Evidence for transcript-specific epigenetic regulation of glucocorticoid-stimulated skeletal muscle 11β-hydroxysteroid dehydrogenase-1 activity in type 2 diabetes

11β-Hydroxysteroid dehydrogenase-1 (11βHSD1) is a bi-directional enzyme, most abundantly expressed in glucocorticoid target tissues such as the liver and adipose [1]. 11βHSD1 displays predominant oxoreductase activity in intact cells, generating the biologically active glucocorticoid cortisol from the inactive form, cortisone [2]. In adipose tissue, several studies show increased 11βHSD1 expression and oxoreductase activity, suggesting that differential elevation of glucocorticoid in this tissue may be involved in the pathogenesis of central obesity [3]. Skeletal muscle (SkM) is a major site of insulin resistance and accounts for the majority of peripheral glucose disposal [4]. We have previously demonstrated that 11βHSD1 is present and biologically active in SkM of both diabetic and nondiabetic subjects [5].

Previous in vitro studies in human myoblasts have demonstrated that addition of glucocorticoid upregulates 11βHSD1 mRNA levels and activity [6]. This is consistent with other studies in a variety of human tissues including skin fibroblasts [7], amnion fibroblasts [8], and omental adipose stromal cells [9] that implicate glucocorticoids in 11βHSD1 upregulation. Our own data in people with type 2 diabetes showed that orally administered dexamethasone increases SkM 11βHSD1 oxoreductase activity [5], but this response was not observed in nondiabetic subjects.

Dexamethasone increases 11βHSD1 mRNA levels and activity in cultured chorionic trophoblasts, an effect that is blocked by the glucocorticoid receptor antagonist RU486 [10]. Co-localization of 11βHSD1 and the glucocorticoid receptor was demonstrated [10]. This indicates that the stimulatory effect of glucocorticoids on 11βHSD1 is likely to be mediated via the glucocorticoid receptor. Glucocorticoids appear to act in an autocrine manner, stimulating the expression of 11βHSD1, which in turn increases conversion of cortisone to active cortisol, a so-called positive feed-forward mechanism [11].

The human genome contains two annotated transcription start sites for the HSD11B1 gene (encoding 11βHSD1), which are potentially regulated in an independent manner in different tissues (Figure 1). However, mechanisms underlying regulation of this gene remain poorly understood. Several studies have demonstrated the importance of the CCAAT/enhancer-binding protein (C/EBP) transcription factors [11–16] in HSD11B1 expression from the proximal P2 promoter. In human A549 lung epithelial cells, C/EBPβ has been shown to mediate the induction of 11βHSD1 by glucocorticoids [13], whereas in human amnion fibroblasts C/EBPα rather than C/EBPβ was involved [11]. However, inhibition of C/EBP is insufficient to completely block glucocorticoid stimulation of 11βHSD1 activity, at least in some cells [11].

DNA methylation is an epigenetic process that plays a pivotal role in the regulation of gene expression in both a temporal and spatial manner throughout development [17]. Methylation of DNA in vertebrates occurs almost exclusively in CpG dinucleotides, often clustered in gene promoter regions. Promoter methylation is generally associated with reduced gene activity. Changes in promoter methylation may occur in a dynamic fashion with measured periodicity of as little as 100 minutes [18, 19]. The observed induced 11βHSD1 enzyme activity following glucocorticoid treatment may therefore potentially be associated with a reduction in HSD11B1 promoter methylation. Each of the two annotated HSD11B1 promoters contains CpG sites, one of which (within the distal P1 promoter) overlaps a conserved glucocorticoid response element (GRE; Figure 1).

In this study, we tested the hypothesis that the increased 11βHSD1 mRNA expression and enzyme activity seen in SkM post dexamethasone treatment is associated with a reduction in DNA methylation at the putative GRE of the HSD11B1 P1 gene promoter in human diabetic subjects.

We have previously demonstrated that oral dexamethasone administered at a dose of 4 mg/day for 4 days increases 11βHSD1 oxoreductase activity in SkM of subjects with type 2 diabetes but not nondiabetic subjects [5]. We have now extended these findings by examining the association of the DNA methylation level in two annotated promoter regions with gene expression levels and enzyme activity in the diabetic group, which is subject to upregulation by dexamethasone. Evidence, primarily from accumulated expressed sequence tag data, suggests that the HSD11B1 has two independent transcription start sites and associated promoters, designated P1 [14] and P2. In the mouse, the P1 promoter predominates in the lung and is independent of C/EBPα, while the P2 promoter predominates in the liver, adipose tissue and brain [14]. At present the predominant transcriptional start site in SkM remains unclear, although our data confirm that both transcripts are present. The P1 promoter region is CpG poor but contains a conserved putative GRE upstream of the transcriptional start site.

In this study we demonstrated that the dexamethasone-induced increase in overall 11βHSD1 mRNA, derived from both P1 and P2 promoters, and enzyme activity is associated with a significantly reduced level of DNA methylation at the putative GRE-associated CpG site in HSD11B1 P1. Further, the extent of methylation reduction correlates significantly with the subsequent increase in 11βHSD1 enzyme activity. While a statistical association does not prove causality, the findings suggest that HSD11B1 expression may be subject to dynamic epigenetic modulation in response to glucocorticoid status. This is further supported by the finding of significant upregulation of the P1 promoter-derived transcript following dexamethasone treatment. The absolute changes in methylation were small, but so were the changes in SkM 11βHSD1 activity in many of the subjects. Indeed in two subjects where a small decrease in 11βHSD1 activity was observed, P1 promoter methylation was increased or unchanged. Such a modest effect size is in accordance with other emerging studies in complex disease that routinely report small changes in DNA methylation in a case–control setting. As an example, Rakyan and colleagues identify small disease-associated methylation differences of 1.3 to 6.6%, which compare well with other multiple referenced studies in complex disease and our data [20]. They point out that this could be ‘the norm for complex disease associated epigenetic variation’ [20].

While methylation of a single CpG site was analyzed in the P1 promoter region, analysis of methylation of the CpG sites within the P2 region did not produce statistically significant changes following dexamethasone. The dose of dexamethasone utilized in this study is within the range used clinically, and completely suppresses the hypothalamic–pituitary adrenal axis, leading to the near absence of circulating cortisol. Dexamethasone induced insulin resistance in both diabetic and nondiabetic subjects and resulted in an elevation in fasting glucose, insulin and HOMA2-IR homeostasis model assessment in the current study.

P2-associated transcription of HSD11B1 via the C/EBP transcription factors has previously been described [11]. The 11βHSD1 P2 promoter contains 10 C/EBP binding sites. The precise role of the different C/EBPs in the regulation of 11βHSD1 appears to be tissue specific. In HepG2 (hepatoma) cells, C/EBPα is the predominant regulator of 11βHSD1 expression, while C/EBPβ is only a weak activator in the absence of C/EBPα [15]. Both C/EBPα and C/EBPβ are required for basal transcriptional activity of 11βHSD1 in the mouse pre-adipocyte cell line 3T3-L1 [12]. Induction of C/EBPα shows a greater increase in basal 11βHSD1 expression, while C/EBPβ is the key factor mediating the increase in 11βHSD1 in response to cyclic AMP stimulation in these cells [12]. In human A549 lung epithelial cells, the stimulatory effect of dexamethasone on 11βHSD1 activity appears to be mediated by two mechanisms. Firstly, 11βHSD1 activity is dependent on the glucocorticoid receptor, since the co-administration of the glucocorticoid receptor antagonist RU486 blocks it; and secondly, the stimulatory effect of dexamethasone is indirect requiring new protein synthesis, since the glucocorticoid upregulation is also blocked by the protein synthesis inhibitor cycloheximide [13]. While a GRE lies between two key C/EBP binding sites in the P2 promoter, mutation of this GRE region did not appear to affect dexamethasone responsiveness [13]. C/EBPβ, but not C/EBPα or C/EBPδ, is a crucial mediator of the increased 11βHSD1 expression and activity following dexamethasone in A549 cells [13]. In contrast, in the human adipocyte cell line PAZ6, C/EBPα but not C/EBPβ or C/EBPδ was induced by dexamethasone [21].

We have shown here that humans administered exogenous dexamethasone exhibit a significant rise in SkM C/EBPβ mRNA levels consistent with the findings of Yang and colleagues [16]. The extent of the increase in C/EBPβ and 11βHSD1 in SkM was considerably less than observed by Sai and colleagues, who showed a 12-fold increase in 11βHSD1 mRNA levels in response to a threefold to fourfold increase in C/EBPβ in A549 lung epithelial cells [13]. Since our data demonstrated an increase in expression of both P1 and P2 promoters, we suggest that there may be a dual mechanism of dexamethasone-induced activation of SkM 11βHSD1 activity; via demethylation of the P1 promoter and through C/EBPβ stimulation of the P2 promoter.

In SkM, the precise mechanism(s) involved in glucocorticoid-induced activation of 11βHSD remain to be characterized. Which promoter (P1 or P2) is used in muscle to transcribe the HSD11B1 gene in response to glucocorticoid was previously not known, although we have now shown that both transcripts are clearly detectable by specific RT-PCR. In vitro reporter experiments aimed at demonstrating functionality of the putative P1-associated GRE in appropriate cell models are required to confirm the functionality of this region in the observed glucocorticoid response. The P2-associated CpG sites are not associated with a putative GRE, suggesting that a reduction in methylation at the P2 promoter of HSD11B1 is not associated with the increased SkM 11βHSD1 activity following dexamethasone administration.

The enzyme 11βHSD type 2 (11βHSD2), which converts cortisol to cortisone, is highly expressed in mineralocorticoid target tissues such as the kidney and colon, but is expressed at low levels in SkM [5]. There is evidence that 11βHSD2 is subject to epigenetic regulation, both at the level of DNA methylation but also of the histone modification profile [22]. Human subjects who developed hypertension when treated with prednisone had increased 11βHSD2 promoter methylation in peripheral blood mononuclear cells [23]. Our previous work demonstrated that SkM 11βHSD2 expression and activity was downregulated in the type 2 diabetic subjects [5]. This downregulation may thus possibly be associated with increased methylation of the 11βHSD2 promoter, but this was not the focus of the present study.

Methylation of DNA is a well-described mechanism of epigenetic modulation of gene expression. DNA methylation had been assumed not to be as dynamic as other epigenetic processes. However, recent studies have demonstrated that highly dynamic DNA methylation changes can occur even during the relatively short period of a single cell cycle in a number of human genes [13, 19]. Other studies have examined epigenetic mechanisms in metabolic pathways in SkM of human subjects. Non-CpG methylation of the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene in myotubes has been associated with reductions in mitochondrial density in subjects with type 2 diabetes [24]. Furthermore, elevated DNA methylation of SkM PGC-1α is observed in normal birth weight nondiabetic subjects overfed a high-fat diet [25]. Our findings support the involvement of DNA methylation at the conserved putative GRE in the P1 11βHSD1 promoter in the regulation of glucocorticoid-induced HSD11B1 gene activation in human SkM. Specifically, we find that the extent of the reduction in methylation of this GRE following dexamethasone correlates with the subsequent increment in SkM 11βHSD1 oxoreductase activity. While the putative GRE in the P1 11βHSD1 promoter may have a lesser role compared with C/EBP-stimulated P2 promoter activation in many tissues, our results suggest that even relatively small changes in methylation may influence the extent of the increment in SkM 11βHSD1 activity. However, this does not preclude the existence of a parallel response involving C/EBPβ action at the P2 promoter. Further functional studies are required to determine the relative contribution of these promoter regions and regulatory mechanisms to the overall regulation of the HSD11B1 gene in SkM.

This study has found a reduction in methylation of the putative GRE within the P1 promoter of HSD11B1 that correlates with gene expression and the increase in 11βHSD1 oxoreductase activity in SkM following dexamethasone treatment. This observation implicates dynamic epigenetic remodeling in the regulation of glucocorticoid action via 11βHSD1.

The experimental protocol was approved by the institutional Human Research Ethics Committee. Fifteen volunteers with type 2 diabetes gave written informed consent for participation. The subjects were treated by diet alone (n = 4) or with oral hypoglycemic agents (metformin alone, n = 2; a sulphonylurea alone, n = 2; or both, n = 7). No patient was using insulin or thiazolidinediones. Exclusion criteria included poorly controlled hypertension (systolic blood pressure ≥160 mmHg and/or diastolic blood pressure ≥90 mmHg), a history of liver or renal disease, and evidence of unstable angina or peripheral vascular disease. Data from 12 of these subjects were included in our previous study examining the effect of oral dexamethasone on 11βHSD1 and 11βHSD2 in diabetic and nondiabetic subjects [5]. Nondiabetic subjects were not included in this study because we had previously shown that the induction of 11βHSD1 oxoreductase activity did not occur in this group.