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. 2013 Oct 28;203(2):345–357. doi: 10.1083/jcb.201211134

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© 2013 Winbanks et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 2. — BMP7-induced skeletal muscle growth is mediated by Smad1/5. (a) rAAV6:Smad6 was applied to C2C12 cells to determine its ability to suppress BMP4-induced BRE-luciferase activity. After administration of vector, cells were treated for 24 h with 3 nM BMP4 and then lysed to assess luciferase activity (*, P < 0.001 vs. control). Figure represents three independent experiments, with each individual experiment performed in triplicate. (b) The coadministration of rAAV6:Smad6 with rAAV6:BMP7 significantly attenuated BMP7-induced hypertrophy in mouse limb muscles, as determined by changes in TA muscle mass (*, P < 0.001 vs. control). Muscles expressing both BMP7 and Smad6 did not differ compared with control muscles when examined 28 d after injection (ND, not different). n = 4–10 per treatment. (c) Image of TA muscles demonstrating BMP7-induced hypertrophy and the inhibitory effect of Smad6 coexpression. Bar, 5 mm. (d) Representative images and myofiber diameter and area quantification demonstrate that BMP7-induced myofiber hypertrophy is significantly suppressed by Smad6 (*, P = 0.01 vs. control; #, P = 0.02 vs. control; n = 3 per treatment). Myofiber diameter is presented as box and whisker plots comprising minimum, lower quartile, median, upper quartile, and maximum values for myofiber diameter. Muscles coexpressing BMP7 and Smad6 were not different from control muscles 28 d after injection (ND, not different). Bar, 100 µm. (e) Smad6 coadministration reduced the phosphorylation of Smad1/5^S463/465 normally induced 28 d after injection of rAAV6:BMP7 alone, as determined by Western blot analysis (*, P < 0.001 vs. control; #, P < 0.05 vs. control; +, P < 0.001 vs. BMP7-treated muscles; n = 5–10 per treatment). (f) Smad6 coadministration reduced the phosphorylation of Akt^S473 (*, P < 0.001 vs. control; #, P = 0.003 vs. BMP7 treatment), mTOR^S2448 (*, P = 0.006 vs. control; #, P = 0.005 vs. BMP7 treatment), S6RP^S235/236 (*, P = 0.04 vs. control; #, P = 0.004 vs. BMP7 treatment), and 4EBP1^T37/46 (*, P = 0.009 vs. control; #, P = 0.004 vs. BMP7 treatment) when compared with the effect of BMP7 alone. n = 4–8 per treatment. Data are presented as means ± SEM.