Transcriptional Co-Activator With PDZ Binding Motif (TAZ) Inhibits Dexamethasone-Induced Muscle Atrophy via mTOR Signalling

Abstract

Background: Glucocorticoid therapy has a beneficial effect in several diseases, but chronic treatment has adverse effects, including muscle atrophy, which refers to the gradual decrease in muscle mass, size and strength. It is important to know how the muscle atrophy occurs, but the underlying mechanism is not yet fully understood. This study shows that dexamethasone decreases levels of the transcriptional co-activator with PDZ binding motif (TAZ), which facilitates dexamethasone-induced muscle atrophy.

Methods: To induce muscle atrophy, C2C12 myotubes were treated with dexamethasone, and mice were fed with water containing dexamethasone. Muscle atrophy was analysed for the expression of myosin heavy chain, MuRF1 and Atrogin-1 using immunofluorescence staining, immunoblot analysis and qRT-PCR. Muscle tissue was analysed by haematoxylin and eosin staining. Adeno-associated virus was used for overexpression of wild-type and mutant TAZ.

Results: TAZ levels decrease in dexamethasone-treated mice (0.36-fold, p < 0.001) and C2C12 myotubes (0.44-fold, p = 0.024). Overexpression of the TAZ mutant, which resists its proteolytic degradation, inhibits dexamethasone-induced muscle atrophy. Atrogin-1 and MuRF1 interact with TAZ and facilitate its degradation in dexamethasone-treated C2C12 myotubes. TAZ mutant stimulates protein synthesis through activation of mTOR signalling via induction of RhebL1 (DEX; Con vs, TAZ4SA: 5.1-fold, p < 0.001) in dexamethasone-treated mice. Ginsenoside Rb3 increases TAZ levels in dexamethasone-treated mice (1.49-fold, p = 0.007) and C2C12 myotubes (1.63-fold, p = 0.01), which stimulates mTOR signalling and inhibits dexamethasone-induced muscle atrophy.

Conclusions: Our results demonstrate a novel regulatory mechanism of dexamethasone-induced muscle atrophy by TAZ, suggesting that stabilisation of TAZ in muscle cells ameliorates the muscle atrophy. These results suggest that TAZ may be a drug target for the dexamethasone-induced muscle atrophy.

Keywords: dexamethasone; ginsenoside; mTOR; muscle atrophy.

FIGURE 1

Dexamethasone reduces TAZ levels in skeletal muscle. (A) Schematic representation of the experimental design for dexamethasone treatment in C2C12 myotubes. DM refers to differentiation media. Bottom panel shows the levels of myosin heavy chain (MHC) by immunofluorescence staining in vehicle‐treated [DEX(−)] and dexamethasone [DEX(+)]‐treated myotubes. Scale bar, 100 μm. The area of MHC was quantified from the immunofluorescence staining (n = 3). (B) Left: The levels of MHC, TAZ, MuRF1, Atrogin‐1, and vinculin in Panel (A) were analysed by immunoblotting. Vinculin was used as a loading control. Right: The levels of MHC, TAZ, MuRF1, and Atrogin‐1 were quantified (n = 3). (C) The transcript levels of Taz, Atrogin‐1 and MuRF1 were analysed by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) in Panel (A) (n = 3). (D) Left: Mice were administered drinking water containing dexamethasone, and body weights were analysed at the indicated time points (n = 5). Right panel depicts the ratio of gastrocnemius (GA) muscle weight to total body weight in the left panel. (E) The levels of MHC, TAZ, MuRF1, Atrogin‐1, and vinculin in Panel (D) were analysed by immunoblotting. Vinculin was used as a loading control. Right: The levels of MHC, TAZ, MuRF1 and Atrogin‐1 were quantified (n = 5). The data are presented as the mean ± SD for (A), (B) and (C) and as the mean ± SEM for (D) and (E). Statistical analysis for all figures was performed using two‐tailed t‐test. Exact p values are provided in the graph, and non‐significant results are not explicitly indicated.

FIGURE 2

TAZ mutant inhibits dexamethasone‐induced muscle atrophy. (A) TAZ wild‐type (TAZ) or mutant (TAZ4SA) overexpressing C2C12 myotubes were treated with either a vehicle [DEX(−)] or dexamethasone [DEX(+)]. The levels of MHC, TAZ and α‐tubulin were analysed by immunoblotting. α‐Tubulin was used as a loading control. Right: The levels of MHC and TAZ were quantified (n = 3) (B) The transcript levels of Atrogin‐1 and MuRF1 were analysed by qRT‐PCR in Panel (A) (n = 3). (C) Control (Con), TAZ or TAZ4SA AAV viruses were introduced into the GA muscle of mice. Following the administration of dexamethasone, the body weights of the subjects were analysed at the indicated time points (n = 5). (D) The ratio of GA muscle weight to total body weight was analysed in Panel (C). (E) Left: Histological examination of the GA muscles using haematoxylin and eosin (H&E) staining in panel (C). Scale bar, 50 μm. Right: Muscle fibre diameters were quantified using the ImageJ software. Each dot on the graph represents the diameter of an individual muscle fibre, and the total number of fibres measured was over 250 per group. Histological analysis was performed on muscle samples from five mice per group. (F) The levels of MHC, TAZ and α‐tubulin in Panel (C) were analysed by immunoblotting. α‐tubulin was used as a loading control. Bottom: The levels of MHC and TAZ were quantified (n = 3). The data are presented as the mean ± SD for (A) and (B) and as the mean ± SEM for (C), (D), (E) and (F). Statistical analysis was performed using appropriate tests: Panels (A) and (B) were analysed using two‐way ANOVA followed by Holm–Sidak's multiple comparisons test. Panel (C) was analysed using two‐way repeated measures ANOVA followed by Tukey's post hoc test for multiple comparisons. Panels (D) and (E) were analysed using one‐way ANOVA followed by Tukey's multiple comparisons test. Exact p values are provided in the graph, and non‐significant results are not explicitly indicated.

FIGURE 3

Atrogin‐1 and MuRF1 interacts with TAZ and induces its degradation. (A) Flag‐tagged TAZ (F‐TAZ) and TAZ4SA (F‐TAZ4SA) overexpressing C2C12 myotubes were treated with either a vehicle [DEX(−)] or dexamethasone [DEX(+)] in the presence of MG132. The cell lysates were subjected to immunoprecipitation with anti‐FLAG antibody, and the immune complexes were analysed by immunoblotting for TAZ, Atrogin‐1 and MuRF1. The right panel shows the quantification of the data presented in the left panel (n = 3). (B) The CRISPR/Cas9 system was employed to deplete endogenous Atrogin‐1. In Atrogin‐1‐depleted C2C12 myotubes (Atrogin‐1 KO), dexamethasone‐induced TAZ degradation was examined by immunoblotting. The right panel shows the quantification of the data in the left panel (n = 3). (C) The CRISPR/Cas9 system was employed to deplete endogenous MuRF1. In MuRF1‐depleted C2C12 myotubes (MuRF1 KO), dexamethasone‐induced TAZ degradation was examined by immunoblotting. The right panel shows the quantification of the data in the left panel (n = 3). All data are presented as the mean ± SD. Statistical analysis was performed using appropriate tests: Panel (A) was analysed using two‐way ANOVA followed by Holm–Sidak's multiple comparisons test, and Panels (B) and (C) were analysed using two‐tailed t‐test. Exact p values are provided in the graph, and non‐significant results are not explicitly indicated.

FIGURE 4

TAZ stimulates mTOR signalling. (A) Left: C2C12 myotubes were treated with either a vehicle [DEX(−)] or dexamethasone [DEX(+)]. The levels of Rheb, RhebL1, phospho‐p70 S6K (p‐p70 S6K), p70 S6K and vinculin were analysed by immunoblotting. Right: The protein levels depicted in the left panel were quantified (n = 3). (B) Left: TAZ‐ and TAZ4SA‐overexpressing C2C12 myotubes were treated with either a vehicle [DEX(−)] or dexamethasone [DEX(+)]. The levels of RhebL1, p‐p70 S6K, p70 S6K and α‐tubulin were analysed by immunoblotting. Right: The protein levels shown in the left panel were quantified (n = 3). (C) Left: The control (Con), TAZ and TAZ4SA AAV viruses were introduced into the GA muscle of mice. Following dexamethasone administration, gastrocnemius muscle was isolated, and the levels of RhebL1, p‐p70 S6K and p70 S6K were analysed by immunoblotting. Right: The protein levels of the left panel were quantified (n = 3). α‐Tubulin in Figure 2F was used for the quantification of RhebL1. The data are presented as the mean ± SD for Panels (A) and (B) and as the mean ± SEM for Panel (C). Statistical analysis was performed using appropriate tests: Panel (A) was analysed using one‐tailed t‐test, Panel (B) was analysed using two‐way ANOVA followed by Holm–Sidak's multiple comparisons test, and Panel (C) was analysed using one‐way ANOVA followed by Tukey's multiple comparisons test. Exact p values are provided in the graph, and non‐significant results are not explicitly indicated.

FIGURE 5

Ginsenoside Rb3 inhibits dexamethasone‐induced TAZ depletion. (A) Left: Schematic representation of the experimental design for the ginsenoside treatment in C2C12 myotubes. DM refers to differentiation media. Ginsenosides Rb3, Rg1 and Rb1 were administered to C2C12 myotubes that had been treated with dexamethasone. The levels of MHC, TAZ, MuRF1, Atrogin‐1 and α‐tubulin levels were analysed by immunoblotting. α‐tubulin was used as a loading control. Right: The levels of MHC, TAZ, MuRF1 and Atrogin‐1 were quantified (n = 3). (B) Left: The levels of MHC was analysed by immunofluorescence staining in C2C12 myotubes treated with a vehicle (Con), dexamethasone (DEX) and dexamethasone plus ginsenoside Rb3 (DEX + Rb3). Scale bar, 100 μm. Right: The MHC‐positive area was quantified from the immunofluorescence staining (n = 3). (C) The transcript levels of Atrogin‐1 and MuRF1 were analysed by qRT‐PCR in Panel (B) (n = 3). (D) Left: The levels of MHC, TAZ, RhebL1, p‐p70 S6K, p70 S6K and α‐tubulin were analysed by immunoblotting in Panel (B). α‐tubulin was used as a loading control. Right: The protein levels of the left panel were quantified (n = 3). All data are presented as the mean ± SD. Statistical analysis for all figures was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. Exact p values are provided in the graph, and non‐significant results are not explicitly indicated.

FIGURE 6

Ginsenoside Rb3 inhibits dexamethasone‐induced muscle atrophy through the induction of TAZ. (A) During dexamethasone‐induced muscle atrophy, ginsenoside Rb3 was administered orally at a dose of 10 mg/kg. Subsequently, the GA muscles from the mice treated with dexamethasone‐only (DEX) and dexamethasone plus ginsenoside Rb3 (DEX + Rb3) were isolated and subjected to analysis. The levels of MHC, TAZ, RhebL1, p‐p70 S6K, p70 S6K and α‐tubulin were analysed by immunoblotting. α‐Tubulin was used as a loading control. Right: The protein levels shown in the left panel were quantified (n = 5). (B) Left: H&E staining of GA muscles in Panel (A). Scale bar, 50 μm. Right: Muscle fibre diameters were quantified using ImageJ software. Each dot on the graph represents the diameter of an individual muscle fibre, and the total number of fibres measured was over 250 per group. Histological analysis was performed on muscle samples from five mice per group. All data are presented as the mean ± SEM. Statistical analysis for all figures was performed using two‐tailed t‐test. Exact p values are provided in the graph, and non‐significant results are not explicitly indicated.