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.
Geriatric Research, Education and Clinical Center, Department of Veterans Affairs Medical Center, Sepulveda, California, 91343, USA. murrayes@ucla.edu
E J
EJ
Murray
G V
GV
Bentley
M S
MS
Grisanti
S S
SS
Murray
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001704:Biopolymers / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000187:drug effects / Q000502:physiology*; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D010281:Parathyroid Hormone / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D015290:Second Messenger Systems / Q000187:drug effects; D019379:Teriparatide / Q000031:analogs & derivatives / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
D001704:Biopolymers; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D014452:Ubiquitins; D019379:Teriparatide; C067713:lactacystin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C035748:parathyroid hormone (1-34)amide; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine