IntroductionMultiple myeloma (MM) almost exclusively develops in the bone marrow and generates devastating bone destruction by osteoclasts (OCs) recruited around MM cells. A marked stimulation of osteoclastic bone resorption causes debilitating clinical symptoms, including intractable bone pain, disabling multiple fractures, and hypercalcemia. The severity of bone disease correlates with the tumor burden and is one of the major parameters in widely used Durie and Salmon clinical staging system. It is of note that the aggressive features of MM bone lesions have significantly contributed to its poor prognosis despite the recent development of intensive chemotherapeutic regimens. 1,2 Therefore, elucidation of the molecular mechanism of bone destruction and tumor progression is essential for the development of effective therapies to improve survival as well as quality of life of patients with MM.Interactions between receptor activator of nuclear factorkappaB (RANK) expressed on the surface of the OC lineage cells and RANK ligand expressed on stromal cells play a key role in the development and activation of OCs, whereas osteoprotegerin, a decoy receptor for RANK ligand secreted from stromal cells, inhibits RANK ligand-RANK signaling. 3-8 MM cells stimulate osteoclastogenesis by triggering a coordinated increase in RANK ligand and decrease in osteoprotegerin in bone marrow (BM) stromal cells. [9][10][11] We and others have demonstrated that osteoclastogenic CC chemokines macrophage inflammatory protein 1␣ (MIP-1␣) and MIP-1␤ are secreted from most of MM cells and play a critical role in the development of MM bone lesions. [12][13][14][15][16][17][18] These chemokines act on MM cells in an autocrine/paracrine fashion and enhance MM cell adhesion to stromal cells through activation of integrins, including very late antigen-4 (VLA-4). The interaction between MM and stromal cells then induces RANK ligand expression by stromal cells, leading to OC differentiation and activation. 12 Almost exclusive development of MM in the BM suggests that the BM microenvironment supports MM cell growth and survival. Among cell components in the BM, roles of stromal cells in MM cell growth and survival have been extensively studied. When cocultured with MM cells, stromal cells are stimulated to produce interleukin 6 (IL-6), which promotes proliferation of MM cells and prevents them from apoptosis induced by anticancer agents. [19][20][21] Other than stromal cells, OCs induced by MM cells are among major cellular components of the BM microenvironment. Administration of inhibitors of osteoclast activity, including bisphosphonates, RANK-Fc, and osteoprotegerin, not only prevented MMinduced bone destruction but also interfered with tumor progression in animal models of MM. 9,22-25 Repeated administration of bisphosphonates has also been reported to reduce the tumor burden without chemotherapy in a portion of patients with MM. 26 These observations raise a possibility that an interaction between OCs and MM Materials and methods ChemicalsThe f...

IntroductionMultiple myeloma (MM) almost exclusively develops and expands in the bone marrow (BM) and generates devastating bone destruction by osteoclasts (OCs). The bone destruction causes debilitating clinical symptoms including intractable bone pain, disabling multiple fractures, and hypercalcemia. The severity of bone disease correlates with the tumor burden and is one of the major parameters in the Durie and Salomon clinical staging system. Furthermore, the aggressive features of MM bone lesions have contributed significantly to its poor prognosis despite the recent development of intensive chemotherapeutic regimens. 1,2 Therefore, elucidation of the molecular mechanism of bone destruction and tumor progression is essential for the development of effective therapies to improve survival as well as quality of life of patients with MM.Interaction between receptor activator of nuclear factor-B (RANK) expressed on the surface of cells of osteoclastic lineage and RANK ligand expressed on stromal cells plays a key role in the development and activation of OCs, whereas osteoprotegerin, a decoy receptor for RANK ligand secreted from stromal cells, inhibits RANK ligand-RANK signaling. 3,4 MM cells stimulate osteoclastogenesis by triggering a coordinated increase in RANK ligand and decrease in osteoprotegerin in the BM. [5][6][7] We and others have demonstrated that osteoclastogenic CC chemokines macrophage inflammatory protein 1␣ (MIP-1␣) and MIP-1␤ are secreted from most MM cells and play a critical role in the development of MM bone lesions. [8][9][10][11][12] These chemokines directly act on MM cells in an autocrine/paracrine fashion and enhance MM cell adhesion to stromal cells through activation of integrins including very late antigen 4. The interaction between MM cells and stromal cells induces RANK ligand expression by stromal cells, leading to OC differentiation and activation. 8 Furthermore, OCs enhance MM cell growth and survival through a cell-to-cell contact-dependent mechanism that is partially mediated by OC-derived interleukin 6 (IL-6) and osteopontin. 13,14 These observations suggest that interactions of MM cells and OCs form a vicious cycle leading to extensive bone destruction and MM cell expansion.Along with enhanced bone resorption, mineralization is impaired in MM bone lesions. Radiographic examinations show radiolucent lesions without calcification known as "punched-out" lesions. Analyses of bone turnover in patients with MM by biochemical bone markers also suggested an imbalance of bone turnover with enhanced bone resorption and suppressed bone formation. 15 However, the mechanisms of impaired bone formation in bone lesions of patients with MM remain poorly understood.A canonical Wingless-type (Wnt) signaling pathway has been shown to play an important role in osteoblast differentiation. Wnts are secreted cysteine-rich glycoproteins, known as regulators of the differentiation of hematopoietic and mesenchymal cells as well as embryonic development. [16][17][18] Wnt proteins bind to the Frizzle...

BackgroundMultiple myeloma (MM) expands almost exclusively in the bone marrow and generates devastating bone lesions, in which bone formation is impaired and osteoclastic bone resorption is enhanced. TGF-β, a potent inhibitor of terminal osteoblast (OB) differentiation, is abundantly deposited in the bone matrix, and released and activated by the enhanced bone resorption in MM. The present study was therefore undertaken to clarify the role of TGF-β and its inhibition in bone formation and tumor growth in MM.Methodology/Principal FindingsTGF-β suppressed OB differentiation from bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and also inhibited adipogenesis from C3H10T1/2 immature mesenchymal cells, suggesting differentiation arrest by TGF-β. Inhibitors for a TGF-β type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-β inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-β inhibition expedited OB differentiation in parallel with suppression of MM cell growth. The anti-MM activity was elaborated exclusively by terminally differentiated OBs, which potentiated the cytotoxic effects of melphalan and dexamethasone on MM cells. Furthermore, TGF-β inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models.Conclusions/SignificanceThe present study demonstrates that TGF-β inhibition releases stromal cells from their differentiation arrest by MM and facilitates the formation of terminally differentiated OBs, and that terminally differentiated OBs inhibit MM cell growth and survival and enhance the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. Therefore, TGF-β appears to be an important therapeutic target in MM bone lesions.

Purpose: Similar to osteoclastogenesis, angiogenesis is enhanced in the bone marrow in myeloma in parallel with tumor progression.We showed previously that myeloma cells and osteoclasts are mutually stimulated to form a vicious cycle to lead to enhance both osteoclastogenesis and tumor growth. The present study was undertaken to clarify whether myeloma cell-osteoclast interaction enhances angiogenesis and whether there is any mutual stimulation between osteoclastogenesis and angiogenesis. Experimental Design: Myeloma cells and monocyte-derived osteoclasts were cocultured, and angiogenic activity produced by the cocultures was assessed with in vitro vascular tubule formation assays and human umbilical vascular endothelial cell (HUVEC) migration and survival. Osteoclastogenic activity was determined with rabbit bone cell cultures on dentine slices. Results: Myeloma cells and osteoclasts constitutively secrete proangiogenic factors, vascular endothelial growth factor (VEGF) and osteopontin, respectively. A cell-to-cell interaction between myeloma cells and osteoclasts potently enhanced vascular tubule formation. Blockade of both VEGF and osteopontin actions almost completely abrogated such vascular tubule formation as well as migration and survival of HUVECs enhanced by conditioned medium from cocultures of myeloma cells and osteoclasts. Furthermore, these factors in combination triggered the production of osteoclastogenic activity by HUVEC. Conclusions: Osteoclast-derived osteopontin and VEGF from myeloma cells cooperatively enhance angiogenesis and also induce osteoclastogenic activity by vascular endothelial cells. These observations suggest the presence of a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion.