Doxycycline-induced inhibition of prolidase activity in human skin fibroblasts and its involvement in impaired collagen biosynthesis

Abstract

Several lines of evidence suggest that doxycycline, a semi-synthetic derivative of tetracycline, may be a useful agent in the treatment of osteoarthritis. It inhibits collagen synthesis and collagenase activity in hypertrophic chondrocytes, slowing the process of collagen turnover. However, the mechanism of doxycycline-induced inhibition of these processes has not been established. We considered prolidase, an enzyme involved in collagen metabolism, as a possible target for the doxycycline-induced inhibition of collagen synthesis. Cultured human skin fibroblasts, specialized for collagen synthesis, were used as model cells. Prolidase [E.C. 3.4.13.9] is a manganese-dependent cytosolic exopeptidase that cleaves imidodipeptides containing C-terminal proline, thus providing large amounts of proline for collagen resynthesis. Enzyme activity is regulated through the β 1 integrin receptor. Therefore, we compared the effect of doxycycline on prolidase activity and expression, collagen biosynthesis, gelatinolytic activity and β 1 integrin expression in 24-h treated cultured human skin fibroblasts. We found that doxycycline induced coordinately inhibition of prolidase activity and collagen biosynthesis (IC 50 at about 150 μg/ml) and gelatinolytic activity in cultured human skin fibroblasts. The inhibitory effect of doxycycline on the processes was not due to the cytotoxicity of this drug, as shown in the cell viability tetrazoline test. However, an inhibitory effect of the drug on DNA synthesis was observed (IC 50 at about 100 μg/ml). The decrease in prolidase activity in fibroblasts treated with doxycycline was not accompanied by any differences in the amount of prolidase or β 1 integrin recovered from these cells, as shown by Western immunoblot analysis. This suggests that the doxycycline-induced down-regulation of prolidase is a post-translational event. The data presented here raise the possibility that the doxycycline-induced decrease in collagen biosynthesis is mostly due to the inhibition of prolidase activity.