TGF-b Inhibition in Myeloma Figure 3. TGF-b inhibition restores OB differentiation and potentiates Smad1 phosphorylation by BMP-2. A. MC3T3-E1 cells were cultured for 14 days in osteogenic media in the absence or presence of conditioned media from RPMI8226 and U266 cells at 20%. SB431542 and Ki26894 were added at 3 and 10 mM to the indicated wells, respectively. B. MC3T3-E1 cells were cultured for 14 days in osteogenic media in the absence or presence of bone marrow plasma from patients with MM at 5%. SB431542 was added at 3 mM to the indicated wells. C. A chimeric firefly luciferase reporter plasmid (Topflash) and a renilla luciferase reporter plasmid (pRL-TK) were cotransfected into MC3T3-E1 cells as described in Materials and methods. The MC3T3-E1 cells were cultured in osteogenic media with rhBMP-2 at 50 ng/ml in the absence or presence of conditioned medium from RPMI8226 cells at 10%. SB431542 was added at 3 mM to the indicated wells. After 12 hours of incubation luiferase reporter activity was measured as renilla luiferase activity. Data were expressed as means +/2 SD for six independent experiments. D. MC3T3-E1 cells were cultured in a- MEM with 1% FBS for 24 hours in 10-cm dishes at 60% confluence. SB431542 (3 mM), rhTGF-b (10 ng/mL), and the two in combination were added to the indicated dishes and the cells were cultured for another 48 hours, after which rhBMP-2 was added at 200 ng/ml (upper). SB431542 (3 mM), rhTGF- b (10 ng/mL), and the two in combination were added to the indicated dishes (lower). Following incubation for 30 minutes, cell lysates were collected. The protein levels of phosphorylated Smad1, Smad1, phosphorylated Smad2 and Smad2 were estimated by Western blotting. b-actin was used as a protein loading control. doi:10.1371/journal.pone.0009870.g003 and survival. Thus, an induction of OB differentiation through TGF-b inhibition may provide a novel approach to ameliorate both bone destruction and tumor progression in MM. Results TGF-b suppresses and TGF-b inhibitors enhance OB differentiation Because OB differentiation from stromal cells is impaired in MM bone lesions, and because TGF-b is activated through enhanced bone resorption, we first examined the effect of TGF-b and its inhibition on OB differentiation. Addition of BMP-2 induced OB differentiation with the formation of mineralized nodules by MC3T3-E1 preosteoblastic cells (Figure 1A) and primary bone marrow stromal cells from a patient with MM (Figure 1B). Interestingly, treatment with inhibitors of the TGF-b type I receptor kinase ALK5, SB431542 and Ki26894, to inhibit endogenous TGF-b increased the formation of mineralized nodules. To simulate TGF-b-rich MM bone lesions, we added TGF-b, and examined OB differentiation in the presence of TGF- b. Addition of TGF-b almost completely suppressed the BMP-2- induced formation of mineralized nodules. However, treatment with SB431542 or Ki26894 restored the mineralization on addition of TGF-b. In addition, SB431542 alone facilitated osteogenesis to achieve mineralized nodule formation by MC3T3-E1 cells even in the absence of BMP-2 (Figure 1C), which may be due to the suppression of endogenous TGF-b produced by MC3T3-E1 cells. Thus, TGF-b suppresses and its inhibition enhances OB differentiation. TGF-b suppresses adipogenic differentiation as well as OB differentiation from immature mesenchymal cells In addition to OB differentiation, BMP-2 can induce the differentiation of mesenchymal stem cells or pluripotent C3H10T1/2 immature mesenchymal cells into adipocytes .[25,26] When C3H10T1/2 cells were cultured in an osteogenic medium with BMP-2, significant numbers of oil red O-positive adipocytes appeared (Figure 2A). Addition of SB431542 to suppress endoge- nous TGF-b actions enhanced adipogenic differentiation by BMP-2. TGF-b almost completely suppressed adipogenic differentiation, which resumed with the addition of SB431542. Also, the mRNA expression of adipogenic differentiation markers including adiopo- nectin and aP2 was induced along with the expression of ALP, a marker for OBs, by BMP-2, which was further up-regulated by TGF-b inhibition (Figure 2B). TGF-b suppressed the expression of both the adipogenic and OB differentiation markers, which was reversed by TGF-b inhibition. Thus, TGF-b suppresses adipogenic as well as osteoblastic differentiation in C3H10T1/2 cells, suggesting immaturity of undifferentiated mesenchymal cells or stromal cells retained by TGF-b. TGF-b inhibition restores and enhances OB differentiation suppressed by MM Because MM cells produce soluble Wnt antagonists and suppress OB differentiation,[5,6,7] we next examined whether TGF-b inhibition can restore OB differentiation suppressed by MM cells. Conditioned media from MM cell lines suppressed the development of mineralized nodules stimulated by BMP-2 (Figure 3A). Addition of SB431542 or Ki26894 restored the modulation in the presence of MM cell conditioned media to a level more than BMP-2 alone, suggesting that TGF-b inhibition antagonizes the suppression of OB differentiation by MM cells. To further investigate the effects of TGF-b inhibition on OB differentiation in the context of the MM bone marrow microen- vironment, we looked at the effect of TGF-b inhibition on the formation of mineralized nodules in the presence of bone marrow plasma collected from three MM patients with extensive bone destruction. Bone marrow plasma from all these MM patients suppressed the development of mineralized nodules (Figure 3B). Treatment with SB431542 restored and enhanced the minerali- zation suppressed by the bone marrow plasma from MM patients. These results suggest that the inhibition of TGF-b restores OB differentiation and bone formation suppressed in MM. Because TGF-b inhibition antagonizes the suppressive effects of MM cell conditioned media on OB differentiation (Figure 3A), we next examined whether TGF-b inhibition influences canonical Wnt signaling suppressed by MM cells, using luciferase reporter assays of the T-cell factor (TCF)/ lymphoid enhancer factor (LEF) transcription factor, a downstream target of a canonical Wnt signaling pathway. Addition of MM cell conditioned media suppressed the TCF/LEF reporter activity in MC3T3-E1 cells (Figure 3C). The TCF/LEF reporter activity remained suppressed after the addition of SB431542 at doses high enough to restore OB differentiation, suggesting that TGF-b inhibition does not affect canonical Wnt signaling down-regulated by MM cells. To further investigate mechanisms of stimulation of mineralized nodule formation by TGF-b inhibition, we next looked at the effect of SB431542 on BMP-2 signaling. Binding of BMP-2 to its cognate receptors enhances phosphorylation of Smad1, a downstream effecter of a canonical BMP-2 signaling pathway. Indeed, addition of BMP-2 induced phosphorylation of Smad1 (Figure 3D). Interestingly, pretreatment with SB431542 further potentiated the phosphorylation of Smad1 by BMP-2. Pretreat- ment with TGF-b inhibited the phosphorylation of Smad1 by BMP-2; however, addition of SB431542 together with TGF-b restored the BMP-2-induced phosphorylation of Smad1. SB431542 abolished the phosphorylation of Smad2 by TGF-b in MC3T3-E1 cells (Figure 3D), which confirmed the specific inhibition of TGF-beta signaling by SB431542. These results are consistent with the notion that TGF-b inhibition potentiates BMP- 2 action and releases OB precursor cells from the blockade of PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9870