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. 2011 Jan 28;286(4):2559-66.
doi: 10.1074/jbc.M110.149153. Epub 2010 Oct 22.

Induction of Sirt1 by mechanical stretch of skeletal muscle through the early response factor EGR1 triggers an antioxidative response

Patricia S Pardo  1 , Junaith S Mohamed, Michael A Lopez, Aladin M Boriek
Affiliations

Induction of Sirt1 by mechanical stretch of skeletal muscle through the early response factor EGR1 triggers an antioxidative response

Patricia S Pardo et al. J Biol Chem. .

Abstract

Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species. The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription. In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging.

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Figures

FIGURE 1.
FIGURE 1.
Mechanical stretch regulates SIRT1 expression in diaphragm muscles. a, SIRT1 RNA content was determined in excised mouse hemidiaphragm muscles (n = 3) subjected to a 30-min stretch (s) and the nonstretched (ns) hemidiaphragm by RT real-time PCR. b, SIRT1 protein was determined on whole protein (80 μg) from 30 min-stretched and nonstretched hemidiaphragms by Western blot. c, SIRT1 protein quantification was performed by densitometry from Western blots and corrected for tubulin (n = 5). Statistical difference between data from nonstretched and stretched diaphragm samples was determined by Student's t test; an asterisk represents p < 0.005. d, C2C12 myoblasts growing on FlexCell plates were transfected with pTA-luc (open boxes) or pTA-Sirt1 promoter (−212) (black boxes) Myoblasts were allowed to differentiate, and luciferase assays were performed on nonstretched (n/s) or stretched myotubes, 2 h (2h) or 4 h after stretch (4h). An asterisk indicates statistically significant difference from data obtained for pTA-luc nonstretched, p < 0.001. Bars indicate means, and error bars represent standard deviations.
FIGURE 2.
FIGURE 2.
EGR1 binds to the Sirt1 promoter and has a stimulatory effect on Sirt1 transcription. a, mouse and human Sirt1 promoter alignment. The initiator element consensus sequence is underlined, and EGR1 canonical binding sites are in boldface and underlined. b, ChIP assays were performed on 30 min stretched (s, black boxes) or nonstretched (ns, empty boxes) hemidiaphragms with an antibodies against EGR1 and RNA polymerase (positive control) and rabbit IgG (negative control). Immunoprecipitated DNA was quantified by real-time PCR, and promoter occupancy was calculated as described under “Experimental Procedures.” c, C2C12 myoblasts were transfected with pTA-luc (black boxes) or pTA-Sirt1 promoter (−212) (open boxes) in the presence of pcDNA or pcDNA-EGR1 (0.2 μg/well), and luciferase assays were performed with Dual Glo Luciferase (Promega) 24 h after transfection. Statistical difference between pcDNA and pcDNA-EGR1 transfected cells was calculated by t test. An asterisk indicates a statistically significant difference (p = 0.002). Bars indicate means, and error bars represent standard deviations.
FIGURE 3.
FIGURE 3.
Mechanical stretch stimulates EGR1 and SIRT1 expression in C2C12 myotubes. C2C12 myotubes grown on flexible-bottomed plates were stretched for 30 min and collected before stretch and at the indicated times after stretch (a, b, and c). EGR1 (a) and SIRT1 (b) RNA content was determined by RT real-time PCR in stretched and nonstretched myotubes. Values were corrected for GAPDH RNA content. Error bars correspond to standard deviation, SD (n = 3). The overall statistically significant difference among groups was estimated by ANOVA, p < 0.001. An asterisk indicate values significant different from nonstretched myotubes by multiple comparison versus control analysis by Holman-Sidak method. c, 80 μg of protein from whole cell extracts obtained from nonstretched and stretched myotubes collected at the indicated times before or during stretch (Stretch) and after (After Stretch) were analyzed by Western blot with EGR1 and SIRT1 antibodies; a tubulin antibody was used to assess approximately equal loading.
FIGURE 4.
FIGURE 4.
EGR1 is necessary for transcriptional activation of Sirt1 by stretch in skeletal muscle cells. a, C2C12 myoblasts growing on FlexCell plates were transfected with pTA-luc (Clontech) or pTA-Sirt1 promoter (−212) wild type or mutant versions, MT1 and MT2 obtained as described under “Experimental Procedures.” Myoblasts were allowed to differentiate for 3 days, and luciferase assays were performed on nonstretched (ns, open boxes) or stretched myotubes, 2 h (s2, black boxes), or 4 h after stretch (s4, dashed boxes). An asterisk indicates statistical significant difference from data obtained for pTA-luc nonstretched, p < 0.001. b, C2C12 myoblasts plated on FlexCell plates were infected with nontarget or shEGR1 lentiviral particles and allowed to differentiate for 3 days and subjected to a 30-min stretch (s, black boxes) or kept without being stretched (ns, open boxes); RNA content was determined in nonstretched myotubes and 3 h after stretch in the stretched myotubes by RT real-time PCR. An asterisk indicate statistical significant difference from data for nonstretched myotubes infected with nontarget viral particles, p = 0.004. Bars represent means, and error bars indicate standard deviations. c, cells were grown in growth medium (GM) for 24 h and transfected with 80 pmol of nonsilencing RNA or small interference RNA specific for EGR1 or SP1 (Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions; after 8 h, media was replaced with differentiation medium (DM); on the second day of differentiation, EGR1, SP1, and SIRT1 protein were examined with specific antibodies by Western blot, and tubulin was used as a loading control.
FIGURE 5.
FIGURE 5.
EGR1 is necessary to increase nucler expression of SIRT1 in stretched skeletal muscle cells. C2C12 myotubes were infected with nontarget lentivirus (no stretch and stretch) or shEGR1 lentivirus, where indicated cells were stretched. Immunostaining was performed with a rabbit polyclonal anti-SIRT1, mouse monoclonal anti-tubulin, Alexa Fluor 488-conjugated anti-rabbit and Alexa Fluor 594 anti-mouse secondary antibodies, and DAPI staining. Images were collected in an Axioscop microscope with a 200× magnification.
FIGURE 6.
FIGURE 6.
Stretch-induced SIRT1 promotes Mn-SOD expression and contributes to ROS scavenging. a, total protein (500 μg) from nonstretched (ns) or stretched myotubes collected immediately after (s0) or 3 h after stretch (s3) were immunoprecipitated with 5 μg of a goat polyclonal FOXO4 antibody (Santa Cruz Biotechnology). 80 μg of total protein (Input) from each sample were analyzed by Western blot with SIRT1 and FOXO4 antibodies. Immunoprecipitates (IP) from each sample (IP FOXO4) were subjected to Western blot with SIRT1 and acetyl-lysine antibodies. b, ChIP assays were performed with FOXO4 (black boxes) and SIRT1 (gray boxes) as specific antibodies and RNA polymerase (RNA pol) antibodies (positive control, open boxes) on nonstretched myotubes (ns), myotubes subjected to a 30-min stretch and harvested immediately (s0) or 3 h after (s3). Immunoprecipitated DNA was analyzed by real-time PCR, and promoter occupancy was estimated as described under “Experimental Procedures.” Statistical significance was calculated by ANOVA. An asterisk indicates data with significant difference from nonstretched samples, p ≤ 0.001. c, C2C12 myoblasts plated on FlexCell plates were infected with nontarget (black boxes) or shEGR1 (empty boxes) lentiviral particles and allowed to differentiate; on the third day of differentiation, myotubes were stretched or kept without being stretched; SOD2 RNA content was determined by RT real-time PCR in nonstretched myotubes (ns) or 3 h after stretch (s). Statistical significance was determined by ANOVA; an asterisk indicates statistical significant difference from data for nonstretched myotubes infected with nontarget viral particles, p < 0.001. d, Mn-SOD activity was determined as described under “Experimental Procedures” in whole cell extracts from nonstretched (ns), stretched myotubes collected immediately (s0) or 4 h after stretch (s4) from samples not treated (black boxes) or treated by addition of 10 mm nicotinamide to the media immediately after stretch (open boxes); an asterisk indicates data with a statistically significant difference from nontreated samples, p < 0.05. e, myoblasts previously infected with nontarget (non-si) or shEGR1 lentiviral particles (si-EGR1) were infected with pBaBe-Puro (non-si and si-EGR1) or pYE-hSir2 retroviral particles (non-si/Sirt1 and si-EGR1/SIRT1) and allowed to differentiate. On the third day of differentiation, cells were stretched by 30 min, and superoxide anion content was determined before stretch and at 1, 2, and 5 h after stretch. Analysis by two-way ANOVA reflected that the difference in mean values among the different treatments is greater than would be expected by chance after allowing for effects of differences by time with p = 0.003, pairwise versus non-si; si-EGR1, p = 0.0048 (significant); non-si/SIRT1, p = 0.0005 (significant); and si-EGR1/SIRT1, p = 0.0725 (not significant). Error bars indicate standard deviations (n = 3).
FIGURE 7.
FIGURE 7.
EGR1 involvement in Sirt1 induction and ROS scavenging after stretch of skeletal muscle cells. Mechanical stretch of myotubes increases superoxide anion production and EGR1 expression. EGR1 binds to the Sirt1 promoter and activates SIRT1 expression. Induced SIRT1 binds to FOXO and promotes FOXO binding to the Sod2 promoter. Stimulation of SOD2 expression by SIRT1 contributes to ROS scavenging after stretch.
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