ASCORBIC-ACID ALTERS COLLAGEN INTEGRINS IN BONE CULTURE

The effects of ascorbic acid on collagen synthesis, mineralization, an d integrins were investigated in a mineralizing organ culture system d erived from 20-day fetal rat parietal bones. A significant dose-depend ent decrease in calcification at 96 h was demonstrated with decreasing concentrations of ascorbic acid (100-0 mu g/ml). No effect on DNA con tent, [H-3]thymidine incorporation, or dry weight was found in control (100 mu g/ml ascorbic acid) bones compared with bones treated with de creased ascorbic acid concentrations (10, 1, and 0 mu g/ml). Collagen synthesis, measured by [H-3]proline incorporation, and alpha 1(I) proc ollagen messenger RNA levels were also unaffected. However, ascorbic a cid produced a dose-dependent decrease in the hydroxyproline content, with a maximal 76.8% decrease in bones without ascorbic acid compared with the control bones with 100 mu g/ml ascorbic acid. Light microscop y of the ascorbic acid-deficient bones revealed a disruption of the os teoblast layer with misshapen osteoblasts and a decrease in the osteoi d seam. The loss of osteoblast organization was also confirmed by anal yzing the integrins for collagen by Northern and Western blot and immu nofluorescence microscopy. A dose-dependent decrease in alpha(2) and b eta(1) integrin messenger RNA levels and in alpha(1), alpha(2), and be ta(1) protein were found in 96-h bone cultures deficient in ascorbic a cid. These integrin subunits mediate the binding of osteoblasts to col lagen. Immunofluorescence microscopy also demonstrated a dose-dependen t decrease in alpha(2) and beta(1) staining of the osteoblast layer. H owever, the protein levels of alpha(3) and alpha(5) subunits were not affected. No beta(5) was detected, whereas only bones cultured without ascorbic acid demonstrated a small decrease in alpha(v) and beta(3) p rotein levels. The alpha(3), alpha(5), alpha(v), and beta(3) subunits are involved in cell binding to extracellular matrix proteins other th an collagen. Thus, the integrins for collagen are down-regulated, prob ably in response to the underhydroxylated collagen fibrils, which caus es a disruption of osteoblast organization leading to a decrease in mi neralization of bone. Integrin assays for specific extracellular prote ins may be useful tools in detecting matrix defects in various metabol ic bone diseases.