Cell adhesion and communication最新文献

The proto-oncogene product p95Vav (Vav) undergoes rapid phosphorylation on tyrosine following stimulation of the T or B cell antigen receptor, and in response to a variety of other cell surface stimuli. Vav contains, among other, a guanine nucleotide exchange factor domain with homology to the Rho/Rac/CDC42 exchange protein Db1. It has been recently shown that Vav is functionally linked to small GTPases of the Rho family, suggesting that it is an activator of Rho GTPases and may participate in regulation of cytoskeletal organization. The present study shows that cell adhesion to fibronectin triggers rapid phosphorylation of Vav on tyrosine in Vav-transfected CHO cells and in Jurkat T cells. Vav phosphorylation is strongly dependent on adhesion and is mediated by beta 1 integrins. Furthermore, Vav overexpression enhances the adhesion-dependent increase in the rate and extent of phosphorylation on focal adhesion kinase and paxillin, and the formation of stress fibers and lamellipodia. In addition, there is a marked increase in the amount of Vav localized to the triton-insoluble fraction following 1 h of incubation on FN. Finally, Vav increases the growth rate of the cells in an adhesion-dependent manner. Our results strongly implicate Vav as a mediator of integrin signal transduction.

Cellular interactions with the extracellular matrix determine to a large extent cell behavior, including cell migration. These interactions take place at specialized cellular structures, the focal adhesions, which have a substrate-specific morphology. To determine the molecular and functional relevance of this observation, the composition of isolated focal adhesions developed by fibroblasts adhering to fibronectin or laminin-1 was analyzed by indirect immunofluorescence and immunoblotting with or without stabilization of the structures by cross-linking. In the absence of cross-linking, integrins, talin, vinculin and, to a lower extent, paxillin remained associated with the focal adhesions formed on both substrates, indicating a tight association of these proteins with the extracellular matrix support. By contrast, alpha-actinin, FAK, and actin were apparently loosely maintained within focal adhesions and were found associated to these structures only after stabilization by cross-linking. Interestingly, although both substrates induced clustering and aggregation of all these proteins, their relative concentration, with the exception of alpha-actinin, was lower within the focal adhesions formed on laminin-1 than in those formed on fibronectin. Moreover, as assessed in migration assays, the locomotory speed of fibroblasts was higher on laminin-1 than on fibronectin. Altogether these results indicate that integrins involved in cellular interactions with fibronectin or laminin-1 trigger the formation of focal adhesion structures which differ by molecular organization, concentration in several adhesion plaque components, and function.

The adhesion of monocytes to endothelium, an early event in atherosclerosis, is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-kappaB; moreover, intracellularly generated oxygen-derived free radicals (ODFR) play a major role in this process. This study evaluated the extent to which troglitazone, an oral antidiabetic agent with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells (HUVECs), induced by different prooxidant signals such as oxidized LDL and tumor necrosis factor-alpha (TNF-alpha). Furthermore we assessed whether the NF-kappaB activation is modulated by the antioxidative effect of troglitazone. Oxidized LDL not only caused a dose-dependent increase of ICAM-1, VCAM-1 and E-selectin (p<0.001), but also synergically increased their TNF-alpha-induced expression (p<0.001). Troglitazone reduced in a dose-dependent manner the expression of VCAM-1, ICAM-1 and E-selectin induced by different amounts of oxidized LDL (p<0.001). The addition of troglitazone to HUVECs significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-alpha alone or in combination with oxidized LDL (p<0.001); this reduction was paralleled by a significant fall in NF-kappaB translocation. The results suggest that troglitazone may have prevented NF-kappaB-mediated adhesion molecule expression by exerting its antioxidant effect on ODFR.

Recently cDNAs coding for cell surface molecules have been isolated from sponges. The molecules for alpha-integrin, galectin, and receptor tyrosine kinase (RTK), obtained from the marine sponge, Geodia cydonium, have been described earlier. In the present study also the cDNA for one putative beta-integrin has been identified from G. cydonium. The deduced aa sequence comprises the characteristic signatures, found in other metazoan beta-integrin molecules; the estimated size is 95,215 Da. To obtain first insights into the molecular events which proceed during autograft fusion, the expressions of these genes were determined on transcriptional and translational level. The cDNAs as well as antibodies raised against the recombinant sponge proteins alpha-integrin, RTK and galectin were used and Northern blot experiments and immunocytochemical analyses have been performed. The results show that transcription of the two subunits of an integrin receptor as well as of the RTK are strongly upregulated after grafting; levels of > 10-fold have been determined in the fusion zone of the grafts after a 10 days incubation. Immunofluorescence studies of sections through the fusion zone support these data. In contrast the transcription of the gene encoding galectin is drastically downregulated after grafting. In a parallel series of experiments the level of the heat-shock protein-70 was determined and it was found that it remained unchanged after grafting. We conclude that integrin subunits and the RTK molecule are involved in self-self recognition of sponge.

Glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) is a mucin-like glycoprotein previously identified on high endothelial venules (HEV) of lymph nodes and also in lactating mammary glands. A specifically glycosilated form of GlyCAM-1 on HEV has been shown to be a ligand for a leukocyte L-selectin, which plays an important role in leukocyte rolling along the inflamed endothelium. Here we report that GlyCAM-1 is also expressed in the cochlea. Immunohistochemistry revealed the lateral wall of the cochlea, tectorial membrane, modiolus, organ of corti, and spiral modiolar vein (SMV) to be strongly stained with polyclonal anti-GlyCAM-1 antibody. Moreover, RT-PCR of the cochlear tissue by the use of specific oligonucleotide primers for rat GlyCAM-1 generated a 378 bp product which was then verified by nucleotide sequencing to represent GlyCAM-1. Electron microscopic investigation revealed the presence of GlyCAM-1 over the entire lumenal surface of the vessels, and the basolateral infoldings in stria vascularis. However, soluble L-selectin or mAb MECA-79 which recognizes a carbohydrate epitope on functional L-selectin ligands bound only to the spiral ligament, tectorial membrane and modiolus. These observations suggest that GlyCAM-1 expressed in the cochlear region is heterogenous in terms of its glycosylation.

Lyme arthritis following infection with Borrelia burgdorferi (B. burgdorferi) is associated with the presence of bacteria in the joint, but the mechanism of persistent infection in the presence of specific antibodies and lymphocytes remains unknown. To investigate how an infection with B. burgdorferi might influence the local immune response in the joint, we examined the expression of cell adhesion molecules, human leucocyte antigens and inducible nitric oxide synthase (iNOS)-1 and -2 in human synovial cells after infection with B. burgdorferi in vitro. Synovial cells are known to influence the function of local immunologic effector cells and play a key role in the pannus formation of erosive arthritis. It has been shown previously that B. burgdorferi can persist in the cytosol of human synovial cells. The expression of the surface molecules ICAM-1, VCAM-1, HLA-class-I and -class-II and the cytosolic production of iNOS-1 and -2 in synovial cells was measured by flow cytometry for up to 5 days after infection with B. burgdorferi. A significant, lasting downregulation of surface ICAM-1 could be demonstrated on synovial cells, whereas no significant changes were seen in the expression of VCAM-1, HLA-class-I and -II, and of iNOS-1 and -2. To determine the biological significance of this downregulation an in vitro adhesion assay using peripheral blood mononuclear cells was developed. After infection with B. burgdorferi a significantly smaller number of mononuclear cells was adhering to the synovial cell monolayer. Adhesion of peripheral mononuclear cells was shown to be in part mediated by ICAM-1 by using a blocking mononuclear antibody against ICAM-1. Downregulation of ICAM-1 on synovial cells due to infection with B. burgdorferi might suppress the local immunosurveillance and might help the bacteria to persist in joint cells in vivo.

Bin1 is a nucleocytoplasmic adaptor protein and tumor suppressor. A novel protein termed Bau was identified through its ability to interact with a region of Bin1 required to inhibit malignant cell transformation by certain oncogenes. Bau is a splice form of Neurabin-I, one of two related F-actin-binding proteins that are proposed to link cadherin-based cell-cell adhesion sites with the growth regulatory kinase p70S6K. Bau lacks actin- and p70S6K-binding domains found in Neurabin-I but includes coiled-coil domains that are part of its central domain as well as additional sequences not found in Neurabin-I. Interaction with Bin1 requires the presence of the U3 region which is alternately spliced in muscle cells. Bau localizes to the nucleus and cytosol. Like Bin1, Bau can suppress oncogene-mediated transformation and inhibit tumor cell growth. We suggest that Bau may link Bin1 to the Neurabin-I/p70S6K system in muscle and other cells, perhaps providing a mechanism to influence adhesion-dependent signals which affect cell fate.

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.

Intercellular adhesion molecule-3 (ICAM-3, CD50), an adhesion receptor of the immunoglobulin superfamily, is suggested to play a key role in adhesive cellular interactions during the initial phase of an immune response. We here provide evidence that ICAM-3 is abundantly expressed by cells of the human mast cell line HMC-1 and, to a lower degree, by purified skin mast cells, as demonstrated by flow-cytometry, ELISA and RT-PCR. ICAM-3 immunoprecipitated from surface biotinylated HMC-1 cells migrates as a broad band of Mr 124,000 by Western blot analysis. We also demonstrate that monoclonal antibodies directed against ICAM-3 are capable of inducing rapid HMC-1 cell aggregation, the extent of which strongly depends on the epitope recognized by the mAb applied. Interestingly, although inhibitable by two of six mAbs against LFA-1, HMC-1 aggregation induced via ICAM-3 appears to be mediated by an adhesive pathway independent of LFA-1. Dermal mast cells are also aggregated with anti-ICAM-3 mAbs, a phenomenon which has not been described before for isolated tissue mast cells. However, this process displays slower kinetics, as compared to HMC-1 cells. That anti-ICAM-3 mAbs are able to mediate biological effects is further illustrated by their capability to increase stimulation-dependent release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 from HMC-1 cells. Taken together, these results indicate that ICAM-3 is not only expressed by immature and mature human mast cells, but also possesses functional relevance and may therefore play a significant role in mast cell associated processes.

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.