Inhibited expression of hematopoietic progenitor kinase 1 associated with loss of jumonji domain containing 3 promoter binding contributes to autoimmunity in systemic lupus erythematosus
Highlights
► HPK1 expression is significantly decreased in CD4+ T cells from SLE patients. ► Reduced JMJD3 leads to increased H3K27me3 at the HPK1 promoter in SLE CD4+ T cells. ► Increased H3K27me3 enrichment results in HPK1 down-regulation in SLE CD4+ T cells. ► Decreased HPK1 contributes to abnormal T cell reactivity in SLE.
Introduction
Autoimmune diseases are multifactorial in their pathogenesis. Contributory factors include: genetic predisposition and environmental factors [1], [2]. Autoimmune diseases are always female-predominant [3], [4], [5]. As a typical chronic autoimmune disease, systemic lupus erythematosus (SLE) is characterized by T cell overactivation and the overproduction of autoantibodies against multiple self antigens [6]. Although the molecular mechanisms that initiate these autoimmune responses in SLE remain unclear, it is widely accepted that epigenetic alterations in the promoters of certain immune-related genes play critical roles in the onset and progression of SLE [7], [8], [9], [10]. The term “epigenetics” refers to stable and heritable changes in gene expression that are not related to changes in the DNA sequence [11], [12]. The major mechanisms of epigenetic regulation include DNA methylation, histone modifications, chromatin modifications, noncoding RNA regulation, and so forth [11], [12], [13], [14]. Among histone modifications, histone H3 lysine 27 trimethylation (H3K27me3) is a hallmark of gene silencing [15], [16], [17], [18], via its binding to Pc protein, which forms part of the polycomb repressive complex 1 (PRC1). PRC1 then blocks the access of transcriptional activation factors and chromatin remodeling factors to DNA, and prevents initiation of transcription by RNA polymerase II. Furthermore, PRC1 is known to associate with histone deacetylases (HDACs, which can inhibit transcription), and PRC1 and H3K27me3 can block positively-acting imprints such as H3K4 methylation [15], [17]. It is well-known that the histone demethylase jumonji domain containing 3 (JMJD3) [19], [20], [21], [22] and the histone methyltransferase enhancer of zeste homolog 2 (EZH2) [23], [24] can both regulate H3K27me3 levels.
Hematopoietic progenitor kinase 1 (HPK1, also known as MAP4K1) is a mammalian Ste20-related serine/threonine protein kinase. It belongs to the germinal center kinase (GCK) family and can be activated by a variety of signal stimuli, such as epidermal growth factor (EGF) [25], prostaglandin E2 (PGE2) [26], transforming growth factor-β (TGF-β) [27], erythropoietin (EPO) [28], and TCR and BCR stimulation [29], [30], [31], [32]. It is also involved in various cellular events, such as MAPK [33], [34], [35], [36], NF-κB [29], [37], [38], [39] and cytokine signalings [28], [40], [41], as well as cellular proliferation and apoptosis [31], [42], [43], [44]. In addition, HPK1 negatively regulates TCR signaling and T cell-mediated immune responses [40], [43]. Shui et al. found that HPK1−/− mice T cells become hyperproliferative in response to stimulation with anti-CD3 and anti-CD28 antibodies, and that these cells can produce more proinflammatory cytokines when immunized with T cell-dependent antigens. Furthermore, T cell-dependent humoral responses in HPK1−/− immunized mice are more vigorous than in controls, and HPK1−/− mice demonstrate even more severe autoimmune phenotypes in an experimental autoimmune encephalomyelitis (EAE) model [43]. The T cell phenotypes observed in these HPK1-deficient mice are reminiscent of those observed in the T cells of patients with SLE, suggesting that HPK1 may play a causative role in SLE pathogenesis.
In then present study, chromatin immunoprecipitation (ChIP) microarray analysis revealed that H3K27me3 enrichment at the HPK1 promoter was significantly higher in CD4+ T cells of patients with SLE than in healthy controls. According to this clue, we set out to further investigate the putative roles of HPK1 in the development of autoimmunity in SLE and epigenetic regulation of its expression. To achieve this, we first confirmed that HPK1 mRNA and protein expressions were significantly decreased in SLE CD4+ T cells. Secondly, we showed that HPK1 negatively regulated CD4+ T cell activation and production of IFNγ and IgG in both healthy and SLE CD4+ T cells. We also observed elevated H3K27me3 enrichment and decreased JMJD3 binding at the HPK1 promoter in SLE CD4+ T cells, with no change in EZH2 binding. By down- and up-regulating JMJD3 expression using JMJD3-siRNA and JMJD3-plasmid, respectively, we demonstrated that changes in HPK1 expression were associated with changes in JMJD3 binding and H3K27me3 enrichment at its promoter. Together, these results provide novel insights into the mechanisms that cause SLE, and suggest a new approach for the treatment of SLE.
Section snippets
Subjects
15 patients with SLE and 15 age- and sex-matched healthy controls were enrolled in this study. Relevant patient information is listed in Table 1. All patients (n = 15, age: 28.60 ± 6.25 years) were recruited from the out-patient clinic and in-patient ward of the Department of Dermatology of the Second Xiangya Hospital, Central South University, China. All patients fulfilled at least 4 of the American College of Rheumatology (ACR) Classification Criteria for SLE [45]. Disease activity was
Increased H3K27me3 enrichment at the HPK1 promoter in SLE CD4+ T cells in the result of ChIP microarrays
We first used ChIP microarray analysis to examine H3K27me3 enrichment at various gene promoters in pooled CD4+ T cell lysates from 5 SLE patients and 5 age- and sex-matched healthy controls. Based on the microarray results, out of 20,832 distinct gene promoters screened, 552 showed a greater than two-fold difference in H3K27me3 enrichment between SLE and control CD4+ T cells. Among these, H3K27me3 enrichment at the HPK1 promoter in SLE CD4+ T cells was approximately five times higher than in
Discussion
In recent years, accumulating evidence has demonstrated that epigenetic alterations play essential roles in the pathogenesis of SLE [7], [8], [9], [10]. In particular, considerable interest has been focused on DNA demethylation, and a growing number of studies indicate that demethylation of regulatory sequences can result in the overexpression of certain immunity-related genes, including perforin (PRF1) [47], CD11a (ITGAL) [48], [49], CD70 (TNFSF7) [48], [50], and CD40 ligand (CD40L) [51], [52]
Acknowledgments
The authors gratefully acknowledge Dr. Charlie Degui Chen and Dr. Rüdiger Arnold for providing plasmids. This work was supported by the National Natural Science Foundation of China (grant 30730083 and 81101194) and the National Basic Research Program of China (973 Plan, grant 2009CB825605)
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