Regulation of osteoblastic differentiation by the proteasome inhibitor bortezomib.

In eukaryotic cells, degradation of most intracellular proteins is carried out by the ubiquitin-proteasome pathway. Recent investigations suggest that bone metabolism is also regulated by this pathway. The clinical efficacy of bortezomib, a 26S proteasome inhibitor used as an anticancer drug, has been linked to an increase in bone formation. In this study, we show that proteasome inhibitors induce expression of osteoblastic differentiation-related genes such as osteocalcin and alkaline phosphatase in C2C12 cells. In contrast, myogenic differentiation is inhibited. Among the proteasome inhibitors tested, bortezomib induced the greatest increase in osteocalcin expression. Although these effects were similar to that of bone morphogenetic protein (BMP) 2, proteasome inhibitors did not induce transcriptional activity of Smad1/4-dependent reporter or BMP2 signaling target gene expression. Transient transfection of osteocalcin promoter-luciferase constructs with bortezomib resulted in an increase in luciferase activity. Mutation of OSE2, but not OSE1, sites of the osteocalcin promoter diminished the bortezomib-induced activity. Also, Runx2 binding activity and protein levels were induced by bortezomib treatment. These results suggest that the bortezomib induces osteoblastic differentiation by modifying the activity of Runx2 and that the function of the proteasome in controlling degradation of differentiation-related transcription factors plays an important role in osteoblast differentiation.

Wasf2: a novel target of intermittent parathyroid hormone administration.

Systemic intermittent administration of parathyroid hormone (PTH) stimulates bone formation in animals and humans, and recombinant human PTH1-34 (teriparatide) is used clinically for the treatment of osteoporosis. In this study, we investigated the regulation of gene expression by intermittent PTH administration in MC3T3-E1 osteoblastic cells. We found that intermittent PTH1-34 administration downregulated Wiskott-Aldrich syndrome protein family member (Wasf) 2 mRNA expression. Wnt inhibitor, IWP-2, and protein kinase C inhibitor, Go6976, inhibited this downregulation. However, continuous PTH did not affect Wasf2 expression. Transfection of Wasf2 siRNA reduced bone sialoprotein (BSP) mRNA expression in a similar manner following intermittent PTH administration in MC3T3-E1 cells. These results identify Wasf2 as a novel target of intermittent PTH administration via the Wnt and phosphoinositide-dependent protein kinase signaling pathways, and the resulting regulation of BSP expression may contribute to the anabolic effects of PTH.

Canonical Wnt signaling activates miR-34 expression during osteoblastic differentiation.

The canonical Wnt signaling pathway is crucial for the regulation of bone mass in humans and for the development of osteoblasts. MicroRNAs (miRs) represent a class of non­coding RNAs, ~22 nucleotides in length, that regulate gene expression by targeting mRNAs for cleavage or translational repression. Several previous studies have demonstrated the involvement of miRNAs in modulating gene expression in osteoblasts and regulating osteoblast differentiation. In the present study, microRNA profiling was conducted using Wnt3a­C2C12 cells; C2C12 cells were transfected with a Wnt3a expression plasmid to activate canonical Wnt signaling. miR­34b­5p and miR­34c were identified to be upregulated by the activation of canonical Wnt signaling in C2C12 cells. Expression of mature miR­34b/c increased from low levels at day 0 to maximum levels at day 28 of MC3T3­E1 cell differentiation. To analyze the effects of these miRNAs on osteoblast differentiation, an antisense inhibitor was transfected into MC3T3­E1 cells and osteoblast­related gene expression was investigated. Knockdown of miR34b/c enhanced osteocalcin mRNA expression; however, alkaline phosphatase mRNA expression and activity were decreased by miR34b/c inhibition. These results indicated that miR­34b/c regulates gene expression by targeting regulators of the osteogenic pathways and thereby contributes to osteoblast differentiation.