The ubiquitin-proteasome system and cellular proliferation and regulation in osteoblastic cells.

The 26S proteasome is the macromolecular assembly that mediates ATP- and ubiquitin-dependent extralysosomal intracellular protein degradation in eukaryotes. However, its contribution to the regulation of osteoblast proliferation and hormonal regulation remains poorly defined. Treating osteoblasts with MG-132 or lactacystin (membrane-permeable proteasome inhibitors) attenuates proliferation. Three proteasome activities (peptidylglutamyl-peptide bond hydrolase-, chymotrypsin-, and trypsin-like) were detected in osteoblasts. Catabolic doses of PTH stim-ulated these activities, and cotreatment with PTH and MG-132 blocked stimulation. The proteasome alpha- and beta-subunits, polyubiquitins, and large ubiquitin-protein conjugates were detected by Western blotting. A 90-min treatment with 10 nM PTH had no effect on the amount of proteasome alpha or beta subunit protein, but increased the relative amount of large ubiquitin-protein conjugates by 200%. MG-132 inhibited deubiquitination of large ubiquitin-protein conjugates. The protein kinase A inhibitor SQ22536 blocked much of the PTH-induced stimulation of MCP activities, while dibutyryl cAMP stimulated it, suggesting that protein kinase A-dependent phosphorylation is important in PTH stimulation of proteasome activities. In conclusion, the ubiquitin-proteasome system is essential for osteoblast proliferation under control and PTH-treated conditions. PTH mediates its metabolic effects on the osteoblast, in part, by enhancing ubiquitinylation of protein substrates and stimulating three major proteasome activities by a cAMP-dependent mechanism.