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Focal adhesion kinase (pp125FAK) is a nonreceptor protein tyrosine kinase transducing signals initiated through integrin activation triggered by cell/extracellular matrix (ECM) interactions. To examine its role in epithelial cell adhesion, proliferation, and differentiation, we have studied pp125FAK expression, activity, and association with paxillin in two canine prostate models in which these functions can be selectively regulated: in vitro by vitronectin (VN) and serum factors, and in vivo by sex steroids. Kinetic studies revealed that the adhesion and spreading of prostatic epithelial cells in primary culture was regulated by serum VN and a natural ECM containing VN produced by prostate cells. While barely detectable in freshly isolated prostate cells, proliferating cells, after 72 h in culture, expressed higher levels of FAK mRNA (8-fold), pp125FAK (50-fold), and paxillin (50-fold). In prostate cells with a reduced growth rate after 2 weeks in culture, we observed a decrease in pp125FAK (4-fold) and its transcript (3-fold), but no change in paxillin. In vivo, both proteins were undetectable in normal and hyperplastic glands composed of a well differentiated epithelium, and in prostates restored by androgen supplementation. In contrast, pp125FAK and paxillin were up-regulated by androgen deprivation (castration) and further increased by estrogen treatment, which yielded metaplastic prostates mostly composed of proliferating basal epithelial cells. Moreover, both proteins were constitutively phosphorylated on tyrosine in the metaplastic prostate, as well as in proliferating cultured cells. Together, these results demonstrate that pp125FAK expression is regulated at the protein and mRNA levels and forms active signaling complexes with paxillin when epithelial cells in contact with ECM proteins are induced to proliferate in vivo and in vitro.

The molecules that mediate metastatic cell homing to specific organ sites remain largely unidentified. As a target organ for metastasis, the liver is a unique environment characterized by fenestrated sinusoidal endothelium, lack of a complete basement membrane, and production of serum components, including fibronectin and vitronectin. We examined a series of marine RAW117 large cell lymphoma variants selected in vivo for liver-colonizing properties (H10 >> L17 > P). Compared with L17 or P cells, the highly liver-colonizing H1O cells expressed much higher levels of surface integrin alphavbeta3, as shown by affinity chromatography, immunoprecipitation, and flow cytometry. H10 cells adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin, and type I collagen. Among the RGD peptides, H10 cells adhered at significantly higher rates to the polymeric RGD peptide (glycyl-arginyl-glycyl-aspartyl-serine)4 than to monomeric RGD peptides. H10 cells were able to spread on immobilized vitronectin with highly polarized morphology but not on fibronectin. In contrast, the poorly liver-metastatic P and L17 cells did not adhere or spread well on vitronectin or fibronectin. H10 cells also migrated toward vitronectin concentration gradients. Blocking cell surface alphavbeta3 molecules with specific anti-beta3 monoclonal antibodies resulted in significant decreases in the adhesion of H10 cells to vitronectin and (glycyl-arginyl-glycyl-aspartyl-serine)4 and significant inhibition of the formation of experimental liver metastases. These data suggest an important role of integrin alphavbeta3 in the metastasis of RAW117 cells to the liver.

Bone sialoprotein (BSP) is a protein highly specific for bone, which contains an arginine-glycine-aspartic acid (RGD) cell attachment sequence involved in osteoclast adhesion to bone matrix via the vitronectin receptor. We have investigated its role in in vitro bone resorption using the well described isolated osteoclast resorption pit assay. BSP significantly stimulates bone resorption in a dose-dependent manner, increasing the overall resorbed area on ivory slices and the number of lacunae at concentrations as low as 50 nM. Neither recombinant osteopontin nor intact vitronectin has any effect on bone resorption, suggesting a specific effect of BSP. The stimulation of bone resorption induced by BSP could be partially explained by an increase in osteoclast adhesion to bone via its RGD sequence, and our results suggest another mechanism of action of BSP that might involve another region of the molecule, such as its acidic sequences. Although BSP stimulates bone resorption in a coculture system of bone marrow cells and osteoblastic cells, it dose dependently inhibits the formation of osteoclast-like cells at equivalent concentrations in this culture model. In conclusion, we provide evidence that BSP plays an important role in the bone resorption process and may regulate bone resorption as well as osteoclast formation.

Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum-bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. Chemotaxis was abolished by a blocking antibody to alphavbeta3 (LM609), whereas haptotaxis was inhibited only by approximately 50%, suggesting involvement of multiple receptors and/or signaling pathways. However, blocking antibodies to alphavbeta5, also present on A2058 cells, did not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by >80%, but did not inhibit haptotaxis. Adhesion and spreading over vitronectin induced the phosphorylation of paxillin on tyrosine. In cells migrating over substratum-bound vitronectin, tyrosine phosphorylation of paxillin increased 5-fold between 45 min and 5 h. Dilutions of anti- alphavbeta3 that inhibited haptotaxis also inhibited phosphorylation of paxillin (by approximately 50%) and modestly reduced cell spreading. In contrast, soluble vitronectin (50-100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin.

Polyamine-brushed substrata for cell culture were designed by solvent casting of polystyrene-graft-polyamine copolymer (SA) on hydrophobically modified glass surface, and adhesion, spreading, and proliferation of bovine aortic endothelial cells (BAEC) on these substrata were evaluated. Adhesion and spreading of BAEC increased with increasing polyamine content in the copolymer. Close correlation was found between cellular spreading and subsequent cell growth; the surface inducing better spreading of adhered cells showed higher endothelial cell growth. Adhesion and spreading of BAEC were significantly influenced by fetal calf serum (FCS)-pretreatment of the SA copolymer surfaces, being increased with increasing polyamine content, whereas on bovine serum albumin (BSA)-preabsorbed surfaces, BAEC adhesion was considerably prevented and eventually no cell spreading was observed. Then, adsorption of cell-adhesion proteins, fibronectin (FN), and vitronectin (VN), out of FCS onto SA copolymer surfaces were evaluated using enzyme immunoassay. Both FN and VN adsorption on SA copolymer surfaces were increased with increasing polyamine content in the copolymer, suggesting a crucial role of these cell-adhesion proteins in BAEC adhesion and subsequent growth behavior on SA copolymer surfaces.

When UMR201 cells, phenotypically preosteoblastic, were placed onto a type I collagen gel, expression of osteopontin (OP) mRNA and protein were strongly upregulated, compared to cells plated onto plastic. This upregulation was dose-dependent, with respect to the concentration of collagen gel, and was observable within hours of cells having attached and spread on the substrate. Retinoic acid (RA) acted synergistically with type I collagen at each concentration to induce a much greater increase in OP mRNA than in cells on plastic. In addition, RA increased the phosphorylation of secreted OP. The exogenous collagen substrate inhibited the growth of UMR201 cells, with the extent and duration of inhibition dependent on the collagen concentration. The effect of type I collagen was specific; plating cells on fibronectin, laminin or vitronectin did not upregulate OP expression. In contrast to the effects on OP expression, the strong RA induction of alkaline phosphatase (ALP) mRNA in cells on plastic was attenuated in cells plated on type I collagen. Growth on type I collagen did not change OP mRNA stability or transcription rate, although there was decreased stability of the ALP mRNA in cells on collagen.

Morphogenesis and tissue repair require appropriate cross-talk between the cells and their surrounding milieu, which includes extracellular components and soluble factors, e.g., cytokines and growth factors. The present work deals with this communication needed for recovery after axotomy in the central nervous system (CNS). The failure of CNS axons to regenerate after axonal injury has been attributed, in part, to astrocyte failure to repopulate the injury site. The goal of this work was to provide an in vitro model to mimic the in vivo response of astrocytes to nerve injury and to find ways to modulate this response and create a milieu that favors astrocyte migration and repopulation of the injury site. In an astrocyte scratch wound model, we blocked astrocyte migration by tumor necrosis factor alpha (TNF-alpha). This effect could not be reversed by astrocyte migration-inducing factors such as transforming growth factor beta 1 (TGF-beta 1) or by any of the tested extracellular matrix (ECM) components (laminin and fibronectin) except for vitronectin (Vn). Vn, added together with TNF-alpha, counteracted the TNF-alpha blockage and allowed a massive migration of astrocytes (not due to cell proliferation) beyond that allowed by Vn only. Heparan sulfate proteoglycans (HSPG) were shown to be involved in the migration. The results may be relevant to regeneration of CNS axons, and may also provide an example that an extracellular component (Vn) can overcome and neutralize a negative effect of a growth factor/cytokine (TNF-alpha) and can act in synergy with other features of this cytokine to promote a necessary function (e.g., cell migration) that is otherwise inhibited.

Whole animal studies have implicated cell adhesion molecules in a diverse array of developmental processes. The present study reports on the morphological effects of RGD-related peptides on the cell surface of various living chick embryonic tissues. We report a novel and characteristic plasma membrane reaction that is caused by treatment with different RGD-peptides. Not only does each peptide evoke a response in certain tissues and not in others, but each brings about a specific type of plasma membrane reaction (bleb). Although the mechanisms are unknown, the specificity of this phenomenon suggests that it could provide a window into new surface interactions in morphogenetic systems.

The effect on phenotypic expression of rabbit vascular smooth muscle cells (SMC) of the interstitial matrix proteins collagen I and fibronectin, the basal lamina proteins collagen IV and laminin, and the serum adhesion protein vitronectin was examined in culture. Experiments were performed in foetal calf serum stripped of fibronectin and vitronectin to eliminate their confounding effects. All the proteins promoted adhesion to the plastic culture dish (in a concentration dependent manner) of SMC freshly isolated from the artery wall. These cells had a high volume density of myofilaments (Vvmyo) in their cytoplasm. Laminin was best at maintaining SMC with a high Vvmyo (Vvmyo = 49.8%) followed by collagen IV (41.7%). Cells plated on vitronectin showed the lowest Vvmyo (31.3%). The results support the concept that the SMC basal lamina has a role in maintaining cells in the high Vvmyo phenotype.

The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects.

The effect on phenotypic expression of rabbit vascular smooth muscle cells (SMC) of the interstitial matrix proteins collagen I and fibronectin, the basal lamina proteins collagen IV and laminin, and the serum adhesion protein vitronectin was examined in culture. Experiments were performed in foetal calf serum stripped of fibronectin and vitronectin to eliminate their confounding effects. All the proteins promoted adhesion to the plastic culture dish (in a concentration dependent manner) of SMC freshly isolated from the artery wall. These cells had a high volume density of myofilaments (Vvmyo) in their cytoplasm. Laminin was best at maintaining SMC with a high Vvmyo (Vvmyo = 49.8%) followed by collagen IV (41.7%). Cells plated on vitronectin showed the lowest Vvmyo (31.3%). The results support the concept that the SMC basal lamina has a role in maintaining cells in the high Vvmyo phenotype.

We studied the interaction between Trypanosoma congolense and bovine aorta endothelial (BAE) cell monolayers. Our findings suggest that trypanosomes adhere predominantly to the flattened, peripheral cell surface domains as well as to filamentous endothelial outgrowths that are present during in vitro cultivation in non-confluent monolayers. Adhesion is mediated exclusively by the flagellum in a distinct geometrical order with respect to the flagellar cytoskeleton. Thus, it is possible to define exactly the trypanosomal cell surface domain involved in the attachment process. After 24-48 h of cultivation on monolayers, trypanosomes start to develop short, filopodia-like flagellar protrusions, which serve as additional elements in assisting parasite attachment. Small filaments (3-5 nm) also serve as cross-links between flagellar and endothelial cell surface membranes. Lectin-gold labeling shows that these cross-links contain sialic acid residues. In vitro assays confirm that sialic acid is involved in the adhesion process, whereas the extracellular matrix (ECM) proteins fibronectin, collagen, laminin and vitronectin are not. The presence of T. congolense exhibits a mitogenic effect on BAE cells.