Synthetic peptide containing Arg-Gly-Asp inhibits bone formation and resorption in a mineralizing organ culture system of fetal rat parietal bones

Abstract

The role of integrins, cell surface receptors involved in cell adhesion to the matrix, was studied in a mineralizing organ culture system. Integrin-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 peptides on bone formation and resorption was studied in the mineralizing organ culture system derived from 18 day fetal rat parietal bones. Addition of 0.1–50 μM GRGDSPK to bones cultured for 4 days inhibited mineralization in a dose-dependent manner as determined by measuring calcium content and % bone/unit area of tissue. A maximal decrease in calcium content and % bone/unit area of 32.5 and 42.9%, respectively, was found with 50 μM GRGDSPK. With 10 and 50 μM GRGDSPK, bone morphology was dramatically altered, with a disruption of osteoblast and mineralized matrix organization. To assess the effect of the peptides on bone resorption, fetal bones were prelabeled in vivo with ⁴⁵Ca and resorption was stimulated in vitro with parathyroid hormone in the presence or absence of the peptide. A significant decrease in ⁴⁵Ca release was found with 10 and 50 μM GRGDSPK. Osteoclast number was also significantly decreased on the bone surface. The peptide was not cytotoxic, since no effect on DNA content, dry weight, or collagen synthesis was found. GRADSP, a control peptide, had no significant effect on mineralization, resorption, or other parameters of bone growth. Visualization of β₁ and α₂ integrin in GRGDSPK-treated bones by indirect immunofluorescence demonstrated a decreased in integrin staining, particularly in the osteoblast layer, compared to control bones and bones treated with GRADSP. The inhibition of bone formation and resorption by an RGD-containing peptide in a mineralizing organ culture system suggests that integrins have an important role in osteoblast and osteoclast-mediated bone remodeling.