Facilitation of Osteoblast Differentiation by ATF-4 Accumulation through Proteasome Inhibition.

Abstract 1823

Poster Board I-849

The proteasome inhibitor bortezomib, a novel anti-myeloma (MM) agent, has recently drawn considerable attention to its anabolic actions on bone formation in patients with MM. Bortezomib was reported to enhance the activity of Runx2/cbfa1, an essential transcription factor for osteoblast (OB) induction, in mesenchymal stem cells to induce OB differentiation. However, because over-expression of Runx2 unexpectedly suppresses terminal OB differentiation or mineralization, there may be critical factors involved in OB differentiation in concert with Runx2 to achieve terminal OB differentiation in the treatment with bortezomib. Proteasome inhibition accumulates a variety of proteins and induces ER stress or unfolded protein response. Among proteins induced by ER stress, activating transcription factor-4 (ATF-4) plays a critical role in OB differentiation. ATF-4 is expressed in osteoprogenitors and preOBs following Runx2, and acts in concert with Runx2 to facilitate terminal maturation of OBs. However, it is unknown whether a change in ATF-4 protein levels plays any role in OB differentiation induced by proteasome inhibition. In the present study, we therefore explored the role of ATF-4 in OB differentiation by proteasome inhibition in Runx2-expressing immature OB lineage cells. Bortezomib dose-dependently increased ATF-4 protein levels in primary bone marrow stromal cells and ST-2 stromal and MC3T3-E1 preosteoblastic cell lines at concentrations higher than 10 nM as early as 3 hours. Because serum bortezomib levels reach around 100 nM (Cmax) with T1/2 of 3 hours after iv injection at therapeutic doses, bortezomib treatment in MM patients is expected to enhance ATF-4 protein levels in OB lineage cells. Interestingly, bortezomib treatment did not change mRNA levels of ATF-4 as well as βTrCP1, E3 ligase for ATF-4. Because translation of ATF-4 mRNA is triggered by ER stress response, it is plausible that the ATF-4 accumulation by bortezomib is mediated by the suppression of proteasomal degradation with subsequent induction of ER stress response. MM cell-derived factors and TGF-β released from bone by enhanced bone resorption suppress OB differentiation in MM bone lesions. Treatment with bortezomib was able to accumulate ATF-4 in the presence of MM conditioned media (CM) or TGF-β to the levels similar to those without MM CM nor TGF-β. Furthermore, bortezomib enhanced promotor activity of osteocalcin, a marker of mature OBs, as well as BMP-2-induced mineralized nodule formation in MC3T3-E1 cells, and these effects of bortezomib were suppressed by ATF-4 siRNA. These results demonstrate that bortezomib treatment accumulate ATF-4, and suggest that the effect of bortezomib on OB differentiation is mediated via an accumulation of ATF-4 protein in OB lineage cells. We have previously demonstrated that OB differentiation is suppressed in MM bone lesions, and that differentiated OBs suppress MM cell growth and survival. Thus, resumption of bone formation by bortezomib may further suppress MM cell growth in concert with its direct anti-MM actions.