Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression

Abstract

Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C₂C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C₂C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids.

Keywords: GRα 5; GRβ 4; MAFbx 7; MuRF1 8; atrophy 6; dexamethasone 9; glucocorticoid receptor α 2; glucocorticoid receptor β 3; myogenesis.

Figure 1

GR expression and responsiveness in C₂C₁₂ myoblasts. (A) Western blot of C₂C₁₂ myoblasts treated with vehicle (Ctrl), dexamethasone (Dex), or insulin (Ins) for 24 h; (B) Quantification of GRα protein expression in response to Dex and Ins; ** p < 0.01 compared to Ctrl; (C) Quantification of GRβ protein expression in response to Dex and Ins; *** p < 0.001 compared to Ctrl. n = 3 experiments. Data expressed as mean ± SEM.

Figure 2

Changes in glucocorticoid receptor and myogenic mRNA expression during myotube formation. C₂C₁₂ myoblasts were induced to differentiate into myotubes starting at ~90% confluence, d0 (day zero). Differentiation was carried out for four days, d1 (day one) through d4 (day four). (A) GRβ. # p ≤ 0.0009 compared to d0; ^$ p < 0.05 compared to d1; (B) GRα. ^# p < 0.01 compared to d0; ^$ p < 0.01 compared to d1; (C) MyoD. ^# p = 0.0179 compared to d0; (D) Myogenin. ^# p < 0.0001 compared to d0, ^$ p < 0.0001 compared to d1, and ^& p < 0.0001 compared to d2 (day two). n = 3 to 6 experiments per time point. Data expressed as mean relative quantification (RQ) ± SEM.

Figure 3

GRβ overexpression reduces dexamethasone responsiveness in C₂C₁₂ myoblasts. (A) Myoblasts overexpressing GRβ (GRβOE) had a significant increase in GRβ mRNA expression compared to vector transfected cells. * p = 0.016; (B) GRα mRNA expression was not altered by GRβ overexpression; (C) GRβ mRNA expression was not altered in GRβOE cells following 2-h dexamethasone (Dex) treatment. **** p < 0.0001 as indicated by brackets; (D) Glucocorticoid-induced leucine zipper (GILZ) mRNA levels showed an abrogated response to Dex in GRβOE myoblasts. **** p < 0.0001 compared to respective Ctrl and as indicated by brackets. n = 3 to 5 experiments. Data expressed as mean RQ ± SEM.

Figure 4

GRβ overexpression increases myogenic mRNA expression and proliferation. (A) MyoD and (B) Myogenin mRNA levels were significantly elevated in GRβOE myoblasts compared to vector. *** p = 0.0008, **** p < 0.0001; (C) PTEN mRNA levels were suppressed in GRβOE myoblasts. * p = 0.017. n = 3 to 6 experiments; (D) An MTT proliferation assay showed that GRβOE myoblast had an increase in proliferation at days 3 and 4 of assessment compared to vector. *** p = 0.0008 at day 3 and p = 0.0001 at day 4 between cells types and the time point indicated. n = 3 experiments per time point. Data expressed as mean RQ ± SEM for real time PCR analysis and mean fold change ± SEM for proliferation.

Figure 4

GRβ overexpression increases myogenic mRNA expression and proliferation. (A) MyoD and (B) Myogenin mRNA levels were significantly elevated in GRβOE myoblasts compared to vector. *** p = 0.0008, **** p < 0.0001; (C) PTEN mRNA levels were suppressed in GRβOE myoblasts. * p = 0.017. n = 3 to 6 experiments; (D) An MTT proliferation assay showed that GRβOE myoblast had an increase in proliferation at days 3 and 4 of assessment compared to vector. *** p = 0.0008 at day 3 and p = 0.0001 at day 4 between cells types and the time point indicated. n = 3 experiments per time point. Data expressed as mean RQ ± SEM for real time PCR analysis and mean fold change ± SEM for proliferation.

Figure 5

GRβ overexpression enhances fusion and myotube formation. (A) Myoblast fusion and myotube formation was assessed in vector and GRβOE cells using myosin heavy chain (MHC) labeling following four days of differentiation. Myotubes were identified as MHC+ cells with a minimum of two nuclei; (B) The fusion index; (C) total nuclei within myotubes; (D) nuclei per myotube; (E) the number of myotubes, were all significantly greater in GRβOE cells. * p < 0.05, and **** p < 0.0001 compared to vector. n = 6 to 9 experiments. 5 to 7 fields were analyzed per experiment by blinded investigators. All images analyzed at 10× magnification. Data expressed as mean ± SEM.

Figure 6

Dexamethasone-induced atrogene expression is reduced in GRβOE myotubes. (A) Foxo3a mRNA was reduced in GRβOE myotubes. * p = 0.025; (B) Muscle atrophy F-box (MAFbx; also known as atrogen-1); (C) muscle ring finger 1 (MuRF1) mRNA response to 24 h of Dex exposure was diminished in GRβOE myotubes. ** p < 0.01 and **** p < 0.0001. n = 3 experiments. Data expressed as mean RQ ± SEM.