CCAAT/enhancer binding protein-delta expression is increased in fast skeletal muscle by food deprivation and regulates myostatin transcription in vitro

Abstract

We recently demonstrated that mRNA levels of three members of the CCAAT/enhancer binding factor (C/EBP) family of transcription factors are increased in skeletal muscle following 12 days of spaceflight. In the present study, we further explored the expression of C/EBP-δ in atrophying fast skeletal muscle by examining its expression in muscle from food-deprived (FD) mice, and investigated its role in regulating the expression of the secreted antigrowth factor myostatin. C/EBP-δ mRNA and protein levels were significantly increased by 2 days of food deprivation in the tibialis anterior (TA) muscle, and expression of both myostatin and C/EBP-δ mRNA during food deprivation was attenuated by injection with the glucocorticoid inhibitor RU486. The increase in myostatin mRNA levels with food deprivation appears to be at least partially transcriptionally driven, since levels of myostatin pre-mRNA were significantly increased in the TA muscle. C/EBP-δ mRNA levels and promoter activity were significantly increased by transfection of C(2)C(12) myotubes with a glucocorticoid receptor construct and 24 h of treatment with the synthetic glucocorticoid dexamethasone. Furthermore, activity of the C/EBP-δ promoter was significantly increased with as little as 1 h of dexamethasone treatment, while activity of the mouse myostatin promoter was only significantly increased with longer treatment periods of 24 h or more. Activity of the myostatin promoter-reporter construct was significantly increased in C(2)C(12) myotubes by cotransfection with expression constructs for C/EBP-α, -β, and -δ, with C/EBP-δ having the greatest effect. The myostatin promoter contains two potential C/EBP binding sequences, a CCAAT box, and a C/EBP binding element (CBE). Mutation of the CCAAT box attenuated basal myostatin promoter activity but potentiated C/EBP-δ-activated myostatin promoter activity in C(2)C(12) myotubes in vitro, while mutation of the CBE abolished glucocorticoid receptor and C/EBP-δ responsiveness. The present results support a model in which glucocorticoid-induced increases in C/EBP-δ expression may contribute to myostatin transcription during atrophic states.

Fig. 1.

CCAAT/enhancer binding factor-δ (C/EBP-δ) expression is increased by food deprivation. A: C/EBP-δ mRNA expression in tibialis anterior (TA) muscle from Fed, food-deprived (FD), and FD + Refed mice. Bars represent means ± SE for n = 4 animals/group *Significantly different from Fed, P < 0.05. †Significantly different from FD + Refed, P < 0.05. B: representative Western blots showing 2 homogenate samples each from Fed TA muscle and FD TA muscle using antibodies to C/EBP-δ and β-actin. All samples for both Fed and FD mice were run on the same gel so as to avoid problems with quantification across gels. Bar graph shows the difference in C/EBP-δ protein expression between Fed and FD TA muscle. Bars represent means ± SE for 3 animals/group.*Significantly different from Fed, P < 0.05.

Fig. 2.

Myostatin (MSTN) pre-mRNA in Fed and FD TA muscle. A: gel shows MSTN pre-mRNA and GAPDH mRNA in TA muscle from Fed and FD mice. Top, gel results from RT-PCR using a exonic primer to amplify MSTN mature mRNA for 3 Fed and 3 FD TA muscle samples; middle, gel results from RT-PCR intronic-exonic primer pair for MSTN to amplify MSTN pre-mRNA for 3 Fed and 3 FD samples; and bottom, exonic-exonic GAPDH primers to amplify processed GAPDH as a control for RNA loading for 3 Fed and 3 FD samples. B: bar graph shows quantification of densitometrically scanned results for MSTN mRNA. C: bar graph shows quantification of densitometrically scanned results MSTN pre-mRNA. In both cases, the values were normalized to GAPDH mRNA. Bars in B and C represent means ± SE for 3 animals/group. *Statistically significantly different from Fed, P < 0.05.

Fig. 3.

Effects of glucocorticoid inhibition on muscle mass (A) C/EBP-δ (B) and MSTN (C) mRNA levels in TA muscle. A: effects of RU486 treatment with and without food deprivation on TA muscle mass. RU486 significantly attenuated the decrease in TA muscle mass with food deprivation. B: effects of RU486 treatment with and without food deprivation on C/EBP-δ mRNA levels in the TA muscle. RU486 treatment had no effect on C/EBP-δ mRNA levels in the Fed TA muscle but significantly attenuated the increase in C/EBP-δ mRNA levels with food deprivation. C: effects of RU486 treatment with and without food deprivation on MSTN mRNA levels in the TA muscle. RU486 treatment had no effect on MSTN mRNA levels in the Fed TA muscle but abolished the increase in MSTN mRNA levels with food deprivation. Bars represent means ± SE for 4 animals/group. *Significantly different from Fed vehicle (Veh)-injected mice, P < 0.05. †Significantly different from FD vehicle injected, P < 0.05. ‡Significantly different from Fed RU486-injected, P < 0.05.

Fig. 4.

Effects of glucocorticoids on C/EBP-δ mRNA levels and promoter activity. A: gel showing the effects of 24 h of vehicle + green fluorescent protein (GFP (left) and glucocorticoid receptor (GR) + 1 μM dexamethasone (DEX) treatment (right) on C/EBP-δ (top) and GAPDH (bottom) mRNA levels. B: quantification of C/EBP-δ mRNA levels. Twenty four hours of GR + DEX treatment significantly increased C/EBP-δ mRNA levels relative to GFP + vehicle controls. Bars represent means ± SE for 5 independent transfections. *Significantly different from GFP vehicle-treated control, P < 0.05. C: mouse C/EBP-δ promoter activity after 24 h of treatment with GFP cotransfection and vehicle alone (GFP vehicle), GFP cotransfection and 1 μM DEX, GR cotransfection and vehicle alone (GR vehicle), or glucocorticoid receptor cotransfection with 1 μM DEX (GR DEX). GR cotransfection with and without DEX cotreatment significantly increased activity of the mouse C/EBP-δ promoter at 24 h effect on mouse MSTN promoter activity with or without DEX cotreatment at either time point. Bars represent means ± SE for 3 independent transfections with 6 wells/transfection. *Significantly different from GFP vehicle-treated control, P < 0.05. †Significantly different from GFP DEX treated, P < 0.05. ‡Significantly different from GR vehicle treated, P < 0.05.

Fig. 5.

Effects of glucocorticoids on activity of the mouse C/EBP-δ and MSTN promoters in C₂C₁₂ myotubes in vitro. Mouse C/EBP-δ promoter activity after 1 h (A) or 6 h (B) of treatment. GR cotransfection with and without DEX cotreatment significantly increased activity of the mouse C/EBP-δ promoter at both 1 and 6 h. Mouse MSTN promoter activity after 1 h (C) or 6 h (D) of treatment with GFP vehicle, GFP DEX, GR vehicle, or GR DEX (see Fig. 4 legend for explanation of groups). GR cotransfection had no significant effect on mouse MSTN promoter activity with or without DEX cotreatment at either time point. Bars represent means ± SE for 2–3 independent transfections with 6 wells/transfection. *Significantly different from GFP vehicle-treated control, P < 0.05. †Significantly different from GFP DEX treated, P < 0.05.

Fig. 6.

Effects of C/EBP cotransfection on MSTN promoter activity in C₂C₁₂ myutubes. A: effects of C/EBP-α, -β, and -δ cotransfection on activity of the ∼1,200 bp mouse MSTN promoter in C₂C₁₂ myutubes . All C/EBP expression constructs significantly increased mouse MSTN promoter activity relative to cotransfection with the GFP control, but C/EBP-δ had the greatest effect. B: effects of C/EBP-δ cotransfection on activity of the ∼1,000 bp human MSTN promoter in C₂C₁₂ myutubes. C/EBP-δ cotransfection also significantly increased activity of the human MSTN promoter. Bars for A and B represent means ± SE for n = 3 independent transfections with 4–6 wells/transfection. *Significantly different from CMV-GFP cotransfected control, P < 0.05. †Significantly different from C/EBP-α cotransfected, P < 0.05. ‡Significantly different from C/EBP-β cotransfected, P < 0.05. C: effects of GR and C/EBP-δ cotransfection on mouse MSTN promoter activity in C₂C₁₂ myutubes. Myotubes were transfected with the mouse MSTN promoter and either GFP alone (GFP GFP), GR (GFP GR), C/EBP-δ (GFP C/EBP-δ), or GR and C/EBP-δ (GR C/EBP-δ). Bars represent means ± SE for n = 3 independent transfections with 4–6 wells/transfection. *Significantly different from GFP GFP cotransfected control, P < 0.05. †Significantly different from GFP GR transfected, P < 0.05.

Fig. 7.

Sequence analysis of the MSTN promoter showing the 2 potential C/EBP binding sites. A: schematic of ∼300 bp of the upstream promoter region of the mouse MSTN gene. The TATA box, SMAD-box-1, and FoxO-box-1 previously identified by us (1) are shown along with the CCAAT box and the CBE (both in bold) identified by sequence analysis in the present study. Sequence alignment of the CCAAT box (B) and CBE (C) showing the conservation of these sites. The consensus CCAAT box sequence reported by Nussinov (27) is shown at the bottom in bold. On the right, the consensus reported by Ryden and Beemon (32) and the revised consensus reported by Osada et al. (28) are shown. Underlined nucleotides for the CCAAT box are ones that do not match the consensus sequence reported by Nussinov (27); underlined nucleotides for the CBE are ones that do not match the consensus sequence reported by Osada et al. (28). D: effects of mutation of the CCAAT box or CBE on basal MSTN promoter activity. Bars represent means ± SE for 3 independent transfections with 4–6 wells/transfection. *Significantly different from wild-type control, P < 0.05. †Significantly different from CCAAT box mutated, P < 0.05. E: effects of mutation of the CCAAT box or CBE on C/EBP-δ-dependent MSTN promoter activity. Bars represent means ± SE for 3 independent transfections with 4–6 wells/transfection. *Significantly different from GFP transfected, P < 0.05. †Significantly different from wild-type CMV-GFP cotransfected, P < 0.05. ‡Significantly different from CCAAT mutation (mut) construct C/EBP-β cotransfected, P < 0.05.