Download Epigenetics, the holy grail in the pathogenesis of

RHEUMATOLOGY
Rheumatology 2015;54:1759–1770
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
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.
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.
! The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]
R EV I E W
Abstract
Nezam Altorok et al.
Pathogenesis of SSc
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 [29–32]. 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 [12–14]. 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 proliferator–activated 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.
1760
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,
www.rheumatology.oxfordjournals.org
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
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.
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
www.rheumatology.oxfordjournals.org
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].
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 [45–48] 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
1761
Nezam Altorok et al.
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.
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
1762
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
www.rheumatology.oxfordjournals.org
Epigenetics of SSc
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 type–specific manner [65–67]. 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 [69–72]. Several miRNAs are regulated by
TGF-b, such as collagens [73–78], 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
www.rheumatology.oxfordjournals.org
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
1763
Nezam Altorok et al.
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
Reference
[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
1764
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].
www.rheumatology.oxfordjournals.org
Epigenetics of SSc
FIG. 1 Schematic overview of the evidence for epigenetic alterations in cellular and molecular pathways in SSc FBs
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.
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].
www.rheumatology.oxfordjournals.org
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.
1765
Nezam Altorok et al.
FIG. 2 Overview of the evidence for epigenetic modification of SSc MVECs
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.
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
1766
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
www.rheumatology.oxfordjournals.org
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.
.
Acknowledgements
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.
References
1 Abraham DJ, Varga J. Scleroderma: from cell and
molecular mechanisms to disease models. Trends
Immunol 2005;26:587–95.
2 Nikpour M, Stevens WM, Herrick AL, Proudman SM.
Epidemiology of systemic sclerosis. Best Pract Res Clin
Rheumatol 2010;24:857–69.
3 Maricq HR, Weinrich MC, Keil JE et al. Prevalence of
scleroderma spectrum disorders in the general population
of South Carolina. Arthritis Rheum 1989;32:998–1006.
4 Mayes MD. Scleroderma epidemiology. Rheum Dis Clin
North Am 2003;29:239–54.
5 Valesini G, Litta A, Bonavita MS et al. Geographical
clustering of scleroderma in a rural area in the province
of Rome. Clin Exp Rheumatol 1993;11:41–7.
6 Silman AJ, Howard Y, Hicklin AJ, Black C. Geographical
clustering of scleroderma in south and west London. Br J
Rheumatol 1990;29:93–6.
7 LeRoy EC, Black C, Fleischmajer R et al. Scleroderma
(systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol 1988;15:202–5.
8 Altorok N, Sawalha AH. Epigenetics in the pathogenesis
of systemic lupus erythematosus. Curr Opin Rheumatol
2013;25:569–76.
9 Javierre BM, Hernando H, Ballestar E. Environmental
triggers and epigenetic deregulation in autoimmune disease. Discov Med 2011;12:535–45.
12 Campbell PM, LeRoy EC. Pathogenesis of systemic
sclerosis: a vascular hypothesis. Semin Arthritis Rheum
1975;4:351–68.
13 Sgonc R, Gruschwitz MS, Dietrich H et al. Endothelial cell
apoptosis is a primary pathogenetic event underlying skin
lesions in avian and human scleroderma. J Clin Invest
1996;98:785–92.
14 Matucci-Cerinic M, Kahaleh B, Wigley FM. Review:
evidence that systemic sclerosis is a vascular disease.
Arthritis Rheum 2013;65:1953–62.
15 Mavalia C, Scaletti C, Romagnani P et al. Type 2 helper
T-cell predominance and high CD30 expression in
systemic sclerosis. Am J Pathol 1997;151:1751–8.
16 Leroy EC. Connective tissue synthesis by scleroderma
skin fibroblasts in cell culture. J Exp Med 1972;135:
1351–62.
17 Ihn H, Yamane K, Kubo M, Tamaki K. Blockade of
endogenous transforming growth factor beta signalling
prevents up-regulated collagen synthesis in scleroderma
fibroblasts: association with increased expression of
transforming growth factor beta receptors. Arthritis
Rheum 2001;44:474–80.
18 Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J
Med 2000;342:1350–8.
19 Mori Y, Chen SJ, Varga J. Expression and regulation
of intracellular SMAD signaling in scleroderma skin fibroblasts. Arthritis Rheum 2003;48:1964–78.
20 Scharffetter K, Lankat-Buttgereit B, Krieg T. Localization
of collagen mRNA in normal and scleroderma skin by
in-situ hybridization. Eur J Clin Invest 1988;18:9–17.
21 Ihn H, Yamane K, Asano Y, Jinnin M, Tamaki K.
Constitutively phosphorylated Smad3 interacts with Sp1
and p300 in scleroderma fibroblasts. Rheumatology 2006;
45:157–65.
22 Asano Y, Ihn H, Yamane K, Kubo M, Tamaki K. Impaired
Smad7-Smurf-mediated negative regulation of TGF-beta
signaling in scleroderma fibroblasts. J Clin Invest 2004;
113:253–64.
23 Kubo M, Czuwara-Ladykowska J, Moussa O et al.
Persistent down-regulation of Fli1, a suppressor of collagen transcription, in fibrotic scleroderma skin. Am J Pathol
2003;163:571–81.
24 Wei J, Ghosh AK, Sargent JL et al. PPARg downregulation
by TGFb in fibroblast and impaired expression and
function in systemic sclerosis: a novel mechanism for
progressive fibrogenesis. PLoS One 2010;5:e13778.
25 Feghali-Bostwick C, Medsger TA Jr, Wright TM. Analysis
of systemic sclerosis in twins reveals low concordance
for disease and high concordance for the presence
of antinuclear antibodies. Arthritis Rheum 2003;48:
1956–63.
26 Favalli E, Ingegnoli F, Zeni S, Fare M, Fantini F.
[HLA typing in systemic sclerosis]. Reumatismo 2001;53:
210–4.
10 Gu YS, Kong J, Cheema GS et al. The immunobiology
of systemic sclerosis. Semin Arthritis Rheum 2008;38:
132–60.
27 Luo Y, Wang Y, Wang Q, Xiao R, Lu Q. Systemic sclerosis:
genetics and epigenetics. J Autoimmun 2013;41:161–7.
11 Varga J, Abraham D. Systemic sclerosis: a prototypic
multisystem fibrotic disorder. J Clin Invest 2007;117:
557–67.
28 Agarwal SK, Reveille JD. The genetics of scleroderma
(systemic sclerosis). Curr Opin Rheumatol 2010;22:
133–8.
www.rheumatology.oxfordjournals.org
1767
Nezam Altorok et al.
29 Radstake TR, Gorlova O, Rueda B et al. Genome-wide
association study of systemic sclerosis identifies CD247
as a new susceptibility locus. Nat Genet 2010;42:426–9.
30 Rueda B, Broen J, Simeon C et al. The STAT4 gene
influences the genetic predisposition to systemic sclerosis
phenotype. Hum Mol Genet 2009;18:2071–7.
31 Ito I, Kawaguchi Y, Kawasaki A et al. Association of a
functional polymorphism in the IRF5 region with systemic
sclerosis in a Japanese population. Arthritis Rheum 2009;
60:1845–50.
32 Dieude P, Guedj M, Wipff J et al. Association between the
IRF5 rs2004640 functional polymorphism and systemic
sclerosis: a new perspective for pulmonary fibrosis.
Arthritis Rheum 2009;60:225–33.
33 Reinhart BJ, Slack FJ, Basson M et al. The 21-nucleotide
let-7 RNA regulates developmental timing in
Caenorhabditis elegans. Nature 2000;403:901–6.
47 Hellman A, Chess A. Gene body-specific methylation on
the active X chromosome. Science 2007;315:1141–3.
48 Miranda TB, Jones PA. DNA methylation: the nuts and
bolts of repression. J Cell Physiol 2007;213:384–90.
49 Lyon MF. Gene action in the X-chromosome of the mouse
(Mus musculus L.). Nature 1961;190:372–3.
50 Fukasawa C, Kawaguchi Y, Harigai M et al. Increased
CD40 expression in skin fibroblasts from patients with
systemic sclerosis (SSc): role of CD40-CD154 in the
phenotype of SSc fibroblasts. Eur J Immunol 2003;33:
2792–800.
51 Lian X, Xiao R, Hu X et al. DNA demethylation of CD40l
in CD4+ T cells from women with systemic sclerosis:
a possible explanation for female susceptibility. Arthritis
Rheum 2012;64:2338–45.
34 Richardson B. Primer: epigenetics of autoimmunity. Nat
Rev Rheumatol 2007;3:521–7.
52 Valentini G, Romano MF, Naclerio C et al. Increased
expression of CD40 ligand in activated CD4+ T lymphocytes of systemic sclerosis patients. J Autoimmun 2000;
15:61–6.
35 Wang Y, Fan PS, Kahaleh B. Association between
enhanced type I collagen expression and epigenetic
repression of the FLI1 gene in scleroderma fibroblasts.
Arthritis Rheum 2006;54:2271–9.
53 Komura K, Fujimoto M, Yanaba K et al. Blockade of CD40/
CD40 ligand interactions attenuates skin fibrosis and
autoimmunity in the tight-skin mouse. Ann Rheum Dis
2008;67:867–72.
36 Qi Q, Guo Q, Tan G et al. Predictors of the scleroderma
phenotype in fibroblasts from systemic sclerosis patients.
J Eur Acad Dermatol Venereol 2009;23:160–8.
54 Selmi C, Feghali-Bostwick CA, Lleo A et al. X chromosome
gene methylation in peripheral lymphocytes from monozygotic twins discordant for scleroderma. Clin Exp
Immunol 2012;169:253–62.
37 Fish JE, Marsden PA. Endothelial nitric oxide synthase:
insight into cell-specific gene regulation in the vascular
endothelium. Cell Mol Life Sci 2006;63:144–62.
38 Romero LI, Zhang DN, Cooke JP et al. Differential
expression of nitric oxide by dermal microvascular
endothelial cells from patients with scleroderma.
Vasc Med 2000;5:147–58.
39 Huang PL, Huang Z, Mashimo H et al. Hypertension in
mice lacking the gene for endothelial nitric oxide synthase.
Nature 1995;377:239–42.
40 Lee PC, Salyapongse AN, Bragdon GA et al. Impaired
wound healing and angiogenesis in eNOS-deficient mice.
Am J Physiol 1999;277(4 Pt 2):H1600–8.
55 Jiang H, Xiao R, Lian X et al. Demethylation of TNFSF7
contributes to CD70 overexpression in CD4+ T cells from
patients with systemic sclerosis. Clin Immunol 2012;143:
39–44.
56 Wang YS, Shu Y, Wang Q et al. Demethylation of ITGAL
(CD11a) Regulatory Sequences in CD4+ T Lymphocytes
of Systemic Sclerosis. 2013. Abstract 2905. American
College of Rheumatology, San Diego.
57 Kondo Y, Shen L, Cheng AS et al. Gene silencing
in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation. Nat Genet 2008;
40:741–50.
41 Wang Y, Kahaleh B. Epigenetic repression of bone morphogenetic protein receptor II expression in scleroderma.
J Cell Mol Med 2013;17:1291–99.
58 Sato S, Fujimoto M, Hasegawa M, Takehara K. Altered
blood B lymphocyte homeostasis in systemic sclerosis:
expanded naive B cells and diminished but activated
memory B cells. Arthritis Rheum 2004;50:1918–27.
42 Lei W, Luo Y, Lei W et al. Abnormal DNA methylation in
CD4+ T cells from patients with systemic lupus erythematosus, systemic sclerosis, and dermatomyositis. Scand
J Rheumatol 2009;38:369–74.
59 Wang Y, Yang Y, Luo Y et al. Aberrant histone modification in peripheral blood B cells from patients with systemic
sclerosis. Clin Immunol 2013;149:46–54.
43 Sawalha AH, Jeffries M, Webb R et al. Defective T-cell
ERK signaling induces interferon-regulated gene expression and overexpression of methylation-sensitive genes
similar to lupus patients. Genes Immun 2008;9:368–78.
44 Sawalha AH, Richardson B. MEK/ERK pathway inhibitors
as a treatment for inflammatory arthritis might result in the
development of lupus: comment on the article by Thiel
et al. Arthritis Rheum 2008;58:1203–4; author reply 4.
60 Kramer M, Dees C, Huang J et al. Inhibition of H3K27
histone trimethylation activates fibroblasts and induces
fibrosis. Ann Rheum Dis 2013;72:614–20.
61 Hemmatazad H, Rodrigues HM, Maurer B et al. Histone
deacetylase 7, a potential target for the antifibrotic treatment of systemic sclerosis. Arthritis Rheum 2009;60:
1519–29.
45 Singer-Sam J, Riggs AD. X chromosome inactivation and
DNA methylation. EXS 1993;64:358–84.
62 Huber LC, Distler JH, Moritz F et al. Trichostatin A prevents the accumulation of extracellular matrix in a mouse
model of bleomycin-induced skin fibrosis. Arthritis Rheum
2007;56:2755–64.
46 Robertson KD. DNA methylation and chromatin –
unraveling the tangled web. Oncogene 2002;21:
5361–79.
63 Matouk CC, Marsden PA. Epigenetic regulation of vascular endothelial gene expression. Circ Res 2008;102:
873–87.
1768
www.rheumatology.oxfordjournals.org
Epigenetics of SSc
64 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97.
65 Saj A, Lai EC. Control of microRNA biogenesis and transcription by cell signaling pathways. Curr Opin Genet Dev
2011;21:504–10.
66 Suzuki HI, Miyazono K. Emerging complexity of microRNA
generation cascades. J Biochem 2011;149:15–25.
67 Krol J, Loedige I, Filipowicz W. The widespread regulation
of microRNA biogenesis, function and decay. Nat Rev
Genet 2010;11:597–610.
68 Vrba L, Garbe JC, Stampfer MR, Futscher BW. Epigenetic
regulation of normal human mammary cell type-specific
miRNAs. Genome Res 2011;21:2026–37.
69 Tijsen AJ, Pinto YM, Creemers EE. Non-cardiomyocyte
microRNAs in heart failure. Cardiovascu Res 2012;93:
573–82.
70 Kato M, Park JT, Natarajan R. MicroRNAs and the
glomerulus. Exp Cell Res 2012;318:993–1000.
71 Wang XW, Heegaard NH, Orum H. MicroRNAs in liver
disease. Gastroenterology 2012;142:1431–43.
72 Vettori S, Gay S, Distler O. Role of microRNAs in fibrosis.
Open Rheumatol J 2012;6:130–9.
73 Nakashima T, Jinnin M, Yamane K et al. Impaired IL-17
signaling pathway contributes to the increased collagen
expression in scleroderma fibroblasts. J Immunol 2012;
188:3573–83.
74 Makino K, Jinnin M, Hirano A et al. The downregulation of
microRNA let-7a contributes to the excessive expression
of type I collagen in systemic and localized scleroderma.
J Immunol 2013;190:3905–15.
75 Kajihara I, Jinnin M, Yamane K et al. Increased accumulation of extracellular thrombospondin-2 due to low
degradation activity stimulates type I collagen expression in scleroderma fibroblasts. Am J Pathol 2012;180:
703–14.
76 Etoh M, Jinnin M, Makino K et al. microRNA-7 downregulation mediates excessive collagen expression in
localized scleroderma. Arch Dermatol Res 2013;305:
9–15.
77 Zhu H, Li Y, Qu S et al. MicroRNA expression abnormalities in limited cutaneous scleroderma and diffuse cutaneous scleroderma. J Clin Immunol 2012;32:514–22.
78 Maurer B, Stanczyk J, Jungel A et al. MicroRNA-29, a key
regulator of collagen expression in systemic sclerosis.
Arthritis Rheum 2010;62:1733–43.
79 Sing T, Jinnin M, Yamane K et al. microRNA-92a expression in the sera and dermal fibroblasts increases in
patients with scleroderma. Rheumatology 2012;51:
1550–6.
80 Honda N, Jinnin M, Kira-Etoh T et al. miR-150 downregulation contributes to the constitutive type I collagen
overexpression in scleroderma dermal fibroblasts via
the induction of integrin beta3. Am J Pathol 2013;182:
206–16.
81 Makino K, Jinnin M, Kajihara I et al. Circulating
miR-142-3p levels in patients with systemic sclerosis.
Clin Exp Dermatol 2012;37:34–9.
82 van Rooij E, Sutherland LB, Thatcher JE et al.
Dysregulation of microRNAs after myocardial infarction
www.rheumatology.oxfordjournals.org
reveals a role of miR-29 in cardiac fibrosis. Proc Natl
Acad Sci USA 2008;105:13027–32.
83 Honda N, Jinnin M, Kajihara I et al. TGF-beta-mediated
downregulation of microRNA-196a contributes to the
constitutive upregulated type I collagen expression in
scleroderma dermal fibroblasts. J Immunol 2012;188:
3323–31.
84 Jiang X, Tsitsiou E, Herrick SE, Lindsay MA. MicroRNAs
and the regulation of fibrosis. FEBS J 2010;277:2015–21.
85 Kahaleh B, Wang W. Decrease activity of DNA demethylase in SSC fibroblast and microvascular endothelial cells:
a possible mechanism for persistent SSC phenotype
[abstract]. Rheumatology 2012;51:ii5e6.
86 Hu B, Gharaee-Kermani M, Wu Z, Phan SH. Essential
role of MeCP2 in the regulation of myofibroblast differentiation during pulmonary fibrosis. Am J Pathol 2011;178:
1500–8.
87 Fabbri M, Garzon R, Cimmino A et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting
DNA methyltransferases 3A and 3B. Proc Natl Acad Sci
USA 2007;104:15805–10.
88 Wang Z, Jinnin M, Kudo H et al. Detection of hairmicroRNAs as the novel potent biomarker: evaluation
of the usefulness for the diagnosis of scleroderma.
J Dermatol Sci 2013;72:134–41.
89 Wang Y, Kahaly OR, Kahaleh B. Down-regulated
microRNA-152 induces aberrant DNA methylation
in scleroderma endothelial cells by targeting DNA
methyltransferase 1 [abstract]. Arthritis Rheum 2010;
62(Suppl 10):1352.
90 Zhu H, Luo H, Li Y et al. MicroRNA-21 in scleroderma
fibrosis and its function in TGF-beta-regulated fibrosisrelated genes expression. J Clin Immunol 2013;33:1100–9.
91 Klip H, Verloop J, van Gool JD et al. Hypospadias in sons
of women exposed to diethylstilbestrol in utero: a cohort
study. Lancet 2002;359:1102–7.
92 Greer JM, McCombe PA. The role of epigenetic mechanisms and processes in autoimmune disorders. Biologics
2012;6:307–27.
93 Lucas A. Programming by early nutrition: an experimental
approach. J Nutr 1998;128(Suppl):401S–6S.
94 Choi SW, Friso S. Epigenetics: a new bridge between
nutrition and health. Adv Nutr 2010;1:8–16.
95 Johnson AB, Denko N, Barton MC. Hypoxia induces
a novel signature of chromatin modifications and
global repression of transcription. Mutat Res 2008;640:
174–9.
96 Chen H, Yan Y, Davidson TL, Shinkai Y, Costa M.
Hypoxic stress induces dimethylated histone H3 lysine 9
through histone methyltransferase G9a in mammalian
cells. Cancer Res 2006;66:9009–16.
97 Jung JE, Lee HG, Cho IH et al. STAT3 is a potential
modulator of HIF-1-mediated VEGF expression in human
renal carcinoma cells. FASEB J 2005;19:1296–8.
98 Herrick AL, Rieley F, Schofield D et al. Micronutrient
antioxidant status in patients with primary Raynaud’s
phenomenon and systemic sclerosis. J Rheumatol 1994;
21:1477–83.
99 Sambo P, Baroni SS, Luchetti M et al. Oxidative stress
in scleroderma: maintenance of scleroderma fibroblast
1769
Nezam Altorok et al.
phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH
oxidase complex pathway. Arthritis Rheum 2001;44:
2653–64.
100 Andersen GN, Caidahl K, Kazzam E et al. Correlation
between increased nitric oxide production and
markers of endothelial activation in systemic sclerosis: findings with the soluble adhesion molecules
E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1. Arthritis Rheum 2000;43:
1085–93.
101 Ogawa F, Shimizu K, Muroi E et al. Serum levels of
8-isoprostane, a marker of oxidative stress, are elevated
in patients with systemic sclerosis. Rheumatology 2006;
45:815–8.
102 Svegliati S, Cancello R, Sambo P et al. Platelet-derived
growth factor and reactive oxygen species (ROS)
1770
regulate Ras protein levels in primary human fibroblasts
via ERK1/2. Amplification of ROS and Ras in
systemic sclerosis fibroblasts. J Biol Chem 2005;280:
36474–82.
103 O’Hagan HM, Wang W, Sen S et al. Oxidative
damage targets complexes containing DNA
methyltransferases, SIRT1, and polycomb members to promoter CpG islands. Cancer Cell 2011;20:
606–19.
104 Zhang R, Kang KA, Kim KC et al. Oxidative stress causes
epigenetic alteration of CDX1 expression in colorectal
cancer cells. Gene 2013;524:214–9.
105 Lim SO, Gu JM, Kim MS et al. Epigenetic
changes induced by reactive oxygen species in
hepatocellular carcinoma: methylation of the Ecadherin promoter. Gastroenterology 2008;135:
2128–40, 2140e1–8.
www.rheumatology.oxfordjournals.org