Skeletal muscle 11beta-HSD1 controls glucocorticoid-induced proteolysis and expression of E3 ubiquitin ligases atrogin-1 and MuRF-1

Abstract

Recent studies demonstrated expression and activity of the intracellular cortisone-cortisol shuttle 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in skeletal muscle and inhibition of 11beta-HSD1 in muscle cells improved insulin sensitivity. Glucocorticoids induce muscle atrophy via increased expression of the E3 ubiquitin ligases Atrogin-1 (Muscle Atrophy F-box (MAFbx)) and MuRF-1 (Muscle RING-Finger-1). We hypothesized that 11beta-HSD1 controls glucocorticoid-induced expression of atrophy E3 ubiquitin ligases in skeletal muscle. Primary human myoblasts were generated from healthy volunteers. 11beta-HSD1-dependent protein degradation was analyzed by [(3)H]-tyrosine release assay. RT-PCR was used to determine mRNA expression of E3 ubiquitin ligases and 11beta-HSD1 activity was measured by conversion of radioactively labeled [(3)H]-cortisone to [(3)H]-cortisol separated by thin-layer chromatography. We here demonstrate that 11beta-HSD1 is expressed and biologically active in interconverting cortisone to active cortisol in murine skeletal muscle cells (C2C12) as well as in primary human myotubes. 11Beta-HSD1 expression increased during differentiation from myoblasts to mature myotubes (p < 0.01), suggesting a role of 11beta-HSD1 in skeletal muscle growth and differentiation. Treatment with cortisone increased protein degradation by about 20% (p < 0.001), which was paralleled by an elevation of Atrogin-1 and MuRF-1 mRNA expression (p < 0.01, respectively). Notably, pre-treatment with the 11beta-HSD1 inhibitor carbenoxolone (Cbx) completely abolished the effect of cortisone on protein degradation as well as on Atrogin-1 and MuRF-1 expression. In summary, our data suggest that 11beta-HSD1 controls glucocorticoid-induced protein degradation in human and murine skeletal muscle via regulation of the E3 ubiquitin ligases Atrogin-1 and MuRF-1.

Figure 1. Regulation of 11beta-HSD1 and differentiation markers during differentiation of C2C12 cells and primary human myoblasts.

Data were normalized to mRNA expression of 16s-RiboProtein. Mean ± SEM of at least three experiments are shown. Relative 11beta-HSD1 mRNA expression in C2C12 (A). Relative Myosin Heavy Chain-1 mRNA expression in C2C12 cells (B). Relative Myf5 mRNA expression in C2C12 cells (C). Relative 11beta-HSD1 mRNA expression in primary human myoblasts (D). Relative Myosin Heavy Chain-1 mRNA expression in primary human myoblasts (E). * p<0.05, ** p<0.01, *** p<0.0001.

Figure 2. Effects of cortisone, dexamethasone (dexa) and inhibition of 11beta-HSD1 by carbenoxolone (Cbx) on 11beta-HSD1 activity in C2C12.

Mean ± SEM of at least three experiments are shown. * p<0.05, ** p<0.01, *** p<0.0001.

Figure 3. Effects of cortisone, dexamethasone (dexa) and inhibition of 11beta-HSD1 by carbenoxolone (Cbx) on protein degradation in C2C12 myotubes.

Protein degradation was measured by determining the rate of release of TCA-soluble proteins radioactively labeled with [³H]-tyrosine into the media. Mean ± SEM of at least three experiments are shown. * p<0.05, ** p<0.01, *** p<0.0001.

Figure 4. Effects of cortisone, dexamethasone (dexa) and inhibition of 11beta-HSD1 by carbenoxolone (Cbx) on Atrogin-1 and MuRF-1 expression.

Relative Atrogin-1 and MuRF-1 mRNA expression in C2C12 (A and B) and in primary human myotubes (C and D). Data are normalized to mRNA expression of 16s-RiboProtein. Mean ± SEM of at least three experiments are shown. * p<0.05, ** p<0.01, *** p<0.0001.