Identification of functional glucocorticoid response elements in the mouse FoxO1 promoter☆
Introduction
FoxO1 is one of a family of four forkhead O transcription factors that is known to be involved in diverse cellular processes that include proliferation, differentiation, regulation of metabolism and control of bone formation [1], [2], [3]. An important determinant of transcriptional activity of FoxO1 is inhibitory phosphorylation by Akt in response to IGF-1 or other growth factors [4], because such phosphorylation excludes FoxOs from the nucleus. In skeletal muscle, expression levels of FoxO1 relate inversely to muscle size [5], and FoxO1 and FoxO3A proteins have been found to determine the expression levels of several negative regulators of muscle mass [6], [7], [8], [9], [10], [11]. Upregulation of FoxO1 expression is a conserved feature of diverse conditions that result in muscle atrophy including paralysis, glucocorticoid administration, and immobilization [12], [13], [14], [15]. In parallel, during muscle atrophy, FoxOs are activated through mechanisms that include reduced Akt activity, thereby driving increased expression of FoxO-regulated genes for muscle atrophy (atrogenes).
Elevated levels of glucocorticoids are one well recognized cause of muscle atrophy. Increases in glucocorticoid levels occur in states of physiologic stress such as starvation, confinement, stroke, or diabetes [16], or through administration of glucocorticoid medications [17]. We have previously reported that FoxO1 expression is upregulated by glucocorticoids in rat skeletal muscle [15]. The glucocorticoid receptor (GR) is a member of the steroid hormone receptor family of nuclear receptors [18]. The classical mechanism of signaling through the GR involves its binding to glucocorticoid response elements in promoters and enhancers of target genes resulting in either repression or upregulation of transcription [18]. We hypothesized that the FoxO1 gene contained one or more GREs through which glucocorticoids determined FoxO1 transcription. The goal of the present study was to test this possibility in cultured myoblasts reporter genes and gel shift assays.
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Cell culture
Mouse C2C12 cells (ATCC, Rockland, MD) were maintained in DMEM containing 10% fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (growth media) at 37 °C in a humidified atmosphere containing 5% carbon dioxide. Differentiation was initiated by replacing growth media with media containing 2% horse serum. Primary cultures of human skeletal muscle myoblasts (Lonza, Walkersville, MD) were maintained following the manufacturer’s recommended procedures.
Results
We began by testing whether dexamethasone upregulated FoxO1 expression in differentiating C2C12 myotubes. Incubation of these cells with dexamethasone increased FoxO1 mRNA by approximately 3-fold and FoxO1 protein by more than 2-fold (Fig. 1A and B). In parallel experiments, primary cultures of human myoblasts were subjected to differentiating conditions and the effects of dexamethasone were examined. FoxO1 protein levels were significantly increased in primary myoblast cultures by nearly
Discussion
The above data support the conclusions that FoxO1 is a glucocorticoid-responsive gene whose expression is regulated by binding of the GR to GREs within the proximal promoter. These GREs are clustered between −800 and approximately −2000 bp upstream of the first codon of the FoxO1 gene and include two GREs that are highly conserved between mice and humans. These conclusions are supported by findings that dexamethasone increased mRNA levels for FoxO1 without altering FoxO1 mRNA degradation, that
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2021, ToxicologyCitation Excerpt :GR is a ligand-dependent transcriptional factor that binds to the glucocorticoid response element region (GRE) of the promoter region of the target gene and recruits various cofactors to regulate the expression of downstream target genes. Qin et al. confirmed that FOXO1 is a glucocorticoid-responsive gene whose expression is regulated by binding of the GR to GREs within the proximal promoter (Qin et al., 2014). GR regulates various processes, such as muscle atrophy, through promoting FOXO1 transcription (Cid-Diaz et al., 2017; Qin et al., 2014, 2010).
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2020, Analytical BiochemistryCitation Excerpt :GREs have been found in promoter regions of MuRF1 and myostatin, thus directly inducing their expression [42,43]. Additionally, FOXO transcription factors can also be directly regulated by GR, as promoters of FoxO genes contain multiple functional GREs [44,45]. FOXO transcription factors then activate expression of MuRF1 and Atrogin-1 by binding to their promoter regions [23,46].
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2016, Seminars in Cell and Developmental BiologyCitation Excerpt :Finally, an additional level of regulation is the induction of FoxO at the transcriptional level. In fact, mRNA levels of FoxO members are induced in cancer cachexia and in many other catabolic conditions, and recent evidence shows that the glucocorticoid receptor is a direct activator of FoxO gene expression [124–126]. Collectively, several studies are consistent in suggesting that FoxO members play a critical role in cancer-mediated protein breakdown.
Redox regulation of FoxO transcription factors
2015, Redox BiologyCitation Excerpt :The study shows how, under conditions of metabolic stress, GR and high levels of intracellular AMP cooperate to induce FoxO3 gene transcription and post-translationally activate FoxO3a protein [167]. Multiple functional GREs were recently detected also in the murine FoxO1 promoter [168]. These experiments, performed in the C2C12 myoblasts, suggested an additional mechanism by which GR stimulates muscle atrophy [168].