Excessive glucocorticoid-induced muscle MuRF1 overexpression is independent of Akt/FoXO1 pathway

Abstract

The ubiquitin-proteasome system (UPS)-dependent proteolysis plays a major role in the muscle catabolic action of glucocorticoids (GCs). Atrogin-1 and muscle-specific RING finger protein 1 (MuRF1), two E3 ubiquitin ligases, are uniquely expressed in muscle. It has been previously demonstrated that GC treatment induced MuRF1 and atrogin-1 overexpression. However, it is yet unclear whether the higher pharmacological dose of GCs induced muscle protein catabolism through MuRF1 and atrogin-1. In the present study, the role of atrogin-1 and MuRF1 in C2C12 cells protein metabolism during excessive dexamethasone (DEX) was studied. The involvement of Akt/forkhead box O1 (FoXO1) signaling pathway and the cross-talk between anabolic regulator mammalian target of rapamycin (mTOR) and catabolic regulator FoXO1 were investigated. High concentration of DEX increased MuRF1 protein level in a time-dependent fashion (P<0.05), while had no detectable effect on atrogin-1 protein (P>0.05). FoXO1/3a (Thr24/32) phosphorylation was enhanced (P<0.05), mTOR phosphorylation was suppressed (P<0.05), while Akt protein expression was not affected (P>0.05) by DEX. RU486 treatment inhibited the DEX-induced increase of FoXO1/3a phosphorylation (P<0.05) and MuRF1 protein; LY294002 (LY) did not restore the stimulative effect of DEX on the FoXO1/3a phosphorylation (P>0.05), but inhibited the activation of MuRF1 protein induced by DEX (P<0.05); rapamycin (RAPA) inhibited the stimulative effect of DEX on the FoXO1/3a phosphorylation and MuRF1 protein (P<0.05).

Keywords: FoXO1; MuRF1; glucocorticoid; muscle cell; protein metabolism.

Figure 1. The time-dependent effect of DEX treatment

The effect of DEX treatment (100 µM for 3, 8, 12, and 24 h) on atrogin-1 (A, C) and MuRF1 (B, D) expressions in C2C12 cells. Results are presented as the mean ± S.E.M. (n≥4); control and DEX treatments were compared at each time point, and means with * are significantly different (P<0.05).

Figure 2. Dex effects on protein degradation regulators and protein synthesis regulators

The effect of DEX treatment (100 µM for 8 h) on mRNA expressions of FoXO1, FoXO3, MuRF1, and atrogin-1 (A), protein expressions of p-FoXO1/3a (B), MuRF1, atrogin-1 (C), p-mTOR and p-Akt (D) in C2C12 cells. Results are presented as the mean ± S.E.M. (n≥5); control and DEX treatments were compared for each gene, and means with * are significantly different (P<0.05).

Figure 3. Effect of proteasome inhibition with EPOX on DEX-induced effects

The effect of DEX (100 µM for 8 h) and EPOX (1.5 μM for 5 h) on protein expressions of MuRF1 (A) and atrogin-1 (B) in C2C12 cells. Results are presented as the mean ± S.E.M. (n≥3); four groups were compared for each gene, and means without a common letter are significantly different (P<0.05).

Figure 4. Effect of GR blockage with RU486 on DEX-induced effects

The effect of DEX (100 µM for 8 h) and RU486 (10 μM for 5 h) on protein expressions of p-FoXO1/3a (A), MuRF1 and atrogin-1 (B) in C2C12 cells. Results are presented as the mean ± S.E.M. (n≥3); four groups were compared for each gene, and means without a common letter are significantly different (P<0.05).

Figure 5. Effect of PI3K blockage with LY294002 on DEX-induced effects

The effect of DEX (100 µM for 8 h) and LY294002 (10 μM for 5 h) on protein expressions of p-FoXO1/3a (A) and MuRF1 (B) in C2C12 cells. Results are presented as the mean ± S.E.M. (n≥3); four groups were compared for each gene, and means without a common letter are significantly different (P<0.05).

Figure 6. Effect of mTOR blockage with RAPA on DEX-induced effects

The effect of DEX (100 µM for 8 h) and RAPA (25 μM for 5 h) on protein expressions of p-FoXO1/3a (A), MuRF1 and atrogin-1 (B) in C2C12 cells. Results are presented as the mean ± S.E.M. (n≥3); four groups were compared for each gene, and means without a common letter are significantly different (P<0.05).

Figure 7. Proposed model of excessive GC action on protein synthesis and protein degradation in C2C12 cells

(↑ increase; ↓ decrease; gray blocks represent unaffected proteins; solid arrows represent findings in this study and dashed arrows represent findings reported previously [–25,38,39]). Excessive GCs stimulated protein degradation through MuRF1, but not atrogin-1. Other pathway that is independent of Akt/FoXO1 pathway may account for the excessive GC-induced MuRF1 activation. There is a cross-talk between anabolic regulator mTOR and catabolic regulator FoXO1-MuRF1.