cAMP-induced phenotypic reversion of adhesion, aggregation, and endocytosis in adhesion-defective CHO cell variants

ADvF11 cells are a CHO adhesion variant which, unlike wild type (WT) cells, are not able to adhere to fibronectin (Fn) coated substrata or to be aggregated by Fn-beads. However, ADvF11 cells bind Fn-beads to the same extent as WT cells, thus suggesting that the defect(s) associated with ADvF11 cells are distal to the initial receptor-ligand binding event (Cheung and Juliano, Exp. Cell Res. 152:127, 1984). In this communication we report that cAMP analogs such as dibutyryl-cAMP (dbcAMP) and 8-bromo-cAMP are able to correct defect(s) associated with ADvF11 cells enabling them to adhere to Fn-coated dishes and to aggregate in the presence of Fn-beads. However, only approximately 40% of ADvF11 cells were found to be responsive to dbcAMP suggesting heterogeneity in the cell population with respect to dbcAMP sensitivity. Further analysis of this partial response led us to isolate a subclone of ADvF11 cells, F11CA11, which is highly responsive to dbcAMP treatment. Induction of Fn-mediated cell adhesion and aggregation in F11CA11 by dbcAMP is both time and dose dependent. Optimal responses were obtained after overnight incubation in alpha-MEM containing, 1% fetal calf serum, 4% bovine serum albumin, 0.5 mM dbcAMP and 0.2 mM methyl-isobutyl-xanthine (MIX), a phosphodiesterase inhibitor. Under these conditions, 70-80% of F11CA11 cells were found to be adherent, compared to 5-7% of untreated F11CA11 cells and 95-100% of WT cells. Aggregation of dbcAMP-MIX treated F11CA11 cells induced by Fn-beads also approached that of WT cells. In addition, treatment with dbcAMP-MIX markedly increased the ability of F11CA11 cells to internalize Fn-beads. The maintenance of the adherent phenotype required the constant presence of dbcAMP-MIX. Removal of dbcAMP-MIX from the incubation medium resulted in return to the original nonadhesive phenotype. Thus, elevation of cAMP levels can dramatically modify the behavior of F11CA11 cells with respect to fibronectin mediated adhesion, aggregation and endocytosis, in effect causing a phenotypic reversion of all three parameters to wild type status. This suggests that the mechanisms for adhesion, aggregation and endocytosis may each involve regulation by cyclic AMP-protein kinase systems.     

Fibronectin-independent adhesion of fibroblasts to the extracellular matrix: mediation by a high molecular weight membrane glycoprotein

Fibroblastic CHO cells readily adhere to fibronectin (Fn) coated substrata. From the parental cell population we have recently selected a series of adhesion variants (ADV cells) that cannot adhere to Fn substrata (Harper and Juliano. 1980. J. Cell. Biol. 87:755-763). However, ADV cells readily adhere to substrata coated with extracellular matrix material (ECM) derived from human diploid fibroblasts by a mechanism that does not involve fibronectin (Harper and Juliano. 1981. Nature (Lond.). 290:136-138). Te Fn-dependent adhesion mechanism of parental cells (type 1 adhesion) and the ECM- dependent adhesion of ADV cells (type II adhesion) can also be discriminated on the basis of their differential sensitivity to proteolysis, with the type II mechanism being far more sensitive. In this communication we report that parental CHO cells possess both type I and type II mechanisms whereas ADV cells possess only the type II mechanism. We also identify a high molecular weight membrane glycoprotein (gp 265) that seems to play a role in type II adhesion. This component is detected by [125I]lactoperoxidase of [3H]borohydride- galactose oxidase labeling of surface proteins in WT and AD cells. Cleavage of gp 265 with low doses of proteases correlates completely with the loss of type II adhesion capacity. Thus CHO cells possess two functionally and biochemically distinct adhesion mechanisms, one involving exogenous Fn and the other mediated by the membrane component gp 265.     

Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L- lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.     

CHO cell aggregation induced by fibronectin-coated beads. Differences between wild-type and adhesion-variant cells

ADvF11 (F11), a Chinese Hamster Ovary (CHO) cell variant, is defective in its ability to adhere to fibronectin (Fn)-coated substrata but will adhere to substrata coated with poly-L-lysine, conA or extracellular matrix (ECM) [1]. We have observed that both F11 and CHO wild-type (WT) cells were able to bind 3H-Fn beads in a similar manner; however, only WT cells and not F11 cells aggregate in the presence of Fn beads. Both cell types aggregated similarly in the presence of lectins. Fn-bead-mediated aggregation was blocked by low temperature and aggregation did not occur when formaldehyde-fixed WT cells were used. Colchicine, tetracaine and cytochalasin B were not effective in blocking aggregation induced by Fn beads. These results suggest that: 1. Both WT and F11 cells have surface membrane-binding sites for Fn. 2. The aggregation defect in F11 cells is distal to the initial interaction between the cell surface and Fn, but proximal to the cytoskeletal rearrangements required for cell adhesion.     

Effects of alcohols on mouse embryonal carcinoma-substrate adhesion

The effects of ethanol and closely related alcohols on the cell-substrate adhesion of embryonal carcinoma cells were studied in microtiter wells using the enzyme cytochemical alkaline phosphatase technique and an ELISA reader. Three embryonal carcinoma cell lines (NF-1, NE and F9) were used. Prior to plating of cells the wells were coated with laminin, fibronectin or collagen type I. NF-1 cells adhered only to laminin; NE adhered to all substrata and uncoated wells equally well; F9 adhered only to fibronectin and laminin coated wells. Ethanol reduced the binding of cells to laminin and collagen type I but did not affect the binding of NE or F9 cells to fibronectin. The effect of ethanols was dose dependent; it lasted as long as an adequate concentration of this alcohol was maintained in vitro, and it was reversible. Other short chain alcohols inhibited the binding of cells to laminin proportionately to their membrane/buffer partition coefficients. These data show that various embryonal carcinoma cells differ with regards to their capacity to adhere to different extracellular matrix components. Cell adhesion to some but not all substrates can be prevented by ethanol and related short chain alcohols. The effects of alcohols on the adhesion of embryonal carcinoma cells to various substrates may be relevant for the elucidation of the fetal alcohol syndrome.     

Effects of alcohols on mouse embryonal carcinoma-substrate adhesion

Summary The effects of ethanol and closely related alcohols on the cell-substrate adhesion of embryonal carcinoma cells were studied in microtiter wells using the enzyme cytochemical alkaline phosphatase technique and an ELISA reader. Three embryonal carcinoma cell lines (NF-1, NE and F9) were used. Prior to plating of cells the wells were coated with laminin, fibronectin or collagen type I. NF-1 cells adhered only to laminin; NE adhered to all substrata and uncoated wells equally well; F9 adhered only to fibronectin and laminin coated wells. Ethanol reduced the binding of cells to laminin and collagen type I but did not affect the binding of NE or F9 cells to fibronectin. The effect of ethanols was dose dependent; it lasted as long as an adequate concentration of this alcohol was maintained in vitro, and it was reversible. Other short chain alcohols inhibited the binding of cells to laminin proportionately to their membrane/buffer partition coefficients. These data show that various embryonal carcinoma cells differ with regards to their capacity to adhere to different extracellular matrix components. Cell adhesion to some but not all substrates can be prevented by ethanol and related short chain alcohols. The effects of alcohols on the adhesion of embryonal carcinoma cells to various substrates may be relevant for the elucidation of the fetal alcohol syndrome.     

Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.     

An antibody that inhibits fibronectin-independent adhesion of fibroblasts to extracellular matrix material

Chinese hamster ovary (CHO) fibroblasts adhere to the extracellular matrix by both fibronectin-dependent and -independent mechanisms (Harper and Juliano, 1981a,b). Previous studies have suggested that a trypsin-sensitive, 265,000-dalton membrane glycoprotein (gp265) is involved in the fibronectin-independent adhesion process. Using a polyclonal antibody against soluble products obtained from trypsin-treated CHO cells, we have been able to further analyze this involvement. This antibody immunoprecipitates a trypsin-sensitive 265,000-dalton protein from detergent-solubilized cells. Incubation of AdvF11, a variant cell line that does not utilize fibronectin for adhesion, with this antibody blocks their adhesion to extracellular matrix material (ECM). The immunoglobulin fraction will also partially block adhesion of the parental cell line to ECM particularly when the ECM is first treated with an antifibronectin antibody. Taken together these results add support for the involvement of gp265 in fibronectin-independent adhesion and provide a methodology for further characterization.     

The integrin-linked kinase regulates cell morphology and motility in a rho-associated kinase-dependent manner

The integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transmission from integrin and growth factor receptors. We have determined that ILK regulates U2OS osteosarcoma cell spreading and motility in a manner requiring both kinase activity and localization. Overexpression of wild-type (WT) ILK resulted in suppression of cell spreading, polarization, and motility to fibronectin. Cell lines overexpressing kinase-dead (S343A) or paxillin binding site mutant ILK proteins display inhibited haptotaxis to fibronectin. Conversely, spreading and motility was potentiated in cells expressing the "dominant negative," non-targeting, kinase-deficient E359K ILK protein. Suppression of cell spreading and motility of WT ILK U2OS cells could be rescued by treatment with the Rho-associated kinase (ROCK) inhibitor Y-27632 or introduction of dominant negative ROCK or RhoA, suggesting these cells have increased RhoA signaling. Activation of focal adhesion kinase (FAK), a negative regulator of RhoA, was reduced in WT ILK cells, whereas overexpression of FAK rescued the observed defects in spreading and cell polarity. Thus, ILK-dependent effects on ROCK and/or RhoA signaling may be mediated through FAK.     

Fibronectin from aged fibroblasts is defective in promoting cellular adhesion

Late passage fibroblasts show decreased cell-substrate adhesion. We provide evidence that the reduced adhesion is due to a defect in the adhesive glycoprotein fibronectin. Late passage cells become more adhesive in culture media that has been conditioned by the growth of early passage cells. Analysis of fibronectins purified from early and late passage cell conditioned media indicates that there are striking differences in their abilities to promote cell adhesion. Young cell fibronectin supports the maximal adhesion of both young and old cells. However, old cells require quantitatively more fibronectin. In contrast, old cell fibronectin is less effective in supporting the adhesion of either cell type. In addition, neither cell type achieves a normal morphology in the presence of old cell fibronectin. The results support the conclusion that the fibronectin released by late passage cells is defective and does not support normal cell-substrate interactions.     

Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle-like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (αVβ3 integrin) and fibronectin (α5β1 integrin) receptors in these changes. Mesangial cells on vitronectin-rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin-coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin-coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin-coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin—but not on vitronectincoated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus-type extracellular matrix contacts. © 1993 Wiley-Liss, Inc.     

Motility of fibronectin receptor-deficient cells on fibronectin and vitronectin: collaborative interactions among integrins.

Cells are capable of adhering to and migrating on protein components of the extracellular matrix. These cell-matrix interactions are thought to be mediated largely through a family of cell surface receptors termed integrins. However, the manner in which individual integrins are involved in cell adhesion and motility has not been fully determined. To explore this issue, we previously selected a series of CHO variants that are deficient in expression of the integrin alpha 5 beta 1, the "classical" fibronectin receptor. Two sets of subclones of these variants were defined which respectively express approximately 20% or 2% of fibronectin receptor on the cell surface when compared to wild-type cells (Schreiner, C. L., J. S. Bauer, Y. N. Danilov, S. Hussein, M. M. Sczekan, and R. L. Juliano. 1989. J. Cell Biol. 109:3157-3167). In the current study, the variant clones were tested for haptotactic motility on substrata coated with fibronectin or vitronectin. Data from assays using fibronectin show that cellular motility of the 20% variants was substantially decreased (30-75% of wild type), while the motility of the 2% variants was nearly abolished (2-20% of wild type). Surprisingly, a similar pattern was seen for haptotactic motility of both 2% and 20% variants when vitronectin was used (approximately 20-30% of wild type). The reduced haptotactic motility of the fibronectin receptor-deficient variant clones on vitronectin was shown not to be due to reduced vitronectin receptor (alpha v beta 3) expression nor to a failure of these variants to adhere to vitronectin substrata. Transfection of the deficient variants with a cDNA for the human alpha 5 subunit resulted in normal levels of fibronectin receptor expression (as a human alpha 5/hamster beta 1 chimera) and restored the motility of the CHO variants on fibronectin and vitronectin. This indicates that expression of the alpha 5 subunit is required for normal haptotactic motility on vitronectin substrata and suggests that the fibronectin receptor (alpha 5 beta 1) plays a cooperative role with vitronectin receptors in cell motility.     

Collagen-like polypeptide poly(Pro-Hyp-Gly) conjugated with Gly-Arg-Gly-Asp-Ser and Pro-His-Ser-Arg-Asn peptides enchances cell adhesion, migration, and stratification

Collagens are widely used in medical applications, including as a scaffold for tissue regeneration. However, animal-derived collagens have several drawbacks, such as low thermal stability, nonspecific cell adhesion, antigenicity, and contamination with pathogenic substances. To overcome these problems, we chemically synthesized the collagen-like polypeptide, poly(prolyl-hydroxyprolyl-glycyl) (poly(Pro-Hyp-Gly)), which forms a collagen-like triple-helical structure and shows biodegradability and biocompatibility. Here, we designed a novel scaffold where fibronectin-derived Gly Arg-Gly-Asp-Ser (GRGDS) and Pro-His-Ser-Arg-Asn (PHSRN) peptides were simultaneously conjugated with poly(Pro-Hyp-Gly). We assessed cell adhesion and migration activities using NIH3T3 cells in the scaffold and stratification ofimmortalized rabbit corneal epithelial cells. Cell adhesion was enhanced in scaffolds with GRGDS, increased with increasing amounts of conjugated GRGDS, and was significantly higher than bovine type I atelocollagen but lower than bovine fibronectin. Interestingly, simultaneous conjugation of GRGDS and PHSRN synergistically enhanced cell migration. Scaffolds containing almost equal amounts of GRGDS and PHSRN showed significantly higher cell migration than bovine type I atelocollagen. Addition of free GRGDS completely inhibited cell migration on the scaffold, whereas addition of free PHSRN partially inhibited cell migration. These results suggest that GRGDS plays a definitive role, and PHSRN plays an additional role, in cell migration. Conjugation of GRGDS resulted in the same level of stratification of rabbit corneal epithelial cells compared with bovine type I atelocollagen and bovine fibronectin. Because the simultaneous conjugation of GRGDS and PHSRN on poly(Pro-Hyp-Gly) enhances cell adhesion, migration, and stratification, it may be a useful scaffold for tissue regeneration.     

Adhesion of B lymphoid (MPC-11) cells to type I collagen is mediated by integral membrane proteoglycan, syndecan.

Differentiating B lymphocytes undergo changes in cell-cell and cell-matrix adhesion that control their movement through a series of distinct microenvironments. The integral membrane proteoglycan, syndecan, is a candidate for mediating B lymphocyte-matrix interactions because it is expressed on B lymphocytes only at times when they associate with matrix, and because syndecan is known to behave as a matrix receptor on simple epithelia. However, syndecan from B lymphocytes is significantly smaller in molecular mass than syndecan from simple epithelia (85 vs 160 kDa) suggesting that syndecan may have distinct functions on these two cell types. Our study was undertaken to determine if syndecan mediates adhesion of B lineage cells to extracellular matrix. The murine myeloma cell line MPC-11 was used because syndecan is the only major heparan sulfate proteoglycan detected on these cells and because they express a form of syndecan almost identical to that found on normal B lymphocytes. Cell binding assays demonstrate that syndecan binds MPC-11 cells to type I collagen. Binding is inhibited by heparin, by pretreatment of cells with heparitinase or by growth of cells before the assay in chlorate, an inhibitor of sulfation. Solid phase assays show that syndecan purified from MPC-11 cells binds to type I collagen but not type IV collagen, laminin, or fibronectin. The interaction of MPC-11-derived syndecan with type I collagen is of relatively high affinity (Kd app = 143 nM) as measured by affinity coelectrophoresis. However, the 160-kDa form of syndecan isolated from epithelial cells has a greater than fourfold higher affinity for type I collagen (Kd app = 31 nM) than does the MPC-11 syndecan, suggesting that different molecular forms of syndecan have distinct ligand binding properties. These results demonstrate that syndecan can mediate B lymphocyte interactions with matrix and suggest that changes in syndecan expression during B cell differentiation are a mechanism for controlling B cell localization within specific microenvironments.     

肝癌细胞-胞外基质粘附性与粘附识别序列的相关性

以微管吸吮技术研究了人肝癌细胞在ＩＶ型胶原／层粘连蛋白（ＬＮ）／纤维连结蛋白（ＦＮ）裱衬表面的粘附性。进一步,用四种人工合成肽精－甘－天冬－丝（ＲＧＤＳ）、甘－精－甘－天冬－苏－脯ＧＲＧＤＴＰ）、酪－异亮－甘－丝－精（ＹＩＧＳＲ０和半胱－天冬－脯－甘－酪－异亮－甘－丝－精（ＣＤＰＧＹＩＧＳＲ）研究了肝癌细胞粘附性对两种粘附识别序列ＲＧＤ和ＹＩＧＳＲ的依赖性。为了归纳和整理实验结果,根据竞争性抑制的     

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.     

Galectin-8 functions as a matricellular modulator of cell adhesion

The interaction of cells with the extracellular matrix regulates cell adhesion and motility. Here we demonstrate that different cell types adhere and spread when cultured in serum-free medium on immobilized galectin-8, a mammalian beta-galactoside-binding protein. At maximal doses, galectin-8 is equipotent to fibronectin in promoting cell adhesion and spreading. Cell adhesion to immobilized galectin-8 is mediated by sugar-protein interactions with integrins, and galectin-8 triggers integrin-mediated signaling cascades including Tyr phosphorylation of focal adhesion kinase and paxillin. Cell adhesion is potentiated in the presence of Mn(2+), whereas it is interrupted in the presence of soluble galectin-8, integrin beta(1) inhibitory antibodies, EDTA, or thiodigalactoside but not by RGD peptides. Furthermore, cells readily adhere onto immobilized monoclonal galectin-8 antibodies, which are equipotent to integrin antibodies in promoting cell adhesion. Cell adhesion to immobilized galectin-8 is partially inhibited by serum proteins, suggesting that complex formation between immobilized galectin-8 and serum components generates a matrix that is less supportive of cell adhesion. Accordingly, cell motility on immobilized galectin-8 readily takes place in the presence of serum. Truncation of the C-terminal half of galectin-8, including one of its two carbohydrate recognition domains, largely abolishes its ability to modulate cell adhesion, indicating that both carbohydrate recognition domains are required to maintain a functional form of galectin-8. Collectively, our findings implicate galectin-8 as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of cell surface integrin receptors. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Because of its dual effects on the adhesive properties of the cells and its association with fibronectin, galectin-8 might be considered a novel type of matricellular protein.     

RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with peptide I. Whereas both peptides promoted RGD-independent cell adhesion, peptide CS1 failed to bind heparin, and exogenous peptide CS1 failed to inhibit peptide I-mediated cell adhesion. The results demonstrate a role for distinct heparin-dependent and -independent cell adhesion determinants on the 33-kD fragment, neither of which are related to the RGD-dependent integrin interaction with fibronectin.     

Retinoic acid effects on an SV-40 large T antigen immortalized adult rat bone cell line

Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against alpha1beta1 integrin. RA up-regulates steady-state levels of alpha1, mRNA without affecting beta1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins.