Rheumatology
Rheumatol Int DOI 10.1007/s00296-015-3266-5
INTERNATIONAL
ORIGINAL ARTICLE - GENES AND DISEASE
Hypermethylation of glucocorticoid receptor gene promoter results in glucocorticoid receptor gene low expression in peripheral blood mononuclear cells of patients with systemic lupus erythematosus Hongbo Chen1 · Junfen Fan1 · Qiyang Shou2 · Lizong Zhang2 · Hongzhen Ma1 · Yongsheng Fan3
Received: 29 October 2014 / Accepted: 1 April 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Our aim was to investigate the relationship between the DNA methylation status of glucocorticoid receptor (GR) gene promoter and mRNA expression level of GRα gene of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). Fifteen newly emerging SLE patients and fifteen healthy controls were enrolled in this study. DNA and total RNA were extracted from the PBMCs of the SLE patients and healthy controls. The DNA methylation status of GR gene promoter 1 of PBMCs was detected through bisulfitesequencing PCR. The mRNA expression of GRα, DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and growth arrest, and DNA damage-induced 45α (GADD45α) of PBMCs was detected using the quantitative real-time polymerase chain reaction method. The mRNA expression of GRα was significantly declined in SLE patients, and the mRNA expression of DNMT1 and GADD45α was significantly elevated in SLE patients. The global methylation status of PBMCs in SLE patients was obviously lower than healthy controls. There were 38, 25, 30, and 49 CpG islands in amplified fragment of GR promoter 1D, 1E, 1F, and 1H, respectively. The overall mean methylation status of the 152 CpG islands of the four promoters was * Yongsheng Fan [email protected] 1
Department of Nephrology, First Affiliated Hospital, Zhejiang Chinese Medicine University, Hangzhou, People’s Republic of China
2
Division of Animal Laboratory Research Center, Zhejiang Chinese Medicine University, Hangzhou, People’s Republic of China
3
Division of Rheumatism Research Institution, Zhejiang Chinese Medicine University, 548#, Binwen RD, Hangzhou 310053, People’s Republic of China
significantly elevated in SLE patients. There was a negative correlation between hypermethylation of GR promoter and GRα mRNA expression in SLE patients. This study demonstrated that hypermethylation of GRα promoter may result in GRα gene low expression in PBMCs of patients with SLE. This study also found that the global methylation status of PBMCs in SLE patients was obviously lower than healthy controls, and it was related to the elevated GADD45α mRNA expression in SLE patients. These conclusions have to be certified by larger-scale clinical studies. Keywords Systemic lupus erythematosus · Glucocorticoid receptor · DNA methylation · Epigenetics
Introduction Systemic lupus erythematosus (SLE) is a systemic autoimmune disease involving multiple organs and systems. Although the detailed etiology is still obscure, both genetics and environmental factors are involved in the pathogenesis of this disease [1–3]. Recent reports have confirmed and extended the earlier work demonstrating the significance of epigenetic mechanisms in the pathogenesis of SLE [4]. Epigenetics is defined as heritable changes in gene expression not due to changes in the DNA sequence. DNA methylation is one important type of epigenetic modification [5]. It mainly occurs at cytosine–guanine dinucleotides. These dinucleotides are concentrated in a variety of repetitive sequences, as well as in regions known as CpG islands, many of which overlap with promoters [6]. The glucocorticoid receptor (GR), a known mediator of glucocorticoid signaling, has been shown to be regulated, in part, by epigenetic mechanism, specifically DNA methylation [7–9]. GR transcription is controlled by nine promoters, each
13
associated with an alternative transcription start site. Seven of these promoters are clustered together in a CpG island [10]. CpG island methylation at promoters is generally associated with transcriptional repression [6]. Some studies found that the expression of glucocorticoid receptor alpha (GRα) in SLE patients was lower than that of healthy controls, which may play an important role during the course of the pathogenesis and treatment for patients with SLE [11–14]. The enzymes that methylate DNA are known as DNA cytosine-5-methyltransferases (DNMTs), the most studied among them being DNMT1. DNMT1 is constitutively expressed and is required to maintain global methylation after DNA replication has taken place [15]. DNMT3a and DNMT3b were also found to be related to DNA methylation. They are able to carry out a maintenance methylation activity similar to DNMT1 [16]. Another DNA demethylation-related gene was the growth arrest and DNA damage-induced 45α (GADD45α) which can promote DNA repair and remove methylation marks, thereby reducing epigenetic gene silencing [17]. In this study, we prepared to examine the methylation status of CpG islands in GR promoter of peripheral blood mononuclear cells (PBMCs) of patients with SLE, and study the correlation between GR gene mRNA expression and methylation status of CpG islands in GR promoter.
Materials and methods Ethics statement This study was approved by the Ethics committee of First Affiliated Hospital of Zhejiang Chinese Medical University, and all the participants had signed the informed consent document. Subjects Fifteen newly emerging SLE patients (mean ± SD age, 28.2 ± 8 years) were recruited from the First Affiliated Hospital of Zhejiang Chinese Medical University. All of the patients fulfilled the 1997 American College of Rheumatology revised criteria for the classification of SLE. The base data of the SLE patients and healthy controls are shown in Table 1. All SLE patients were steroid-naive. Lupus disease activity was assessed using the SLE Disease Activity Index (SLEDAI). The gender, age, and SLEDAI score are shown in Table 2. Fifteen healthy controls (mean ± SD age, 27.9 ± 9 years) were recruited from healthy volunteers. Real‑time quantitative polymerase chain reaction PBMCs were separated by Ficoll-Paque from the fifteen patients with SLE and fifteen healthy controls. Real-time
13
Rheumatol Int Table 1 Base data of SLE patients and healthy controls SLE patients
Healthy controls
p value
BP (mmHg) WBC (109/L) Hemoglobin (g/dL) PLT (109/L) Scr (mmol/L) BUN (mmol/L) Alb (g/L)
153 ± 38/85 ± 26 3.2 ± 2.4 12.1 ± 2.7 206 ± 79 75.1 ± 31.5 7.6 ± 4.4 41 ± 8.1
131 ± 25/67 ± 19 4.7 ± 3.1 13.5 ± 1.5 235 ± 91 61.5 ± 19.8 6.2 ± 3.2 47 ± 9.5
0.05 >0.05 >0.05 >0.05 >0.05 >0.05
Urine protein positive
13/15
0/15
Rheumatol Int DOI 10.1007/s00296-015-3266-5
INTERNATIONAL
ORIGINAL ARTICLE - GENES AND DISEASE
Hypermethylation of glucocorticoid receptor gene promoter results in glucocorticoid receptor gene low expression in peripheral blood mononuclear cells of patients with systemic lupus erythematosus Hongbo Chen1 · Junfen Fan1 · Qiyang Shou2 · Lizong Zhang2 · Hongzhen Ma1 · Yongsheng Fan3
Received: 29 October 2014 / Accepted: 1 April 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Our aim was to investigate the relationship between the DNA methylation status of glucocorticoid receptor (GR) gene promoter and mRNA expression level of GRα gene of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). Fifteen newly emerging SLE patients and fifteen healthy controls were enrolled in this study. DNA and total RNA were extracted from the PBMCs of the SLE patients and healthy controls. The DNA methylation status of GR gene promoter 1 of PBMCs was detected through bisulfitesequencing PCR. The mRNA expression of GRα, DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and growth arrest, and DNA damage-induced 45α (GADD45α) of PBMCs was detected using the quantitative real-time polymerase chain reaction method. The mRNA expression of GRα was significantly declined in SLE patients, and the mRNA expression of DNMT1 and GADD45α was significantly elevated in SLE patients. The global methylation status of PBMCs in SLE patients was obviously lower than healthy controls. There were 38, 25, 30, and 49 CpG islands in amplified fragment of GR promoter 1D, 1E, 1F, and 1H, respectively. The overall mean methylation status of the 152 CpG islands of the four promoters was * Yongsheng Fan [email protected] 1
Department of Nephrology, First Affiliated Hospital, Zhejiang Chinese Medicine University, Hangzhou, People’s Republic of China
2
Division of Animal Laboratory Research Center, Zhejiang Chinese Medicine University, Hangzhou, People’s Republic of China
3
Division of Rheumatism Research Institution, Zhejiang Chinese Medicine University, 548#, Binwen RD, Hangzhou 310053, People’s Republic of China
significantly elevated in SLE patients. There was a negative correlation between hypermethylation of GR promoter and GRα mRNA expression in SLE patients. This study demonstrated that hypermethylation of GRα promoter may result in GRα gene low expression in PBMCs of patients with SLE. This study also found that the global methylation status of PBMCs in SLE patients was obviously lower than healthy controls, and it was related to the elevated GADD45α mRNA expression in SLE patients. These conclusions have to be certified by larger-scale clinical studies. Keywords Systemic lupus erythematosus · Glucocorticoid receptor · DNA methylation · Epigenetics
Introduction Systemic lupus erythematosus (SLE) is a systemic autoimmune disease involving multiple organs and systems. Although the detailed etiology is still obscure, both genetics and environmental factors are involved in the pathogenesis of this disease [1–3]. Recent reports have confirmed and extended the earlier work demonstrating the significance of epigenetic mechanisms in the pathogenesis of SLE [4]. Epigenetics is defined as heritable changes in gene expression not due to changes in the DNA sequence. DNA methylation is one important type of epigenetic modification [5]. It mainly occurs at cytosine–guanine dinucleotides. These dinucleotides are concentrated in a variety of repetitive sequences, as well as in regions known as CpG islands, many of which overlap with promoters [6]. The glucocorticoid receptor (GR), a known mediator of glucocorticoid signaling, has been shown to be regulated, in part, by epigenetic mechanism, specifically DNA methylation [7–9]. GR transcription is controlled by nine promoters, each
13
associated with an alternative transcription start site. Seven of these promoters are clustered together in a CpG island [10]. CpG island methylation at promoters is generally associated with transcriptional repression [6]. Some studies found that the expression of glucocorticoid receptor alpha (GRα) in SLE patients was lower than that of healthy controls, which may play an important role during the course of the pathogenesis and treatment for patients with SLE [11–14]. The enzymes that methylate DNA are known as DNA cytosine-5-methyltransferases (DNMTs), the most studied among them being DNMT1. DNMT1 is constitutively expressed and is required to maintain global methylation after DNA replication has taken place [15]. DNMT3a and DNMT3b were also found to be related to DNA methylation. They are able to carry out a maintenance methylation activity similar to DNMT1 [16]. Another DNA demethylation-related gene was the growth arrest and DNA damage-induced 45α (GADD45α) which can promote DNA repair and remove methylation marks, thereby reducing epigenetic gene silencing [17]. In this study, we prepared to examine the methylation status of CpG islands in GR promoter of peripheral blood mononuclear cells (PBMCs) of patients with SLE, and study the correlation between GR gene mRNA expression and methylation status of CpG islands in GR promoter.
Materials and methods Ethics statement This study was approved by the Ethics committee of First Affiliated Hospital of Zhejiang Chinese Medical University, and all the participants had signed the informed consent document. Subjects Fifteen newly emerging SLE patients (mean ± SD age, 28.2 ± 8 years) were recruited from the First Affiliated Hospital of Zhejiang Chinese Medical University. All of the patients fulfilled the 1997 American College of Rheumatology revised criteria for the classification of SLE. The base data of the SLE patients and healthy controls are shown in Table 1. All SLE patients were steroid-naive. Lupus disease activity was assessed using the SLE Disease Activity Index (SLEDAI). The gender, age, and SLEDAI score are shown in Table 2. Fifteen healthy controls (mean ± SD age, 27.9 ± 9 years) were recruited from healthy volunteers. Real‑time quantitative polymerase chain reaction PBMCs were separated by Ficoll-Paque from the fifteen patients with SLE and fifteen healthy controls. Real-time
13
Rheumatol Int Table 1 Base data of SLE patients and healthy controls SLE patients
Healthy controls
p value
BP (mmHg) WBC (109/L) Hemoglobin (g/dL) PLT (109/L) Scr (mmol/L) BUN (mmol/L) Alb (g/L)
153 ± 38/85 ± 26 3.2 ± 2.4 12.1 ± 2.7 206 ± 79 75.1 ± 31.5 7.6 ± 4.4 41 ± 8.1
131 ± 25/67 ± 19 4.7 ± 3.1 13.5 ± 1.5 235 ± 91 61.5 ± 19.8 6.2 ± 3.2 47 ± 9.5
0.05 >0.05 >0.05 >0.05 >0.05 >0.05
Urine protein positive
13/15
0/15
