TGF-b Inhibition in Myeloma Figure 1. TGF-b suppresses and TGF-b inhibitors enhance OB differentiation. MC3T3-E1 cells (A) and primary bone marrow stromal cells (B) were cultured for 14 and 28 days, respectively, in 24-well culture plates in a-MEM containing 10% FBS supplemented with b-glycerophosphate and ascorbic acid (osteogenic medium). rhBMP-2, SB431542, Ki26894 and rhTGF-b were added at 50 ng/mL, 3 mM, 10 mM and 5 ng/mL to the indicated wells, respectively. After culturing, mineralized nodules were visualized by von Kossa staining. C. MC3T3-E1 cells were cultured for 21 days in osteogenic media in the absence of BMP-2. SB431542 was added at 3 mM to the indicated wells. Mineralized nodules were visualized by von Kossa staining. The data with mineralized nodule formation were quantified by densitometric analyses. doi:10.1371/journal.pone.0009870.g001 cells.[14,15,16] Therefore, there is a possibility that induction of OB differentiation in stromal cells not only prevents bone loss and resumes bone formation in MM bone lesions, but also may perturb MM growth enhanced by stromal cells. TGF-b, a potent inhibitor of terminal OB differentiation and mineralization,[17,18,19] is produced by OBs and osteocytes, and abundantly deposited in bone matrices in a latent form.[20] It is released from bone matrices through bone resorption[21] and activated by acids and matrix metalloproteinases secreted from OCs.[22,23,24] Because osteoclastic bone resorption is enhanced in MM, TGF-b appears to be abundant and active in MM bone lesions, and may play an important role in bone formation impaired by MM. Therefore, the present study was undertaken to explore whether an inhibition of TGF-b enhances OB differen- tiation suppressed by MM, and whether an enhancement of OB differentiation affects MM cell growth and survival. We demon- strate herein that a blockade of TGF-b actions releases stromal cells from their differentiation arrest by MM, and that terminally differentiated OBs inhibit MM cell growth and survival and potentiate responsiveness to anti-MM agents. These results suggest that suppression of OB differentiation by MM not only accelerates bone loss but also creates a MM niche to enhance MM growth Figure 2. TGF-b suppresses adipogenic differentiation as well as OB differentiation from immature mesenchymal cells. A. C3H10T1/2 immature mesenchymal cells were cultured in osteogenic medium for 7 days. 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 for 7days, the cells were stained with oil red O. Representative images of oil red O staining are shown. B. C3H10T1/2 cells were harvested after culturing for 2 days. rhBMP-2, SB431542 and rhTGF-b were added at 50 ng/mL, 3 mM and 5 ng/mL to the indicated wells, respectively. mRNA expression of markers for adipogenic and osteoblastic differentation were analyzed by RT-PCR. The primers used were as follows: Mouse adioponectin sense 59-AGGGTGAGACAGGAGATGTTGGAA-39 and antisense 59-CAGAGGCCTGGTCCA- CATTCTTTT-39. Mouse aP2 sense 59-TCTCACCTGGAAGACAGCTCCTCCTCG-39 and antisense 59-TTCCATCCAGGCCTCTTCCTTTGGCTC-39. Mouse ALP sense 59-CACTCAGGGCAATGAGGTCACATC-39 and antisense 59-TTCAGTGCGGTTCCAGACATAGTG-39. doi:10.1371/journal.pone.0009870.g002 PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9870