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First published online 30 January 2007
doi: 10.1242/jcs.03372

Journal of Cell Science 120, 692-701 (2007)
Published by The Company of Biologists 2007
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TACE release of TNF-{alpha} mediates mechanotransduction-induced activation of p38 MAPK and myogenesis

Mei Zhan1,*, Bingwen Jin1,*, Shuen-Ei Chen1, James M. Reecy2 and Yi-Ping Li1,{ddagger}

1 Department of Medicine, Baylor College of Medicine, One Baylor Plaza-520B, Houston, TX 77030, USA
2 Department of Animal Science, Iowa State University, Ames, IA 50011, USA


Figure 1
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  		Fig. 1. Static stretch activates the p38, ERK1/2 and JNK MAPKs, as well as AKT. C2C12 myoblasts were seeded in six-well Bioflex® plates and allowed to proliferate to ~85% confluence. Stretch was conducted in fresh growth medium for the indicated time periods. Time-matched, non-stretched myoblasts were used as controls. Whole cell extracts of myoblasts were prepared and analyzed by western blotting using antibodies against the phosphorylated form of (A) p38 (43 kDa), (C) ERK1/2 (42/44 kDa), (B) JNK (46/54 kDa) and (D) AKT (60 kDa). Total levels of the kinases were also monitored by western blot analysis. Representative blots from three independent experiments are shown. Arrows indicate bands of phosphorylated JNK. The optical density (OD) of corresponding bands, detected by western blot, was measured and analyzed with the paired t-test, by comparison of stretched samples with time-matched controls (n>=3). * indicates a statistical significance at P<0.01.

 

Figure 2
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  		Fig. 2. Static stretch activates myogenic differentiation markers. C2C12 myoblasts were stretched for the indicated time periods as described in Fig. 1. Whole cell extracts were prepared and subjected to western blot analysis for phosphorylated MEF2C and total MEF2C (46 kDa), myogenin (37 kDa) and p21 (21 kDa). Total MEF2C also serves as a control for the sample loading quality of myogenin and p21 blotting.

 

Figure 3
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  		Fig. 3. Stretch activation of p38 and myogenic differentiation markers are blocked by a TNF-{alpha}-neutralizing antibody. C2C12 myoblasts were stretched for the indicated periods in growth medium that contained an antibody that neutralizes TNF-{alpha} or pre-immune IgG (5 µg/ml). Western blot analysis was performed to determine phosphorylation of p38, ERK1/2, JNK and AKT (A), as well as MEF2C phosphorylation and the expression of myogenin and p21 (B). Levels of total p38 or beta-actin are shown as the loading control.

 

Figure 4
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  		Fig. 4. Stretch activation of myogenic differentiation markers is mediated by p38. C2C12 myoblasts were pre-incubated (30 minutes) and stretched for the indicated periods in growth medium that contained the p38 inhibitor SB203580 (5 µM) or the same volume of DMSO (1 µl/ml), which was used as vehicle for SB203580. Western blot analysis was conducted to determine MEF2C phosphorylation and the expression level of myogenin and p21. beta-actin was monitored as the loading control.

 

Figure 5
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  		Fig. 5. Stretch stimulates MHC expression in a TNF-{alpha}- and p38-dependent manner. (A) Stretch stimulates MHC expression. C2C12 myoblasts were stretched for 6 hours and then incubated for an additional 24 or 48 hours without stretch. MHC (200 kDa) expression in myoblasts was compared with that of non-stretched controls by western blot analysis. (B) Stretch stimulation of MHC expression requires TNF-{alpha} and p38. C2C12 myoblasts were stretched for 6 hours in culture medium that contained the TNF-{alpha} neutralizing antibody (5 µg/ml), pre-immune IgG (5 µg/ml), SB-203580 (5 µM), or the DMSO vehicle (1 µl/ml), following a 30-minute pre-incubation period. Myoblasts were then incubated for an additional 48 hours without stretch. Western blot analysis was performed to evaluate MHC expression.

 

Figure 6
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  		Fig. 6. Stretch activation of p38 requires TNF-{alpha} receptor-mediated signaling. Primary myoblast cultures were prepared from wild-type (WT) or p55–/– p75–/– mice (KO). Myoblasts were seeded in six-well Bioflex® plates and allowed to proliferate to ~65% confluence. Stretch was conducted in fresh growth medium for 6 hours. Time-matched, non-stretched myoblasts were used as controls. Whole cell extracts of myoblasts were prepared and analyzed by western blotting for levels of the phosphorylated form and the total levels of the kinases tested. The optical density of the phosphorylated form of each kinase detected western blotting was normalized to the respective total level and analyzed by paired t-test to compare control and stretched myoblasts for each type of myoblasts (n=3). *P<0.05; **P<0.01.

 

Figure 7
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  		Fig. 7. Stretch activation of myogenin expression requires TNF-{alpha} receptor-mediated signaling. Primary myoblasts were stretched as described in Fig. 6. Myogenin expression was determined by western blot analysis.

 

Figure 8
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  		Fig. 8. TACE mediates stretch activation of p38. (A) Stretch activates TACE. C2C12 myoblasts were stretched for 30 minutes and lysed in RIPA buffer. The cleavage rate of a peptide that contained the TACE cleavage site found in pro-TNF-{alpha} by 5 µg of the cell lysate was determined as described in Materials and Methods. Data from three independent experiments were analyzed by paired t-test, and * indicates a difference at P<0.01. (B) Stretch activation of p38 is dependent on TACE activity. C2C12 myoblasts were pre-incubated for 30 minutes and stretched for 2 hours in growth medium that contained TAPI (5 µM) or an equal volume of DMSO (1 µl/ml) used as vehicle for TAPI. Myoblasts were then processed for western blot analysis of p38 activation. (C) Stretch activation of p38 is dependent on TACE expression. C2C12 myoblasts were transfected with a TACE siRNA duplex or a scrambled siRNA duplex as control. After 40 hours incubation, myoblasts were stretched for 2 hours, and p38 activation and TACE expression were evaluated by western blot analysis. Both the pro-TACE (upper band, 110 kDa) and processed TACE (lower band, 90 kDa) are shown. Results from three independent experiments were expressed in arbitrary unit and analyzed using a paired t-test comparing stretched samples with non-stretched samples in each group (control, scrambled siRNA and TACE siRNA). *Significant difference (P<0.01).

 

Figure 9
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  		Fig. 9. Stretch-conditioned medium activates p38 via TACE-released TNF-{alpha}. (A) Stretch-conditioned medium activates p38. C2C12 myoblasts were stretched for 2 hours. The medium in the stretched myoblast culture was collected, cleared of floating cells by centrifugation, and transferred into plates that contained unstretched C2C12 myoblasts for a 15 or 30 minutes incubation. Medium transferred from unstretched myoblasts was used as control. Activation of p38 in the myoblasts was then evaluated by western blot analysis. (B) TACE activity is responsible for p38 activation by stretch-conditioned medium. C2C12 myoblasts were preincubated with or without TAPI (5 µM) or an equal volume of DMSO (1 µl/ml) and stretched for 2 hours. The media were then transferred into plates that contained unstretched C2C12 myoblast cultures and incubated for 15 minutes. Western blot analysis was carried out to evaluate p38 activation. (C) TNF-{alpha} released into stretch-conditioned medium is responsible for p38 activation. C2C12 myoblasts were stretched for 2 hours in the presence of TNF-{alpha}-neutralizing antibody or pre-immune IgG (5 µg/ml). The media were then transferred to unstretched C2C12 myoblast cultures and incubated for 15 minutes. Western blot analysis was carried out to evaluate p38 activation. (D) Stretch-conditioned medium activates p38 via TNF-{alpha} receptor-mediated signaling. Culture medium from C2C12 myoblasts that were stretched for 2 hours was transferred to WT or p55–/– p75–/– (KO) primary myoblasts and incubated for 15 minutes. Activation of p38 in the myoblasts was then evaluated by western blot analysis

 

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© The Company of Biologists Ltd 2007