SYNTHETIC PEPTIDE-CONTAINING ARG-GLY-ASP INHIBITS BONE-FORMATION AND RESORPTION IN A MINERALIZING ORGAN-CULTURE SYSTEM OF FETAL-RAT PARIETAL BONES

The role of integrins, cell surface receptors involved in cell adhesio n to the matrix, was studied in a mineralizing organ culture system. I ntegrin-mediated cell attachment to matrix proteins has been shown to depend partially on the amino acid sequence Arg-Gly-Asp (RGD), present in the extracellular matrix proteins. Therefore, the effect of RGD pe ptides on bone formation and resorption was studied in the mineralizin g organ culture system derived from 18 day fetal rat parietal bones. A ddition of 0.1-50 mu M GRGDSPK to bones cultured for 4 days inhibited mineralization in a dose-dependent manner as determined by measuring c alcium content and % bone/unit area of tissue. A maximal decrease in c alcium content and % bone/unit area of 32.5 and 42.9%, respectively, w as found with 50 mu M GRGDSPK. With 10 and 50 mu M GRGDSPK, bone morph ology was dramatically altered, with a disruption of osteoblast and mi neralized matrix organization. To assess the effect of the peptides on bone resorption, fetal bones were prelabeled in vivo with Ca-45 and r esorption was stimulated in vitro with parathyroid hormone in the pres ence or absence of the peptide. A significant decrease in Ca-45 releas e was found with 10 and 50 mu M GRGDSPK. Osteoclast number was also si gnificantly decreased on the bone surface. The peptide was not cytotox ic, since no effect on DNA content, dry weight, or collagen synthesis was found. GRADSP, a control peptide, had no significant effect on min eralization, resorption, or other parameters of bone growth. Visualiza tion of beta(1) and alpha(2) integrin in GRGDSPK-treated bones by indi rect immunofluorescence demonstrated a decreased in integrin staining, particularly in the osteoblast layer, compared to control bones and b ones treated with GRADSP. The inhibition of bone formation and resorpt ion by an ROD-containing peptide in a mineralizing organ culture syste m suggests that integrins have an important role in osteoblast and ost eoclast-mediated bone remodeling.