Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro

Abstract

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic beta subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor-1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.

Figure 1

Proteasome inhibitors stimulate bone formation in vitro and in vivo. (a–e) Histologic sections of cultured murine neonatal calvarial bones treated with either (a) media alone, (b) lactacystin (2.5 μM), (c) PS1 (0.1 μM), (d) epoxomicin (0.01 μM), or (e) eponemycin (0.5 μM). (f–i) Histologic sections, BFR, and MAR of murine proximal tibia from mice treated daily for 5 days and sacrificed 16 days later. (f) Vehicle, (g) PTH (0.08 mg/kg/day subcutaneously), (h) PS1 (2 mg/kg/day subcutaneously), (i) epoxomicin (0.1 mg/kg/day intraperitoneally). Values in parentheses are percentage of change from vehicle-treated controls. BV/TV bone volume/tissue volume. *P < 0.05.

Figure 2

Proteasome inhibitors stimulate bone formation in murine calvariae. (a–e) Histological sections of calvariae from mice injected over the right side of the calvarium three times a day for 5 days with either (a and e) vehicle control, (b) PS1 at 0.1 mg/kg/day, (c) PS1 at 1 mg/kg/day, (d) PS1 at 5 mg/kg/day, (f) epoxomicin at 0.05 mg/kg/day, (g) epoxomicin at 0.1 mg/kg/day, and (h) epoxomicin at 0.5 mg/kg/day. Arrows indicate width of new bone.

Figure 3

Proteasome inhibition correlates with bone formation. The stimulation of bone formation activity of these different proteasome inhibitors in calvarial bone organ cultures correlates strongly with the ability of these compounds to inhibit the activity of the proteasome. ED₅₀ is the effective dose required to either inhibit 50% of the proteasomal activity or stimulate 50% of the bone formation activity.

Figure 4

Effect of proteasome inhibition on BMP-2 protein expression and bone formation. (a) Luciferase activity of cell lysates of 2T3 cells transfected with murine BMP-2 promoter (–2712/+165) operatively linked to firefly luciferase cDNA and treated with either simvastatin (dashed line), PS1 (dotted line), or epoxomicin (solid line). (b) Effects of PS1 (50 and 100 nM) on mRNA expression of BMP-2, BMP-4, and BMP-6 in FRC cells. FRC cells were treated with PS1 (50 and 100 nM) for 6 hours and cultured for 48 hours. The mRNA expression of BMP-2, BMP-4, and BMP-6 was detected by Northern blot analysis and quantitated by the PhosphoImager analysis method. PS1 increased BMP-2 mRNA expression 2.4-fold and 3.5-fold at concentrations of 50 and 100 nM, but had no significant effect on BMP-4 and BMP-6 mRNA expression. (c) Correlation of BMP-2 protein production from Hu09 human osteoblastic cells and inhibitory activity on the proteasome among proteasome inhibitors of different types. (d) Correlation of BMP-2 protein production from Hu09 human osteoblastic cells and bone formation activity (as assessed by effects on calvarial bone organ cultures).

Figure 5

Noggin inhibits bone formation induced by proteasome inhibitors. Histologic sections of cultured murine neonatal calvarial bones treated with (a) media alone, (b) noggin (2 μg/ml), (c) epoxomicin (20 nM), (d) epoxomicin (20 nM) and noggin (2 μg/ml), (e) rhBMP-2 (100 ng/ml), (f) rhBMP-2 (100 ng/ml) and noggin (2 μg/ml), (g) aFGF (50 ng/ml), (h) aFGF (50 ng/ml) and noggin (2 μg/ml).

Figure 6

Proteasome inhibitors inhibit the proteolytic processing of Gli3 protein. The 293 cells were transfected with expression plasmid of Flag-tagged FL-Gli3 and treated with 200 μM IBMX in the presence or absence of different concentrations of PS1 (a) and epoxomicin (b) (1, 10, and 100 nM). IBMX induced Gli3 degradation and production of trGli3. PS1 reversed IBMX-induced Gli3 degradation in a dose-dependent manner.

Figure 7

Truncated Gli3 inhibits BMP-2 gene transcription through specific response elements. (a and b) Different amounts of expression plasmids of an FL-Gli3 and trGli3 (62.5, 125, and 250 ng/ml) were cotransfected with the BMP-2 promoter (–2712/+165-Luc) in C2C12 cells (24-well culture plates). FL-Gli3 slightly enhanced BMP-2 promoter activity (a), while trGli3 inhibited BMP-2 promoter activity in a dose-dependent manner (b) (n = 4). *P < 0.05, ANOVA, followed by the Dunnett test. (c) A diagram showing deletion constructs of the BMP-2 promoter, the putative Gli-binding sites in –310/–150 region of BMP-2 promoter, and mutations created in these putative Gli-binding sites (GBS). (d) Expression plasmid of trGli3 was cotransfected with deletion constructs of BMP-2 promoter. trGli3 significantly inhibited promoter activity in constructs –2712/+165, –1803/+165, –839/+165, and –310/+165, but not the construct –150/+165 (n = 3). *P < 0.05, unpaired t test. (e) Expression plasmid of trGli3 was cotransfected with BMP-2 promoter (–310/+165-Luc) constructs with mutations in three putative Gli-binding sites. The inhibitory effect of trGli3 on BMP-2 promoter activity was reversed when these Gli-binding sites were mutated. n = 5; #P < 0.05, unpaired t test.

Figure 8

The stimulatory effects of proteasome inhibitors on BMP-2 expression are dependent on Gli3 processing. C2C12 and 2T3 cells were transfected with trGli3 (200 ng/well, 24-well plates) and treated with different concentrations of PS1 (a and c) and epoxomicin (b and d) (0–80 nM). Overexpression of trGli3 reduced basal level of BMP-2 promoter activity and completely blocked PS1- and epoxomicin-induced BMP-2 promoter activity (n = 3) in C2C12 (a and b) and 2T3 (c and d) cells. *P < 0.05, compared with nontreatment control; #P < 0.05, compared with empty vector-transfected control; two-way ANOVA followed by the Dunnett test.

Figure 9

Calvarial tissues were transfected with empty vector or expression plasmid of trGli3 and treated with or without epoxomicin (20 nM) and insulin (100 μg/ml). Changes in new bone formation and ALP activity in the medium were examined. Expression of trGli3 completely abolished epoxomicin-induced new bone formation (a and c) and ALP activity (d). In contrast, expression of trGli3 had no significant effects on insulin-induced bone formation (b and c) and ALP activity (d). *P < 0.05, unpaired t test, compared with the group without epoxomicin treatment. #P < 0.05, unpaired t test, compared with the group without insulin treatment.