## 8 Proteasome Inhibitors and Osteoblast Functions
### Bortezomib  
Early preclinical studies showed the ability of PIs to induce OB differentiation via multiple mechanisms in different models. [PERSON] et al. firstly showed that PIs, such as epoxomicin, proteasome inhibitors-1, and lactacystin, promoted bone formation in mice  
Figure 2: Biological consequences of proteasome inhibition in MM bone microenvironment. PIs increase the activity of Runx2 in pre-OBs with a consequent increased expression of collagen I, OC, OPN, and ALP through a Wnt-independent stabilization of \(\beta\)-catenin. Bortezomib also increases BMP-2 expression in osteoblastic cells that, in turn, increase the levels of the transcription factor Runx2, enhancing bone formation. The treatment with carfilizomib enhances the activity of Smads and AP1 transcription factors, promoting early OB progenitors’ commitment. The bone anabolic effect of PI treatment, in particular bortezomib and carfilizomib, has been correlated with Notch1 and Dkk-1 inhibition with subsequent enhancement of OB formation. Bortezomib and kazomib promote the ER stress, mainly through the activation of IRE1\(\alpha\)/XBP1s and PERK-ATF4 signaling pathways leading to increased expression of OB markers. The anabolic effect of kazomib is also associated with the activation of SHH pathway by inducing the nuclear translocation of Gili in MSCs and OB differentiation. MM cells and high doses of glucocorticoids induce autophagic death in osteocytes by modulating the levels of two autophagic markers LC3 and p62. Bortezomib blunts osteocyte death by reducing LC3 and increasing p62 protein levels. Bortezomib also potentiates the effect of intermittent PTH on osteocyte viability. Additionally, bortezomib stabilize the transmembrane glycoprotein E11, leading to increased osteoblast-osteocyte transition. in a dose-dependent manner [73]. The same study attributed this effect to the increased expression of BMP-2 in OBs following PIs administration. Afterwards, by using a neonatal mouse calvarial organ culture, [PERSON] et al. confirmed the ability of bortezomib to induce new bone formation via BMP2 stimulation [74]. Additionally, they reported that bortezomibib is a potent inhibitor of Dkk1, a known antagonist of Wnt signaling, at both mRNA and protein levels, thus pointing out the capability of bortezomib to regulate Wnt signaling in bone [74]. In line with these results, [PERSON] and coworkers demonstrated that bortezomib induced the OB phenotype in primary human MSCs with no effects on the number or viability of mature OBs by increasing the activity of Runx2 and the expression of osteoclastcin and type I collagen [75]. Noteworthy, MM patients who responded to bortezomib treatment had a significantly higher number of Runx2-positive OBs compared with the non-responder ones [75]. The authors supposed that either bortezomib has a direct effect on OB differentiation or the removal of malignant cells from the BM may recover the ability of precursors cells to differentiate into OBs. Interestingly, by using SCID-rab mice engrafted with primary MM cells, it has been demonstrated that the antimyeloma response of bortezomib was associated with increased bone mineral density (BMD), OB numbers, and reduced number of OCLs in both myelomatous and non-myelomatous implanted bones [76]. This anabolic effect was not seen in mice treated with melphalan, suggesting that the effect of bortezomib on skeletal homeostasis is not due to a reduced tumor burden [76]. These results have been further validated in a 5T2 MM mouse model [77] and C57 BL/6 mice [78]. Despite the increasing evidence supporting the bone-protection effect of PIs, the underlying molecular mechanism remains poorly understood. Only recently, a possible mechanism that connects proteasome inhibition and osteogenic differentiation has been proposed. [PERSON] et al. showed that while increasing osteogenesis, bortezomib induces G\({}_{0}\)/G\({}_{1}\) phase cell cycle arrest in murine BM MSCs by inducing p2I\({}^{\text{Cip1}}\) and p27\({}^{\text{Kip1}}\) expression [79].