Smad3 induces atrogin-1, inhibits mTOR and protein synthesis, and promotes muscle atrophy in vivo

Abstract

Myostatin, a member of the TGF superfamily, is sufficient to induce skeletal muscle atrophy. Myostatin-induced atrophy is associated with increases in E3-ligase atrogin-1 expression and protein degradation and decreases in Akt/mechanistic target of rapamycin (mTOR) signaling and protein synthesis. Myostatin signaling activates the transcription factor Smad3 (Small Mothers Against Decapentaplegic), which has been shown to be necessary for myostatin-induced atrogin-1 expression and atrophy; however, it is not known whether Smad3 is sufficient to induce these events or whether Smad3 simply plays a permissive role. Thus, the aim of this study was to address these questions with an in vivo model. To accomplish this goal, in vivo transfection of plasmid DNA was used to create transient transgenic mouse skeletal muscles, and our results show for the first time that Smad3 expression is sufficient to stimulate atrogin-1 promoter activity, inhibit Akt/mTOR signaling and protein synthesis, and induce muscle fiber atrophy. Moreover, we propose that Akt/mTOR signaling is inhibited by a Smad3-induced decrease in microRNA-29 (miR-29) expression and a subsequent increase in the translation of phosphatase and tensin homolog (PTEN) mRNA. Smad3 is also sufficient to inhibit peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) promoter activity and to increase FoxO (Forkhead Box Protein, Subclass O)-mediated signaling and the promoter activity of plasminogen activator inhibitor 1 (PAI-1). Combined, this study provides the first evidence that Smad3 is sufficient to regulate many of the events associated with myostatin-induced atrophy and therefore suggests that Smad3 signaling may be a viable target for therapies aimed at preventing myostatin-induced muscle atrophy.

Figure 1.

The effect of Smad3 on Smad-mediated transcriptional activity in skeletal muscle. Mouse TA muscles were cotransfected with LacZ as a control, Smad3 or Smad3 D408H, an SBE firefly (FF) luciferase reporter (SBE FF), and pRL-SV40 Renilla (Ren) luciferase reporter. At 72 hours after transfection, the muscles were collected and FF and Ren luciferase activities were measured by a dual-luciferase assay. Measurements of the relative light units produced by FF luciferase were normalized to that produced by Ren luciferase, and this ratio was expressed as a percentage of the values obtained from the LacZ-transfected muscles. Values are the mean + SEM; n = 3–6 per group. *, Significantly different from the values obtained in LacZ-transfected and Smad3 D408H-transfected muscles.

Figure 2.

The effect of Smad3 on the activity of the atrogin-1, MuRF1, and PGC1α promoters in skeletal muscle. A, Mouse TA muscles were cotransfected with LacZ as a control or Smad3, an atrogin-1 promoter firefly (FF) luciferase reporter (Atrogin-1 FF), and pRL-SV40 Renilla luciferase reporter (Ren). B, Mouse TA muscles were cotransfected with LacZ, Smad3 or c.a.-FoxO3, a MuRF1 promoter firefly luciferase reporter (MuRF1 FF), and Ren. C, Mouse TA muscles were cotransfected with LacZ or Smad3, a PGC1α promoter FF luciferase reporter (PGC1α FF), and Ren. At 72 hours after transfection, the muscles were collected and FF and Ren luciferase activities were measured by a dual-luciferase assay. Measurements of the relative light units produced by FF luciferase were normalized to that produced by Ren luciferase, and this ratio was expressed as a percentage of the values obtained from the LacZ-transfected muscles. Values are the mean + SEM; n = 5–10 per group. *, Significantly different from the values obtained in LacZ-transfected muscles; #, significantly different from the values obtained in LacZ- and Smad3-transfected muscles.

Figure 3.

Smad3 activates the PAI-1 promoter, inhibits the miR-29 promoter, and activates the PTEN 3′-UTR and FoxO response element reporters in skeletal muscle. Mouse TA muscles were cotransfected with LacZ as a control or Smad3, pRL-SV40 Renilla (Ren) luciferase reporter, and the PAI-1 promoter (A), miR-29b/c promoter firefly (B), PTEN 3′-UTR (C), or FoxO response element (FoxO RE FF) firefly (FF) (D) luciferase reporters. At 72 hours after transfection, the muscles were collected and FF and Ren luciferase activities were measured by a dual-luciferase assay. Measurements of the relative light units produced by FF luciferase were normalized to that produced by Ren luciferase, and this ratio was expressed as a percentage of the values obtained from the LacZ-transfected muscles. Values are the mean + SEM; n = 3–10 per group. *, Significantly different from the values obtained in LacZ-transfected muscles.

Figure 4.

Smad3 is sufficient to inhibit mTOR signaling in skeletal muscle. Mouse TA muscles were cotransfected with GFP as a control or Smad3 and GST-tagged p70 (GST p70). At 72 hours after transfection, the muscles were collected and subjected to Western blot analysis with the indicated antibodies. The phosphorylated to total protein ratio for GST P-p70(T389) was calculated and expressed as a percentage of the values obtained in the GFP control samples. Values are the mean + SEM; n = 4–6 per group. *, Significantly different from the values obtained in GFP-transfected muscles.

Figure 5.

Smad3 is sufficient to inhibit protein synthesis and induce atrophy in skeletal muscle. Mouse TA muscles were transfected with LacZ as a control or HA-tagged Smad3. At 7 days after transfection, mice were injected with puromycin 30 minutes before the collection of the muscles. Muscles were then subjected to IHC for rates of protein synthesis (puromycin, red), laminin (blue), and LacZ or the HA tag (green). A and B, Representative merged images of anti-LacZ (A) and anti-HA (B) and antilaminin and antipuromycin signals. D and E, Grayscale images of the signal for puromycin shown in A and B, respectively. C and F, Fiber CSA (C) and puromycin staining intensity (F) in LacZ-transfected and HA-tagged Smad3-transfected fibers, expressed relative to the mean value obtained in nontransfected (control) fibers from the same section. Values are the mean + SEM; n = 153–460 fibers per group from 6 independent samples per group. *, Significantly different from the values obtained in LacZ-transfected fibers.

Figure 6.

A summary of the proposed mechanism by which Smad3 inhibits Akt/mTOR signaling and induces decreased protein synthesis and increased protein degradation in skeletal muscle in vivo. Based on this study, and other previous studies, we propose that Smad3 inhibits miR-29 expression, which leads to increase PTEN mRNA translation and then inhibition of Akt and mTOR signaling. Our results also show that Smad3 is sufficient to activate the atrogin-1 promoter and increase FoxO transcriptional activity. In addition, Smad3 activates the PAI-1 promoter and decreases the activity of the PGC1α promoter. Combined, these Smad3-induced events may result in increased protein degradation, reduced protein synthesis, increased fibrosis, impaired mitochondrial function, and increased energy and oxidative stress.