Identification of functional glucocorticoid response elements in the mouse FoxO1 promoter

https://doi.org/10.1016/j.bbrc.2014.06.080Get rights and content

Highlights

  • Dexamethasone (Dex) increased FoxO1 expression in C2C12 cells and primary myoblasts.

  • The mouse FoxO1 promoters contained GREs that conferred Dex responsiveness to reporter gene.

  • The FoxO1 promoter containing either of two highly conserved GREs bound recombinant GR.

  • We conclude that the FoxO1 promoter contains functional GREs.

Abstract

Glucocorticoids stimulate muscle atrophy through a cascade of signals that includes activation of FoxO transcription factors which then upregulate multiple genes to promote degradation of myofibrillar and other muscle proteins and inhibit protein synthesis. Our previous finding that glucocorticoids upregulate mRNA levels for FoxO1 in skeletal muscle led us to hypothesize that the FoxO1 gene contains one or more glucocorticoid response elements (GREs). Here we show that upregulation of FoxO1 expression by glucocorticoids requires the glucocorticoid receptor (GR) and binding of hormones to it. In cultured C2C12 myoblasts dexamethasone did not alter FoxO1 mRNA stability. Computational analysis predicted that the proximal promoter of the FoxO1 gene contained a cluster of eight GRE half sites and one highly conserved near-consensus SRE; the cluster is found between −800 and −2000 bp upstream of the first codon of the FoxO1 gene. A reporter gene constructed using the first 2 kb of the FoxO1 promoter was stimulated by dexamethasone. Removal of a 5′ domain containing half of the GREs reduced reporter gene activity and removal of all GREs in this region ablated activation by dexamethasone. Restriction fragments of the cluster of 8 upstream GREs bound recombinant GR in gel shift assays. Collectively, the data demonstrate that the proximal promoter of the FoxO1 gene contains multiple functional GREs, indicating that upregulation of FoxO1 expression by glucocorticoids through GREs represents an additional mechanism by which the GR drives glucocorticoid-mediated muscle atrophy. These findings are also relevant to other physiological roles of FoxO1 such as regulation of hepatic metabolism.

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.

Section snippets

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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    Grant support: Department of Veterans Affairs Rehabilitation Research and Development Service B9212C, B0687R and B1313R.

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