title: Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides.
abstract: 3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.
authors.affiliation: Laboratoire de Biologie du Tissu Conjonctif, Faculté de Médecine, Université Paris XII, Créteil, France.
authors.forename: V
authors.initials: V
authors.lastname: Groult
authors.forename: W
authors.initials: W
authors.lastname: Hornebeck
authors.forename: P
authors.initials: P
authors.lastname: Ferrari
authors.forename: J M
authors.initials: JM
authors.lastname: Tixier
authors.forename: L
authors.initials: L
authors.lastname: Robert
authors.forename: M P
authors.initials: MP
authors.lastname: Jacob
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology*; D004549:Elastin / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012867:Skin / Q000166:cytology; D014333:Tropoelastin / Q000378:metabolism; D019096:Vitronectin
chemical_list: D005353:Fibronectins; D006023:Glycoproteins; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D011956:Receptors, Cell Surface; D014333:Tropoelastin; D019096:Vitronectin; C054901:elastin-binding proteins; D004549:Elastin; C058161:glycyl-arginyl-glycyl-aspartyl-seryl-proline; C058347:glycyl-arginyl-glycyl-glutamyl-seryl-proline; C046617:arginyl-glycyl-aspartyl-serine