The glucocorticoid receptor and FOXO1 synergistically activate the skeletal muscle atrophy-associated MuRF1 gene

Abstract

The muscle specific ubiquitin E3 ligase MuRF1 has been implicated as a key regulator of muscle atrophy under a variety of conditions, such as during synthetic glucocorticoid treatment. FOXO class transcription factors have been proposed as important regulators of MuRF1 expression, but its regulation by glucocorticoids is not well understood. The MuRF1 promoter contains a near-perfect palindromic glucocorticoid response element (GRE) 200 base pairs upstream of the transcription start site. The GRE is highly conserved in the mouse, rat, and human genes along with a directly adjacent FOXO binding element (FBE). Transient transfection assays in HepG2 cells and C(2)C(12) myotubes demonstrate that the MuRF1 promoter is responsive to both the dexamethasone (DEX)-activated glucocorticoid receptor (GR) and FOXO1, whereas coexpression of GR and FOXO1 leads to a dramatic synergistic increase in reporter gene activity. Mutation of either the GRE or the FBE significantly impairs activation of the MuRF1 promoter. Consistent with these findings, DEX-induced upregulation of MuRF1 is significantly attenuated in mice expressing a homodimerization-deficient GR despite no effect on the degree of muscle loss in these mice vs. their wild-type counterparts. Finally, chromatin immunoprecipitation analysis reveals that both GR and FOXO1 bind to the endogenous MuRF1 promoter in C(2)C(12) myotubes, and IGF-I inhibition of DEX-induced MuRF1 expression correlates with the loss of FOXO1 binding. These findings present new insights into the role of the GR and FOXO family of transcription factors in the transcriptional regulation of the MuRF1 gene, a direct target of the GR in skeletal muscle.

Fig. 1.

Schematic of the muscle RING finger 1 (MuRF1) promoter and sequence alignment of the proximal regulatory regions. A: promoter sequences from mouse, rat, and human MuRF1 [5,000 base pairs upstream of the transcription start site (+1) through the first exon] were downloaded from the Ensembl database (www.ensembl.org) and aligned using the ClustalW algorithm. Approximate positions of potential transcription factor binding sites are indicated in the schematics of the MuRF1 promoter at top and/or boxed in the alignment at bottom. The forkhead transcription factor class O (FOXO) forkhead binding site (G/A)TAAA(T/C)AA (black ovals), glucocorticoid response element (GRE) (A/T)GAACANNNTGTTC(A/T) (hatched rectangle), CCAAT/enhancer-binding protein (C/EBP) TT(G/T)NGNAA (◊), NF-κB consensus binding sequence GGG(G/A)N(C/T)(C/T)(C/T)CC (gray hexagons), and muscle-specific E-box CANGTG (MyoD, etc.); N represents any nucleotide. The sequence alignments for ∼400 base pairs upstream and 26 base pairs downstream of the rat, mouse, and human MuRF1 promoters are shown below the corresponding promoter schematics. Identical sequences for the indicated regions are highlighted in black. Arrow indicates transcription start site. B: comparison of a consensus GRE and perfect palindrome GRE and putative GREs from the mouse (mMuRF1), rat (rMuRF1), and human MuRF1 (hMuRF1) promoters. Arrows indicate half-sites.

Fig. 2.

Dexamethasone (DEX)-activated glucocorticoid receptor (GR) induces the MuRF1 proximal promoter. A: HepG2 cells were transfected with luciferase reporter constructs (Luc) containing varying lengths of the MuRF1 (pGL3-MuRF1) promoter, an SV40-Renilla luciferase reporter construct, and a mouse GR expression vector (pSG5-GR). Cells were treated with or without 1 μM DEX for 24 h. Luciferase activities in cell extracts were normalized to Renilla luciferase activity to control for transfection efficiency. Numbers on the y-axis indicate distance from the transcription start site fused to the reporter pGL3-Basic, a promoterless LUC vector. B: the isolated MuRF1 GRE supports DEX-mediated transcriptional activation of a heterologous promoter. The DEX regulation of the thymidine kinase (TK) promoter was tested with 0 (control), 1 (1XGRE), 3 (3XGRE), and 4 (4XGRE) inserted GREs. The arrows below the indicated GRE constructs labeled on the y-axis depict the orientation of each individual GRE oligonucleotide. In A and B, fold induction was obtained by dividing values from DEX-treated samples by the mean of values from matched untreated samples. Each condition was done in triplicate, and error bars reflect SD.

Fig. 3.

FOXO transcription factor induction of the MuRF1 promoter. A: HepG2 cells were transfected with 500-base pair MuRF1-promoter-LUC constructs, SV40-Renilla luciferase reporter construct, and expression vectors for murine FoxO1, FoxO3A, or FoxO4 (pcDNA3-FoxO1, pcDNA3-FoxO3A, or pcDNA3-FoxO4) or pcDNA3 alone (dash). Luciferase activities in cell extracts were normalized to Renilla luciferase activity to control for variations in transfection efficiency. Each point was done in triplicate; error bars reflect SD. B: the isolated MuRF1 FOXO binding element (FBE) supports FOXO3a- and FOXO4- but not FOXO1-mediated transcriptional activation of a heterologous promoter. FOXO regulation of the TK promoter was tested by inserting a single FBE and cotransfecting HepG2 cells with the control pcDNA3 vector alone (dash) or each FOXO expression vector as in A. C: concatamerized FOXO binding elements from the MuRF1 promoter (FBE-4X) and Daf-16 binding elements (DBE-6X) support transcriptional activation of the TK promoter by FOXO1. Open bars, normalized luciferase values from the cells transfected with the TK-Luc-4XFBE and TK-Luc-6XDBE in the absence of exogenous FOXO1 expression; black bars, normalized luciferase values from the TK-Luc-4XFBE- and TK-Luc-6XDBE-transfected cells in the presence of exogenous FOXO1 expression. Data were processed as in A and B.

Fig. 4.

DEX-activated GR synergizes with FOXO1 to potently induce the MuRF1 promoter. A: HepG2 cells were transfected with the pGL3-MuRF1 promoter (−500) reporter construct, SV40-Renilla luciferase reporter construct, pSG5-GR, and/or pcDNA3-FOXO1, pcDNA3-FoxO3A, or pcDNA3-FOXO4. Cells were treated with or without 1 μM DEX for 24 h. Firefly luciferase activities in cell extracts were normalized to Renilla luciferase activity to control for variations in transfection efficiency. Fold induction was obtained by dividing values from DEX-treated samples by the mean of values from matched untreated samples. Each point was done in triplicate, and errors reflect SD. B: HepG2 cells were transfected with pGL3-MuRF1 promoter (−500), SV40-Renilla, and pBluescript as filler DNA (control) and either GR (GR) or GR and FoxO1 (GR + FoxO1) expression vectors. Twenty-four hours posttransfection, cells were treated with the indicated DEX concentration and incubated overnight. Each point was done in triplicate. C and D: mutation of either the GRE or the FBE is sufficient to abolish DEX-induced MuRF1 promoter activity. HepG2 cells were transfected with either the wild-type MuRF1 promoter construct (−500) or MuRF1 promoter constructs that have either the GRE mutated (GRE-Mut) or the FBE mutated (FBE-Mut) as shown in C in combination with expression vectors for FoxO1 and/or GR as indicated. The cells where then treated and assayed for luciferase activity as in A.

Fig. 5.

DEX-induced MuRF1 expression is inhibited in mutant GR homodimerization mutant (GR^dim) mice vs. wild-type GR^+/+ mice. A: GR^+/+ and GR^dim mice were treated with DEX in their drinking water for 8 days, weighed (top left), and killed for determination of splenocyte number (top right) as well as gastrocnemius (bottom left) and tiblialis anterior (bottom right) weights. Open bars, vehicle treated; black bars, DEX treated. GR^+/+, n = 8; GR^dim, n = 7. B: Northern analysis of MuRF1, FOXO1, and FOXO3a expression in gasctocnemius muscle from DEX-treated GR^+/+ and GR^dim mice. B, top: control, 6-h-, and 24-h-treated mice. B, bottom: control, 3-day-, and 8-day-treated mice. Only results from 4 of the 8-day-treated GR^+/+ and GR^dim mice are presented. rpL32 expression is shown as a loading control below each set of Northerns. Bars represent means ± SE. C: effect of wild-type GR and the homodimerization mutant GR on DEX induction of the MuRF1 promoter in a transient transfection assay. HepG2 cells were transfected with the pGL3-MuRF1 promoter (−500) reporter construct, SV40-Renilla luciferase reporter construct (control), pcDNA3.1-GRwt (GR) or pcDNA3.1-GR^dim (GR^dim), and/or pcDNA3-FoxO1 (FOXO1). Cells were treated with or without 1 μM DEX for 24 h. Firefly luciferase activities in cell extracts were normalized to Renilla luciferase activity to control for variations in transfection efficiency. Fold induction was obtained by dividing values from DEX-treated samples by the mean of values from matched untreated samples. Each point was done in triplicate, and errors reflect SD.

Fig. 6.

DEX induces the MuRF1 promoter in differentiated C₂C₁₂ myotubes. A: C₂C₁₂ myoblasts were transfected with a reporter construct containing 500 base pairs of the MuRF1 promoter fused to the secreted alkaline phosphatase (SEAP) gene. The myoblasts were then differentiated by switching to low-serum medium followed by treatment with 1 μM DEX over a period of 3 days. The medium was sampled every 24 h to measure for SEAP activity. Conditions were done in triplicate and SEAP numbers normalized with β-galactosidase to correct for variations in transfection efficiency. Each point was done in triplicate, and errors reflect SD. B: mutation of either the GRE or the FBE is sufficient to abolish DEX-induced MuRF1 promoter activity in C₂C₁₂ cells. C₂C₁₂ myoblasts were transfected with either the wild-type MuRF1 promoter construct (−500) or MuRF1 promoter constructs that have either GRE-Mut or FBE-Mut SEAP constructs, differentiated, and treated with 1 μM DEX over a period of 3 days. The medium was sampled every 24 h to measure for SEAP activity as in A. Each time point was done in triplicate and normalized with β-galactosidase to correct for variations in transfection efficiency. C: Northern blot analysis of C₂C₁₂ cells differentiated for 48 h and then treated with DEX (10 μM), IGF-I (20 ng/ml), or DEX (10 μM) + IGF-I (20 ng/ml) for 24, 48, and 72 h. Ligand was refreshed every 24 h. D: IGF-I potently inhibits DEX-induced activation of the MuRF1 promoter. C₂C₁₂ myotubes were treated for 24 h with either 1 μM DEX, 20 ng/ml IGF-I, or 1 μM DEX and 20 ng/ml IGF-I followed by sampling of the medium for SEAP activity, as described in A. Fold induction was obtained by dividing values from DEX-treated samples by the mean of values from matched untreated samples. Each point was done in triplicate, and errors reflect SD.

Fig. 7.

GR and FOXO1 associate directly with the MuRF1 promoter in C₂C₁₂ myotubes. A: C₂C₁₂ myotubes were treated with 1 μM DEX for 0 or 60 min (1 10-cm plate/time point), and cross-linked chromatin was immunoprecipitated with normal rabbit IgG, an anti-FOXO1 antibody, or one of two anti-GR antibodies (P20 or M20). After reversal of cross-links, immunoprecipitated MuRF1 promoter fragments were detected by PCR using primers flanking the predicted GRE and FOXO sites in the mouse MuRF1 promoter, followed by agarose gel electrophoresis. PCRs of a no-template control (NTC) and input DNAs are shown at left. B and C: C₂C₁₂ myotubes were treated with vehicle (open bars), 1 μM DEX, 20 ng/ml R3-IGF-I, or 1 μM DEX + 20 ng/ml R3-IGF-I for 15 min (black bars) or 30 min (gray bars) before harvest. Four plates for each treatment were processed in parallel as in A. Values (fold enrichment) are expressed as the mean MuRF1 promoter copies immunoprecipitated with either the anti-GR antibody (B) or anti-FOXO1 antibody (C) divided by that immunoprecipitated by a nonspecific antibody (anti-HA), as determined by quantitative PCR using the same primer pairs as in A. Error bars reflect SE.