The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S-GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S-GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand.

We have shown that when chondrocytes are isolated by collagenase digestion of hyaline cartilage from growth plate, nasal, and epiphyseal cartilages of bovine fetuses they rapidly elaborate an extracellular matrix in culture. Only growth plate chondrocytes can calcify this matrix as ascertained by incorporation of 45Ca2+, detection of mineral with von Kossa's stain and electron microscopy. There is an extremely close direct correlation between 45Ca2+ incorporation in the first 24 h of culture and the content of the C-propeptide of type II collagen, measured by radioimmunoassay, at the time of isolation and during culture. Moreover, growth plate cells have an increased intracellular content of the C-propeptide per deoxyribonucleic acid and, during culture, per hydroxyproline (as a measure of helical collagen) compared with nasal and epiphyseal chondrocytes. In growth plate chondrocytes 24,25-dihydroxycholecalciferol (24,25-[OH]2D3), but not 1,25-dihydroxycholecalciferol alone, stimulates the net synthesis of the C-propeptide and calcification; proteoglycan net synthesis is unaffected. Together, these metabolites of vitamin D further stimulate C-propeptide net synthesis but do not further increase calcification stimulated by 24,25-(OH)2D3. These observations further demonstrate the close correlation between the C-propeptide of type II collagen and the calcification of cartilage matrix.