Cellular internalization of proinsulin C-peptide.

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37 degrees C for up to 1 h, and was reduced at 4 degrees C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action.

Carcinoembryonic antigen-related cell adhesion molecule 1 on murine dendritic cells is a potent regulator of T cell stimulation.

Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1alpha, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4(+) T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.

The characteristic cellular organization and CEACAM1 expression in the junctional epithelium of rats and mice are genetically programmed and not influenced by the bacterial microflora.

BACKGROUND: The epithelial cell adhesion molecule CEACAM1 exhibits an interesting dynamic expression during tooth development. It is first expressed in the reduced enamel epithelium, its expression then increases in the orally faced reduced epithelium and the overlying oral epithelium that then fuse to give rise to the junctional epithelium. The expression of CEACAM1 remains at high levels in the junctional epithelium, in contrast to the surrounding oral sulcular epithelium which shows much lower expression levels. We investigated if the high expression levels of CEACAM1 and the loosely organized cells characteristic of the junctional epithelium are genetically programmed or result from bacterial infiltration. METHODS: Oral tissues from germ-free rats and mice and animals with conventional bacterial flora were analyzed by transmission electron microscopy and immunohistochemical staining for CEACAM1. RESULTS: The junctional epithelium of both germ-free and conventional animals was identical with respect to both CEACAM1 expression and morphology. Also the presence of leukocytes was the same in both types of animals. CONCLUSIONS: The results indicate that the characteristic morphology and the high expression levels of CEACAM1 in the junctional epithelium are genetically programmed and not a result of bacterial infiltration. This suggests that CEACAM1 has an important role for the structural integrity of the junctional epithelium. This conclusion was supported by the observation that the junctional epithelium does not express any E-cadherin, which is another abundant epithelial cell adhesion molecule.

The tumor growth-inhibiting cell adhesion molecule CEACAM1 (C-CAM) is differently expressed in proliferating and quiescent epithelial cells and regulates cell proliferation.

The homophilic cell adhesion molecule CEACAM1 (C-CAM, BGP, CD66a) occurs as two coexpressed isoforms, CEACAM1-L and CEACAM1-S, in epithelia, endothelia, and leukocytes. CEACAM1-L can inhibit tumor growth; this effect is influenced by CEACAM1-S. To characterize the growth regulatory properties of CEACAM1, we analyzed the expression patterns of the isoforms, and here we demonstrate that both the expression levels and the S:L isoform ratios differ in proliferating and quiescent rat epithelial cells. Quiescent prostate NbE cells expressed more CEACAM1 than quiescent bladder NBT-II cells, a pattern that correlated with the expression levels in the parental tissues. In contrast, both the expression levels and the isoform ratios were strikingly similar in proliferating NbE and NBT-II cells, showing that a particular CEACAM1 expression pattern is compatible with cell proliferation. However, in confluent cells, CEACAM1 seemed to exert inhibitory effects on cell proliferation. Addition of anti-CEACAM1 antibodies to quiescent, confluent cells caused decreased expression of the cyclin-dependent kinase inhibitor, p27Klp1, stimulated growth factor-dependent DNA synthesis, and altered the S:L isoform ratio toward the ratio characteristic of proliferating cells. Taken together, our data suggest that CEACAM1 contributes to contact inhibition of cell proliferation in confluent cells but allows proliferation when expressed at different isoform ratios.

The cell adhesion molecule C-CAM is a substrate for tissue transglutaminase.

C-CAM, a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family, appears as two co-expressed isoforms, C-CAM-L and C-CAM-S, with different cytoplasmic domains, that can form homodimers in epithelial cells. In addition, C-CAM-L has been found in large molecular weight forms suggesting posttranslational, covalent modification. Here we have investigated the possibility that the cytoplasmic domain of C-CAM-L can act as a transglutaminase substrate. Glutathione S-transferase fusion proteins of the cytoplasmic domains of rat and mouse C-CAM-L as well as free cytoplasmic domains, released by thrombin cleavage from the fusion proteins, were converted into covalent dimers by tissue transglutaminase. These results demonstrate that the cytoplasmic domains of rat and mouse C-CAM-L are substrates for tissue transglutaminase, and lend support to the notion that higher molecular weight forms of C-CAM-L are formed by transglutaminase modification.

Characterization of protein kinase C-mediated phosphorylation of the short cytoplasmic domain isoform of C-CAM.

C-CAM is a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family. Two co-expressed isoforms, C-CAM-L and C-CAM-S, are known, having different cytoplasmic domains both of which can be phosphorylated in vivo. Here we have characterized the PKC-mediated phosphorylation of the short cytoplasmic domain isoform, C-CAM-S. Phorbol myristyl acetate induced phosphorylation of C-CAM-S in transfected CHO cells. Using synthetic peptides and Edman degradation we identified Ser449 as the PKC-phosphorylated amino acid residue. Binding experiments with modified peptides indicated that this phosphorylation decreases the ability of the cytoplasmic domain of C-CAM-S to bind calmodulin.

Dynamic expression of E-cadherin in ameloblasts and cementoblasts in mice.

During embryonic development, E-cadherin mediates intercellular adhesion in a variety of epithelia in a spatio-temporal pattern. We have analyzed the distribution of this cell adhesion molecule in the mouse during odontogenesis, at both mRNA and protein levels, in the mandibular first molars and incisors. E-cadherin was strongly expressed at the bell stage by the cells of the dental organ, and by the pre-secretory ameloblasts and the cells of stratum intermedium at the early mineralization stage. At the onset of enamel secretion, E-cadherin disappeared from the apical pool of the ameloblasts and was later absent from the post-secretory ameloblasts. E-cadherin was also found in Hertwig's root sheath and later in the cells producing acellular cementum. These findings indicate that E-cadherin may be involved in the polarization of the ameloblasts and in the early stages of cementogenesis.

CEA adhesion molecules: multifunctional proteins with signal-regulatory properties.

The carcinoembryonic antigen family comprises a large number of complex molecules, several of which possess cell adhesion activities. The primordial adhesion molecules of this family are the cell-cell adhesion molecules (C-CAMs), which have been found to be multifunctional, signal-regulatory proteins. C-CAMs inhibit tumor growth, interact with calmodulin, protein tyrosine kinases and protein tyrosine phosphatases, and are subject to specific dimerization reactions. These new insights indicate that C-CAMs are important regulators of cellular functions.

Spatiotemporal expression of C-CAM in the rat placenta.

We investigated the expression of the immunoglobulin superfamily cell adhesion molecule, C-CAM, in developing and mature rat placenta. By immunohistochemical staining at the light microscopic level, no C-CAM-expression was seen before Day 9 of gestation, when it appeared in the trophoblasts of ectoplacental cones. On Day 10.5, spongiotrophoblasts and invasive trophoblasts around the maternal vessels of the decidua basalis were stained positively. On Day 12.5, C-CAM was detected in the spongiotrophoblasts of the junctional layer, but labyrinth trophoblasts and secondary giant trophoblasts were not stained. On Day 17.5, C-CAM was found only in the labyrinth and lacunae of the junctional layer. At this stage, both the labyrinth cytotrophoblasts of the maternal blood vessels and the endothelial cells of the embryonic capillaries were strongly stained. Placental tissues from gestational Days 12.5 and 17.5 were analyzed by immunoelectron microscopy to determine the location of C-CAM at the subcellular level. On Day 12.5, positive staining of the spongiotrophoblasts was observed, mainly on surface membranes and microvilli between loosely associated cells. On Day 17.5, staining was found primarily on the microvilli of the maternal luminal surfaces of the labyrinth cytotrophoblasts, and both on the luminal surface and in the cytoplasm of endothelial cells of the embryonic vessels. RT-PCR analysis and Southern blotting of the PCR products revealed expression of mRNA species for both of the major isoforms, C-CAM1 and C-CAM2. Immunoblotting analysis of C-CAM isolated from 12.5-day and 14.5-day placentae showed that it appeared as a broad band with an apparent molecular mass of 110-170 kD. In summary, C-CAM was strongly expressed in a specific spatiotemporal pattern in trophoblasts actively involved in formation of the placental tissue, suggesting an important role in placental development. In the mature placenta, C-CAM expression was confined to the trophoblastic and endothelial cells lining the maternal and embryonic vessels, respectively, suggesting important functions in placental physiology.

Evidence for regulated dimerization of cell-cell adhesion molecule (C-CAM) in epithelial cells.

C-CAM is a Ca(2+)-independent cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily. Addition of chemical cross-linkers to isolated rat liver plasma membranes, intact epithelial cells and purified preparations of C-CAM stabilized one major C-CAM-containing product whose apparent molecular mass was approximately twice that of the C-CAM monomer. The failure to detect additional proteins after cleavage of the cross-linked species demonstrated that C-CAM exists as non-covalently linked dimers both in solution and on the cell surface. Dimerization occurred to the same extent in adherent monolayers and in single cell populations, indicating that dimer formation was the result of cis-interactions within the membranes of individual cells. Using isoform-specific anti-peptide antibodies, both C-CAM1 and C-CAM2 were found to be involved in dimerization, forming predominantly homo-dimeric species. Both calmodulin and Ca2+ ionophore modulated the level of dimer formation, suggesting a role for regulated self-association in the functional activity of C-CAM.

Homophilic intercellular adhesion mediated by C-CAM is due to a domain 1-domain 1 reciprocal binding.

The cell adhesion molecule C-CAM belongs to the immunoglobulin superfamily and is expressed in epithelia, vessel endothelia, and hematopoietic cells. Differential splicing gives rise to different isoforms, of which the major two are C-CAM1 and C-CAM2, which both have four Ig-like domains in their extracellular portions, but differ in their cytoplasmic domains. Two different allelic variants of C-CAM, named a and b, occur in the rat. The adhesive binding mechanism(s) of C-CAM is not known in detail. Evidence for both homophilic and heterophilic binding has been presented, and different species and splice variants of C-CAM have shown differences in temperature and cation dependence when expressed in different cell types. Here, we have analyzed the binding mechanism of rat C-CAM2a that was expressed in CHO cells. In this system C-CAM2a-mediated adhesion was calcium- and temperature-independent. C-CAM2a-transfected cells did not adhere to nontransfected cells, demonstrating that the binding was homophilic. Cells transfected with C-CAM2a in which the N-terminal Ig-domain (D1) was deleted did not aggregate, and cells with intact C-CAM2a could not bind to these cells. This was in contrast to cells that were transfected with C-CAM2a in which the fourth Ig-like domain (D4) had been deleted; they both aggregated and bound to cells with intact C-CAM2a. Thus, C-CAM2a mediates intercellular adhesion of CHO cells by a homophilic mechanism, in which the D1 domain binds reciprocally to a D1 domain on an opposed C-CAM molecule.

Calmodulin binds to specific sequences in the cytoplasmic domain of C-CAM and down-regulates C-CAM self-association.

C-CAM is a cell adhesion molecule belonging to the immunoglobulin supergene family and is known to mediate calcium-independent homophilic cell-cell binding. Two major isoforms, C-CAM1 and C-CAM2, which differ in their cytoplasmic domains, have been identified. Previous investigations have demonstrated that both cytoplasmic domains can bind calmodulin in a calcium-dependent reaction. In this investigation, peptides corresponding to the cytoplasmic domains of C-CAM were synthesized on cellulose membranes and used to map the binding sites for 125I-labeled calmodulin. Both C-CAM1 and C-CAM2 had one strong calmodulin-binding site in the membrane-proximal region. Those binding regions were conserved in C-CAM from rat, mouse, and man. In addition, C-CAM1 from rat and mouse contained a weaker binding site in the distal region of the cytoplasmic domain. Biosensor experiments were performed to determine rate and equilibrium constants of the C-CAM/calmodulin interaction. An association rate constants of 3.3 x 10(5) M-1 s-1 and two dissociation rate constants of 2.2 x 10(-2) and 3.1 x 10(-5) s-1 were determined. These correspond to equilibrium dissociation constants of 6.7 x 10(-8) and 9.4 x 10(-11) M, respectively. In dot-blot binding experiments, it was found that binding of calmodulin causes a down-regulation of the homophilic self-association of C-CAM. This suggests that calmodulin can regulate the functional activity of C-CAM.

Simultaneously synthesized peptides on continuous cellulose membranes as substrates for protein kinases.

Sets of peptides with defined sequences, each on a separate spot, were synthesized simultaneously on continuous cellulose membranes (SPOTs membranes), which were originally designed for epitope studies. The applicability of the membrane-bound peptides as substrates for protein kinases was tested using protein kinase A, protein kinase C and casein kinases I and II as model enzymes. We found that the peptide-membrane complexes can serve as kinase substrates. Our results suggest that membrane-bound peptides offer a new potential for the investigation of substrate specificity of protein kinases. An advantage to this method is that there is no need for substrate identification and separation, which is required with high-volume random peptide libraries. Membrane-bound peptides may even form a basis for kinase assays with peptides lacking multiple basic amino acids, required for separation of the substrates in conventional assays. Problems connected with protein kinase substrate specificity can be investigated in any laboratory using the rapid and inexpensive SPOTs technique, as neither costly apparatus nor special experience in peptide synthesis is necessary.

Cell adhesion activity of the short cytoplasmic domain isoform of C-CAM (C-CAM2) in CHO cells.

C-CAM is a Ca(2+)-independent rat cell adhesion molecule belonging to the CEA gene family of the immunoglobulin superfamily. Two major isoforms that differ in the length of their cytoplasmic domains exist. In previous studies it has been reported that only the long isoform (C-CAM1) but not the short isoform (C-CAM2) can mediate adhesion. However, in the mouse, isoforms with both long and short cytoplasmic domains have been reported to have adhesive activity. In order to analyze this apparent conflict we transfected C-CAM1 or C-CAM2 into CHO Pro5 cells and examined their adhesive phenotype in an aggregation assay. We found that in this cellular system both C-CAM1 and C-CAM2 could mediate cell-cell adhesion in a Ca(2+)-independent and temperature-independent way. The results suggest that the cellular environment is important for the activity of C-CAM isoforms.

IL-4-induced B cell migration involves transient interactions between beta 1 integrins and extracellular matrix components.

IL-4 has previously been shown to stimulate motile responses in murine B lymphocytes. This was studied as acquisition of motile morphology and migration through filters in microchemotaxis chambers. In this paper, we investigated IL-4-stimulated migration of B cells into gels of native collagen fibers, which may be a more physiologically relevant assay. When IL-4 was present in the gel and/or in the medium above, B cells were able to invade the collagen gel. Migration was dependent on the dose of IL-4 and was optimal after 45 h of incubation. It appeared that IL-4 acted by inducing both chemokinesis and chemotaxis. Fibronectin (FN) was found to be an important factor for B cell locomotion, since low concentrations of FCS or FN in the gel matrix greatly improved migration. B cell locomotion was inhibited by antibodies specific for beta 1, alpha 4 and alpha 5 integrins, indicating the presence of integrin-extracellular matrix (ECM) interactions in lymphocyte motility responses. Migration was not associated with an up-regulation of beta 1, alpha 4 or alpha 5 integrins. The adhesion between substrate and cells is likely to be of low affinity, since IL-4-stimulated, as well as non-stimulated B cells, did not adhere to ECM-coated culture wells. Our data suggest that transient interactions between integrins and the ECM matrix may favour B cell migration.

Localization of NCAM on NCAM-B-expressing cells with inhibited migration in collagen.

The extracellular matrix is a key element in neuronal development and tumour invasion, providing a substratum which sustains the adhesion and migration of cells. In order to study interactions between the neural cell adhesion molecule (NCAM) and collagen, we transfected mouse L cells with cDNA encoding the human transmembrane NCAM isoform of 140 kDa (NCAM-B). An L-cell/collagen type I system was used to study the influence of NCAM expression on in vitro invasion. We here report that migration of NCAM-expressing cells in collagen was inhibited compared to that of NCAM-negative cells transfected with the empty vector. Immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold electron microscopy using anti-human NCAM antibodies demonstrated a heterogeneous distribution of NCAM on the plasma membrane of transfected L cells grown on collagen. NCAM was preferentially located at the surface of broad cytoplasmic protrusions and slender extensions, some of which were facing the collagen. This was in contrast to the homogeneous surface distribution of NCAM on cells grown on plastic. These data suggest that NCAM and collagen type I interact, and that this might lead to the migration inhibition of NCAM-expressing cells.

C-CAM expression in odontogenesis and tooth eruption.

The distribution of the cell adhesion molecule C-CAM was analyzed during tooth development, eruption and formation of the junctional epithelium in rat molars by immunohistochemistry and in situ hybridization. C-CAM could not be detected during odontogenesis until the late bell stage; then only the mRNA was demonstrated in the odontoblasts and ameloblasts. Prior to eruption, a local increase in C-CAM (mRNA and protein) was observed in the reduced enamel epithelium. During eruption, high C-CAM levels were seen in the fusion zone between the oral epithelium and the reduced enamel epithelium. In the adult rat, C-CAM remained strongly expressed in the junctional epithelium. Our study indicates that C-CAM may play a role in odontogenesis and during formation of the epithelial structures involved in tooth eruption and formation of the junctional epithelium.

Expression of E-cadherin during craniofacial development.

The cell adhesion molecule E-cadherin is assumed to play an important role in organogenesis and histogenesis. We have analyzed the presence of E-cadherin during normal and disturbed craniofacial development with respect to palate and tooth formation by immunohistochemistry using a rat monoclonal antibody (DECMA-1) and by in situ hybridization using an oligonucleotide probe. Cleft palate was induced by retinoic acid (RA) treatment of pregnant dams. Normal and RA-treated fetuses of gestational days 14-18 were examined. E-cadherin was present in epithelia of both ectodermal and endodermal origin, including developing teeth and epithelia of the palate as well as respiratory and oral epithelia. The expression level of E-cadherin increased with age and differentiation. In normal fetuses, at day 18, the expression was higher in the epithelia of the oral cavity than in the forming nasal cavity. The expression pattern of E-cadherin implies that this molecule has a role during normal development of the epithelia of the craniofacial complex.