TGF-b Inhibition in Myeloma Figure 6. TGF-b inhibition facilitates terminal OB maturation to suppress MM growth and survival at earlier time points. A. MC3T3-E1 cells were cultured in osteogenic media for the indicated periods. rhBMP-2, SB431542 and rhTGF-b were added at 50 ng/mL, 3 mM and 5 ng/mL to the indicated wells, respectively. After culturing, mineralized nodules were visualized by von Kossa staining. B. MC3T3-E1 cells were cultured in osteogenic media for induction of OB differentiation. rhBMP-2 and SB431542 were added at 50 ng/mL and 3 mM to the indicated wells, respectively. After culturing for 3, 6 and 9 days, the cells were washed, and subjected to the measurement of ALP activity (top panels) and von Kossa staining for assessing mineralized nodule formation (middle panels). The cells were also cocultured in quadruplicate with 5TGM1 cells for 3 days. Viable 5TGM1 cells were counted. Percent changes from the baseline are shown (bottom panels). Data are expressed as means +/2 SD. *, p,0.05. doi:10.1371/journal.pone.0009870.g006 can not only ameliorate destructive bone lesions, but also disrupt the MM niche to suppress MM growth. Furthermore, TGF-b is a multi-functional cytokine which suppresses normal hematopoie- sis[42] and dendritic cell differentiation[43] but enhances angiogenesis[44] as well as osteoclastogenesis.[45] Therefore, the effect of TGF-b inhibition may extend beyond amelioration of destructive bone lesions and tumor growth and improve other MM-associated clinical features. It is intriguing that OBs which have matured enough to form mineralized nodules suppress the proliferation of MM cells in sharp contrast to their precursor, stromal cells, which support MM cell growth and survival. The production of IL-6, a major stromal cell-derived growth and anti-apoptotic factor for MM cells, was found to be markedly decreased in terminally differentiated OBs (91.265.7 and 15.162.7 pg/mL for 2 days in culture supernatants of untreated and terminally differentiated MC3T3-E1 cells, respectively). Recently, decorin has been identified among OB- derived factors responsible for the suppression of MM cell growth and survival. [46]Profiles of protein production during OB differentiation by a proteome analysis may help identify the OB signature responsible for MM growth suppression. Materials and Methods Ethics Statement All procedures involving human specimens were performed under written informed consent according to the Declaration of Helsinki and the protocol approved by the Institutional Review Board for human protection in University of Tokushima (#240). All experiments with animals were performed according to the guidelines for animal protection in University of Tokushima, and approved by the Institutional Review Board for animal protection. Reagents TGF-b type I receptor kinase activin-like kinase 5 (ALK5) inhibitors, SB431542 and Ki26894, were obtained as follows. SB431542 was purchased from Tocris Bioscience (Ellisville, MO). Its IC50 for ALK5 is 94 nM.[47] Ki26894 was a gift from Kyowa Hakko Kirin Co. Ltd. (Japan), and is suitable for experiments in vivo.[29] The following reagents were purchased from the indicated manufacturers: recombinant human (rh) bone morphogenetic protein (BMP)-2 and rhTGF-b from R&D Systems (Minneapolis, MN); b-glycerophosphate, melphalan, dexamethasone, rh biglycan, rh decorin and rabbit anti-b-actin polyclonal antibody from Sigma (St. Louis, MO); ascorbic acid from Wako Pure Chemical (Osaka, Japan); rh soluble RANK ligand from Pepro Tech (Rocky Hill, NJ); rabbit anti-Smad1 and rabbit anti-Smad2 from Millipore (Billerica, MA); and anti-phosphorylated Smad1, anti-phosphorylated Smad2 and horseradish peroxidase–conjugated anti-mouse IgG from Cell Signaling (Beverly, MA). Cells and cultures Human MM cell lines, RPMI8226 and U266, were obtained from the American Type Culture Collection (Rockville, MD). The human MM cell line KMS-12 was obtained from Health Science Research Resources Bank (Osaka, Japan). The MM cell line INA6 was kindly provided by Dr. Renate Burger (University of Kiel, Kiel, Germany). The mouse MM cell line 5TGM1 was a gift from Dr. Gregory R. Mundy (Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, TN). A mouse preosteoblastic cell line, MC3T3-E1, and an immature mesenchymal cell line, C3H10T1/2 were obtained from RIKEN Bioresource Center (Tsukuba, Japan). Human bone marrow mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifusion from heparinized bone marrow blood drawn from patients with MM. MM cells were further purified from bone marrow mononuclear cells with positive selection using CD138 (Syndecan-1) microbeads and the Miltenyi magnetic cell-sorting system (Miltenyi Biotec, Auburn, CA) according to instructions. Primary stromal cells were isolated from bone marrow aspirates and cultured as previously described.[5] Cells were cultured in Minimal Essential Medium Eagle-Alpha Modification (a-MEM; Sigma) with 10% heat- inactivated fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS) or 1% FBS as a serum-reduced condition, 100 units/mL penicillin(Sigma) and 100 mg/mL streptomycin (Sigma). Con- ditioned media were collected from MM cell lines cultured at 56105 cells/mL for 2 days. Bone marrow plasma was obtained after the centrifusion of heparinized bone marrow blood drawn from patients with MM. OB differentiation MC3T3-E1 pre-osteoblastic cells and bone marrow-derived stromal cells were cultured in 24-well culture plates in an osteogenic medium, a-MEM containing 10% FBS, 10 mM b- glycerophosphate and 50 mg/mL ascorbic acid, as previously described.[5] The medium was replaced every 3 days. Alkaline phosphatase (ALP) activity was determined using an ALP activity assay kit (Wako Pure Chemical) according to the manufacturer’s instructions. For analyzing mineralized nodules, the cells were fixed with 10% neutral-buffered formalin (Wako Pure Chemical) and visualized by von Kossa staining as described.[5] Adipogenic differentation C3H10T1/2 cells were cultured in an osteogenic medium with BMP-2. On day 7 of adipogenic induction, cells were washed, fixed and stained for intracellular lipid inclusion bodies with 0.5% oil red O (Sigma) in isopropanol:distilled water (60:40) for 30 minutes. RT-PCR Total RNA was extracted from cells using TRIZOL reagent (Gibco BRL, Rockville, MD). For reverse transcription-polymer- ase chain reaction (RT-PCR), 2 mg of total RNA was reverse- transcribed with Superscript II (Gibco) in a 20-ml reaction solution. One tenth of the RT-PCR product was used for subsequent PCR analysis with 24–30 cycles of 95uC for 30 seconds, 58uC for 30 seconds, and 72uC for 30 seconds. PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9870