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B Obrink

Publications and source records attributed to B Obrink.

At least 19 recordsLinked to original sources
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

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.

Animals↗
PubMed1996

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.

Adenosine Triphosphatases↗
PubMed1996

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.

Adenosine Triphosphatases↗
PubMed1996

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.

Adenosine Triphosphatases↗
PubMed1995

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.

Adenosine Triphosphatases↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Ameloblasts↗
PubMed1994

C-CAM expression in the developing rat central nervous system.

C-CAM, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, can mediate intercellular adhesion by homophilic, Ca(2+)-independent binding. Immunohistochemical analysis of adult rat tissues has demonstrated that C-CAM is expressed in various epithelia, vessel endothelia, and hematopoietic cells. By molecular cloning and sequence analysis several isoforms differing both in the extracellular and the cytoplasmic domains have been found. Here we have analyzed the expression of C-CAM in the developing rat central nervous system. No neuronal expression was observed, but biochemical and immunohistochemical analyses demonstrated that C-CAM becomes expressed in the microvessels from embryonic day E-13; the intensity of the staining increased through day E-15 and then gradually decreased during the perinatal and early postnatal period. The expression of C-CAM in the walls of the microvessels was confirmed by in situ hybridization. Immunoelectron microscopy showed that C-CAM was localized both to the abluminal surface of the endothelial cells and to cellular processes of primordial pericytes where these two cell types are in contact with each other. No staining was found on the luminal endothelial cell surfaces or inter-endothelial cell contact areas. During the perinatal period C-CAM also became expressed on the opposite side of the pericytes and on other cells, possibly astrocytes, in contact with these areas of the pericytes. These observations suggest that C-CAM may be involved in heterotypic, homophilic adhesion between endothelial cells, pericytes and astrocytes, and in maturation of the vessel walls.

Adenosine Triphosphatases↗
PubMed1994

Distribution of C-CAM in developing oral tissues.

C-CAM is a cell surface glycoprotein that is involved in cell adhesion and may play a role in histogenesis and organogenesis. It is a member of the carcinoembryonic antigen (CEA) gene family, which is a subfamily of the immunoglobulin gene superfamily. We have analyzed the expression of C-CAM during normal and disturbed craniofacial development in the mouse by immunohistochemistry and in situ hybridization. Developmental disturbances were induced by retinoic acid (RA) treatment of pregnant mice. Normal and malformed fetuses were examined on days 14, 15, 16, 17 and 18 of gestation. The expression of C-CAM was detected first at day 16. With age, the signal became gradually stronger. C-CAM was detected in the epithelia of both ectodermal and mesodermal origin, including oral and respiratory epithelia, epithelia of the developing vessels, glands and their ducts. In the RA-treated fetuses, the expression of C-CAM was higher in the epithelium of the oral cavity than in that of the nasal cavity, with a distinct borderline between differentiating nasal and oral epithelium of the palatal shelves. However, the submucosal nasal glands and ducts showed higher expression than oral glands in both normal and RA-treated mice. The expression of C-CAM did not differ significantly between control and RA-treated animals. The presence of C-CAM in all proliferating craniofacial epithelia indicates that this molecule may play an important role in development.

Adenosine Triphosphatases↗
PubMed1994

Differential regulation of C-CAM isoforms in epithelial cells.

C-CAM is a Ca(2+)-independent cell adhesion molecule (CAM) that mediates intercellular adhesion of isolated rat hepatocytes. It is widely distributed in epithelia, where its presence both at lateral cell borders and on apical cell surfaces suggests that it may have diverse biological functions. Two major isoforms, C-CAM1 and C-CAM2, which differ in the lengths of their cytoplasmic domains, have been identified. The lack of suitable in vitro systems has so far prevented a detailed study of the physiological role of C-CAM in epithelia. We now report on the identification, biochemical characterization and functional analysis of C-CAM isoforms in the established epithelial cell line NBT II, derived from a chemically induced carcinoma of rat bladder. C-CAM in NBT II cells is a 110-115 kDa cell surface glycoprotein located predominantly at sites of cell-cell contact but also present on the apical cell surface. Northern blotting analysis revealed the presence of both C-CAM1 and C-CAM2, with the major transcripts for both isoforms present within the 4.0 kb size range. The dissociation of NBT II cell colonies by anti-C-CAM antibodies indicated that at least one function of C-CAM in these cells is to mediate intercellular adhesion. The maintenance of extensive cell-cell contacts and the expression of C-CAM at the contact sites in cells grown in low Ca2+ medium suggested that, like its counterpart in hepatocytes, C-CAM in NBT II cells may be a Ca(2+)-independent cell-cell adhesion molecule. The co-localization and coordinate reorganization of both C-CAM and actin by anti-C-CAM antibodies indicated that these two proteins were associated and suggested that interactions with the cytoskeleton may be important for the regulation of C-CAM function. The specific upregulation of C-CAM1 in cells induced to undergo epithelial to mesenchymal-like transitions (EMT) by the serum substitute Ultroser G suggested that C-CAM isoforms are important modulators of the adhesive properties of these cells.

Actins↗
PubMed1993

Transmembrane neural cell-adhesion molecule (NCAM), but not glycosyl-phosphatidylinositol-anchored NCAM, down-regulates secretion of matrix metalloproteinases.

During embryogenesis interactions between cells and extracellular matrix play a central role in the modulation of cell motility, growth, and differentiation. Modulation of matrix structure is therefore crucial during development; extracellular matrix ligands, their receptors, extracellular proteinases, and proteinase inhibitors all participate in the construction, maintenance, and remodeling of extracellular matrix by cells. The neural cell-adhesion molecule (NCAM)-negative rat glioma cell line BT4Cn secretes substantial amounts of metalloproteinases, as compared with its NCAM-positive mother cell line BT4C. We have transfected the BT4Cn cell line with cDNAs encoding the human NCAM-B and -C isoforms. We report here that the expression of transmembrane NCAM-B, but not of glycosyl-phosphatidylinositol-linked NCAM-C, induces a down-regulation of 92-kDa gelatinase (matrix metalloproteinase 9) and interstitial collagenase (matrix metalloproteinase 1), indicating that cellular expression of the recognition molecule NCAM regulates the metabolism of the surrounding matrix.

Animals↗
PubMed1993

Evidence for calmodulin binding to the cytoplasmic domains of two C-CAM isoforms.

C-CAM (cell-CAM 105) is a transmembrane cell adhesion molecule, belonging to the immunoglobulin superfamily. It is expressed in epithelia, vessel endothelia and leukocytes, and mediates intercellular adhesion in rat hepatocytes by homophilic binding. Two major isoforms (C-CAM1 and C-CAM2) that differ in their cytoplasmic domains occur. A previous study demonstrated that C-CAM can bind calmodulin in a Ca(2+)-dependent manner. In this study we have expressed the cytoplasmic domains of C-CAM1 and C-CAM2 in fusion proteins and measured calmodulin binding by a gel overlay assay, using 125I-labelled calmodulin. Our results indicate that the cytoplasmic domains of both C-CAM1 and C-CAM2 can bind calmodulin.

Adenosine Triphosphatases↗
PubMed1993

Different isoforms and stock-specific variants of the cell adhesion molecule C-CAM (cell-CAM 105) in rat liver.

C-CAM is a cell adhesion molecule of the immunoglobulin superfamily with homophilic binding properties. Here we used the polymerase chain reaction to isolate clones of C-CAM from a rat liver cDNA library. Sequence analyses identified two major isoforms, C-CAM1 and C-CAM2, which differed in their 3' ends. C-CAM2 lacked a sequence of 53 nucleotides that was present in C-CAM1. This causes a frame shift and new stop codons, which gives rise to cytoplasmic domains of different sizes in the two isoforms (10 versus 71 amino-acid residues). In addition, all the clones had a different nucleotide and deduced amino-acid sequence (variant b) in the most N-terminal of the four immunoglobulin-like domains, compared to a previously published C-CAM sequence (variant a). Northern-blot analyses with specific oligonucleotide probes demonstrated that two different rat stocks expressed either variant a or variant b. Northern-blot analyses of rat liver and lung also showed that at least five different C-CAM transcripts are produced. Two major mRNA size classes of 4.0 kb and 6.0 kb, and one minor class of 3.0 kb were found. Both the 4.0-kb and 3.0-kb messenger classes reacted with two different probes that could distinguish between C-CAM1 and C-CAM2, while the 6.0-kb population only reacted with the probe selective for C-CAM1. Taken together these data demonstrate the existence of four different protein-coding sequences of rat liver C-CAM (C-CAM1 a and b, and C-CAM2 a and b). We suggest that both allelic variation and alternative splicing may contribute to the isoform-expression pattern of C-CAM in rats.

Amino Acid Sequence↗
PubMed1992

Decreased expression of E-cadherin and increased invasive capacity in EBV-LMP-transfected human epithelial and murine adenocarcinoma cells.

The EBV-encoded membrane protein LMP is one of 9 viral proteins regularly expressed in virally immortalized B lymphocytes. It is expressed in EBV-carrying lymphoblastoid cell lines of normal origin and in the majority of the poorly differentiated nasopharyngeal carcinomas, but not in Burkitt lymphomas. LMP has been reported to transform rodent fibroblasts, to inhibit epithelial differentiation and to alter morphology and cytokeratin expression in an in vitro immortalized human keratinocyte cell-line, RHEK-I. We now report that an LMP-transfected mouse mammary carcinoma line, SHG, exhibits a similar morphological change to that previously described in the LMP-transfected RHEK-I. In the LMP-transfected RHEK-I and SHG cells, we observed a decreased expression of the calcium-dependent adhesion molecule E-cadherin. The LMP-transfected RHEK-I cells were capable of invading type-I collagen gels while the control cells were not. The LMP-transfected SHG cells showed a significantly higher invasive capacity than the original cell line.

Adenocarcinoma↗
PubMed1992

C-CAM (Cell-CAM 105) is a calmodulin binding protein.

C-CAM (Cell-CAM 105) is a transmembrane cell adhesion molecule belonging to the immunoglobulin superfamily. It mediates intercellular adhesion of rat hepatocytes and occurs in various isoforms in several epithelia, vessel endothelia and leukocytes. We now report that purified liver C-CAM interacts specifically with calmodulin. Binding was observed both when 125I-labeled C-CAM was used in a dot-blot assay and when 125I-labeled calmodulin was used in a gel overlay assay. Experiments with protease-generated peptides indicated that calmodulin bound to the cytoplasmic domain of C-CAM. Analyses of whole liver membranes demonstrated that C-CAM is one of five major proteins that bind calmodulin in a calcium-dependent manner.

Adenosine Triphosphatases↗