The Majority of Osteoclasts Require mRNA and Protein Synthesis for Bone Resorption in Vitro

Abstract

Mithramycin is an mRNA synthesis inhibitor that has been used to decrease bone resorption in patients with humoral hypercalcemia and Paget′s disease. During studies on the mechanism of action of mithramycin it became clear that the compound has a direct inhibitory effect on osteoclastic bone resorption in the in vitro bone slice assay. At concentrations of 0.1 - 100 nM mithramycin directly inhibited osteoclastic bone resorption dose-dependently up to 66 ± 5% at 100 nM (mean ± SEM, 3 expts.). Another mRNA synthesis inhibitor, actinomycin D (0.1 - 100 nM) and the protein synthesis inhibitor, cycloheximide (0.1 - 10 μM), also dose-dependently inhibited osteoclastic bone resorption by 78 ± 7% at 100 nM and 76 ± 7% at 10 μM, respectively. Mithramycin and actinomycin D at 100 nM did not affect osteoclast survival on bone slices and were therefore not cytotoxic at the concentrations used. Mithramycin (100 nM) and cycloheximide (10 μM) both slightly decreased osteoctast cytoplasmic spreading. Addition of 100 nM mithramycin 6 hr after osteoclast adhesion to bone slices still inhibited subsequent resorption by 50%, indicating a continued but lesser requirement for mRNA synthesis during bone resorption. These results show that ∼75% of osteoclasts obtained from neonatal rat long bones are activated by adhesion to mineralized bone surfaces and require mRNA and protein synthesis in order to resorb bone in vitro.