TGF-b Inhibition Restores Terminal Osteoblast Differentiation to Suppress Myeloma Growth Kyoko Takeuchi1, Masahiro Abe1*, Masahiro Hiasa2, Asuka Oda1, Hiroe Amou1, Shinsuke Kido1, Takeshi Harada1, Osamu Tanaka1, Hirokazu Miki1, Shingen Nakamura1, Ayako Nakano 1 , Kumiko Kagawa 1 , Kenichiro Yata1, Shuji Ozaki3, Toshio Matsumoto1 1 Department of Medicine and Bioregulatory Sciences, University of Tokushima Graduate School of Medicine, Tokushima, Japan, 2 Department of Biomaterials and Bioengineering, University of Tokushima Graduate School of Oral Sciences, Tokushima, Japan, 3 Division of Transfusion Medicine, Tokushima University Hospital, Tokushima, Japan Abstract Background: Multiple 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-b, 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-b and its inhibition in bone formation and tumor growth in MM. Methodology/Principal Findings: TGF-b 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-b. Inhibitors for a TGF-b type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-b inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-b 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-b inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models. Conclusions/Significance: The present study demonstrates that TGF-b 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-b appears to be an important therapeutic target in MM bone lesions. Citation: Takeuchi K, Abe M, Hiasa M, Oda A, Amou H, et al. (2010) TGF-b Inhibition Restores Terminal Osteoblast Differentiation to Suppress Myeloma Growth. PLoS ONE 5(3): e9870. doi:10.1371/journal.pone.0009870 Editor: Lin Mei, Medical College of Georgia, United States of America Received June 9, 2009; Accepted January 29, 2010; Published March 25, 2010 Copyright: 2010 Takeuchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grants-in-aid for Scientific Research (A) to T.M. and (C) to M.A., and for the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Science and Sports of Japan, and a Grant-in-aid for Cancer Research (17-16) to M.A. from the Ministry of Health, Labor and Welfare of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: masabe@clin.med.tokushima-u.ac.jp Introduction Multiple myeloma (MM) develops and expands almost exclusively in the bone marrow and generates devastating bone lesions. In typical destructive bone lesions in patients with MM, bone formation is impaired along with an enhancement of osteoclastic bone resorption. We and others have demonstrated that MM cells enhance osteoclastogenesis by MIP-1 and RANK ligand,[1,2,3,4] while suppressing osteoblast (OB) differentiation from their precursors, stromal cells, via the secretion of soluble Wnt antagonists from MM cells,[5,6,7] stromal cells and OBs.[8,9] Thus induced osteoclasts (OCs) as well as stromal cells with defective OB differentiation in turn enhance MM cell growth and survival.[10,11] Furthermore, OCs stimulate angiogenesis in concert with MM cells.[12] These MM cell-induced cell types in MM bone lesions, namely OCs, vascular endothelial cells and stromal cells, create a microenvironment suitable for MM cell growth and survival, which can be called as a ‘‘MM niche’’. [13] Because such a skewed cellular microenvironment protects MM cells from apoptosis induced by chemotherapeutic agents as well as immunotherapy, there is a need to target and disrupt the MM niche to improve the efficacy of present therapeutic modalities against MM progression as well as MM bone disease. Bone marrow stromal cells with defective OB differentiation are a major component of the MM niche, which produce various growth and anti-apoptotic factors for MM cells including IL-6, IGF-1, SDF-1a and VEGF while expressing RANK ligand to stimulate osteoclastogenesis. Importantly, the adhesion of MM cells to stromal cells as well as their extracellular matrices (ECM) confers cell adhesion-mediated drug resistance (CAM-DR) in MM PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9870