title: Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain.
abstract: Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KS-PGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was absorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind. Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 3T3 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects*; D002478:Cells, Cultured; D002807:Chondroitin / Q000031:analogs & derivatives*; D011508:Chondroitin Sulfate Proteoglycans / Q000494:pharmacology*; D003094:Collagen / Q000494:pharmacology; D003871:Dermatan Sulfate / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005353:Fibronectins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011509:Proteoglycans / Q000494:pharmacology*; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000378:metabolism
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D005353:Fibronectins; D006025:Glycosaminoglycans; D011509:Proteoglycans; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; C032649:dermatan sulfate proteoglycan; D003871:Dermatan Sulfate; D002807:Chondroitin; D003094:Collagen