Bortezomib impairs osteosclerosis development in TPO^high mice through OPG inhibition. (A) Adherent murine stromal cell layers were observed by light microscopy. The scale bar is 50 μm. (B) IL-1α stimulates OPG secretion by stromal cells. OPG levels were quantified in murine stromal cell culture supernatants, with or without IL-1α stimulation for 24 hours, using an ELISA. Results are presented as the mean ± SEM of 5 different adherent stromal cell layers. Results of statistical analysis with the Wilcoxon test are as follows: stimulated (IL-1α 5 ng/mL) versus unstimulated (baseline); *P < .05. (C-D) Bortezomib decreases OPG levels in both plasma and extracellular fluids of marrow in TPO^high mice regardless of dosage. OPG levels were quantified in (C) plasma and in (D) extracellular fluids using an ELISA. Results in plasma and in extracellular fluids are presented as the mean ± SEM of 12 (except for the “1 mg/kg” group that included only 6 surviving mice at week 8) and of 3 animals per experimental group, respectively (similar results were obtained in 3 other animals per experimental group at week 16 after engraftment, except for the “1 mg/kg” group). Results of statistical analysis with the Wilcoxon test are as follows: treated (bortezomib 0.25 mg/kg, 0.5 mg/kg, and 1 mg/kg) versus untreated (vehicle) mice; *P < .05. Bzb indicates bortezomib. (E-H) Bortezomib dramatically impairs osteosclerosis development in TPO^high mice after 12 weeks of treatment (16 weeks after engraftment). Representative histologic sections of femora stained by hematoxylin and eosin from untreated (vehicle) TPO^high mice (E-F) show osteosclerosis development with accumulation of bone trabeculae in the medullar cavity (arrow). Bortezomib treatment (0.5 mg/kg) impairs considerably new bone formation (G-H). Original magnification × 100 (F, H), × 25 (E, G).