461

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion, undergoes extensive alternative splicing, resulting in isoforms with 1-4 Ig-like extracellular domains (ECDs) with either long or short cytoplasmic tails. We have previously shown that CEACAM1-4L (4 ECDs with a long cytoplasmic domain) formed glands with lumena in humanized mammary mouse fat pads in NOD/SCID mice. In order to identify the key residues of CEACAM1-4L that play essential roles in lumen formation, we introduced phosphorylation mimic (e.g., Thr-457 or Ser-461 to Asp) or null mutations (Thr-457 or Ser-461 to Ala) into the cytoplasmic domain of CEACAM1-4L and tested them in both the in vivo mouse model and in vitro Matrigel model of mammary morphogenesis. MCF7 cells stably expressing CEACAM1-4L with the single mutation T457D or the double mutant T457D+S461D, but not the null mutants induced central lumen formation in 3D Matrigel and in humanized mammary fat pads. However, the single phosphorylation mimic mutation S461D, but not the null mutation blocked lumen formation in both models, suggesting that S461 has inhibitory function in glandular lumen formation. Compared to our results for the -4S isoform (Chen et al., J. Biol. Chem, 282: 5749-5760, 2008), the T457A null mutation blocks lumen formation for the -4L but not for the -4S isoform. This difference is likely due to the fact that phosphorylation of S459 (absent in the -4L isoform) positively compensates for loss of T457 in the -4S isoform, while S461 (absent in the -4S isoform) negatively regulates lumen formation in the -4L isoform. Thus, phosphorylation of these key residues may exert a fine control over the role of the -4L isoform (compared to the -4S isoform) in lumen formation.     

Carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin

CEA cell adhesion molecule 1 (CEACAM1), a type 1 transmembrane and homotypic cell adhesion protein belonging to the carcinoembryonic antigen (CEA) gene family and expressed on epithelial cells, is alternatively spliced to produce four major isoforms with three or four Ig-like ectodomains and either long (CEACAM1-L) or short (CEACAM1-S) cytoplasmic domains. When murine MC38 (methylcholanthrene-induced adenocarcinoma 38) cells were transfected with human CEACAM1-L and stimulated with sodium pervanadate, actin was found to co-localize with CEACAM1-L at cell-cell boundaries but not in untreated cells. When CEACAM1-L was immunoprecipitated from pervanadate-treated MC38/CEACAM1-L cells and the associated proteins were analyzed by two-dimensional gel analysis and mass spectrometry, actin and tropomyosin, among other proteins, were identified. Whereas a glutathione S-transferase (GST) fusion protein containing the l-isoform (GST-Cyto-L) bound poorly to F-actin in a co-sedimentation assay, the S-isoform fusion protein (GST-Cyto-S) co-sedimented with F-actin, especially when incubated with G-actin during polymerization (K(D) = 7.0 microm). Both GST-Cyto-S and GST-Cyto-L fusion proteins bind G-actin and tropomyosin by surface plasmon resonance studies with binding constants of 0.7 x 10(-8) and 1.0 x 10(-7) m for GST-Cyto-L to G-actin and tropomyosin, respectively, and 3.1 x 10(-8) and 1.3 x 10(-7) m for GST-Cyto-S to G-actin and tropomyosin, respectively. Calmodulin or EDTA inhibited binding of the GST-Cyto-L fusion protein to G-actin, whereas calmodulin and G-actin, but not EDTA, stimulated binding to tropomyosin. A biotinylated 14-amino acid peptide derived from the juxtamembrane portion of the cytoplasmic domain of CEACAM1-L associated with both G-actin and tropomyosin with K(D) values of 1.3 x 10(-5) and 1.8 x 10(-5) m, respectively. These studies demonstrate the direct interaction of CEACAM1 isoforms with G-actin and tropomyosin and the direct interaction of CEACAM1-S with F-actin.     

Role of calpain-9 and PKC-δ in the apoptotic mechanism of lumen formation in CEACAM1 transfected breast epithelial cells

CEACAM1-4S (carcinoembryonic antigen-related cell adhesion molecule 1) is a type I membrane protein with a short (12-amino acid) cytoplasmic tail. Wild type CEACAM1-4S-transfected MCF7 cells form glands with lumena when grown in 3D culture, while null mutations of two putative phosphorylation sites (T457A and S459A) in the cytoplasmic domain fail to undergo lumen formation. When gene chip analysis was performed on mRNA isolated from both wild type and T457A,S459A mutated CEACAM1-4S-transfected MCF7 cells grown in 3D culture, calpain-9 (CAPN9) was identified out of over 400 genes with a > 2 log 2 difference as a potential inducer of lumen formation. Inhibition of CAPN9 expression in MCF7/CEACAM1-4S cells by RNAi or by calpeptin or PD150606 inhibited lumen formation. Transfection of CAPN9 into wild type MCF7 cells restores lumen formation demonstrating that calpain-9 may play a critical role in lumen formation. Additionally, we demonstrate that the apoptosis related kinase, PKC-δ, is activated by proteolytic cleavage during lumen formation exclusively in wild type CEACAM1-4S-transfected MCF7 cells grown in 3D culture and that lumen formation is inhibited by either RNAi to PKC-δ or by the PKC-δ inhibitor rottlerin.     

Phosphorylation of CEACAM1 Molecule by Calmodulin Kinase IID in a Three-dimensional Model of Mammary Gland Lumen Formation

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1), a transmembrane protein, expressed on normal breast epithelial cells is down-regulated in breast cancer. Phosphorylation of Thr-457 on the short cytoplasmic domain isoform (CEACAM1-SF) that is predominant in normal epithelial cells is required for lumen formation in a three-dimensional model that involves apoptosis of the central acinar cells. Calmodulin kinase IID (CaMKIID) was selected as a candidate for the kinase required for Thr-457 phosphorylation from a gene chip analysis comparing genes up-regulated in MCF7 cells expressing wild type CEACAM1-SF compared with the T457A-mutated gene (Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749–5760). Up-regulation of CaMKIID during lumen formation was confirmed by analysis of mRNA and protein levels. CaMKIID was able to phosphorylate a synthetic peptide corresponding to the cytoplasmic domain of CEACAM1-SF and was covalently bound to biotinylated and T457C-modified peptide in the presence of a kinase trap previously described by Shokat and co-workers (Maly, D. J., Allen, J. A., and Shokat, K. M. (2004) J. Am. Chem. Soc. 126, 9160–9161). When cell lysates from wild type-transfected MCF7 cells undergoing lumen formation were incubated with the peptide and kinase trap, a cross-linked band corresponding to CaMKIID was observed. When these cells were treated with an RNAi that inhibits CaMKIID expression, lumen formation was blocked by over 90%. We conclude that CaMKIID specifically phosphorylates Thr-457 on CEACAM1-SF, which in turn regulates the process of lumen formation via apoptosis of the central acinar cells.     

Mutation analysis of the short cytoplasmic domain of the cell-cell adhesion molecule CEACAM1 identifies residues that orchestrate actin binding and lumen formation

CEACAM1-4S (carcinoembryonic antigen cell adhesion molecule 1, with 4 ectodomains and a short, 12-14 amino acid cytoplasmic domain) mediates lumen formation via an apoptotic and cytoskeletal reorganization mechanism when mammary epithelial cells are grown in a three-dimensional model of mammary morphogenesis. We show by quantitative yeast two-hybrid, BIAcore, NMR HSQC and STD, and confocal analyses that amino acids phenylalanine (Phe(454)) and lysine (Lys(456)) are key residues that interact with actin orchestrating the cytoskeletal reorganization. A CEACAM1 membrane model based on vitamin D-binding protein that predicts an interaction of Phe(454) at subdomain 3 of actin was supported by inhibition of binding of actin to vitamin D-binding protein by the cytoplasmic domain peptide. We also show that residues Thr(457) and/or Ser(459) are phosphorylated in CEACAM1-transfected cells grown in three-dimensional culture and that mutation analysis of these residues (T457A/S459A) or F454A blocks lumen formation. These studies demonstrate that a short cytoplasmic domain membrane receptor can directly mediate substantial intracellular signaling.     

Cell–Cell Adhesion Molecule CEACAM1 is Expressed in Normal Breast and Milk and Associates with β1 Integrin in a 3D Model of Morphogenesis

CEA cell adhesion molecule-1 (CEACAM1) is a cell-cell adhesion molecule that, paradoxically, is expressed in an apical location in normal breast epithelium. Strong lumenal membrane staining is observed in 100% of normal glands (11/11), low in atypical hyperplasia (2/6), high in cribiform ductal carcinoma in situ (DCIS) (8/8), but low in other types of DCIS (2/15). Although most invasive ductal carcinomas express CEACAM1 (21/26), the staining pattern tends to be weak and cytoplasmic in tumours with minimal lumena formation (grades 2-3), while there is membrane staining in well-differentiated tumours (grade 1). The 'normal' breast epithelial line MCF10F forms acini with lumena in Matrigel with apical membrane expression of CEACAM1. MCF7 cells that do not express CEACAM1 and fail to form lumena in Matrigel, revert to a lumen forming phenotype when transfected with the CEACAM1-4S but not the -4L isoform. CEACAM1 directly associates with and down-regulates the expression of beta1-integrin. Immuno-electron microscopy reveals numerous vesicles coated with CEACAM1 within the lumena, and as predicted by this finding, CEACAM1 is found in the lipid fraction of breast milk. Thus, CEACAM1 is a critical molecule in mammary morphogenesis and may play a role in the absorption of the lipid vesicles of milk in the infant intestinal tract.     

Homophilic adhesion and CEACAM1-S regulate dimerization of CEACAM1-L and recruitment of SHP-2 and c-Src

Carcinoembryonic antigen (CEA)–related cell adhesion molecule 1 (CAM1 [CEACAM1]) mediates homophilic cell adhesion and regulates signaling. Although there is evidence that CEACAM1 binds and activates SHP-1, SHP-2, and c-Src, knowledge about the mechanism of transmembrane signaling is lacking. To analyze the regulation of SHP-1/SHP-2/c-Src binding, we expressed various CFP/YFP-tagged CEACAM1 isoforms in epithelial cells. The supramolecular organization of CEACAM1 was examined by cross-linking, coclustering, coimmunoprecipitation, and fluorescence resonance energy transfer. SHP-1/SHP-2/c-Src binding was monitored by coimmunoprecipitation and phosphotyrosine-induced recruitment to CEACAM1-L in cellular monolayers. We find that trans-homophilic CEACAM1 binding induces cis-dimerization by an allosteric mechanism transmitted by the N-terminal immunoglobulin-like domain. The balance of SHP-2 and c-Src binding is dependent on the monomer/dimer equilibrium of CEACAM1-L and is regulated by trans-binding, whereas SHP-1 does not bind under physiological conditions. CEACAM1-L homodimer formation is reduced by coexpression of CEACAM1-S and modulated by antibody ligation. These data suggest that transmembrane signaling by CEACAM1 operates by alteration of the monomer/dimer equilibrium, which leads to changes in the SHP-2/c-Src–binding ratio.     

Carcinoembryonic antigen-related cell adhesion molecule 1 expression and signaling in human, mouse, and rat leukocytes: evidence for replacement of the short cytoplasmic domain isoform by glycosylphosphatidylinositol-linked proteins in human leukocytes

Carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1), the primordial carcinoembryonic Ag gene family member, is a transmembrane cell adhesion molecule expressed in leukocytes, epithelia, and blood vessel endothelia in humans and rodents. As a result of differential splicing, CEACAM1 occurs as several isoforms, the two major ones being CEACAM1-L and CEACAM1-S, that have long (L) or short (S) cytoplasmic domains, respectively. The L:S expression ratios vary in different cells and tissues. In addition to CEACAM1, human but not rodent cells express GPI-linked CEACAM members (CEACAM5-CEACAM8). We compared the expression patterns of CEACAM1-L, CEACAM1-S, CEACAM6, and CEACAM8 in purified populations of neutrophilic granulocytes, B lymphocytes, and T lymphocytes from rats, mice, and humans. Human granulocytes expressed CEACAM1, CEACAM6, and CEACAM8, whereas human B lymphocytes and T lymphocytes expressed only CEACAM1 and CEACAM6. Whereas granulocytes, B cells, and T cells from mice and rats expressed both CEACAM1-L and CEACAM1-S in ratios of 2.2-2.9:1, CEACAM1-S expression was totally lacking in human granulocytes, B cells, and T cells. Human leukocytes only expressed the L isoforms of CEACAM1. This suggests that the GPI-linked CEACAM members have functionally replaced CEACAM1-S in human leukocytes. Support for the replacement hypothesis was obtained from experiments in which the extracellular signal-regulated kinases (Erk)1/2 were activated by anti-CEACAM Abs. Thus, Abs against CEACAM1 activated Erk1/2 in rat granulocytes, but not in human granulocytes. Erk1/2 in human granulocytes could, however, be activated by Abs against CEACAM8. We demonstrated that CEACAM1 and CEACAM8 are physically associated in human granulocytes. The CEACAM1/CEACAM8 complex in human cells might accordingly play a similar role as CEACAM1-L/CEACAM1-S dimers known to occur in rat cells.     

Functional interaction between the pro-apoptotic DAP3 and the glucocorticoid receptor

The widely expressed adhesion receptor CEACAM1 is a member of the carcinoembryonic antigen (CEA) family within the immunoglobulin (Ig) superfamily of glycoproteins. While the expression of transmembrane isoforms has been described in detail, only little is known about soluble isoforms. By RT-PCR characterization of rat pheochromocytoma PC12 and mammary adenocarcinoma MTC cell lines, two novel splice variants, designated CEACAM1-4C1 and CEACAM1-4C2, lacking the transmembrane region, were identified. In addition, we demonstrate the expression of transmembrane CEACAM1-4L and CEACAM1-4S with a truncated cytoplasmic domain. The C-termini of CEACAM1-4C2 and CEACAM1-L are identical, which allowed the specific in vitro and in vivo detection of the soluble CEACAM1-4C2 protein by an antiserum generated against the CEACAM1-L cytoplasmic part. Functionally, soluble CEACAM1 could inhibit CEACAM1-mediated aggregation of CHO cells. In conclusion, our data define a new mechanism for the appearance of functionally active rat CEACAM1 protein in body fluids.     

Distinct mechanisms of internalization of Neisseria gonorrhoeae by members of the CEACAM receptor family involving Rac1- and Cdc42-dependent and -independent pathways

Opa adhesins of pathogenic Neisseria species target four members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. CEACAM receptors mediate opsonization-independent phagocytosis of Neisseria gonorrhoeae by human granulocytes and each receptor individually can mediate gonococcal invasion of epithelial cells. We show here that gonococcal internalization occurs by distinct mechanisms depending on the CEACAM receptor expressed. For the invasion of epithelial cell lines via CEACAM1 and CEACAM6, a pathogen-directed reorganization of the actin cytoskeleton is not required. In marked contrast, ligation of CEACAM3 triggers a dramatic but localized reorganization of the host cell surface leading to highly efficient engulfment of bacteria in a process regulated by the small GTPases Rac1 and Cdc42, but not Rho. Two tyrosine residues of a cytoplasmic immune receptor tyrosine-based activating motif of CEACAM3 are essential for the induction of phagocytic actin structures and subsequent gonococcal internalization. The granulocyte-specific CEACAM3 receptor has properties of a single chain phagocytic receptor and may thus contribute to innate immunity by the elimination of Neisseria and other CEACAM-binding pathogens that colonize human mucosal surfaces.     

The transmembrane domain of CEACAM1-4S is a determinant of anchorage independent growth and tumorigenicity

CEACAM1 is a multifunctional Ig-like cell adhesion molecule expressed by epithelial cells in many organs. CEACAM1-4L and CEACAM1-4S, two isoforms produced by differential splicing, are predominant in rat liver. Previous work has shown that downregulation of both isoforms occurs in rat hepatocellular carcinomas. Here, we have isolated an anchorage dependent clone, designated 253T-NT that does not express detectable levels of CEACAM1. Stable transfection of 253-NT cells with a wild type CEACAM1-4S expression vector induced an anchorage independent growth in vitro and a tumorigenic phenotype in vivo. These phenotypes were used as quantifiable end points to examine the functionality of the CEACAM1-4S transmembrane domain. Examination of the CEACAM1 transmembrane domain showed N-terminal GXXXG dimerization sequences and C-terminal tyrosine residues shown in related studies to stabilize transmembrane domain helix-helix interactions. To examine the effects of transmembrane domain mutations, 253-NT cells were transfected with transmembrane domain mutants carrying glycine to leucine or tyrosine to valine substitutions. Results showed that mutation of transmembrane tyrosine residues greatly enhanced growth in vitro and in vivo. Mutation of transmembrane dimerization motifs, in contrast, significantly reduced anchorage independent growth and tumorigenicity. 253-NT cells expressing CEACAM1-4S with both glycine to leucine and tyrosine to valine mutations displayed the growth-enhanced phenotype of tyrosine mutants. The dramatic effect of transmembrane domain mutations constitutes strong evidence that the transmembrane domain is an important determinant of CEACAM1-4S functionality and most likely by other proteins with transmembrane domains containing dimerization sequences and/or C-terminal tyrosine residues.     

Role of CEACAM1 and CEACAM20 in an In Vitro Model of Prostate Morphogenesis

CEACAM20, a novel member of the CEACAM1 gene family with expression limited to the lumen of small intestine, testes, and prostate, is co-expressed with CEACAM1 in adult prostate tissue and down-regulated to the same extent as CEACAM1 in prostate cancer. Since prostate cancer often involves loss of epithelial lumen formation, we hypothesized that CEACAM20 and CEACAM1 play important roles in lumen formation of normal prostate epithelium. When prostate cells were grown on Matrigel as a source of extracellular matrix (ECM), they differentiated into acinar structures with single tubules and well-defined lumina closely resembling embryonic prostate organoids. Confocal microscopic analysis revealed restriction of CEACAM20 to acini and CEACAM1 to tubule structures, respectively. Inhibition of CEACAM1 with antibodies or soluble CEACAM1 or antisense oligonucleotides inhibited tubule formation by over 50% while the remaining tubules were stunted. Inhibition of CEACAM20 with antisense oligonucleotides completely inhibited tubule formation and stunted the growth of acini. We conclude that CEACAM20 and CEACAM1 not only mark the lumina of adult prostate tissue but also play a critical role in the vitro generation of prostate organoids.     

Clustering-induced signaling of CEACAM1 in PC12 cells

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an Ig-like transmembrane protein, functions in cell adhesion, angiogenesis and epithelial cell morphogenesis, and has been identified as a tumor suppressor. For all of these functions, CEACAM1 requires signaling capabilities. However, the mechanisms of CEACAM1-mediated signaling are only poorly understood. Here we characterized for the first time CEACAM1 expression and signaling in the neuroendocrine rat pheochromocytoma PC12 cell line. Stimulation of CEACAM1 by ligation on the cell surface with antibodies induced formation of large CEACAM1 clusters and a rapid and transient CEACAM1 tyrosine dephosphorylation. Functionally, this dephosphorylation correlated with a reduced association between CEACAM1 and the tyrosine phosphatase SHP2. Clustering also stimulated binding of CEACAM1 to the actin cytoskeleton, measured by a partial translocation of CEACAM1 into the insoluble fraction after detergent extraction. Both tyrosine dephosphorylation and interaction with the cytoskeleton were sensitive to neuronal differentiation of PC12 cells. The first detected downstream activation of the mitogen-activated protein kinases ERK1 and ERK2, but not of JNK or p38, describes a novel target of CEACAM1-mediated signaling and contributes to the understanding of how CEACAM1 regulates cellular function.     

Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits proximal TCR signaling by targeting ZAP-70

The long cytoplasmic tail (CT) isoforms of carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1) are expressed on activated human T cells and possess two ITIM motifs in the CT. These isoforms of CEACAM1 are inhibitory for T cell responses initiated by the TCR/CD3 complex with the inhibition dependent upon the ITIMs of CEACAM1 and Src homology 2 domain-containing phosphatase 1 (SHP-1). However, the mechanism by which this inhibition occurs in T cells is unknown. We demonstrate here that the Src family kinase, Lck, and the ability of CEACAM1 to bind homophilically are required for the ITIM phosphorylation of CEACAM1 that is a prerequisite for CEACAM1 association with SHP-1. We further show that CEACAM1 associates with and recruits SHP-1 to the TCR/CD3 complex leading to decreased phosphorylation of CD3-zeta and ZAP-70 and consequently decreased activation of the elements downstream of ZAP-70. This is physiologically relevant because extinction of SHP-1 expression or blockade of homophilic binding by CEACAM1 using a Fab that specifically recognizes the homophilic binding region of human CEACAM1 increases the cytolytic function initiated by the TCR/CD3 complex. These studies show that long CT isoforms of CEACAM1 orchestrate an inhibitory program that abrogates extremely proximal events downstream of the TCR/CD3 complex by focusing on the activation of ZAP-70.     

Carcinoembryonic antigen-related cellular adhesion molecule 1 isoforms alternatively inhibit and costimulate human T cell function

Carcinoembryonic Ag-related cellular adhesion molecule 1 (CEACAM1) represents a group of transmembrane protein isoforms that consist of variable numbers of extracellular Ig-like domains together with either a long cytoplasmic (cyt) tail containing two immunoreceptor tyrosine-based inhibitory motifs or a unique short cyt tail. Although CEACAM1 has been reported to be expressed on the surface of T lymphocytes upon activation, its roles in T cell regulation are controversial due to the lack of functional characterization of each individual CEACAM1 isoform. We thus cotransfected Jurkat T cells with CEACAM1 isoform-encoding constructs and an IL-2 promoter-bearing plasmid or a small interference RNA targeting src homology domain 2 containing phosphatase 1. In a luciferase reporter assay and through measurements of cytokine secretion (IL-2, IL-4, and IFN-gamma), CEACAM1 containing either a long or a short cyt tail inhibited or costimulated, respectively, TCR/CD3 complex plus CD28 mediated activation with the inhibitory functions of the long cyt tail dominating. The inhibitory function of CEACAM1, was dependent upon src homology domain 2 containing phosphatase 1 activity, required both tyrosine residues within the immunoreceptor tyrosine-based inhibitory motif domains of the cyt tail and was mediated through the mitogen-activated protein kinase pathway. CEACAM1-mediated inhibition could be functionally reconstituted by incubation of PBMC with either a CEACAM1-specific mAb or CEACAM1-Fc fusion protein in the presence of an allogeneic or mitogenic stimulus, respectively. These studies indicate that the long and short cyt tails of CEACAM1 serve as inhibitory and costimulatory receptors, respectively, in T cell regulation.     

CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor

CEA-related cell adhesion molecule 1 (CEACAM1) exhibits angiogenic properties in in vitro and in vivo angiogenesis assays. CEACAM1 purified from granulocytes and endothelial cell media as well as recombinant CEACAM1 expressed in HEK293 cells stimulate proliferation, chemotaxis, and capillary-like tube formation of human microvascular endothelial cells. They increase vascularization of chick chorioallantoic membrane and potentiate the effects of vascular endothelial growth factor (VEGF)165. VEGF165 increases CEACAM1 expression both on the mRNA and the protein level. VEGF165-induced endothelial tube formation is blocked by a monoclonal CEACAM1 antibody. These data suggest that CEACAM1 is a major effector of VEGF in the early microvessel formation. Since CEACAM1 is expressed in tumor microvessels but not in large blood vessels, CEACAM1 may be a target for the inhibition of tumor angiogenesis.     

Targeted disruption of the Ceacam1 (MHVR) gene leads to reduced susceptibility of mice to mouse hepatitis virus infection

The CEACAM1 glycoproteins (formerly called biliary glycoproteins; BGP, C-CAM, CD66a, or MHVR) are members of the carcinoembryonic antigen family of cell adhesion molecules. In the mouse, splice variants of CEACAM1 have either two or four immunoglobulin (Ig) domains linked through a transmembrane domain to either a short or a long cytoplasmic tail. CEACAM1 has cell adhesion activity and acts as a signaling molecule, and long-tail isoforms inhibit the growth of colon and prostate tumor cells in rodents. CEACAM1 isoforms serve as receptors for several viral and bacterial pathogens, including the murine coronavirus mouse hepatitis virus (MHV) and Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis in humans. To elucidate the mechanisms responsible for the many biological activities of CEACAM1, we modified the expression of the mouse Ceacam1 gene in vivo. Manipulation of the Ceacam1 gene in mouse embryonic stem cells that contained the Ceacam1a allele yielded a partial knockout. We obtained one line of mice in which the insert in the Ceacam1a gene had sustained a recombination event. This resulted in the markedly reduced expression of the two CEACAM1a isoforms with four Ig domains, whereas the expression of the two isoforms with two Ig domains was doubled relative to that in wild-type BALB/c (+/+) mice. Homozygous (p/p) Ceacam1a-targeted mice (Ceacam1aDelta4D) had no gross tissue abnormalities and were viable and fertile; however, they were more resistant to MHV A59 infection and death than normal (+/+) mice. Following intranasal inoculation with MHV A59, p/p mice developed markedly fewer and smaller lesions in the liver than +/+ or heterozygous (+/p) mice. The titers of virus produced in the livers were 50- to 100-fold lower in p/p mice than in +/p or +/+ mice. p/p mice survived a dose 100-fold higher than the lethal dose of virus for +/+ mice. +/p mice were intermediate between +/+ and p/p mice in susceptibility to liver damage, virus growth in liver, and susceptibility to killing by MHV. Ceacam1a-targeted mice provide a new model to study the effects of modulation of receptor expression on susceptibility to MHV infection in vivo.     

Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (− 8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as − 5 to − 3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased over time. QRT-PCR analysis of the anti-CEACAM1 treated ES cells revealed a significant decrease in the expression of Ceacam1, Pecam1, Tie-1, and Flk-1, while VE-Cad and Tie-2 expression were unaffected. These results suggest that the expression and signaling of CEACAM1 may affect the expression of other factors known to play critical roles in vasculogenesis. Furthermore this 3D model of vasculogenesis in an environment of extracellular matrix may be a useful model for comparison to existing models of angiogenesis.