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The proteasome inhibitor bortezomib may increase osteoblast-related markers in multiple myeloma (MM) patients; however, its potential osteoblastic stimulatory effect is not known. In this study, we show that bortezomib significantly induced a stimulatory effect on osteoblast markers in human mesenchymal cells without affecting the number of osteoblast progenitors in bone marrow cultures or the viability of mature osteoblasts. Consistently we found that bortezomib significantly increased the transcription factor Runx2/Cbfa1 activity in human osteoblast progenitors and osteoblasts without affecting nuclear and cytoplasmatic active ␤-catenin levels. IntroductionMultiple myeloma (MM) is a plasma cell malignancy characterized by the high capacity to induce osteolytic bone lesions. 1 Hystomorphometric studies showed that osteoblast formation and function are profoundly impaired in MM patients and critical in the bone lesion development. [1][2][3] Several mechanisms are involved in MMinduced osteoblast suppression 1,4,7 including the production of Wnt inhibitors as DKK-1 or sFRP-2 5,6 or the impairment of osteoblast formation and differentiation through the block of the critical osteoblast transcription factor Runx2/Cbfa1. 7 In turn, osteoblastic cells also regulate myeloma cell growth 8,9 and the increase of bone formation in mice results in a reduction of tumoral burden. 10 Recent data suggest that ubiquitin-proteasome pathway, which is the major cellular degradative system and therapeutic target in myeloma cells, 11 also regulates osteoblast differentiation. [12][13][14] The ubiquitin-proteasome pathway can modulate the BMP-2 expression, 12 which can induce osteoblast differentiation through the Wnt signaling 13 and regulates the proteolytic degradation of the osteoblast transcription factor Runx2/ Cbfa1. 14 Recently, Garrett et al 12 demonstrated that proteasome inhibitors as PS1 and epoximicin stimulate bone formation in neonatal murine calvarial bones and in vivo in mice. 12 A strong correlation between the capacity of these compounds to inhibit proteasomal activity in osteoblasts and their bone-forming activity was also demonstrated. 12 Preliminary observations obtained in MM patients treated with the proteasome inhibitor bortezomib show an increase of serum bone-specific alkaline phosphatase and other osteoblast related markers suggesting a potential osteoblast stimulatory effect of this drug. [15][16][17][18] Currently it is not known whether the proteasome inhibitor bortezomib may have a direct effect on osteoblast differentiation and formation in vitro in human cultures and in vivo in MM patients. Patients, materials, and methods DrugsBortezomib was purchased from Janssen-Cilag (Milan, Italy). For in vitro studies, the drug was reconstituted in DMSO at a stock concentration of 50 mM, and this stock was diluted in medium just before use, so that the concentration of DMSO never exceeded 0.1%. The proteasome inhibitors MG-132 and MG-262 were purchased from BIOMOL International (Plymouth Meeting, PA; DBA srl, M...

IntroductionAngiogenesis has a critical role in the pathophysiology and progression of multiple myeloma (MM) supporting the growth and survival of MM cells. [1][2][3][4][5] The angiogenic process in MM is sustained mainly by the overexpression of proangiogenic factors directly by MM cells including VEGF, 6 angiopoietin-1 (ANG-1), 7 osteopontin (OPN), . 9 Nevertheless, the molecular mechanisms underlying the regulation of angiogenesis in MM have not been completely elucidated.The new candidate tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of tumor growth and angiogenesis through the association with NF-B. ING4 is a nuclear factor expressed in all normal tissues and markedly reduced in glioblastoma cells and head and neck squamous cell carcinoma, with levels inversely correlated with tumor grade. 10,11 Inhibition of ING4 expression strongly promotes the growth of glioma cells in vivo, whereas its overexpression leads to growth inhibition through ING4's capability to interact with p65 subunit of NF-B. 10 Interestingly, it has been also shown that tumors lacking ING4 showed increased vascularization compared with ING4-expressing tumors. 12 Moreover ING4 down-regulated the angiogenic-related molecules including IL-8 and the hypoxia inducible factor-1␣ (HIF-1 ␣) activity in hypoxic condition through the involvement of HIF prolyl hydroxylase 2 (HPH-2) 10,13 In turn, the role of hypoxia has been recently highlighted in the promotion of angiogenesis. 14 The expression of ING4 by MM cells, as well as its potential role in MM-induced angiogenesis, has never been investigated. In this study, we evaluated the expression of ING4 in malignant MM cells and the potential relationship between ING4 and the production of proangiogenic molecules by MM cells in normoxic and hypoxic conditions, as well as its relationship with the "in vitro and in vivo" angiogenesis. Submitted February 15, 2007; accepted September 4, 2007. Prepublished online as Blood First Edition paper, September 11, 2007; DOI 10.1182 DOI 10. /blood-2007 The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on August 29, 2018. by guest www.bloodjournal.org From Patients, materials, and methods Cells and cell culture conditionsCell lines. Human myeloma cell lines (HMCLs) XG-6, XG-1, and JJN3 were obtained from Dr Bataille (Nantes, France). U266 was obtained from the American Type Culture Collection (Rockville, MD). OPM2 and RPMI-8226 were purchased from DSM (Braunschweig, Germany). ARP-1 and H929 were generously received from Dr Shaughnessy's laboratory (Little Rock, AR).Cell cultures. HMCLs were incubated in RPMI medium at 10% FCS (Invitrogen Life Technologies, Milan, Italy) and maintained with or without IL-6 (3 ng/mL; Endogen Woburn, MA). In a series of experiments, HMCLs were incubated with the HPH-2 in...

Bone destruction in multiple myeloma is characterized both by markedly increased osteoclastic bone destruction and severely impaired osteoblast activity. We reported that interleukin-3 (IL-3) levels are increased in bone marrow plasma of myeloma patients compared with healthy controls and that IL-3 stimulates osteoclast formation. However, the effects of IL-3 on osteoblasts are unknown. Therefore, to determine if IL-3 inhibits osteoblast growth and differentiation, we treated primary mouse and human mar-