Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide of malignancy

Abstract

The effect of human recombinant cystatin C, a cysteine proteinase inhibitor, on bone resorption in vitro was evaluated. Bone resorption was assessed by analyzing the release of ⁴⁵Ca and ³H from mouse calvarial bones prelabeled in vivo by injections with ⁴⁵Ca or [³H]proline, respectively. In 24 h cultures, cystatin C (50 μg/ml) significantly inhibited the release of ⁴⁵Ca and ³H stimulated by parathyroid hormone (PTH, 15 nmol/liter) or parathyroid hormone-related peptide of malignancy (PTHrP, 15 nmol/liter). The degree of inhibition caused by cystatin C in these 24 h cultures was similar to that caused by calcitonin (30 ng/ml). The inhibitory effect of cystatin C on ⁴⁵Ca release induced by PTH was sustained in 96 h cultures, whereas the initial inhibition caused by calcitonin was transient. Cystatin C, 10–100 μg/ml, caused a dose-dependent inhibition of PTH (15 nmol/liter), and PTHrP (15 nmol/liter) stimulated ⁴⁵Ca release. Addition of 50 μg/ml of cystatin C to mouse bone cultures inhibited the release of ⁴⁵Ca induced by PTH and PTHrP at a wide range of submaximal and maximal concentrations of hormones (0.01–10 nmol/liter). No effect of cystatin C on ⁴⁵Ca release in dead bones could be observed, nor did the inhibitor decrease the release of calcium in control bones. The inhibition by cystatin C on PTH-induced mineral mobilization was reversible. Cystatin C (1–100 μg/ml) did not affect protein synthesis or mitotic activities in mouse calvarial bones as assessed by the incorporation of I'HIproline and [³H]thymidine, respectively. These data show that cystatin C is a potent inhibitor of mineral mobilization and matrix degradation in cultured bones stimulated to resorb by PTH and PTHrP and that this effect is not due to general cytotoxicity.