Myostatin augments muscle-specific ring finger protein-1 expression through an NF-kB independent mechanism in SMAD3 null muscle

Abstract

Smad (Sma and Mad-related protein) 2/3 are downstream signaling molecules for TGF-β and myostatin (Mstn). Recently, Mstn was shown to induce reactive oxygen species (ROS) in skeletal muscle via canonical Smad3, nuclear factor-κB, and TNF-α pathway. However, mice lacking Smad3 display skeletal muscle atrophy due to increased Mstn levels. Hence, our aims were first to investigate whether Mstn induced muscle atrophy in Smad3(-/-) mice by increasing ROS and second to delineate Smad3-independent signaling mechanism for Mstn-induced ROS. Herein we show that Smad3(-/-) mice have increased ROS levels in skeletal muscle, and inactivation of Mstn in these mice partially ablates the oxidative stress. Furthermore, ROS induction by Mstn in Smad3(-/-) muscle was not via nuclear factor-κB (p65) signaling but due to activated p38, ERK MAPK signaling and enhanced IL-6 levels. Consequently, TNF-α, nicotinamide adenine dinucleotide phosphate oxidase, and xanthine oxidase levels were up-regulated, which led to an increase in ROS production in Smad3(-/-) skeletal muscle. The exaggerated ROS in the Smad3(-/-) muscle potentiated binding of C/EBP homology protein transcription factor to MuRF1 promoter, resulting in enhanced MuRF1 levels leading to muscle atrophy.

Figure 1.

Smad3^−/− myoblasts have increased ROS production and AOE activity. A, Representative images showing ROS production at 48 hours during differentiation in WT and Smad3^−/− primary myoblasts, probed with the CM-H2DCFDA fluorescent probe. The images were taken using a Leica upright microscope at ×10 magnification. Increased fluorescence (green) intensity is directly proportional to increased ROS production in myoblasts. Scale bar, 100 μm (n = 3). B (i), Smad3^−/− primary myoblasts were treated for 48 hours during differentiation in the presence of ROS cell signaling inhibitors and ROS content was measured using the CM-H2DCFDA probe using a fluorescent multilabel plate reader. The data are expressed as percentage increase or decrease in ROS production; **, P < .01; ***, P < .001; ****, P < .0001 (n = 3). B (ii), ROS production in Smad3^−/− primary myoblasts treated for 48 hours during differentiation in the presence of ROS cell signaling inhibitors using the CM-H2DCFDA probe. The fluorescence was viewed as mentioned in panel A. Scale bar, 100 μm (n = 3). mRNA expression of TNF-α (C) and Nox1 (D) in WT and Smad3^−/− primary myoblasts in differentiation medium for 48 hours. *, P < .05; ***, P < .001 (n = 3). XO production assay (E) and enzyme assays for CAT (F), GPx (G) and GSR (H) were performed on protein lysates from differentiating WT and Smad3^−/− primary myoblasts at indicated time points. *, P < .05; **, P < .01; ***, P < .001; ****. P < .0001 (n = 3).

Figure 2.

Loss of Mstn relieves oxidative stress in Smad3^−/− muscle. A (i), Representative gel showing protein carbonylation in protein lysates of gastrocnemius muscle from WT, Smad3^−/−, Mstn^−/−. and double-KO mice as detected using the Oxyblot assay kit. A (ii), Corresponding densitometric analysis of the gel showing the percentage increase or decrease in carbonylation over and above WT muscle (*, P < .05; **, P < .01); ^^, P < .01: double-KO muscle as compared with Smad3^−/− muscle. α-Tubulin was used as an internal control for equal protein loading on the gel (n = 3). Representative graph showing mRNA expression of TNF-α (B) and Nox1 (C) in gastrocnemius muscle from WT, Smad3^−/−, Mstn^−/−, and double-KO mice. *, P < .05; **, P < .01: when compared with the WT muscle; ^^, P < .01; ^^^, P < .001: when compared to Smad3^−/− muscle (n = 3). Representative graph showing enzyme assays for XO (D), SOD (E), and GSR (F) performed on whole-muscle lysates of quadriceps muscle from WT, Smad3^−/−, Mstn^−/−, and double-KO mice. *, P < .05; **, P < .01: when compared with the WT muscle; ^^, P < .01: when compared to Smad3^−/− muscle (n = 3).

Figure 3.

Smad3 is needed for IKKα-mediated activation of NF-κB (p65) by Mstn. A (i), Representative Western blot analysis of NF-κB (p65), p-NF-κB (p65), p-IκB-α, IκB-α, and IKKα protein levels in whole-muscle lysates, nuclear (NE) and cytoplasmic extracts (CE) obtained from biceps femoris muscle from WT (lane 1), Smad3^−/− (lane 2), Mstn^−/− (lane 3), and double-KO (lane 4) mice. Corresponding densitometric analysis of p-NF-κB (p65) (ii), p-IκB-α (iii), and IKKα (iv) protein levels. α-Tubulin was used as an internal control for equal protein loading on the gel. *, P < .05; **, P < .01: when compared to the WT muscle; ^, P < .05: when compared with Smad3^−/− muscle (n = 3). B (i), Western blot analysis of NF-κB (p65), p-NF-κB (p65), p-IκB-α, IκB-α, and IKKα protein levels in whole-cell lysates, and nuclear (NE) and cytoplasmic extracts (CE) obtained from C2C12 cells treated with CCM (lane 1), CMM (lane 2), SIS3 and CCM (lane 3), and SIS3 and CMM (lane 4) for 48 hours during differentiation. Corresponding densitometric analysis of p-IκB-α (ii) and IKKα (iii) protein levels. α-Tubulin was used as an internal control for equal protein loading on the gel. *, P < .05: when compared with CCM-treated cells; ^, P < .05: when compared with CMM-treated cells (n = 2).

Figure 4.

ROS is generated through p38 and ERK MAPK pathways in Smad3^−/− muscle. Western blot analysis of p-p38 MAPK (A), p38 α/β (A), p-TAK1 (A), TAK1 (A), p-MKK3/6 (A), p-SAPK/JNK (B), SAPK/JNK (B), p-SEK1/MKK4 (B), p-ERK1/2 (C), ERK1/2 (C), and p-MEK1/2 (C) protein levels in protein lysates obtained from gastrocnemius muscle from WT (lane 1), Smad3^−/− (lane 2), Mstn^−/− (lane 3), and double-KO (lane 4) mice. α-Tubulin was used as an internal control for equal protein loading on the gels (n = 3). mRNA expression of TNF-α (D) and Nox1 (E) in shSmad3 C2C12 cells pretreated for 2 hours with SB203580 or for 1 hour U0126 followed by 48-hour treatment with CCM or CMM in differentiation medium. The values are expressed as fold change when compared with respective shControl C2C12 cells. ##, P < .01: when compared with CCM-treated shControl C2C12 cells; **, P < .01; ***, P < .001: when compared with CCM-treated shSmad3 C2C12 cells; ^^, P < .01; ^^^, P < .001: when compared with CMM-treated shSmad3 C2C12 cells (n = 2). SAPK, stress-activated protein kinase.

Figure 5.

Absence of Smad3 led to the up-regulation of IL-6 in skeletal muscle. A (i), Representative graph showing mRNA expression of IL-6 in gastrocnemius muscle from WT, Smad3^−/−, Mstn^−/−, and double-KO mice. *, P < .05; **, P < .01; ****, P < .0001: when compared with the WT muscle; ^, P < .05: when compared with Smad3^−/− muscle (n = 3). A (ii), Western blot analysis of IL-6 protein level in gastrocnemius muscle from WT (lane 1), Smad3^−/− (lane 2), Mstn^−/− (lane 3), and double-KO (lane 4) mice. α-Tubulin was used as an internal control for equal protein loading on the gel (n = 3). B, Western blot analysis of IL-6 protein in C2C12 myoblasts treated with CCM (lane 1), CMM (lane 2), SIS3+CMM (lane 3), and SIS3+CMM (lane 4) during differentiation for 48 hours. α-Tubulin was used as an internal control for equal protein loading on the gel (n = 2).

Figure 6.

Enhanced binding of CHOP mediates MuRF1 up-regulation in Smad3^−/− mice. A, Representative graph showing promoter-luciferase reporter activity in C2C12 myoblasts transfected with either pGL4.10 and pcDNA3 empty vectors or pGL4.10-MuRF1P and pcDNA3 or pGL4.10-MuRF1P and pcDNA3-CHOP, together with the control Renilla luciferase vector pRL-TK. (***, P < .001; ****, P < .0001: when compared with pGL4.10 and pcDNA3; ^^^^, P < .0001: when compared with pGL4.10-MuRF1P and pcDNA3; n = 3). B, EMSA was performed using nuclear extracts from WT and Smad3^−/− biceps femoris muscle. (i) Left panel, representative gel showing increased CHOP binding in Smad3^−/− muscle as indicated by the shifted band in lane 3 (lane 1, oligo only; lane 2, WT, Lane 3-Smad3^−/−). EMSA was also performed with nuclear extracts from WT and Smad3^−/− biceps femoris muscle preincubated with CHOP-specific antibody. (ii) Right panel, representative gel showing the diminished band intensity of CHOP-specific band. Also, the disappearance of the shifted band in the nuclear extracts incubated with ×500 concentration of competitor oligos is observed. (lane 1,oligo only; lane 2, WT; lane 3, Smad3^−/−; lane 4,WT with CHOP antibody; lane 5, Smad3^−/− with CHOP antibody; lane 6, WT with ×500 competitor oligos; lane 7,Smad3^−/− with ×500 competitor oligos). C, Representative agarose gel image showing the binding of CHOP to MuRF1 promoter (lanes 7 and 8), as assessed by ChIP, in C2C12 myoblasts treated with SIS3 for 48 hours during differentiation. The relative amounts of the input DNA in both untreated (lane 1) and SIS3 treated (lane 2) myoblasts were also assessed. Both no antibody (No Ab) (lanes 3 and 4) and isotype-specific IgG (lanes 5 and 6) controls are shown. D, Representative graph showing mRNA expression of Chop in gastrocnemius muscle from WT, Smad3^−/−, Mstn^−/−, and double-KO mice. *, P < .05; **, P < .01: when compared with the WT muscle; ^^^^, P < .0001: when compared with Smad3^−/− muscle (n = 3). E, Western blot analysis (i) and densitometric analysis (ii) of CHOP and MuRF1 protein levels in gastrocnemius muscle from WT (lane 1), Smad3^−/− (lane 2), Mstn^−/− (lane 3), and double-KO (lane 4) mice. α-Tubulin was used as an internal control for equal protein loading on the gel; **, P < .01: when compared with the WT muscle; ^, P < .05; ^^, P < .01: when compared with Smad3^−/− muscle (n = 3). F, Western blot analysis of CHOP protein levels in whole-cell lysates, nuclear (NE) and cytoplasmic extracts (CE), and MuRF1 levels in whole-cell lysates obtained from untreated C2C12 myoblasts (Control) (lane 1) and SIS3 treated (for 48 hours during differentiation) myoblasts (lane 2). α-Tubulin was used as an internal control for equal protein loading on the gel (n = 2). Ab, antibody.

Figure 7.

Proposed Smad3-independent mechanisms behind ROS production by Mstn. In the absence of Smad3, Mstn induces TNF-α and IL-6 to activate p38 and ERK MAPK to promote Nox- and XO-mediated induction of ROS. The excessive ROS leads to increased CHOP levels and up-regulation of MuRF1 transcription. The increased CHOP protein levels also induce ROS production, and the enhanced ROS would further lead to increased Mstn production. NADPH, nicotinamide adenine dinucleotide phosphate.