Differences in aggregation properties and levels of the neural cell adhesion molecule (NCAM) between islet cell types

Cells within rat islets of Langerhans are typically organized as a core of B-cells, surrounded by the other cell types. When mixed in culture, primary islet cells and insulinoma (RIN2A) cells form aggregates where B-cells are centrally located, surrounded by non-B-cells, while RIN-cells segregate as the outermost layer. To gain insight into the molecular basis underlying this nonrandom cellular organization, the aggregation properties of the three cell populations were studied. Isolated islet cells were separated into B-cells and non-B-cells by autofluorescence-activated cell sorting (FACS). In a short-term aggregation assay, primary B-cell aggregation in the absence of calcium was only 19 +/- 3.7%, compared to the 67 +/- 2.9% seen in the presence of calcium (mean +/- SEM; P less than 0.001; n = 7). By contrast, non-B-cell aggregation and RIN cell aggregation in the absence of calcium (62 +/- 2 and 66 +/- 2%, respectively) were only slightly less than with calcium (70 +/- 3 and 76 +/- 3%). The surface density of the Ca2(+)-independent neural CAM (NCAM) was therefore measured by flow cytometry and found to be 2.64 +/- 0.82-fold higher in non-B-cells, compared to that in B-cells (P less than 0.01; n = 3). Even higher levels were found on RIN cells. In the three cell types, NCAM-140 was the only molecular form detected by immunoblotting. In conclusion, differences in the calcium dependency of aggregation and in the levels of NCAM are demonstrated among islet B-cells, non-B-cells, and RIN cells. Because cell-cell adhesion is crucial for the maintenance of adult tissue, these aggregation specificities might contribute to the concentric segregation of islet cell types in culture and to the nonrandom distribution of cells within rat islets.     

The roles of cadherins and nectins in interneuronal synapse formation

Cadherins are Ca(2+)-dependent intercellular adhesion molecules (CAMs) and they play key roles in the intercellular junctions of a wide variety of cells, including interneuronal synapses. Nectins are Ca(2+)-independent immunoglobulin-like CAMs and they are also involved in the organization of various types of intercellular junctions, including interneuronal synapses, either in cooperation with or independently of cadherins. Intercellular adhesion through nectins induces activation of Cdc42 and Rac small G proteins, leading to a reorganization of the actin cytoskeleton, gene expression, and cell polarization.     

The roles of nectins in cell adhesions: cooperation with other cell adhesion molecules and growth factor receptors

Nectins are Ca(2+)-independent Ig-like cell adhesion molecules (CAMs) which homophilically and heterophilically interact in trans with nectins and form cell-cell adhesion. This cell-cell adhesion is involved in the formation of many types of cell-cell junctions such as adherens junctions, tight junctions, and synaptic junctions, cooperatively with other CAMs such as cadherins and claudins. Nectins transduce signals cooperatively with integrin alpha(v)beta(3), and regulate formation of cell-cell junctions. In addition, nectin interacts in cis with PDGF receptor and regulates its signaling for anti-apoptosis. Furthermore, nectin interacts in trans with nectin-like molecule-5 (Necl-5) and regulate cell movement and proliferation. We describe cooperative roles of nectins with other CAMs and growth factor receptors.     

Modulation of specific protein expression in teratocarcinoma cell aggregates by antibodies affecting cell-cell interactions

Decompacting Fab were used as tools to modify cell interactions during embryoid body formation and differentiation of the embryonal carcinoma (EC) PSMB. Two kinds of Fab were used: anti-uvomorulin Fab which recognize only the adhesion glycoprotein uvomorulin and Fab from an anti-EC serum which recognizes a series of molecules including uvomorulin. Both types of Fab led to decompaction and formation of loose aggregates with abnormal deposition of extracellular matrix (ECM) components. Anti-uvomorulin Fab did not affect the pattern of embryoid body differentiation. In contrast Fab anti-EC induced a differentiation of most of the cells in the aggregate into an endoderm-like phenotype. This effect was observed only when anti-EC Fab were added to aggregates and not to monolayer cultures.     

Identification of a putative cell adhesion domain of uvomorulin.

A rat monoclonal antibody (DECMA-1) selected against the murine cell adhesion molecule uvomorulin blocks both the aggregation of mouse embryonal carcinoma cells and the compaction of pre-implantation embryos. However, decompacted embryos eventually become recompacted in the presence of DECMA-1 and form blastocysts composed of both trophectoderm and inner cell mass. DECMA-1 also disrupts confluent monolayers of Madin-Darby canine kidney (MDCK) epithelial cells. DECMA-1 recognizes uvomorulin in extracts from mouse and dog tissues. Protease digestion of mouse and dog uvomorulin generated core fragments including one of 26 kd which reacted with DECMA-1. The same 26-kd fragment is recognized by anti-uvomorulin monoclonal antibodies which have been obtained from other laboratories and which dissociate MDCK cell monolayers and block the formation of the epithelial occluding barrier. This 26-kd fragment therefore seems to be involved in the adhesive function of uvomorulin.     

Classical cadherins.

Cadherins represent a gene family of Ca(2+)-dependent cell adhesion molecules (CAMs) identified during development and in adult organs. They generally mediate cell-cell adhesion by homotypic interaction, although heterotypic binding between different cadherin molecules is possible. Molecular cloning and sequence comparison has led to the characterization of a highly homologous group of 'classical' cadherins and more distantly related members, together composing a gene superfamily. The classical cadherins are transmembrane glycoproteins which exhibit, in addition to the structural homologies, a very similar overall protein topology. Protein sequence comparison has led to the identification of domains of common functional importance. The cytoplasmic domains of cadherins associate with peripheral cytoplasmic proteins termed catenin alpha, beta and gamma with molecular weights of 102, 88 and 80 kDa respectively. This complex formation seems to regulate the adhesive function of cadherins, most likely by connecting cadherins with actin microfilaments. Possible implications of catenins for cadherin function are discussed.     

Biosynthesis of the cell adhesion molecule uvomorulin (E-cadherin) in Madin-Darby canine kidney epithelial cells

The Ca(2+)-dependent cell adhesion molecule uvomorulin is a transmembrane glycoprotein that functions at the cell surface to regulate epithelial cell recognition and adhesion. We have investigated the temporal and spatial regulation of uvomorulin biosynthesis and cell surface expression in Madin-Darby canine kidney epithelial cells. We show that uvomorulin is synthesized as a precursor polypeptide (Mr 135,000) that is core glycosylated in the endoplasmic reticulum. The precursor is processed to the mature polypeptide (Mr 120,000) shortly after addition of complex carbohydrate groups in the late Golgi complex, but prior to delivery of the polypeptide to the cell surface. However, glycosylation is not required for either efficient processing of the precursor or transport of uvomorulin to the cell surface. At the cell surface, uvomorulin is turned over rapidly (t1/2 approximately 5 h). Induction of Ca(2+)-dependent cell-cell contact results in rapid localization of cell surface uvomorulin to regions of contact and an increase in the proportion of uvomorulin that is insoluble in buffers containing Triton X-100. These results indicate several regulatory steps in the biosynthesis and cell surface expression of uvomorulin in epithelial cells.     

The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.

We have determined the amino acid sequence of the Ca2+-dependent cell adhesion molecule uvomorulin as it appears on the cell surface. The extracellular part of the molecule exhibits three internally repeated domains of 112 residues which are most likely generated by gene duplication. Each of the repeated domains contains two highly conserved units which could represent putative Ca2+-binding sites. Secondary structure predictions suggest that the putative Ca2+-binding units are located in external loops at the surface of the protein. The protein sequence exhibits a single membrane-spanning region and a cytoplasmic domain. Sequence comparison reveals extensive homology to the chicken L-CAM. Both uvomorulin and L-CAM are identical in 65% of their entire amino acid sequence suggesting a common origin for both CAMs.     

Molecular organization of the uvomorulin-catenin complex

The Ca(2+)-dependent cell adhesion molecule uvomorulin is a member of the cadherin gene family. Its cytoplasmic region complexes with structurally defined proteins termed alpha-, beta-, and gamma-catenins. Here we show that A-CAM (N-cadherin), another member of this gene family, also associates with catenins suggesting that this complex formation may be a general property of the cadherins. For uvomorulin it has been found that this association with catenins is of crucial importance for the adhesive function, but little is known about the molecular organization of the uvomorulin-catenin complex. Using a combination of biochemical analyses we show that a single complex is composed of one molecule of uvomorulin, one or two molecules of beta-catenin, and one molecule of alpha-catenin. Furthermore, beta-catenin seems to interact more directly with uvomorulin. In pulse-chase experiments beta-catenin is already associated with the 135-kD uvomorulin precursor molecule but the assembly of the newly synthesized alpha-catenin into the complex is only detected around the time of endoproteolytic processing.     

Calcium-dependent cell adhesion molecules

The adhesive function of Ca2(+)-dependent CAMS has in the past been studied only indirectly, mainly using immunological techniques. The molecular cloning and information about the primary structure of several CAMs has been an important step in a more detailed molecular analysis. If there is a homophilic interaction between CAMs of neighbouring cells, an important question concerns the specificity of each CAM-mediated adhesiveness. Has each CAM a unique specificity and can this specificity be linked to a defined amino acid sequence? It will be important to elucidate the molecular mechanism of how each CAM interacts with the other. The experiments of Volk et al. (1987) suggest that an interaction of two different CAMs can occur. Since during development a given cell can express more than one CAM such an heterophilic interaction could play some regulatory role. Alternative splicing mechanisms or different protein forms during development or on different cell types have not yet been observed for Ca2(+)-dependent CAMs. However, uvomorulin is assumed to have a slightly different function during development and in adult tissues. During development uvomorulin is involved in the condensation, the pattern formation, and the sorting out of cells. In these processes the uvomorulin-mediated adhesiveness should be controlled, since cells reorganize and migrate during development. For the maintenance of the histoarchitecture in adult tissues uvomorulin might act more as a glue. This argues for the existence of mechanisms to regulate the strength of adhesiveness, and the cytoplasmic domain might be involved in these processes. The association of the cytoplasmic domain of uvomorulin with catenins could be an important observation in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

The neural cell adhesion molecule L1 is distinct from the N-CAM related group of surface antigens BSP-2 and D2.

The neural cell adhesion molecule L1 and the group of N-CAM related molecules, BSP-2 and D2 antigen, are immunochemically distinct molecular species. The two groups of surface molecules are also functionally distinct entities, since inhibition of Ca2+-independent adhesion among early post-natal mouse cerebellar cells by Fab fragments of both antibodies are at least additive, when compared with equal concentrations of the individual antibodies.     

Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function

We show that a synthetic peptide corresponding to the sequence of one putative Ca2+ binding motif of the cell adhesion molecule uvomorulin is able to complex Ca2+. This function is abolished if the first Asp in the peptide is replaced by Lys. Accordingly, we expressed in L cells mutant uvomorulin with a replacement of Asp to Lys or Ala. Mutant protein was resistant to Ca2+/trypsin under mild conditions but became susceptible at or near the site of replacement at higher concentrations, leaving the remaining Ca2+ binding domains protected. Remarkably, in cell aggregation assays both mutant uvomorulins failed to mediate cell adhesiveness, demonstrating that a single amino acid substitution in one Ca2+ binding site inactivates the adhesive function.     

ET-1 inhibits B-16 murine melanoma cell migration by decreasing K(+) currents

Cell migration is mediated by ion channels and transporters, and plays crucial roles in a variety of physiological and pathological processes. Previously, our studies have shown that a Ca(2+)-regulated K(+) current exists in B-16 murine melanoma cells, and that endothelin-1 (ET-1) inhibits the K(+) current via a PKC-dependent pathway. In the present study, patch-clamp whole-cell recording and transwell migration assays were used to examine the effects of ET-1 on B-16 murine melanoma cell migration. ET-1 (100 nM in the injection pipette and 10 nM in the incubation medium) decreased the K(+) current amplitude by 33.0 +/- 2.5% and inhibited migration of B-16 cells by 57.4 +/- 9.4%. Similarly, the Ca(2+)-regulated K(+) channel blockers, BaCl(2) and quinidine, decreased the K(+) current by 20.5 +/- 1.0% and 36.6 +/- 1.2%, respectively, and slowed migration of B-16 melanoma cells by 37.1 +/- 8.6% and 42.7 +/- 8.8%, respectively. The effect of ET-1 on the K(+) current and cell migration was simulated by ET-3. In contrast, the K(+) channel opener, diclofenac, increased the K(+) current by 128.8 +/- 11.7%, 257.4 +/- 35.8% at concentrations of 1 and 5 mM, respectively. Likewise, the migration of B-16 murine melanoma cells dramatically increased by 75.6 +/- 12.7% in the presence of 100 microM diclofenac in incubation medium. Furthermore, the ET-1- and ET-3-induced inhibition of K(+) current and migration was abrogated by diclofenac. In the presence of diclofenac, ET-1 only reduced the K(+) current amplitude by 10.6 +/- 1.1%, and slowed B-16 cell migration by only 10.8 +/- 8.9%. The results suggest that the K(+) channel-dependent migration of B-16 melanoma cells is modulated by ET-1. Cell Motil.     

The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex

The role of the epithelial adhesion molecule uvomorulin in the formation of the epithelial junctional complex in the Madin-Darby canine kidney (MDCK) cell line was investigated. Experiments were carried out to determine whether specific inhibition of uvomorulin function would interfere selectively with the formation, stability, or function of the apical zonula adherens (ZA) and zonula occludens (ZO), or whether it would interfere with all forms of intercellular contact including the desmosomes. The effects of blocking antibodies and Fab fragments to uvomorulin on the formation of the junctional complex was examined with a Ca2+ switch assay for de novo junction assembly. The formation of the ZO, the ZA, and the desmosomes was assayed by fluorescence staining with an antibody to the tight junction-specific protein ZO-1, with rhodamine-phalloidin for ZA-associated actin filaments, and with an anti-desmoplakin antibody, respectively. Under different conditions and times of antibody treatment the extent of inhibition of the formation of each of the junctional elements was very similar. The ability of the cells to eventually overcome the inhibitory effect of the antibodies and form junctions correlated with the reappearance of uvomorulin at the regions of cell-cell contact. Therefore uvomorulin seems to mediate an early adhesion event between epithelial cells that is a prerequisite for the assembly of all elements of the junctional complex. In contrast, the transepithelial electrical resistance of confluent, well-established monolayers of MDCK cells grown on filters was not greatly affected by treatment with the various antibodies or Fab fragments. A small transient decrease in resistance observed with the polyclonal alpha-uvomorulin IgG may be due to a more subtle modulation of the junctional complex.     

HCO(3)(-) ions modify the role of PKC isoforms in the modulation of rat mast cell functions

PKC and the intracellular calcium signal are two well-known intracellular signaling pathways implicated in the induction of mast cell exocytosis. Both signals are modified by the presence or absence of HCO(3)(-) ions in the external medium. In this work, we studied the regulation of the exocytotic process by PKC isozymes and its relationship with HCO(3)(-) ions and PKC modulation of the calcium entry. The calcium entry, induced by thapsigargin and further addition of calcium, was inhibited by PMA, a PKC activator, and enhanced by 500 nM GF109203X, which inhibits Ca(2+)-independent PKC isoforms. PMA inhibition of the Ca(2+) entry was reverted by 500 and 50 nM GF109203X, which inhibit Ca(2+)-independent and Ca(2+)-dependent isoforms, respectively, and G?6976, a specific inhibitor of Ca(2+)-dependent PKCs. Thus, activation of Ca(2+)-dependent and Ca(2+)-independent PKC isoforms inhibit Ca(2+) entry in rat mast cells, either in a HCO(3)(-)-buffered or a HCO(3)(-)-free medium. PMA, GF109203X, G?6976 and rottlerin, a specific inhibitor of PKC delta, were also used to study the role of PKC isoforms in the regulation of exocytosis induced by thapsigargin, ionophore A23187 and PMA. The results demonstrate that Ca(2+)-dependent PKC isoforms inhibit exocytosis in a HCO(3)(-)-dependent way. Moreover, Ca(2+)-independent PKC delta was the main isoform implicated in promotion of Ca(2+)-dependent mast cell exocytosis in the presence or absence of HCO(3)(-). The role of PKC isoforms in the regulation of mast cell exocytosis depends on the stimulus and on the presence or absence of HCO(3)(-) ions in the medium, but it is independent of PKC modulation of the Ca(2+) entry.     

Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells

The integrity of the endothelial layer, which lines the entire cavity of the vascular system, depends on tight adhesion of the cells to the underlying basement membrane as well as to each other. It has been previously shown that such interactions occur via membrane receptors that determine the specificity, topology, and mechanical properties of the surface adhesion. Cell-cell junctions between endothelial cells, in culture and in situ, involve both Ca(2+)-dependent and -independent mechanisms that are mediated by distinct adhesion molecules. Ca(2+)- dependent cell-cell adhesion occurs mostly via members of the cadherin family, which locally anchor the microfilament system to the plasma membrane, in adherens junctions. Ca(2+)-independent adhesions were reported to mainly involve members of the Ig superfamily. In this study, we performed three-dimensional microscopic analysis of the relative subcellular distributions of these two endothelial intercellular adhesion systems. We show that cadherins are located at adjacent (usually more apical), yet clearly distinct domains of the lateral plasma membrane, compared to PECAM-1. Moreover, cadherins were first organized in adherens junctions within 2 h after seeding of endothelial cells, forming multiple lateral patches which developed into an extensive belt-like structure over a period of 24 h. PECAM-1 became associated with surface adhesions significantly later and became progressively associated with the cadherin-containing adhesions. Cadherins and PECAM-1 also differed in their detergent extractability, reflecting differences in their mode of association with the cytoskeleton. Moreover, the two adhesion systems could be differentially modulated since short treatment with the Ca2+ chelator EGTA, disrupted the cadherin junctions leaving PECAM-1 apparently intact. These results confirm that endothelial cells possess distinct intercellular contact mechanisms that differ in their spatial and temporal organization as well as in their functional properties.     

Both Ca2+ -dependent and -independent pathways are involved in rat hepatic stellate cell contraction and intrahepatic hyperresponsiveness to methoxamine

In chronic liver injury, hepatic stellate cells (HSCs) have been implicated as regulators of sinusoidal vascular tone. We studied the relative role of Ca(2+)-dependent and Ca(2+)-independent contraction pathways in rat HSCs and correlated these findings to in situ perfused cirrhotic rat livers. Contraction of primary rat HSCs was studied by a stress-relaxed collagen lattice model. Dose-response curves to the Ca(2+) ionophore A-23187 and to the calmodulin/myosin light chain kinase inhibitor W-7 served to study Ca(2+)-dependent pathways. Y-27632, staurosporin, and calyculin (inhibitors of Rho kinase, protein kinase C, and myosin light chain phosphatase, respectively) were used to investigate Ca(2+)-independent pathways. The actomyosin interaction, the common end target, was inhibited by 2,3-butanedione monoxime. Additionally, the effects of W-7, Y-27632, and staurosporin on intrahepatic vascular resistance were evaluated by in situ perfusion of normal and thioacetamide-treated cirrhotic rat livers stimulated with methoxamine (n = 25 each). In vitro, HSC contraction was shown to be actomyosin based with a regulating role for both Ca(2+)-dependent and -independent pathways. Although the former seem important, an important auxiliary role for the latter was illustrated through their involvement in the phenomenon of "Ca(2+) sensitization." In vivo, preincubation of cirrhotic livers with Y-27632 (10(-4) M) and staurosporin (25 nM), more than with W-7 (10(-4) M), significantly reduced the hyperresponsiveness to methoxamine (10(-4) M) by -66.8 +/- 1.3%, -52.4 +/- 2.7%, and -28.7 +/- 2.8%, respectively, whereas in normal livers this was significantly less: -43.1 +/- 4.2%, -40.2 +/- 4.2%, and -3.8 +/- 6.3%, respectively. Taken together, these results suggest that HSC contraction is based on both Ca(2+)-dependent and -independent pathways, which were shown to be upregulated in the perfused cirrhotic liver, with a predominance of Ca(2+)-independent pathways.     

Homotypic and heterotypic Ca(++)-independent cell adhesion activities of biliary glycoprotein, a member of carcinoembryonic antigen family, expressed on CHO cell surface.

Homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, biliary glycoprotein a (BGPa), have been examined. CHO cells transfected with the cDNA for BGPa, CEA, non-specific cross-reacting antigen (NCA) and CGM6 have been used. The BGPa producers showed both homotypic and heterotypic adhesion to CEA and NCA producers. However, they hardly adhered to CGM6 producers. Calcium ion was not required for BGPa-mediated homotypic and heterotypic cell adhesion as well as for the adhesions of other members of CEA family. The results strongly suggested that BGPa may play some important roles through Ca(++)-independent cell adhesion activities.