陆前进
表观遗传学机制与T细胞基因调控及其在系统性红斑狼疮(SLE)发病机理中的作用
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- 姓名:陆前进
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学术头衔:
博士生导师
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学科领域:
皮肤病学
- 研究兴趣:表观遗传学机制与T细胞基因调控及其在系统性红斑狼疮(SLE)发病机理中的作用
陆前进,男,1962年出生,1987年至1995年:湖南医科大学附二医院主治医师,主要从事SLE患者前列腺素代谢与骨代谢的研究。1997年至1999年:湖南医科大学附二医院副教授,医学实验中心主任。主要从事反基因定向阻断自身抗体IgG2q抗原决定簇治疗SLE的研究。1999年至2002年:美国密执安大学医学中心(university of michigan medical center)博士后主要从事DNA甲基化与自身免疫病的研究。2002年至2005年:美国密执安大学医学中心研究员。主要从事DNA甲基化与T细胞基因调控及其在SLE发病机理中的作用研究。2005年至今:中南大学湘雅二医院表观遗传学研究中心主任,中南大学湘雅二医院皮肤科教授、博士生导师,皮肤性病学教研室副主任。主要从事DNA甲基化、组蛋白修饰与T细胞基因调控及其在SLE发病作用的研究。
多年来一直从事皮肤病与性病的临床与科研工作,在自身免疫病的发病机理研究方面有较深的学术造诣。先后负责承担国家自然科学基金项目和美国NIH课题。主要从事表观遗传学机制与T细胞基因调控及其在系统性红斑狼疮(SLE)发病机理中的作用,取得了原创性的研究成果。首次提出了DNA低甲基化市导致SLE T细胞具有自身反应性的关键所在。T细胞甲基化敏感性基因调控序列低甲基化可作为SLE早期生物学标记和治疗目标。先后在Blood等国际高水平杂志发表论文12篇,被SCI收录期刊引用70多次。本人在表观遗传学机制与自身免疫病这一研究领域已成为主要学术带头人之一。
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陆前进, Qianjin Lu, *† Ailing Wu, * and Bruce C. Richardson*‡
The Journal of Immunology, 2005, 174: 6212-6219,-0001,():
-1年11月30日
Exposing genetically predisposed individuals to certain environmental agents is believed to cause human lupus. How environmental agents interact with the host to cause lupus is poorly understood. Procainamide and hydralazine are drugs that cause lupus in genetically predisposed individuals. Understanding how these environmental agents cause lupus may indicate mechanisms relevant to the idiopathic disease. Abnormal T cell DNA methylation, a repressive epigenetic DNA modification, is implicated in procainamide and hydralazine induced lupus, as well as idiopathic lupus. Procainamide is a competitive DNA methyltransferase (Dnmt) inhibitor, hydralazine inhibits ERK pathway signaling thereby decreasing Dnmt expression, and in lupus T cells decreased ERK pathway signaling causing a similar Dnmt decrease. T cells treated with procainamide, hydralazine, and other Dnmt and ERK pathway inhibitors cause lupus in mice. Whether the same genetic regulatory elements demethylate in T cells treated with Dnmt inhibitors, ERK pathway inhibitors, and in human lupus is unknown. CD70 (TNFSF7) is a B cell costimulatory molecule overexpressed on CD4 lupus T cells as well as procainamide and hydralazine treated T cells, and contributes to excessive B cell stimulation in vitro and in lupus. In this report we identify a genetic element that suppresses CD70 expression when methylated, and which demethylates in lupus and in T cells treated with Dnmt and ERK pathway inhibitors including procainamide and hydralazine. The results support a model in which demethylation of specific genetic elements in T cells, caused by decreasing Dnmt expression or inhibiting its function, contributes to drug-induced and idiopathic lupus through altered gene expression.
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陆前进, Qianjin Lu and Bruce Richardson
Biol. Proced. Online 2004; 6(1): 189-203,-0001,():
-1年11月30日
Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoidspecific ITGAL (CD11a) and PRF1 (perforin) expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5' flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail.
PHA,, phytohemagglutinin, 5-azaC,, 5-azacytidine, dC,, deoxycytosine, SAM,, Sadenosylmethionine, MeCP2,, methyl-CpG-binding protein 2
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陆前进, Mariana J. Kaplan, * Qianjin Lu, * Ailing Wu, * John Attwood, * and Bruce Richardson*†
The Journal of Immunology, 2004, 172: 3652-3661,-0001,():
-1年11月30日
Inhibiting DNA methylation in CD4+ T cells causes aberrant gene expression and autoreactive monocyte/macrophage killing in vitro, and the hypomethylated cells cause a lupus-like disease in animal models. Similar decreases in T cell DNA methylation occur in idiopathic lupus, potentially contributing to disease pathogenesis. The genes affected by DNA hypomethylation are largely unknown. Using DNA methylation inhibitors and oligonucleotide arrays we have identified perforin as a methylation-sensitive gene. Our group has also reported that DNA methylation inhibitors increase CD4+ T cell perforin by demethylating a conserved methylation-sensitive region that is hypomethylated in primary CD8+ cells, which express perforin, but is largely methylated in primary CD4+ cells, which do not. As lupus T cells also have hypomethylated DNA and promiscuously kill autologous monocytes/macrophages, we hypothesized that perforin may be similarly overexpressed in lupus T cells and contribute to the monocyte killing. We report that CD4+ T cells from patients with active, but not inactive, lupus overexpress perforin, and that overexpression is related to demethylation of the same sequences suppressing perforin transcription in primary CD4+ T cells and demethylated by DNA methylation inhibitors. Further, the perforin inhibitor concanamycin A blocks autologous monocyte killing by CD4 lupus T cells, suggesting that the perforin is functional. We conclude that demethylation of specific regulatory elements contributes to perforin overexpression in CD4+ lupus T cells. Our results also suggest that aberrant perforin expression in CD4+lupus T cells may contribute to monocyte killing.
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陆前进, Kurt Oelke, Qianjin Lu, Derek Richardson, Ailing Wu, Chun Deng, Samir Hanash, and Bruce Richardson
ARTHRITIS & RHEUMATISM Vol.50, No.6, June 2004, pp 1850-1860,-0001,():
-1年11月30日
Objective. Generalized DNA hypomethylation contributes to altered T cell function and gene expression in systemic lupus erythematosus (SLE). Some of the overexpressed genes participate in the disease process, but the full repertoire of genes affected is unknown. Methylation-sensitive T cell genes were identified by treating T cells with the DNA methyltransferase inhibitor 5-azacytidine and comparing gene expression with oligonucleotide arrays. CD70, a costimulatory ligand for B cell CD27, was one gene that reproducibly increased. We then determined whether CD70 is overexpressed on T cells treated with other DNA methylation inhibitors and on SLE T cells, and determined its functional significance. Methods. Oligonucleotide arrays, real-time reverse transcription–polymerase chain reaction, and flow cytometry were used to compare CD70 expression in T cells treated with 2 DNA methyltransferase inhibitors (5-azacytidine and procainamide) and 3 ERK pathway inhibitors known to decrease DNA methyltransferase expression (U0126, PD98059, and hydralazine). The consequences of CD70 overexpression were tested by coculture of autologous T and B cells with and without anti-CD70 and measuring IgG production by enzymelinked immunosorbent assay. The results were compared with those of T cells from lupus patients. Results. SLE T cells and T cells treated with DNA methylation inhibitors overexpressed CD70 and overstimulated B cell IgG production. The increase in IgG synthesis was abrogated by anti-CD70. Conclusion. SLE T cells and T cells treated with DNA methyltransferase inhibitors and ERK pathway inhibitors overexpress CD70. This increased B cell costimulation and subsequent immunoglobulin overproduction may contribute to drug-induced and idiopathic lupus.
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【期刊论文】DNA Methylation and Chromatin Structure Regulate T Cell Perforin Gene Expression 1
陆前进, Qianjin Lu, * Ailing Wu, * Donna Ray, * Chun Deng, † John Attwood, * Samir Hanash, * Matthew Pipkin, ‡ Mathias Lichtenheld, ‡ and Bruce Richardson*§
The Journal of Immunology, 2003, 170: 5124-5132.,-0001,():
-1年11月30日
Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4+ and CD8+ T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4 and CD8 cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at-1 kb and the distal promoter at-0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5- flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.
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陆前进, Chun Deng, Qianjin Lu, Zhiyong Zhang, Tharaknath Rao, John Attwood, Raymond Yung and Bruce Richardson
ARTHRITIS & RHEUMATISM Vol.48, No.3, March 2003, pp 746-756,-0001,():
-1年11月30日
Objective. To determine whether hydralazine might decrease DNA methyltransferase (DNMT) expression and induce autoimmunity by inhibiting extracellular signal-regulated kinase (ERK) pathway signaling. Methods. The effect of hydralazine on DNMT was tested in vitro using enzyme inhibition studies, and in vivo by measuring messenger RNA (mRNA) levels and enzyme activity. Effects on ERK, c-Jun N-terminal kinase, and p38 pathway signaling were tested using immunoblotting. Murine T cells treated with hydralazine or an ERK pathway inhibitor were injected into mice and anti-DNA antibodies were measured by enzyme-linked immunosorbent assay. Results. In vitro, hydralazine did not inhibit DNMT activity. Instead, hydralazine inhibited ERK pathway signaling, thereby decreasing DNMT1 and DNMT3a mRNA expression and DNMT enzyme activity similar to mitogen-activated protein kinase kinase (MEK) inhibitors. Inhibiting T cell ERK pathway signaling with an MEK inhibitor was sufficient to induce anti–double-stranded DNA antibodies in a murine model of drug-induced lupus, similar to the effect of hydralazine. Conclusion. Hydralazine reproduces the lupus ERK pathway signaling abnormality and its effects on DNMT expression, and inhibiting this pathway induces autoimmunity. Hydralazine-induced lupus could be caused in part by inducing the same ERK pathway signaling defect that occurs in idiopathic lupus.
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陆前进, John Tra a, Tadashi Kondo a, Qianjin Lu b, Rork Kuick a, Samir Hanash a, Bruce Richardson b, c, *
Mechanisms of Ageing and Development 123(2002)1487-1503,-0001,():
-1年11月30日
Hypermethylation of CpG islands, resulting in the inactivation of tumor suppressor genes, is an early event in the development of some malignancies. Recent studies suggest that this abnormal methylation may be a function of aging. The number of CpG islands that methylate with age is unknown. We used restriction landmark genome scanning (RLGS) to approximate the extent to which CpG islands change methylation status during aging. Comparison of more than 2000 loci in T lymphocytes isolated from newborn, middle age, and elderly people revealed that 29 loci (~1%)changed methylation status during aging, with 23 increasing methylation, and six decreasing. The same subset also changed methylation status with age in the esophagus, lung, and pancreas, but in variable directions. Virtual genome scanning identified one of these loci as a member of the forkhead family, recently implicated in aging, and another as an EST fragment. The methylation status of both correlated with level of expression. Confirming studies in multiple tissues from normal and DNMT1+/- mice demonstrated only one age dependent change in the methylation of more than 2000 loci, occurring in liver and kidney. These results indicate that the methylation status of the majority of CpG islands in both mice and humans is tightly controlled during aging, and that changes are infrequent and in humans confined to a specific subset of genes.
DNA methylation, Aging, T cells
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【期刊论文】Age-dependent DNA methylation changes in the ITGAL (CD11a) promoter
陆前进, Zhiyong Zhang, Chun Deng, Qianjin Lu, Bruce Richardson
Mechanisms of Ageing and Development 123(2002)1257-1268,-0001,():
-1年11月30日
DNA methylation patterns change with age in a complex fashion, typically with an overall decrease in genomic deoxymethylcytosine (dmC) content, but with local increases in some promoters that contain GC-rich sequences known as CpG islands. While the consequences of age-dependent CpG island methylation have recently been studied in organs such as the colon, less is known about the functional significance of the progressive hypomethylation of promoters lacking CpG islands, and the significance of age-dependent changes in T cell DNA methylation is completely unexplored. We asked if age-dependent DNA hypomethylation might contribute to overexpression of the T cell ITGAL gene, which encodes CD11a, a subunit of LFA-1. CD11a mRNA increased with age as well as with experimentally induced DNA hypomethylation. This increase correlated with hypomethylation of sequences flanking the ITGAL promoter in vitro and in aging. 'Patch' methylation of the region suppressed promoter function. DNA methyltransferases 1 and 3a also decreased with aging. These results indicate that hypomethylation of regions flanking the ITGAL promoter may increase CD11a expression, and suggest that age-dependent hypomethylation of promoters lacking CpG islands, perhaps due to decreased DNA methyltransferase expression, may be one mechanism contributing to increased T cell gene expression with aging.
DNA methylation, ITGAL, LFA-1, T lymphocytes, Aging
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【期刊论文】Effect of DNAmethylation and chromatin structure on ITGAL expression
陆前进, Qianjin Lu, Donna Ray, David Gutsch, and Bruce Richardson
DNAMETHYLATIONAND ITGAL EXPRESSION BLOOD, 15 JUNE 2002 VOLUME 99, NUMBER 12,-0001,():
-1年11月30日
LFA-1 (CD11a/CD18, αLβ2) is an integrinexpressed in a tissue-specific fashionand is important in inflammatory and immune responses. Promoter analysis has identified transcription factors that may be involved in CD11a expression, but the mechanisms contributing to its tissuespecific expression are incompletely characterized. In this report we have asked if DNA methylation and/or chromatin structure could contribute to tissue-specific CD11a expression. Bisulfite sequencing was used to compare methylation patterns in the promoter and 5; flanking regions of the ITGAL gene, encoding CD11a, in normal human T cells, which express LFA-1, and fibroblasts, which do not. The region was found to be heavily methylated in fibroblasts but not T cells, and methylation correlated with an inactive chromatin configuration as analyzed by deoxyribonuclease 1 sensitivity. Patch methylation of the promoter region revealed that promoter activity was methylation-sensitive but that methylation of the 5' flanking regions more than 500 base pairs 5' to the transcription start site could also suppress promoter function. Treating fibroblasts with a DNA methylation inhibitor decreased ITGAL promoter methylation and increased CD11a messenger RNA. The results thus indicate that methylation and chromatin structure may contribute to the tissue-specific expression of CD11a.
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【期刊论文】Demethylation of ITGAL (CD11a) Regulatory Sequences in Systemic Lupus Erythematosus
陆前进, Qianjin Lu, Mariana Kaplan, Donna Ray, Doreen Ray, Sima Zacharek, David Gutsch, and Bruce Richardson
ARTHRITIS & RHEUMATISM Vol.46, No.5, May 2002, pp 1282-1291,-0001,():
-1年11月30日
Objective. Inhibition of T cell DNA methylation causes autoreactivity in vitro and a lupus-like disease in vivo, suggesting that T cell DNA hypomethylation may contribute to autoimmunity. The hypomethylation effects are due, in part, to overexpression of lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18). Importantly, T cells from patients with active lupus have hypomethylated DNA and overexpress LFA-1 on an autoreactive subset, suggesting that the same mechanism could contribute to human lupus. The present study investigated the nature of the methylation change that affects LFA-1 expression in vitro and in human lupus. Methods. Bisulfite sequencing was used to determine the methylation status of the ITGAL promoter and flanking regions in T cells from lupus patients and healthy subjects, and in T cells treated with DNA methylation inhibitors. "Patch" methylation of promoter sequences in reporter constructs was used to determine the functional significance of the methylation changes. Results. Hypomethylation of specific sequences flanking the ITGAL promoter was seen in T cells from patients with active lupus and in T cells treated with 5-azacytidine and procainamide. Patch methylation of this region suppressed ITGAL promoter function.
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