Epigenetics, the holy grail in the pathogenesis of systemic sclerosis.

RHEUMATOLOGY

Rheumatology 2015;54:17591770 doi:10.1093/rheumatology/keu155 Advance Access publication 16 April 2014

New pathways in the pathogenesis of SSc

Epigenetics, the holy grail in the pathogenesis of systemic sclerosis Nezam Altorok1,2, Nawaf Almeshal2, Yongqing Wang1 and Bashar Kahaleh1,2 Abstract

Key words: epigenetic, scleroderma, DNA methylation, histone modifications, endothelial cells, fibroblasts, fibrosis, nitric oxide synthase, friend leukaemia integration 1 transcription factor, microRNA.

Introduction Scleroderma (systemic sclerosis, SSc) is a complex multisystem autoimmune disease characterized by three pathological hallmarks: vascular damage, activation of the immune system as demonstrated by the presence of disease-specific autoantibodies and excessive deposition of collagen in the skin and internal organs [1]. SSc is characterized by a striking female predominance, with females accounting for >80% of SSc cases [2], higher frequency among African Americans and Hispanics compared with Caucasians [3, 4] and geographic clustering [5, 6]. These prevalence characteristics suggest potential hormonal, genetic and environmental influences

1 Division of Rheumatology and Immunology and 2Department of Internal Medicine, University of Toledo Medical Center, Toledo, OH, USA

Submitted 31 December 2013; revised version accepted 25 February 2014 Correspondence to: Bashar Kahaleh, Division of Rheumatology and Immunology, Department of Internal Medicine, University of Toledo Medical Center, 3000 Arlington Avenue, Mailstop 1186, Toledo, OH 43614, USA. E-mail: [email protected]

on disease pathogenesis. Depending on the extent of skin involvement, SSc is categorized as lcSSc if skin thickening is confined to the face and the extremities distal to the elbows and knees, whereas SSc is categorized as dcSSc if skin thickening involves areas proximal to the elbows and knees, including the trunk [7]. There is overwhelming evidence supporting the concept that the epigenome serves to coordinate the unique gene expression cascade in each cell type through a highly developed regulatory system. Moreover, there is growing evidence to indicate that environmental factors participate in modulating the epigenome and in disease predisposition. Therefore epigenetics is considered to be the link between a range of environmental factors and disease predisposition and perpetuation [8]. However, until now the exact environmental epigenetic factors in this linkage have remained largely uncharacterized, with some exceptions (e.g. certain drugs, ultraviolet light, exposure to silica, toxic oil, infection, smoking and diet) [9]. The aetiology of SSc remains unclear despite vigorous research efforts in the field. However, there is considerable evidence that epigenetic alterations may contribute to SSc pathogenesis [10]. In this review we explore the evidence that supports the role of epigenetic alterations in the development and pathogenesis of SSc.

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R EV I E W

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The objective of this review is to present evidence that supports the central role of epigenetic regulation in the pathogenesis of SSc. SSc is a complex autoimmune disease characterized by immune activation, fibrosis of the skin and internal organs and obliterative vasculopathy affecting predominantly the microvessels. Remarkable progress has been made in the past few years emphasizing the importance of epigenetic modifications in the pathogenesis of many disorders, including SSc. Current evidence demonstrates alterations in DNA methylation, histone code modifications and changes in microRNA (miRNA) expression levels in SSc cells. Recent reports have described the differential expression of numerous regulatory miRNAs in SSc, mainly in SSc fibroblasts, a number of which are important in TGF-b pathways and downstream signalling cascades. While studies to date have revealed the significant role of epigenetic modifications in the pathogenesis of SSc, the causal nature of epigenetic alterations in SSc pathogenesis remains elusive. Additional longitudinal and comprehensive epigenetic studies designed to evaluate the effect of environmental epigenetic factors on disease pathogenesis are needed.

Nezam Altorok et al.

Genetics of SSc

The pathogenesis of SSc involves a complex interplay between vascular damage, inflammation and autoimmunity, and progressive tissue fibrosis [11].

Genetic factors clearly contribute to the pathogenesis of SSc, as demonstrated by the identification of multiple genetic susceptibility loci in SSc patients. However, the cumulative effect size of these loci accounts for only a small fraction of disease heritability (estimated as only 0.008%) [25]. Moreover, the low concordance rates in SSc monozygotic twins (4.2%), which are not different from the rates seen in dizygotic twins (5.6%) [25], do not support a prominent role for genetic predisposition among patients with SSc. The majority of susceptibility loci in SSc are located in the major histocompatibility system (or the associated HLA gene family). For instance, variants in HLA antigens (A23, B18 and DR11) have been reported in SSc patients and appear to be associated with more severe clinical features [26]. Several other HLA-related polymorphisms also have been linked to SSc [27]. Associated singlenucleotide polymorphisms outside the HLA region also have been observed among SSc patients in several linkage and association studies (including CTGF, STAT4, IRF5, BANK1, FAM167A, TBX21, TNFSF4, HGF, C8orf13-BLK, KCNA5, PTPN22, NLRP1, CD226, IL2RA, IL12RB2, TLR2, HIF1A, SOX5, and CD247) (reviewed in [27] and [28]). Of particular interest, SLE, RA and SSc share several susceptibility genes, such as IRF5, STAT4 and CD247 [2932]. These observations underscore the common origin of these autoimmune diseases.

Vascular damage It is generally accepted that the most fundamental and earliest pathological lesion in SSc is related to abnormalities in microvascular endothelial cell (MVEC) function and structure [1214]. SSc vasculopathy is associated with progressive endothelial damage, reduction in the number of capillaries, thickening of arteriolar walls and eventually obliterative vasculopathy, leading ultimately to organ failure.

Autoimmunity Histopathological skin sections, especially in the early stages of SSc, are characterized by prominent cellular infiltration—primarily of CD4+ T cells [15]. Overexpression of cellular adhesion molecules in the vessels and interstitium facilitates the accumulation of inflammatory cells and provides the means for direct cellular contact of lymphocytes with MVECs and fibroblasts (FBs). This interaction is believed to result in MVEC and FB activation, which manifests as vascular dysfunction and tissue fibrosis, respectively.

Definition of epigenetics Tissue fibrosis It is well established now that dermal SSc FBs differ from normal FBs in several ways: (i) SSc FBs produce more collagen than dermal FBs isolated from healthy subjects [16]; (ii) SSc FBs display characteristics of myofibroblasts, including the expression of alpha smooth muscle actin (a-SMA), a marker of myofibroblasts; and (iii) SSc FBs display a persistently activated phenotype characterized by excessive production of collagen, increased proliferation and decreased apoptosis in vitro [10]. Moreover, SSc FBs exhibit increased responsiveness to and production of cytokines and chemokines—in particular, TGF-b [17, 18] and cytokines in the TGF-b downstream signalling cascades [19], platelet-derived growth factor (PDGF) and IL-1 [10]. Of particular interest, FBs are frequently detected near small blood vessels surrounded by inflammatory cellular infiltrate in the early stages of SSc [20]. This observation underscores the important interplay between vascular injury, inflammation and FB activation. Collagen gene expression and transcription in FBs are modulated by several profibrotic cytokines and transcription factors, including transcription factors Sp-1 [21] and Smad3 [19]; p300/CREB binding protein [21]; inhibitory factors, including Smad7 [22], friend leukaemia integration 1 (Fli-1) [23], peroxisome proliferatoractivated receptor [24], and p53; and other factors [11]. There is convincing evidence suggesting that dysregulation of these factors in SSc FBs results in enhanced collagen expression.

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The field of epigenetics has evolved during the last decade to become an indispensable area of research in mechanisms involved in human disease pathogenesis. Epigenetics is defined as heritable changes in gene expression that do not involve alterations in DNA sequence. Epigenetic mechanisms are important in controlling the patterns of gene expression and are essential for cell differentiation. The main mechanisms of epigenetic gene regulation are DNA methylation and histone modification. These mechanisms interact with each other in modulating chromatin architecture and in turn permit gene transcription or repression. More recently, microRNAs (miRNAs) have been identified as a major contributor to gene expression through post-transcriptional mechanisms [33] and thus were added to the epigenetic machinery.

DNA methylation in SSc DNA methylation is the most widely studied epigenetic mechanism in autoimmune diseases and is considered the core epigenetic control mechanism. DNA methylation involves the modification of the fifth carbon in cytosine residues of CpG dinucleotide by the addition of a methyl group. In general, CpG dinucleotide methylation near the gene regulatory regions is a repressive mark associated with transcriptional suppression. DNA methylation is established during development by the de novo DNA methyltransferases (DNMTs) DNMT3a and DNMT3b,

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Pathogenesis of SSc

Epigenetics of SSc

while an existing methylation pattern is maintained and thus inherited in proliferating cells by DNMT1 [34]. There is emerging evidence suggesting that alteration of DNA methylation profiles at global or gene-specific levels may contribute to SSc pathogenesis. We will explore first the evidence supporting DNA methylation dysregulation in SSc FBs, MVECs and lymphocytes.

Fibroblasts

Microvascular endothelial cells Endothelial nitric oxide (NO) synthase (eNOS)derived NO is a key factor in the regulation of microvascular functions. NO has important vasodilatory, antithrombotic, antiplatelet, and anti-oxidation properties [37]. Of note, NOS3 (the gene encoding eNOS) expression is reduced in SSc MVECs [38]. Moreover, endothelial NOS3-null mice are characterized by systemic and pulmonary hypertension, impaired wound healing and angiogenesis, and impaired mobilization of stem and endothelial progenitor cells, leading to failure of neovascularization [39, 40]. Data from our lab suggest that heavy methylation of the CpG sites in the promoter region of NOS3 leads to gene repression and that the addition of 5-azacytidine leads to normalization of NOS3 overexpression. Another endothelial gene that is regulated by epigenetic control is bone morphogenic protein receptor II (BMPRII). BMPs are members of the TGF-b superfamily of proteins that coordinate cell proliferation, differentiation and survival. The latter is particularly true for MVECs, where


Lymphocytes In contrast to MVECs and FBs, the data suggest that there is global hypomethylation of SSc CD4+ T cells, which is similar to the findings observed in SLE [42]. Therefore the expression of DNMT1 is significantly decreased in SSc CD4+ T cells, which correlates with global DNA hypomethylation in the cells [42]. Theoretically, global DNA hypomethylation of CD4+ T cells may alter gene-specific expression and reactivation of endoparasitic sequences, such as Line1 retrotransposable elements, which may contribute to autoimmunity [18]. The observation of divergence in global methylation between FBs and MVECs (increased methylation) and CD4+ T cells (reduced methylation) is intriguing and suggests that there is a distinct methylation profile in different cells involved in SSc pathogenesis. The regulatory molecular mechanism responsible for the difference in global methylation pattern in different cells is unclear, but defects in the extracellular signalregulated kinase (ERK) signalling pathway, which regulates DNA methylation in T cells, are documented in SLE and animal models of autoimmunity [43, 44]. Therefore it is plausible that the same defect in the ERK signalling pathway may explain global hypomethylation of CD4+ T cells in SSc. Overall, it is clear that epigenetic modifications should be investigated in a single cell type at a time rather than investigating heterogeneous cell populations (i.e. skin biopsies or whole tissue samples) in order to understand the unique epigenetic alteration in each specific cell type. Interestingly, there appears to be an association between SSc susceptibility, gender and epigenetics that may explain the female gender bias in the disease. DNA methylation has been implicated in X chromosome inactivation [4548] in order to maintain a balance among genes encoded on the X chromosome in males and females [49]. Female SSc patients exhibit demethylation of promoter sequences of CD40LG on the inactive X chromosome in CD4+ T cells, indicating impaired maintenance of DNA methylation, which naturally silences one X chromosome

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At the global level, our lab [35] has demonstrated increased expression levels of epigenetic maintenance mediators in SSc FBs. In addition, increased expression of DNAMT1 in cultured SSc FBs has also been reported and confirmed by others [36]. Moreover, levels of methylCpG DNA binding protein 1 (MBD-1), MBD-2, and methyl-CpG binding protein 2 (MeCP-2) were noted to be significantly elevated in SSc FBs compared with healthy FBs. These observations highlight the ability of cultured FBs to maintain SSc phenotype over multiple generations by cellular epigenetic inheritance. SSc is characterized by persistently activated FBs, leading to excessive production of collagen and other extracellular matrix components. FLi-1 is a transcription factor encoded by the FLI1 gene, which is a negative regulator of collagen production by FBs. The expression level of Fli-1 is significantly reduced in SSc fibrotic skin and explanted SSc FBs compared with healthy controls [23], suggesting that the reduced level of Fli-1 may be responsible for elevated collagen synthesis and accumulation in patients with SSc. We reported the existence of heavy methylation of CpG sites in the promoter region of FLI1 in SSc FBs [35]. Furthermore, exposure of SSc FBs to 5-azacytidine, a universal demethylating agent (DNMT1 inhibitor), led to restoration of FLI1 expression and resulted in reduced type I collagen production in vitro. This observation was the first evidence to demonstrate that epigenetic modifications may mediate the fibrotic phenotype of SSc FBs.

BMP signalling through BMPRII favours MVEC survival and apoptosis resistance. We have recently examined the expression of BMPRII in SSc MVECs and demonstrated a significant decrease in expression levels in MVECs as well as in freshly processed skin biopsies compared with healthy controls [41]. We also observed enhanced SSc MVEC responses to apoptotic signals, including serum starvation and oxidation injury. Sequencing the BMPRII promoter region after bisulphite conversion demonstrated heavily methylated CpG sites in SSc MVECs. BMPRII expression levels were normalized by the addition of 5-azacytidine, and the impaired SSc MVEC apoptotic response to serum starvation and oxidation injury was restored to levels comparable to MVECs in the control group. These data suggest that epigenetic repression of BMPRII may play a central role in MVEC vulnerability to apoptosis and that impaired BMPRII signalling in SSc MVECs may contribute to the pathogenesis of SSc vasculopathy [41].


Histone modification in SSc The nucleosome is the basic subunit of chromatin, comprised of 146 bp of DNA wrapped around an octamer of proteins consisting of two copies of each of the four core histones: H2A, H2B, H3 and H4. Each histone subtype can be modified by several post-translational

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modifications. The two main histone modifications are acetylation and methylation. Histone acetylation is usually associated with transcriptional activity, while deacetylation of terminal lysine residues contributes to the silencing of transcription. The effect of histone methylation depends on the position of lysine; for instance, histone H3 lysine 4 (H3K4) methylation enhances gene expression, while H3K27 tri-methylation (H3K27me3) is a repressive event [57]. Histone modifications and DNA methylation are mechanistically linked since methyl binding domain (MBD) protein recruitment after DNA methylation binds to methylated cytosines and in turn recruits histone deacetylases (HDACs). Modifications of the histone code represent post-translational processes that modulate chromatin architecture and therefore provide access for transcriptional machinery and transcription factors to gene-regulatory regions. Activation of B cells is important in the pathogenesis of SSc [58]. Wang et al. [59] recently demonstrated global histone H4 hyperacetylation and global histone 3 lysin 9 (H3K9) hypomethylation associated with down-regulation of HDAC2 and HDAC7 in B cells from SSc patients, and all favour a permissive chromatin architecture for gene expression, therefore it is hypothetically possible that these changes in the chromatin lead to overexpression of the autoimmune-related genes. We examined histone H3 and H4 acetylation in the promoter region of the FLI1 gene in SSc and normal FBs and found a significant reduction in the acetylated forms of H3 and H4 in SSc FBs [35]. Histone deacetylation is a repressive mark for target gene expression, which could contribute to the repression of FLI1, as discussed earlier. Moreover, H3K27me3, which is a potent repressor of target gene transcription, is increased in SSc FBs in comparison with controls [60]. Kramer et al. [60] demonstrated that inhibition of H3K27me3 stimulates the release of collagen in SSc FBs and in a bleomycin-induced experimental fibrosis model. The description of histone code modifications in FBs and B cells in SSc led to remarkable interest in using HDAC inhibitors, such as trichostatin (TSA), which is already available for the treatment of myelodysplastic disease. Of interest, treatment with TSA attenuated expression of collagen I in dermal SSc FBs [35] and reduced the accumulation of collagens, extracellular matrix and fibronectin in SSc FBs [61] and in an animal model of skin fibrosis [62]. Similarly, after treatment of SSc MVECs with TSA, acetylation of histones H3 and H4 at the NOS3 promoter region increased concomitantly with enhanced NOS expression [63]. These observations introduce the consideration that epigenetic modulators should be considered in the treatment of SSc. Still, the concern here is that the effects of these modulators are diffuse in the epigenome and that a yet unknown off-site effect may limit their usefulness.

miRNA in SSc miRNAs are genome-encoded, non-coding RNA molecules that mediate the post-transcriptional regulation of



in healthy women [47]. As a consequence of that, reactivation of genes that are typically suppressed in the inactive X chromosomes of female SSc patients could arguably contribute to the observed predominance of SSc among females. Of note, CD40L plays an important role in B cell activation, fibrosis and expression of adhesion molecules on endothelial cells. CD40L expression is increased in dermal SSc FBs obtained from clinically involved skin [50], in CD4+ T cells, especially from female patients with SSc [51, 52], and in tight-skin mouse models [53]. Additional evidence to support reactivation of the normally silenced X chromosome in SSc comes from Selmi et al. [54], who compared genome-wide methylation profiles in a relatively small study using peripheral blood mononuclear cells (PBMCs) from monozygotic twins discordant and concordant for SSc (n = 7 and n = 1, respectively). The interpretation of the results is limited by the use of a heterogeneous cell population (PBMCs), a small sample size and the absence of total expression profiles. However, the power of such a study design (i.e. using monozygotic twins) offers the unique advantage of eliminating the risk of genetic confounders. Interestingly, this study demonstrated consistent differences between investigated twins only in genes located on the X chromosome. The data suggest that DNA demethylation and subsequent reactivation of the silenced X chromosome in female SSc patients may explain the susceptibility of women to SSc. Co-stimulation of B and/or T cells is crucial for development of an appropriate immune response by inducing proliferation of the co-stimulated cells. Defective costimulation has been shown to contribute to the pathogenesis of several autoimmune diseases, such as SLE and RA. CD70 is one of the best-characterized co-stimulatory molecules expressed on activated B and T cells. Recently Jiang et al. [55] demonstrated that CD70 is overexpressed in SSc CD4+ T cells and that demethylation of the CD70 promoter region contributes to the overexpression of CD70 in CD4+ T cells. CD11a (also known as ITGAL) is another molecule that is involved in co-stimulatory signalling by adhesive interactions between T cells, dendritic cells and B cells. Recently it was shown that CD11a is overexpressed in SSc CD4+ T cells and that the promoter region of CD11a is hypomethylated [56]. Overall, increased expression of CD70 and CD11a has been identified as playing a central role in the pathogenesis of several autoimmune diseases, but it remains to be shown whether CD70 and CD11a signalling is in fact involved in SSc pathogenesis to the same extent that these molecules are involved in the pathogenesis of other autoimmune diseases.

Epigenetics of SSc


SSc, and these levels correlate with the severity of SSc [81]. Overall, it remains unclear at this stage whether miRNAs could be clinically useful as biomarkers of disease activity or prognosis, although the idea is appealing.

Epigenetic triggers in SSc In most cases the nature of the specific stimuli that trigger epigenetic modifications among patients with SSc remain uncharacterized but may include external factors (e.g. diet, chemicals, exposure to silica, toxins and drugs) and internal factors (e.g. ageing, sex hormones, hypoxia and oxidation injury). The difficulty in identifying the environmental epigenetic trigger(s) is complicated by the fact that while some epigenetic effects are manifested in the generation of patients that are exposed to the modifying agent, in other cases it appears that disease may occur one or two generations after the exposure [91, 92].

Diet and nutrition Nutritional sources may provide the methyl donors (methionine, choline) and co-factors (folic acid, vitamin B12 and pyridoxal phosphate) essential for DNA and histone methylation. It is now well recognized that susceptibility to chronic disease is influenced by persistent adaptations to prenatal and early postnatal nutrition [93]. Furthermore, there are also reports of diet-induced epigenetic changes in the adult state [94]. It has been proposed that epigenetic links between nutrition and autoimmunity may well contribute to the epidemiology of several autoimmune diseases. However, while studies in animals using arbitrarily chosen dietary elements tend to support this proposal, human data from real-life clinical settings or randomized clinical trials remain inconclusive at present [92].

Hypoxia In general, hypoxia decreases global transcriptional activity and has a major effect on cellular phenotype; the hypoxia-inducible factor (HIF) transcription paradigm is an ancient eukaryotic response that allows cells to adapt to changes in oxygen supply or availability. Evidence suggests that epigenetic pathways are also relevant in the adaptation to hypoxia [95]. Hypoxia is shown to induce a global decrease in H3K9 acetylation in various cells as a possible consequence of HDAC up-regulation [96], while acetylated H3K9 is enriched at the promoter regions of hypoxia-activated genes, such as VEGF [97]. The effects of hypoxia on global levels of DNA methylation are just beginning to be studied. Future genome-wide mapping of specific acetyl and methyl histone modifications, histone demethylases, histone density and DNA methylation in hypoxic cells will be necessary to fully understand their importance in transcriptional regulation and the formation of distinct hypoxia-mediated epigenetic signatures of hypoxia-regulated genes.

Oxidation Oxidative stress and high levels of reactive oxygen species (ROS) have been both directly and indirectly

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multiple target gene expression [64]. It is generally accepted that miRNAs target the 30 -untranslated region of messenger RNA (mRNA) by base pairing, causing degradation or translational repression of mRNA. Like the other epigenetic mechanisms, miRNAs are expressed in a tissue-specific and cell typespecific manner [6567]. Of particular interest, epigenetic modifications, such as DNA and histone methylation and histone deacetylation, also participate in modulation of miRNA transcription [68]. This finding documents the existence of cross-talk among the various components of the epigenetic machinery. Recent reports suggest that miRNAs are key elements in the pathogenesis of SSc; several miRNAs are differentially expressed in different cell types, or even in serum and hair shaft samples from SSc patients compared with healthy subjects, an observation noted in other fibrotic diseases [6972]. Several miRNAs are regulated by TGF-b, such as collagens [7378], matrix metalloproteinase [79], integrins [80, 81], and Smad signalling pathways [77, 82, 83], and their predicted target genes are involved in matrix repair and remodelling. Elevated expressions of profibrotic miRNAs or reduced expressions of antifibrotic miRNAs are likely to be important in the development of fibrosis among patients with SSc [84]. Table 1 summarizes some of these reports. Maurer et al. [78] reported underexpression of miR-29 in skin FBs from both diffuse and limited SSc, as well as FBs from a bleomycin-induced skin fibrosis mouse model. They also demonstrated that induced expression of miR-29 in SSc FBs reduces the expression of collagen. Moreover, stimulation of normal skin FBs with profibrotic mediators, such as TGF-b and PDGF-B, reduces the level of miR-29. Of significant interest, the authors showed that the stimulatory effects of TGF-b and PDGF-B on collagen synthesis could be reduced by rescuing cells with miR-29 and that the down-regulation of miR-29 leads to further up-regulation of TGF-b and PDGF-B. Taken together, this report provides the first evidence that miRNAs may play an integral role in the pathogenesis of fibrosis among patients with SSc. Zhu et al. [77] demonstrated that miR-21 was one of several miRNAs up-regulated in SSc FBs and that TGF-b regulated the expression of miR-21 as well as other fibrosis-related genes. It appears that the target of miR-21 is Smad7, since overexpression of miR-21 in SSc FBs decreases levels of Smad7, whereas knockdown of miR-21 increases Smad7 expression [79, 90]. Therefore miR-21 seems to exert a profibrogenic effect by negatively regulating Smad7. Some miRNA expression levels seem to correlate with features of SSc. For instance, miR-92a levels correlate with the presence of telangiectasia but do not correlate with disease activity [79]. On the other hand, miRNAs miR-21, miR-31, miR-146, miR-145, miR-29b, and others do correlate with disease activity. Moreover, the serum level of miR-92a is higher in SSc patients compared with members of a control group [79]. Also, serum levels of miR-142 are elevated in patients with


TABLE 1 Summary of key epigenetic alterations observed in SSc Gene/pathway

Epigenetic defect

Cell typea

Target/consequence

DNA methylation FLI1 DNMT1

Hypermethylation Overexpressed

FBs FBs, MVECs

DNMT1 DNA demethylase activity MBD1

Down-regulated Down-regulated

CD4+ T cells MVECs

Overexpression of collagen genes in SSc FBs. Increased expression of Dnmt1 could be contributing to hypermethylation of certain genes, such as FLI1. Global hypomethylation in CD4+ T cells. Hypermethylation and repression of FLI1.

Overexpressed

FBs

CD40L

Hypomethylation

CD4+ T cells

CD70 (TNFSF7)

Hypomethylation

CD4 T cells

ACTA

Hypomethylation

Human alveolar FBs

BMPRII

Hypermethylation

MVECs

NOS

Hypermethylation

MVECs

Reduced

FBs

H3K27me3

Increased

FBs, murine dermal FBs

Global H4 acetyl ation, H3K methylation

Increased H4 acetylation, decreased H3K methylation

B cells

FLI1 H3 and H4 acetylation TGF-b

Reduced

FBs

Unclear

FBs

Down-regulated

FBs, murine dermal FBs Serum

Histone modification H3, H4 acetylation

miRNA miR-29 miR-142

Overexpressed

miR-196a

Down-regulated

miR-21

Overexpressed

miR-31 miR-145 miR-146 miR-152 miR-503 miR-7

Overexpressed Down-regulated Overexpressed Down-regulated Overexpressed Overexpressed in SSc, down-regulated in LSc Down-regulated Overexpressed Down-regulated Down-regulated

miR let-7a miR-92-a miR-150 miR-129-5p

FBs, serum, and hair shafts. Skin tissue, FBs, murine dermal FBs Skin tissue, FBs Skin tissue, FBs Skin tissue, FBs MVECs Skin tissue, FBs FBs, skin, serum

FBs, serum FBs, serum FBs, serum FBs

[35] [35, 36, 85]

[42] [35, 85]

Interference with transcriptional machinery, recruitment of HDACs. Unfavourable chromatin structure. Co-stimulatory molecule, role is not clear in SSc. Co-stimulatory molecule, role is not clear in SSc. Lung FBs exhibit significantly lower levels of DNA methylation of ACTA promoter, not clear if demethylation of ACTA is a prerequisite for FB activation. Failure of the inhibitory mechanism for cell proliferation and induction of apoptosis. Reduced NOS activity in MVECs. Increased expression of proinflammatory and vasospastic genes.

[35]

Unfavourable chromatin structure for target gene expression. May contribute to inhibition of collagen suppressor genes and therefore collagen deposition. Favour target gene expression in B cells. Could be contributing to activation of genes in the immune system and antibody production. Repression of FLI1, therefore overexpression of collagen genes. HDAC inhibitor prevents SSc-related tissue fibrosis by reducing collagen I and fibronectin in dermal SSc FBs.

[35]

Antifibrotic factor, putative target is type 1 collagen. Unclear, could be involved in regulating the expression of integrin aV. Putative target is type I collagen.

[77, 78, 87]

Profibrotic factor, target SMAD7.

[77, 85]

Putative target is type 1 collagen. Putative target is SMAD3. Putative target SMAD4. Overexpression of DNMT1. Unclear, putative target SMAD7. Target type 1 collagen.

[77] [77] [77] [89] [77] [75, 76]

Unclear, putative target is type 1 collagen. Unclear, probably inhibit MMP-1. Induction of integrin b3. Unclear, putative target is type 1 collagen.

[74] [79] [80] [73]

[51] [55] [86]

[41] [63]

[60]

[59]

[35] [62]

[81] [83, 88]

a

Human cells in origin, unless otherwise specified. ACTA: actin, alpha 2, smooth muscle, aorta gene; BMPRII: bone morphogenetic protein type II receptor; ECM: extracellular matrix; FLI1: friend leukaemia virus integration gene; DNMT1: DNA (cytosine-5-)-methyltransferase 1; FB: fibroblast; H3K27me3: tri-methylation of histone H3 on lysine 27; LSc: localized scleroderma; MBD1: methyl-CpG-binding domain protein 1; MVEC: microvascular endothelial cells; MeCP2: methyl CpG binding protein 2; MMP-1: matrix metalloproteinase 1; NOS: nitric oxide synthetase; SMAD: intracellular proteins that transduce extracellular signals from TGF-b ligands; HDACs: histone deacetylases.

implicated in SSc pathogenesis [98, 99]. SSc patients exhibit significant evidence of oxidative stress, which is shown by abnormalities of NO and NOS and by increased levels of oxidative biomarkers [100, 101]. Moreover, high

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levels of ROS production by FBs, independent of inflammatory stimuli, suggest that this cell type is the endogenous source for oxidative stress among patients with SSc [102].



+

Reference

Epigenetics of SSc

FIG. 1 Schematic overview of the evidence for epigenetic alterations in cellular and molecular pathways in SSc FBs

There is growing interest in the involvement of oxidative stress in the epigenetic regulation of gene expression, and specifically in controlling DNA methylation. Oxidative damage induces the formation of large silencing complexes containing DNMTs that localize at certain genes and induce gene silencing, as clearly seen in cancerspecific aberrant DNA methylation and transcriptional silencing [103]. ROS-induced oxidative stress has been shown to silence the tumour suppressor caudal type homeobox 1 (CDX1) through epigenetic regulation and may therefore be associated with the progression of colorectal cancer [104]. ROS have also been shown to induce the up-regulation of Snail expression, leading to the methylation of CpG sites in the E-cadherin promoter that is believed to lead to cellular acquisition of migratory properties and tumour metastasis [105].


Conclusion We have explored in this review several studies that confirm substantial epigenetic modification in SSc, particularly in FBs (Fig. 1), MVECs (Fig. 2) and in B and T cells. Although the aetiology of SSc is indeterminate, there is a minor (albeit significant) genetic component to the disease, and there is epigenetic variation in pathways involved in SSc pathogenesis, such as TGF-b and downstream pathways. Therefore it appears that SSc pathogenesis is the result of complex interactions between genetic susceptibility, environmental exposure and epigenetic modifications. Studies focusing on a single cytokine or pathway without accounting for the epigenetic factors are not likely to be productive in delineating the pathogenesis of SSc.

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There is good evidence that hypermethylation of the promoter region of FLI1 leads to repression of Fli-1, which is a transcription factor with an inhibitory function on collagen gene expression. Therefore epigenetic repression of FLI1 may play an important role in collagen deposition and tissue fibrosis. Cellular injury (due to viruses, autoantibodies, hypoxia, oxidation, toxins, etc.) causes activation of the TGF-b signalling pathway. This pathway is further activated by miRNAs through up-regulation of profibrotic molecules, such as Smad3 and Smad4, or by down-regulation of anti-fibrotic molecules, such as Smad7, that contribute to increased collagen synthesis and extracellular matrix (ECM) expansion. miRNAs also modulate collagen gene expression; for instance, underexpression of miR-196a, Let-7a and miR-29 are examples of post-transcriptional modification of collagen genes. FB: fibroblasts; Fli-1: friend leukaemia integration-1; miRNA: microRNA.


FIG. 2 Overview of the evidence for epigenetic modification of SSc MVECs

Unlike genetic mutations, epigenetic changes are reversible and amenable to modifications in dividing cells. Translating this knowledge into therapeutics for patients with SSc is highly anticipated, as there is an unmet need for disease-modifying therapies to treat this serious disease. To achieve this goal, we need to identify the spectrum of epigenetic alterations across the genome in all the cells involved in the pathogenesis of SSc. The use of available epigenetic modifiers (HDAC inhibitors, DNMT inhibitors and even miRNAs) may be limited by the potential off-site effects. We believe that the most promising approach should be directed at specific epigenetic modifications and that epigenetic editing should be directed in a genespecific or pathway-specific manner. In this review we presented persuasive evidence that supports the important contribution of epigenetic

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regulation in the emergence of the vascular, fibrotic and immune SSc phenotype. Further discoveries of epigenetic alteration in the disease will undoubtedly be found by many investigators for some time to come. Still, the controlling mechanism that activates the specific fibrotic, vascular and immune epigenomic regulation—the holy grail—is completely unknown and currently remains elusive and inaccessible. To advance the field forward, more studies are needed to fully characterize the epigenome in specific cell types and across all cells that are involved in the pathogenesis of SSc. The use of heterogeneous groups of cells to study epigenetic alteration in the disease should be discouraged. Furthermore, the functional characterization and understanding of the biologic significance of the numerous miRNAs that are differentially expressed in SSc are



This is a schematic presentation showing DNA hypermethylation and repression of key genes in SSc MVECs, which is maintained by up-regulation of DNMT1 expression in MVECs. Methylated CpG sites in gene regulatory regions interfere with the binding of transcription factors (TFs) and contribute to an unfavourable chromatin structure for gene expression. Hypermethylation of the promoter region of bone morphogenic protein receptor II (BMPRII) and NOS3, and consequently underexpression of these genes in MVECs, leads to a cascade of events characterized by EC apoptosis, vasoconstriction, recruitment of inflammatory cells, oxidation injury and ultimately activation of fibroblasts. MVEC: microvascular endothelial cells.

Epigenetics of SSc

urgently needed. A deeper understanding of the pathogenic environment, epigenomic control and reprogramming may represent the final strategy in the prevention and/or treatment of SSc. Rheumatology key messages SSc is the result of complex interaction between genetic susceptibility and environmental epigenetic factors. . There is evidence for epigenetic alterations in key pathways in the pathogenesis of SSc. . Oxidation injury and hypoxia might be the trigger for epigenetic alterations in SSc. .

This work was supported by start-up research funding from the University of Toledo, School of Medicine. Disclosure statement: The authors have declared no conflicts of interest.

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