Modulation of human fibroblast gap junction intercellular communication by hyaluronan

The composition of the extracellular matrix changes during dermal repair. Initially, hyaluronan (HA) concentration is high, however, by day 3, HA is eliminated. HA optimizes collagen organization within granulation tissue. One possible mechanism of HA modulation of collagen packing is through the promotion of gap junction intercellular communication (GJIC). Gap junctions are gated channels that allow rapid intercellular communication and synchronization of coupled cell activities. The gap junction channel is composed of connexin (Cx) proteins that form a gated channel between coupled cells. HA is reported to enhance Cx43 expression in transformed fibroblasts. GJIC was quantified by the scrape loading technique and reported as a coupling index. The coupling index for human dermal fibroblasts was 4.6 +/- 0.2, while the coupling index for fibroblasts treated with HA more than doubled to 10.6 +/- 0.7. By Western blot analysis no differences were appreciated in the protein levels of Cx43 or beta-catenin, a protein involved in the translocation of Cx to the cell surface. By immuno-histology Cx43 and beta-catenin were evenly distributed throughout the cell in controls, but in cells treated with HA these proteins were co-localized to the cell surface. Coupled fibroblasts are reported to enhance the organization of collagen fibrils. It is proposed that HA increases the accumulation of Cx43 and beta-catenin on the cell surface, leading to greater GJIC and enhanced collagen organization.     

Genome-wide analysis of gene expression in human embryonic tooth germ

While numerous genes that play important regulatory roles during tooth development in mice have been identified, little is known about gene expression profile and their function during human odontogenesis. To unveil expression profile of odontogenic genes in humans, we conducted genome-wide gene expression analysis by microarray assays to analyze differential gene expression between tooth germ and lip tissue from 11-week old human fetuses. We identified 167 genes that are strongly expressed in the cap stage tooth germ as compared to the lip tissue. Among them, 145 genes were further identified by gene ontology enrichment analysis that are highly represented in multiple gene ontology classes, include extracellular components, sequence-specific DNA binding proteins, Wnt-protein binding molecules, system development, organogenesis, and cell differentiation. Sixty-seven genes that are known to be associated with mammalian tooth development and tooth abnormalities were identified. Real-time PCR was further employed to validate microarray data. Moreover, in situ hybridization assay demonstrated tooth type specific expression of ISL1 and BARX1 in the incisor, canine, and molar respectively, consistent with microarray results. Our results represent a set of reliable data that could provide a solid base for future elaboration of molecular mechanisms underlying human tooth development.     

Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ.

The distribution of the matrix protein fibronectin was studied by indirect immunofluorescence in differentiating mouse molars from bud stage to the stage of dentin and enamel secretion, and compared to that of collagenous proteins procollagen type III and collagen type I. Fibronectin was seen in mesenchymal tissue, basement membranes, and predentin. The dental mesenchyme lost fibronectin staining when differentiating into odontoblasts. Fibronectin was not detected in mineralized dentin. Epithelial tissues were negative except for the stellate reticulum within the enamel organ. Particularly intense staining was seen at the epithelio-mesenchymal interface between the dental epithelium and mesenchyme. Fibronectin may here be involved in anchorage of the mesenchymal cells during their differentiation into odontoblasts. Procollagen type III was lost from the dental mesenchyme during odontoblast differentiation but reappeared with advancing vascularization of the dental papilla. Similarly, procollagen type III present in the dental basement membrane during the bud and cap stages disappeared from the cuspal area along with odontoblast differentiation. Weak staining was seen in predentin but not in mineralized dentin. The staining with anti-collagen type I antibodies was weak in dental mesenchyme but intense in predentin as well as in mineralized dentin.     

In vitro adhesion of mouse fetal germ cells to extracellular matrix components

Mouse primordial germ cells (PGCs) isolated from the dorsal mesentery and gonadal ridges of 10.5–12.5 days post coitum (dpc) embryos showed a progressively increasing adhesiveness to laminin and fibronectin coated substrates, whereas type I collagen and various glycosaminoglycans (hyaluronic acid, heparin and chondroitinsulphates) were poor adhesive substrates. At later stages germ cells appeared to lose their adhesiveness to fibronectin and laminin substrates; the ability to adhere to laminin decreased very rapidly in male and slowly in female germ cells. Oocytes and prospermatogonia from 15.5 dpc fetal gonads showed poor adhesiveness to all substrates tested. PGC adhesion to laminin and fibronectin substrates did not require calcium but was markedly trypsin sensitive. Antibodies against the fibronectin receptor of CHO fibroblasts and short peptides containing the Arg-Gly-Asp sequence greatly reduced PGC adhesion to fibronectin. Following adhesion to laminin or fibronectin, most PGCs did not exhibit a morphology typical of motile cells, but remained spherical. A significant proportion (about 30%) of oocytes from 13.5–14.5 dpc embryos appeared, however, able to spread and elongate following attachment to laminin. The results support the hypothesis that mouse PGCs may utilize laminin and/or fibronectin as adhesive substrates during migration and gonad colonization, but indicate that additional factors are probably required to promote PGC motility. In addition, our data provide indirect evidence that binding sites for specific components of extracellular matrix are present in PGCs, and that their expression may be developmentally regulated.     

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms

Summary A chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of ³H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of ³H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations. The reverse experiment, in which unlabelled quail hypoblast and possibly some mesoblast have been combined with a tritiated host deprived of its hypoblast, also shows the transfer of label from the host to the cellular surface of the graft. A two-way exchange of glucosamine-containing molecules thus occurs in the blastoderm. It is hypothesized that: (1) low molecular weight compounds, macromolecular material, and/or catabolic products, are exchanged between the different germ layers during gastrulation; (2) the components of the extracellular matrix turn over and are continuously changing; (3) this transfer is a possible mechanism of transmission for developmental or inductive signals during embryonic development. The present results also demonstrate the participation of underlying tissue in the biosynthesis of basement membrane components of an epithelium.     

Atypical endplate miniature potentials in the frog neuromuscular junction after modification of the intercellular matrix and osmotic exposures]

In frog cutaneous-pectoris muscles the frequency of slowly rising atypical miniature endplate potentials (MEPPs) was significantly enhanced after collagenase (0.1%) treatment. Treatment with trypsin, hyaluronidase, hyper- and hypoosmotic solutions caused no changes in slowly rising MEPP (frequency in muscle fibers with intact acetylcholinesterase (AChE). Inhibition of AChE caused appearance of giant MEPPs. Acceleration of acetylcholine diffusion from synaptic cleft after treatment with hyaluronidase decreased giant MEPP frequency demonstrating their dependence upon nonhydrolyzed acetylcholine in synaptic cleft. The relation between slowly rising MEPPs and activity of synaptic Schwann cells in discussed.     

PKC inhibition increases gap junction intercellular communication and cell adhesion in human neuroblastoma

Gap junction intercellular communication and cell–cell adhesion are essential for maintaining a normal cellular phenotype, including the control of growth and proliferation. Loss of either cell–cell adhesion or communication is common in cancers, while restoration of function is associated with tumor suppression. Protein kinase C (PKC) isozymes regulate a broad spectrum of cellular functions including growth and proliferation, and their overexpression has been correlated with carcinogenesis. Consequently, PKC inhibitors are currently undergoing clinical trials as an anti-cancer agents although the precise cellular alterations induced by PKC inhibitors remain to be elucidated. In the current study, the effects of PKC inhibitors on cell interactions were investigated using human neuroblastoma (IMR32, SKNMC, and SHSY-5Y) cell lines. An analysis of intercellular communication revealed an increase in gap junctional coupling with PKC inhibition. The observed increase in coupling was not associated with a change in Connexin43 distribution or an alteration of phosphorylation status of the protein. There was also an increase in cell–cell adhesion with PKC inhibitor treatment as indicated by a cell aggregation assay. Therefore, the growth suppressive abilities of PKC inhibition on tumors may be due to the cancer suppressive effects of increased gap junction intercellular communication and cell–cell adhesion.     

Protective effect of corticosteroids on intercellular junction

Actions of adrenocortical hormones on intercellular communication in salivary gland cells of Chironomus plumosus were studied by means of electrophysiological measurements. Glucocorticoids have an effect maintaining junctional communication, while mineralocorticoids have an effect blocking it. When glucocorticoids in their proper concentration are previously applied to the cells, they can prevent the block of junctional communication which should be produced by EGTA, lipase and trypsin. Mineralocorticoids prevail in competitive action between glucocorticoids and mineralocorticoids on junctional communication. The interaction between glucocorticoids and uncoupling agents, and between glucocorticoids and mineralocorticoids on junctional communication were discussed at the level of the cell membrane.     

Alpha-catenin: at the junction of intercellular adhesion and actin dynamics

Alpha-catenin has often been considered to be a non-regulatory intercellular adhesion protein, in contrast to beta-catenin, which has well-documented dual roles in cell-cell adhesion and signal transduction. Recently, however, alpha-catenin has been found to be important not only in connecting the E-cadherin-beta-catenin complex to the actin cytoskeleton, but also in coordinating actin dynamics and inversely correlating cell adhesion with proliferation. As the number of alpha-catenin-interacting partners increases, intriguing new connections imply even more complex regulatory functions for this protein.     

Molecular components of the adherens junction

Adherens junctions serve to couple individual cells into various arrangements required for tissue structure and function. The central structural components of adherens junctions are transmembrane adhesion receptors, and their associated actin-binding/regulatory proteins. The molecular machineries that organize these adhesion receptor complexes into higher order junction structures, and the functional consequences of this junctional organization will be discussed.     

Conversion of midbodies into germ cell intercellular bridges

Whereas somatic cell cytokinesis resolves with abscission of the midbody, resulting in independent daughter cells, germ cell cytokinesis concludes with the formation of a stable intercellular bridge interconnecting daughter cells in a syncytium. While many proteins essential for abscission have been discovered, until recently, no proteins essential for mammalian germ cell intercellular bridge formation have been identified. Using TEX14 as a marker for the germ cell intercellular bridge, we show that TEX14 co-localizes with the centralspindlin complex, mitotic kinesin-like protein 1 (MKLP1) and male germ cell Rac GTPase-activating protein (MgcRacGAP) and converts these midbody matrix proteins into stable intercellular bridge components. In contrast, septins (SEPT) 2, 7 and 9 are transitional proteins in the newly forming bridge. In cultured somatic cells, TEX14 can localize to the midbody in the absence of other germ cell-specific factors, suggesting that TEX14 serves to bridge the somatic cytokinesis machinery to other germ cell proteins to form a stable intercellular bridge essential for male reproduction.     

Molecular components of the adherens junction

Adherens junctions serve to couple individual cells into various arrangements required for tissue structure and function. The central structural components of adherens junctions are transmembrane adhesion receptors, and their associated actin-binding/regulatory proteins. The molecular machineries that organize these adhesion receptor complexes into higher order junction structures, and the functional consequences of this junctional organization will be discussed.     

Immunoinhibition of human fertilization in vitro by antibodies to the cumulus oophorus intercellular matrix

Rabbit polyvalent antiserum raised against the solubilized cumulus matrix was a powerful inhibitor of human fertilization in vitro, affecting sperm-zona pellucida interaction. Both sperm binding to, and penetration of, the zona pellucida were severely impaired by the anti-cumulus matrix antiserum, whereas no effects of this antiserum on cumulus matrix solubilization or penetration of zona-free human eggs were evident. Moreover, the anti-cumulus antiserum partly neutralized the acrosome reaction-inducing activity of the cumulus matrix. These results warrant further research into the functional specificity of different cumulus matrix components and into the effects of the respective antibodies on reproductive function as a possible lead to a new approach to contraceptive vaccine development.     

Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots

Three rat hybridoma cell lines have been isolated which produce monoclonal antibodies identifying a noduleenhanced, soluble component of Pisum sativum root nodules. These antibodies each recognized a protease-sensitive band (M_r 95K) on SDS-polyacrylamide gels. The 95K antigen was resolved by isoelectric focusing into acidic and neutral components which were separately detected by AFRC MAC 236 and MAC 265 respectively. The third antibody (MAC 204) reacted with both acidic and neutral components through an epitope that was sensitive to periodate oxidation. These monoclonal antibodies were used for immunogold localizations at light and electron microscopic levels. In each case, the antigen was shown to be present in the matrix that surrounds the invading rhizobia in infection threads and infection droplets, as well as in the intercellular spaces between plant cell walls of nodules and also of uninfected roots. By contrast, a fourth monoclonal antibody, AFRC JIM 5, labelled a pectic component in the walls of infection threads, and JIM 5 was also found to label the middle lamella of plant cell walls, especially at three-way junctions between cells. The composition and structure of the infection thread lumen is thus comparable to that of an intercellular space.     

Production and utilization of extracellular matrix components by human melanocytes

Normal human melanocytes were separated from keratinocytes and maintained in culture using KGM medium supplemented with 12-O-tetradecanoylphorbol acetate and cholera toxin. The melanocytes were examined for the production of extracellular matrix molecules including fibronectin, laminin, and thrombospondin and for the utilization of these molecules in adhesion and motility assays. Melanocytes produced significant amounts of fibronectin as indicated by biosynthetic labeling/immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). Fibronectin was expressed on the surface of these cells. Laminin was also produced by melanocytes and expressed on the cell surface. The amount of laminin produced was significantly less than the amount of fibronectin. In contrast, melanocytes did not produce measurable thrombospondin as indicated by biosynthetic labeling/immunoprecipitation. Only traces of thrombospondin were detected by ELISA and no surface fluorescence was observed. When examined in adhesion and motility assays, melanocytes were found to utilize fibronectin for both processes. Laminin also stimulated adhesion but it was much less effective than fibronectin. Thrombospondin did not stimulate either attachment and spreading or motility. The pattern of extracellular matrix molecule production and utilization by melanocytes is significantly different from that shown previously for human epidermal keratinocytes (J. Varani et al., 1988, J. Clin. Invest. 81, 1537). These differences may underlie the differences with which the two cell types interact with basement membranes in vivo.