Ebook Epigenetics and dermatology: Part 2

C H A P T E R

12

Epigenetics and Systemic
Sclerosis
Nezam Altorok1 and Amr H. Sawalha2,3
1

Division of Rheumatology, Department of Internal Medicine, University of
Toledo Medical Center, Toledo, OH 2Division of Rheumatology,
Department of Internal Medicine, University of Michigan, Ann Arbor, MI
3
Center for Computational Medicine and Bioinformatics, University of
Michigan, Ann Arbor, MI

12.1 INTRODUCTION
Scleroderma is a term that encompasses most forms of thickened and
sclerotic skin, including both localized (morphea, linear scleroderma,
etc.) and systemic sclerosis (SSc) (limited, diffuse) variants. SSc is a complex multisystem autoimmune disease that is characterized by three
pathological hallmarks: activation of the immune system, vascular
injury, and fibrosis of the skin and internal organs [1]. There are two
major subsets of SSc: diffuse cutaneous (dcSSc) and limited cutaneous
(lcSSc) that are distinguished by the extent of skin thickening; lcSSc is
characterized by skin thickening that is confined to the extremities distal
to the elbows and knees with or without facial involvement, whereas
dcSSc is characterized by skin thickening that involves areas proximal
to the elbows and knees, including the trunk [2]. Besides the extent of
skin involvement, the two subsets of SSc have different patterns of
organ involvement, autoantibody profiles, and survival rates. For
instance, patients with lcSSc are at risk for developing subcutaneous calcinosis, telangiectasia, malabsorption, digital ulcers, and pulmonary

hypertension, whereas patients with dcSSc are at high risk for interstitial lung disease, renal failure, diffuse gastrointestinal disease, and

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© 2015 Elsevier Inc. All rights reserved.


250
TABLE 12.1

12. EPIGENETICS AND SYSTEMIC SCLEROSIS

Examples of Environmental Agents Linked to SSc

Occupational
exposures

Welding; silica dusts; toxic oil; xenobiotics; pesticides; ultraviolet
light exposure; organic solvents; epoxy resins; benzene;
trichloroethylene; xylene; urea formaldehyde; and vinyl chloride

Infectious agents

Human cytomegalovirus

Diet

L-Tryptophan

Drugs

Methysergide; pentazocine; cocaine; talc; heroin; bleomycin;
ethosuximide; vitamin K; and amphetamines

myocardial involvement. Anti-topoisomerase I (Scl-70) and anti-RNA
polymerase antibodies are common in dcSSc, and anti-centromere antibodies are more common in the lcSSc subset.
Despite significant efforts, the identity of the initial trigger(s) of SSc
remains a major challenge. Current hypotheses suggest a possible infectious or perhaps chemical agent that activates the immune system that,
in turn, causes vascular injury/dysfunction and persistent activation of
fibroblasts [3]. The end product of this interaction is deposition of collagens and extracellular matrix (ECM) glycoproteins in organs, which
cause organ damage and dysfunction.
Over the last few years it became evident that substantial epigenetic aberrancies are present in SSc. These findings stem from candidate gene and epigenome-wide studies and are supported by the
striking geographic clustering of SSc [4]. These observations suggest
a role for an epigenetic program in the pathogenesis of SSc, driven
by epigeneticÀenvironmental factors. The environmental factors that
are involved in the pathogenesis of SSc are by large uncharacterized.
However, epidemiological and experimental data have linked a
number of occupational exposures to the development of SSc
(Table 12.1).
In this chapter, we briefly discuss the pathogenesis of SSc; we then
explore evidence for epigenetic aberrancies in DNA methylation, histone code, and altered expression of microRNAs (miRNAs) across different cell types that are involved in the pathogenesis of SSc.

12.2 PATHOGENESIS OF SSc
The current paradigm suggests that the pathogenesis of SSc is based
upon a complex interaction between activation of the immune system
and vascular damage, in association with fibroblast activation, which
leads to progressive tissue fibrosis [5].

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251

1. Activation of the immune system
SSc is a connective tissue disorder that is characterized by
chronic deregulation of the immune system. It appears that the
most prominent effect of immune system deregulation occurs in
the early phases of SSc, based on the observation that there are
significant inflammatory cell infiltrates in the skin in the early
phases of SSc [6]. In addition, there is significant upregulation of
growth factors and cytokines in skin and sera samples,
respectively, from patients with SSc [7]. Moreover, SSc is
characterized by the presence of disease-specific circulating
autoantibodies. These observations suggest that activation of the
immune system is a key feature in SSc.
a. T lymphocytes in SSc
T lymphocytes contribute to the pathogenesis of SSc.
Although the total number of peripheral blood T lymphocytes
in SSc is not increased and in fact may be lower than that in
healthy people, there is evidence for activation of circulating
T lymphocytes in SSc [8]. In addition, there is evidence for
T-lymphocyte infiltration in lung and skin tissues in the
early phases of SSc [9].
b. B lymphocytes
B cells, among other immune cells, are activated in SSc, as
manifested by the presence of circulating antibodies,

hypergammaglobulinemia, stimulation of polyclonal B cells,
and overexpression of CD 19 molecules on naı¨ve and memory
B lymphocytes [10]. Although the number of naı¨ve B cells is
increased in SSc, the number of memory B cells is reduced, but
they are activated [11]. Human B lymphocytes are a source of
transforming growth factor-β (TGF-β) and express receptors for
TGF-β [12], and B lymphocytes secrete IL-6. TGF-β and IL-6
may activate fibroblasts and induce upregulation of collagen
production.
c. Other immune cells
In SSc, several cell types contribute to activation of the immune
system; for example, dendritic cells, macrophages, and natural
killer cells play an important role in the production of type I
interferon, which is upregulated in SSc [13].
2. Vascular injury/dysfunction
Vascular damage occurs early in the course of SSc as suggested by
the presence of Raynaud’s phenomenon. There is evidence for
abnormal microvascular endothelial cell (MVEC) function and
structure in SSc [14]. MVEC dysfunction leads to a host of changes in
the blood vessels, including obliterative vasculopathy, that eventually
results in a state of chronic tissue ischemia [3].

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12. EPIGENETICS AND SYSTEMIC SCLEROSIS

3. Role of fibroblasts in SSc

Fibroblasts play an important role in the pathogenesis of SSc,
especially considering that fibroblasts are the most proximate cell for
collagen production. In comparison to normal fibroblasts, SSc
fibroblasts produce more collagen [15] and are characterized by
increased proliferation and decreased apoptosis in vitro [16]. Moreover,
SSc fibroblasts exhibit increased responsiveness to TGF-β [17], and in
response overexpress α-smooth muscle actin (α-SMA), which is a
marker of myofibroblasts. Additionally, fibroblasts play a role in
activation of the immune system via production of numerous cytokines
and chemokines and upregulation of adhesion and costimulatory
molecules. Fibroblasts are frequently detected near small blood vessels
surrounded by inflammatory cellular infiltrate in the early stages of SSc
[18]. These observations highlight an important role for fibroblasts in
the pathogenesis of SSc that goes beyond collagen production and
expansion of ECM, to involve activation of the immune system.
The TGF-β signaling pathway is the most potent stimulus for
myofibroblast differentiation as demonstrated by a robust fibrotic
response upon exposure of fibroblasts to TGF-β, along with
upregulation of matrix gene expression, and myofibroblast
transformation [19]. Other fibrotic pathways are also important in SSc,
such as the Wnt/β-catenin, Hedgehog, and JaggedÀNotch signaling
pathways. Collagen gene transcription in fibroblasts is modulated by
several profibrotic cytokines and transcription factors. Friend leukemia
integration-1 (Fli-1) is one of the transcription factors that repress
expression of collagen [20]. Fli-1 is among the transcription factors that
are underexpressed in SSc fibroblasts. SMAD3 is a profibrotic factor in
the TGF-β downstream signaling cascade [21], whereas SMAD7 is an
inhibitory factor that modulates TGF-β signaling [22]. There is
convincing evidence suggesting that deregulation of these factors and
pathways in SSc fibroblasts results in an imbalance that favors

increased collagen expression and tissue fibrosis.

12.3 GENETIC FACTORS IN SSc
Genome-wide and candidate-gene association studies have identified
several genetic susceptibility loci in SSc (PTPN22, STAT4, IRF5, TNFSF4,
SOX5, CD247, TBX21, CTGF, BANK1, FAM167A, HGF, C8orf13-BLK,
KCNA5, NLRP1, CD226, IL2RA, IL12RB2, TLR2, and HIF1A, as well as
several loci in the HLA region) [23,24]. However, it appears that genetic
factors account for a small proportion of SSc heritability [25].
Concordance rate calculations between twin pairs help in identifying the

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253

respective contributions of genetics and the environment in disease pathogenesis. Studies have demonstrated low concordance rates in SSc monozygotic twins, which are not different from the rates seen in dizygotic
twins (B5%) [25]. Indeed, these observations underscore a prominent
role for epigeneticÀenvironmental factors in the pathogenesis of SSc.

12.4 EPIGENETIC ABERRANCIES IN SSc
In general, epigenetic mechanisms regulate several aspects of chromatin structure and function, including regulation of the chromatin configuration and accessibility of the transcriptional machinery to gene
regulatory regions. In this section, we will explore the evidence supporting the role of aberrancies in the three epigenetic programs (DNA methylation, histone code modification, and altered expression of miRNAs)
involved in the pathogenesis of SSc.
1. Fibroblasts
It is not surprising to see that most of the studies that have
evaluated epigenetics in SSc used dermal fibroblasts, because the
skin is the most common tissue involved in SSc and is easily

accessible for biopsy.
a. DNA methylation aberrancies in fibroblasts
The evidence is expanding regarding the role of DNA
methylation aberrancies in the pathogenesis of SSc. Genome-wide
methylation studies and studies that evaluated candidate-gene
DNA methylation have provided new insights into the role of
DNA methylation in the pathogenesis of SSc.
1. Altered DNA methylation maintenance factors in SSc
Similarly to the situation with other autoimmune diseases,
the molecular mechanism by which DNA methylation is
regulated in patients with SSc is still elusive, but there is
evidence of altered levels of epigenetic maintenance
mediators—specifically, increased expression levels of DNMT1
in cultured SSc fibroblasts, increased expression of methyl-CpG
DNA-binding protein 1 (MBD-1), MBD-2, and methyl-CpGbinding protein 2 (MeCP-2) in SSc fibroblasts [26].
Theoretically, these observations may explain the ability of
cultured fibroblasts to maintain SSc phenotype over multiple
generations by cellular epigenetic inheritance.
2. TGF-β signaling pathway
TGF-β is considered one of the master-regulators of fibrosis.
It is generally accepted that activation of the TGF-β signaling
pathway in SSc leads to a cascade of fibroblast activation [27]

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12. EPIGENETICS AND SYSTEMIC SCLEROSIS

and promotes the transition of fibroblasts and precursor cells
toward persistently activated fibroblast phenotype, and
upregulation of collagen and ECM [28]. Genome-wide DNA
methylation studies have shed light on altered DNA
methylation in genes that are important in activation of the
TGF-β signaling pathway. For instance, ITGA9, which encodes
for α integrin 9, is hypomethylated and overexpressed in SSc
fibroblasts compared to controls [29]. Integrins are a family of
transmembrane receptors that bind extracellularly to the ECM
and intracellularly to the cytoskeleton, thereby “integrating” the
extracellular environment with the cell interior to control cell
behavior [30]. There is an interesting bidirectional interaction
between integrins and TGF-β signaling in fibrosis, with TGF-β
inducing integrin expression and several integrins directly
controlling TGF-β activation including regulation of TGF-β
downstream signaling pathway components [31]. Upregulation
of integrins has been demonstrated in SSc fibroblasts [32À34]
and lung fibroblasts from patients with lung fibrosis [35]. There
is evidence that integrins contribute to fibroblast activation,
persistent myofibroblast phenotype [36], and activation of TGFβ in fibrotic diseases [37]. Moreover, in the same study,
ADAM12 was hypomethylated and overexpressed in SSc
fibroblasts. ADAM12 contributes to the process of fibrosis
through enhancing TGF-β signaling [38À41]. Thus, in light of
these observations, there appears to be a role for DNA
methylation in upregulation of ITGA9 and ADAM12 that in
turn contributes to persistent activation of the TGF-β pathway,
which leads to tissue fibrosis in SSc.
3. Epigenetic aberrancies in transcription factors that are involved in
collagen gene expression
As set forth, there is an imbalance between profibrotic and

antifibrotic factors in SSc. There is evidence that levels of Fli-1,
which is a transcription factor encoded by the FLI1 gene, are
significantly reduced in SSc fibrotic skin and cultured SSc
fibroblasts compared with healthy controls [20]. Fli-1 is a
negative regulator of collagen production by fibroblasts.
Therefore, it appears that reduced levels of Fli-1 may be
responsible for increased collagen synthesis and accumulation
in patients with SSc. Of interest, studies have demonstrated
heavy methylation of the promoter region of FLI1 in SSc
fibroblasts [26]. Indeed, exposure of SSc fibroblasts to 5azacytidine (5-AZA), a universal demethylating agent (DNMT1
inhibitor), resulted in reduced type I collagen production
in vitro. These observations demonstrate that DNA methylation

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255

aberrancies contribute to excessive collagen production in SSc
fibroblasts. It is difficult to draw conclusions regarding the
potential for 5-AZA as a treatment modality in fibrosis based
on this evidence, as other profibrotic factors could be
overexpressed due to the global demethylation effect of 5-AZA
on the genome, and, hence, there is a risk of paradoxical
activation of the fibrotic process or autoimmunity.
Furthermore, there is evidence for DNA methylation
aberrancies in genes encoding transcription factors that are
indirectly involved in collagen production. RUNX1 and RUNX2

are transcription factors that induce expression of SOX5 and
SOX6, which leads to the induction of type II collagen
expression [42,43]. RUNX3, another member of the RUNX
family, is also likely to contribute to collagen synthesis in
association with RUNX2 [44]. Hypomethylation of RUNX1,
RUNX2, and RUNX3 associated with overexpression of at least
RUNX3 in dcSSc and lcSSc has been established [29]. These
data indicate that alteration of DNA methylation could affect
expression of transcription factors that play a role in collagen
production by SSc fibroblasts.
4. DNA methylation aberrancies in collagen and ECM-protein encoding
genes
Tissue fibrosis is the most prominent clinical manifestation
in SSc. Fibrosis is the result of excessive production of collagen
and ECM components, or defective remodeling of the ECM.
Studies have confirmed hypomethylation and overexpression of
two collagen genes (COL23A1, COL4A2) in dcSSc and lcSSc
fibroblasts compared to control fibroblasts [29], in addition to
hypomethylation of several collagen genes in each subset
separately [29]. Moreover, TNXB was hypomethylated in dcSSc
and lcSSc fibroblasts [29]. TNXB encodes a member of the
tenascin family of ECM glycoproteins, which are involved in
matrix maturation [45].
5. The Wnt/β-catenin signaling pathway
There is an increasing interest in the role of the Wnt/
β-catenin signaling pathway as one of the profibrotic pathways
in SSc. Studies have demonstrated persistent activation of the
Wnt/β-catenin pathway as demonstrated by localization of
β-catenin in fibroblast-like cells present in affected tissues [46].
Moreover, stimulation of normal fibroblasts with Wnt ligands

results in β-catenin-mediated expression of collagen and other
matrix genes, and enhanced myofibroblast differentiation and
increased cell migration as expected in SSc [47,48]. In SSc,
canonical Wnt signaling is activated by overexpression of Wnt

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12. EPIGENETICS AND SYSTEMIC SCLEROSIS

proteins and by downregulation of the endogenous Wnt
antagonists. The intensity and duration of Wnt/β-catenin
signaling is normally tightly regulated by endogenous inhibitors.
There is evidence of reduced expression of the endogenous Wnt
antagonists, DKK1 and SFRP1, due to hypermethylation of the
promoter region of DKK1 and SFRP1 in SSc fibroblasts [49]. On
the other hand, there is evidence of hypomethylation of genes
representative of the Wnt/β-catenin pathway in SSc—specifically,
we demonstrated recently hypomethylation of CTNNA2 and
CTNNB1 in dcSSc fibroblasts, and CTNNA3 and CTNND2 in lcSSc
fibroblasts compared to control fibroblasts [29]. These findings
suggest that DNA methylation aberrancies contribute to
decreased expression of Wnt antagonists and increased
expression of Wnt ligands and probably contribute to chronic
activation of Wnt/β-catenin pathway signaling in SSc.
6. Cadherins
Cadherins are a group of transmembrane glycoproteins that
mediate calcium-dependent homophilic cell-to-cell adhesion at

adherens junctions [50]. Microarray studies have demonstrated
overexpression of CDH11, which encodes cadherin-11, in
fibroblasts from patients with SSc [51,52]. Moreover, Cdh11deficient mice developed less fibrosis in bleomycin-induced
fibrosis [53]. There is evidence for hypomethylation of CDH11 in
dcSSc fibroblasts in comparison to fibroblasts from healthy controls
[29]. It is possible that hypomethylation of CDH11 contributes to its
overexpression, which facilitates the differentiation of resident
tissue fibroblasts into myofibroblasts in SSc.
7. The methylome in dcSSc versus lcSSc fibroblasts
Recently, a genome-wide DNA methylation study
demonstrated an interesting difference in DNA methylation
aberrancies between dcSSc and lcSSc subsets in reference to
healthy fibroblasts. The study demonstrated 3528 differentially
methylated CpG sites in SSc, of which there were only 203
(B6%) CpG sites differentially methylated in both dcSSc and
lcSSc. This finding suggests an interesting divergence of the
DNA methylome at the genome-wide level between dcSSc and
lcSSc that reflects heterogeneity at the epigenome level in
scleroderma subsets [29]. Therefore, it is prudent to evaluate
DNA methylation aberrancies and probably other epigenetic
mechanisms in SSc in subset-specific approaches.
b. Histone modification aberrancies in SSc fibroblasts
We have discussed DNA hypermethylation and repression of
FLI1 in SSc fibroblasts early in this chapter. It is interesting to note
that there is also significant reduction of histone H3 and H4

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257

acetylation in the promoter region of the FLI1 gene in SSc
fibroblasts compared to healthy fibroblasts [26]. Moreover,
trimethyl histone H3 on lysine 27 (i.e. H3K27me3), which is a
potent repressor mark for target gene transcription, is increased in
SSc fibroblasts in comparison with controls [54]. Altogether, these
observations indicate that there are defects in the histone code in
SSc, and that cross-talk between DNA methylation and histone
modification changes can be involved in the pathogenesis of SSc,
as demonstrated by FLI1 repression in SSc fibroblasts.
c. miRNA expression aberrancies in fibroblasts
Briefly, miRNAs are small noncoding RNAs (generally 19À25
nucleotides in length) that play important regulatory roles mainly
by cleavage or translational repression of targeted mRNAs. Many
miRNAs are reported to be differentially expressed in SSc,
suggesting that miRNA dysregulation plays a role in the
pathogenesis of SSc.
1. miRNA regulation of the TGF-β signaling pathway
TGF-β mediates fibrosis positively by activating its
downstream mediators, SMAD2 and SMAD3, but negatively
via its inhibitory factor SMAD7. miR-21, which is upregulated
in SSc fibroblasts [55], targets SMAD7. Overexpression of miR21 in SSc fibroblasts decreases levels of SMAD7, whereas
knockdown of miR-21 increases SMAD7 expression [56,57].
Therefore, miR-21 probably exerts a profibrotic effect by
negatively regulating SMAD7 in SSc fibroblasts.
Altered expression of several other miRNAs in SSc with
putative targets in the TGF-β downstream pathway (such as miR145, miR-146, and miR-503) has also been demonstrated
(Table 12.2).

2. miRNAs directly target collagen genes in SSc
miR-29 underexpression was reported in skin fibroblasts from
patients with SSc, as well as fibroblasts from the mouse model of
bleomycin-induced skin fibrosis [65]. It was demonstrated that
induced expression of miR-29 in SSc fibroblasts reduces the
expression of its target genes, and collagen type I and type III.
Other potential targets for miRNA-29 include profibrotic molecules
such as platelet-derived growth factor B (PDGF-B) and
thrombospondin. Of significant interest, the stimulatory effects of
TGF-β and PDGF-B on collagen synthesis were reduced by
inducing the expression of miR-29 [65]. On the other hand,
downregulation of miR-29 leads to further upregulation of TGF-β
and PDGF-B. Taken together, these data argue for an antifibrotic
role of miR-29 in SSc.

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TABLE 12.2

Gene/pathway

12. EPIGENETICS AND SYSTEMIC SCLEROSIS

Summary of Key Epigenetic Aberrancies Reported in SSc

Epigenetic defect

Cell type

Putative target/
mechanism of
action in SSc

References

DNA METHYLATION
COL4A2yz,
COL23A1yz,
COL8A1y,
COL16A1y,
COL29A1y,
COL1A1z,
COL6A3z,
COL12A1z

Hypomethylation

Fibroblasts

Likely contributes to
overexpression of
collagen genes

[29]

PAX9yz

Hypomethylation

Fibroblasts

Hypomethylation of
PAX9 may
contribute to the
process of fibrosis
by overexpression
of pro-α 2 chain of
type I collagen

[29]

TNXByz

Hypomethylation

Fibroblasts

Unclear; possible
overexpression of
ECM glycoproteins

[29]

ITGA9yz

Hypomethylation

Fibroblasts

Hypomethylation of
ITGA9 contributes
to ITGA9
overexpression in
SSc. ITGA9 plays an
integral role in
myofibroblast
differentiation and
activation of TGF-β
signaling pathway

[29]

RUNX1yz,
RUNX2yz,
RUNX3yz

Hypomethylation

Fibroblasts

Indirectly induce
expression of type II
collagen by
increasing
expression of SOX5
and SOX6

[29]

ADAM12yz

Hypomethylation

Fibroblasts

ADAM12
overexpression in
SSc contributes to
fibrosis by inducing
TGF-β signaling
pathway

[29]

(Continued)

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12.4 EPIGENETIC ABERRANCIES IN SSc

TABLE 12.2

(Continued)
Putative target/
mechanism of

action in SSc

References

Gene/pathway

Epigenetic defect

Cell type

CTNNA2y,
CTNNB1y
CTNNA3z,
CTNND2z

Hypomethylation

Fibroblasts

Unclear;
hypomethylation of
these genes that are
components of the
embryonic Wnt/
β-catenin pathway
may be contributing
to the observed
recapitulation of
Wnt/β-catenin
pathway in SSc

[29]

DKK1, SFRP1

Hypermethylation

Fibroblasts,
PBMCs

Reduced expression
of Wnt/β-catenin
antagonists

[49]

PDGFCy

Hypomethylation

Fibroblasts

A profibrotic factor
that is
overexpressed in
SSc

[29]

CDH11y

Hypomethylation

Fibroblasts

Overexpression of
CDH11 induces
myofibroblast
differentiation

[29]

FLI1y

Hypermethylation

Fibroblasts

Overexpression of
collagen genes in
SSc

[26]

BMPRIIy

Hypermethylation

MVECs

Failure of the
inhibitory
mechanism for cell
proliferation and
induction of
apoptosis

[59]

NOS3

Hypermethylation

MVECs

Reduced NOS
activity in MVECs;
increased
expression of
proinflammatory
and vasospastic
genes

[60]

CD40L

Hypomethylation

CD41 T

cells

Costimulatory
molecule

[61]

CD70
(TNFRSF7)

Hypomethylation

CD41 T
cells

Costimulatory
molecule

[62]
(Continued)

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12. EPIGENETICS AND SYSTEMIC SCLEROSIS

TABLE 12.2

(Continued)

Gene/pathway

Epigenetic defect

Cell type

CD11a (ITGAL)

Hypomethylation

CD41 T
cells

Putative target/
mechanism of
action in SSc
Involved in
costimulatory
signaling

References
[63]

HISTONE MODIFICATIONS
H3K27me3

Increased

Fibroblasts,
murine
dermal
fibroblasts

May contribute to
inhibition of
collagen suppressor
genes and,
therefore, collagen
deposition

[54]

Global H4
acetylation,
H3K
methylation

Increased H4
acetylation,
decreased H3K
methylation

B cells

Favors target gene
expression in B
cells; could be
contributing to

activation of genes
in the immune
system and
antibody production

[64]

FLI1 H3 and H4
acetylationy

Reduced

Fibroblasts

Repression of FLI1;
therefore,
overexpression of
collagen genes

[26]

microRNA
y

miR-29

Downregulated

Fibroblasts,
murine

dermal
fibroblasts

Antifibrotic factor;
probable target is
type I and type III
collagen

[55,65,66]

miR-21y

Overexpressed

Skin tissue,
fibroblasts,
murine
dermal
fibroblasts

Profibrotic factor;
targets SMAD7.
Upregulates
canonical and
noncanonical TGF-β
signaling pathways

[55,56]

miR-142yz

Overexpressed

Serum

Seems to be
involved in
regulating the
expression of
integrin αV

[67]

miR-196ayz

Downregulated

Fibroblasts,
serum, and
hair shafts

Predicted target is
type I collagen

[68,69]

(Continued)

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261

12.4 EPIGENETIC ABERRANCIES IN SSc

TABLE 12.2

(Continued)

Gene/pathway

Epigenetic defect

Cell type

Putative target/
mechanism of
action in SSc

miR-145y

Downregulated

Skin tissue,
fibroblasts

Predicted target is
SMAD3

[55]

miR-146y

Overexpressed

Skin tissue,
fibroblasts

Predicted target is
SMAD4

[55]

miR-152

Downregulated

MVECs

Predicted target is
DNMT1

[70]

miR-503y

Overexpressed

Skin tissue,
fibroblasts

Predicted target is
SMAD7

[55]

miR-7

Overexpressed

Fibroblasts,
skin, serum

Predicted target is
type I collagen

[71]

miR let-7a

Downregulated

Fibroblasts,
serum

Predicted target is
type I collagen

[72]

miR-92-ay

Overexpressed

Fibroblasts,
serum

Predicted target
MMP-1

[57]

miR-150y

Downregulated

Fibroblasts,
serum

Predicted target is
integrin β3

[73]

miR-129-5p

Downregulated

Fibroblasts

Predicted target is
type I collagen

[74]

References

The study design and analysis allows for distinction between ydcSSc and zlcSSc; yzthe finding was
reported in both dcSSc and lcSSc.
BMPRII, bone morphogenetic protein type II receptor; DNMT1, DNA (cytosine-5-)-methyltransferase 1;
ECM, extracellular matrix; H3K27me3, trimethylation of histone H3 on lysine 27; MBD1, methyl-CpGbinding domain protein1; MVEC, microvascular endothelial cells; MMP-1, matrix metalloproteinase 1;
NOS, nitric oxide synthetase; PBMCs, peripheral blood mononuclear cells; SMAD, intracellular
proteins that transduce extracellular signals from TGF-β ligands; TGF-β, transforming growth factor-β.
Reproduced and modified with permission [58].

Moreover, several studies have demonstrated
downregulation of other antifibrotic miRNAs (such as miR196a, miR let-7a, and miR-129-5p), or increased expression of
profibrotic miRNAs (such as miR-7) in SSc fibroblasts. The
putative target for the aforementioned miRNAs is probably
type I collagen [68,71,72,74] (Table 12.2).
2. MVECs
a. DNA methylation alterations in nitric oxide synthesis
It has been demonstrated that there are intrinsic defects in the
mechanism of nitric oxide (NO) production by MVECs isolated
from SSc patients [75]. NO is a potent vasodilator and an inhibitor

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12. EPIGENETICS AND SYSTEMIC SCLEROSIS

of smooth muscle cell growth. Also, NO has antithrombotic,
antiplatelet, and antioxidation properties [76]. NO is produced
partly by MVEC by the action of nitric oxide synthase (NOS).
There is evidence for underexpression of NOS3, the gene encoding
endothelial NOS in SSc-MVECs, and that the promoter region of
NOS3 is hypermethylated in SSc-MVEC compared to controls [60].
This finding indicates that the epigenetic program contributes to
MVEC dysfunction in SSc.
b. DNA methylation in MVEC apoptosis
Enhanced MVEC apoptosis is one of the pathogenic
manifestations of SSc. It has become apparent that MVEC
apoptosis could be an initial element in the pathogenesis of SSc,
and that MVEC apoptosis may even precede the onset of the
fibrotic stage [77]. Bone morphogenetic proteins (BMPs) are a
group of proteins that constitute morphogenetic signals and
orchestrate tissue architecture through coordinating cell survival
and differentiation. BMP signaling through bone morphogenic
protein receptor II (BMPRII) favors MVEC survival and apoptosis
resistance. There is evidence for reduced expression of BMPRII in
SSc-MVECs in comparison with healthy controls which could be
related to heavy methylation of the promoter region of BMPRII in
SSc-MVECs compared to healthy controls [59]. In the same study
[59], treatment of SSc-MVECs with 5-AZA normalized BMPRII
expression levels and restored SSc-MVEC response to apoptosis to
normal levels. Therefore, it seems that DNA methylation may play
a role in MVEC response to apoptosis in SSc.
c. miRNA aberrant expression in MVEC

Most of the studies that evaluated miRNA expression in SSc
have focused on dermal fibroblasts; very few studies have
evaluated the extent of aberrant miRNA expression in SSc-MVEC.
It appears that miR-152 is downregulated in SSc-MVEC and the
target for miR-152 is DNMT1 [70]. Forced expression of miR-152 in
MVEC led to increased expression levels of DNMT1, whereas
inhibition of miR-152 expression led to enhanced DNMT1
expression and lower expression levels of NOS3. These data
indicate that miR-152 plays a role in the SSc-MVEC phenotype
probably through DNA methylation.
3. Lymphocytes
a. DNA methylation aberrancies in T lymphocytes
It has been established that DNA methylation is a natural
physiological process to maintain inactivation of one X
chromosome in order to keep a balance among genes encoded
on the X chromosome in males and females [78]. CD40L is a
costimulatory molecule that is expressed predominantly on the

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263

surface of activated T cells. The main function of CD40L is to
regulate B-cell function by engaging CD40 on the B-cell
surface. Studies have demonstrated increased expression of
CD40L, which is encoded on the X chromosome, in female SSc
patients, associated with demethylation of the promoter region

of CD40L on the inactive X chromosome in CD41 T cells.
Moreover, there was no difference in CD40L expression
between male patients with SSc and male controls [61]. The
same observation of hypomethylation and overexpression of
CD40L was reported in SLE [79]. These data indicate that there
are defects in the epigenetic program, which leads to
reactivation of genes that are located on the naturally silenced
X chromosome in female patients with autoimmune diseases
like SLE and SSc, which may explain female predominance in
autoimmune diseases.
The CD70/CD27 axis has gained interest in autoimmune
diseases because of its capacity to regulate immunity versus
tolerance. CD70 is another costimulatory molecule that is
expressed on activated lymphocytes and plays an important role
in regulating B- and T-cell activation. CD70 is overexpressed in
SSc CD41 T cells, and there is evidence that demethylation of the
CD70 promoter region contributes to the overexpression of CD70
in CD41 T cells [62]. Overall, these data suggest that DNA
methylation aberrancies contribute to overexpression of
costimulatory molecules, but it remains to be seen whether CD40L
and CD70 signaling are involved in the pathogenesis of SSc to the
same extent that these molecules are involved in the pathogenesis
of other autoimmune diseases.
b. Histone code modification in B lymphocytes
Activation of the immune system is one of the pathological
features of SSc. B cells play a special role in the pathogenesis of
SSc, as suggested by the presence of disease-specific circulating
autoantibodies in SSc. Very little is known about epigenetic
aberrancies in SSc B lymphocytes. However, there is an evidence
that B cells from patients with SSc are characterized by global H4

hyperacetylation and global H3 lysine 9 (H3K9) hypomethylation,
associated with downregulation of histone deacetylase 2 (HDAC2)
and HDAC7 compared to B cells from healthy controls [64]. The
aforementioned modifications of the histone code favor
permissive chromatin architecture for gene expression. It is not
clear at this stage what could be the effect of these changes on
B-lymphocyte function, but it is suggested that this histone code
in SSc B-lymphocytes might enhance overexpression of
autoimmunity-related genes in SSc [64].

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12. EPIGENETICS AND SYSTEMIC SCLEROSIS

12.5 WHAT MIGHT TRIGGER EPIGENETIC
DYSREGULATION IN SSc?
If the contribution of epigenetic aberrancies to pathogenesis of autoimmune diseases, like SSc, is becoming increasingly clear, the trigger(s)
that induce the defects in the epigenetic program are less so. We will
present in this section some theories about the triggers that are largely
driven by observational studies in SSc as well as data from epigenetic
deregulation in general.
Much of the current interest about epigenetics and human diseases
stems from the idea that the modifications in the epigenetic program
are sensitive to environmental factors. However, the environmentalÀ
epigenetic triggers remain largely uncharacterized with few exceptions.
One of the central obstacles hampering progress in identifying the
environmentalÀepigenetic triggers in general is complicated by the temporality and causality issue; where epigenetic changes take place prior to

the onset of disease, even in some cases, it appears that disease may
occur one or two generations after the exposure [80,81]. With regard to
triggers of epigenetic deregulation in SSc, it seems that external factors
(e.g., exposure to organic solvents, silica exposure, UV light, toxins, diet,
drugs, and infective factors, particularly human cytomegalovirus)
(Table 12.1), and internal factors (e.g., hypoxia, oxidative stress, aging,
and sex hormones) could be possible candidates [58].
a. Occupational exposures
The observation of geographical clustering of SSc and the
epidemiological studies that linked SSc to exposure to occupational
agents suggest that the environment plays a role in predisposition to
SSc in susceptible hosts. However, the “causality” inference of
occupational exposure in the pathogenesis of SSc is challenging; in
most cases, it is hard to identify a single occupational agent due to the
complexity of our environment, which is characterized by exposure to
numerous chemical and toxic agents every day. Also, the “pathogenic
environment” in epigenetics has not yet been characterized. Moreover,
the timing of environmental exposure is difficult to identify, which
makes recall of exposure even more difficult. These factors, in addition
to the retrospective caseÀcontrol design of the studies that have
reported a link between SSc and specific occupational agents, make the
identification of the environmental trigger of SSc a challenge.
b. Diet and nutrition
While there is so far very little evidence to suggest that a
particular diet is specifically linked to predisposition to SSc, there is
a piece of evidence that the susceptibility to chronic disease is
influenced by persistent adaptations to prenatal and early postnatal

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265

nutrition [82]. The nutritional element in predisposing to SSc is not
clear, but abnormalities in the availability of methyl donors
(methionine and choline) and cofactors (folic acid, vitamin B12, and
pyridoxal phosphate) may contribute to aberrancies in DNA and
histone methylation. It is also possible that the observed geoepidemiology of SSc may be linked to undefined dietary patterns.
c. Hypoxia
The observation that Raynaud’s phenomenon is usually the
earliest clinical manifestation in the vast majority of patients with SSc
[83], and that Raynaud’s phenomenon usually precedes onset of the
fibrotic phase by several months or years in patients with SSc is
intriguing. Raynaud’s phenomenon is characterized by vasospasm
and reduced tissue perfusion to the distal extremities that causes
intermittent tissue hypoxia and possible endothelial injury which
perpetuate vascular dysfunction. Interestingly, tissue fibrosis starts in
the distal extremities in most patients with SSc, which is the same
site of reduced tissue perfusion in Raynaud’s phenomenon. Hypoxia
itself is a potent stimulus for the synthesis of collagen and its crosslinking enzyme lysyl hydroxylase, fibronectin, and fibrogenic
cytokines [84]. Therefore, these observations suggest the hypothesis
that transient tissue hypoxia due to decreased blood flow related to
Raynaud’s phenomenon might be the trigger for fibrosis through an
epigenetic mechanism. Indeed, it has been established that hypoxia
decreases global transcriptional activity and has a major effect on
cellular phenotype through different mechanisms that include the
hypoxia-inducible factor (HIF) transcription paradigm in eukaryotic
cells through HIF-1, which is a critical transcription regulator of a

majority of genes in response to hypoxia [85]. There is evidence that
epigenetic pathways are also relevant in the adaptation to hypoxia
[86]. Hypothetically, hypoxia evokes the anaerobic metabolism
pathways which lead to lower levels of acetyl-CoA; therefore, it is
possible that hypoxia may lead to a global decrease in histone
acetylation levels [87]. Also, it seems that hypoxia may also induce
HDAC upregulation, which induces a global decrease in H3K9
acetylation in various cells [88].
d. Oxidation
SSc is an oxidative stress state based on the observations that there
are abnormalities in the NO/NOS axis and the presence of increased
levels of oxidative biomarkers in SSc [89,90]. Oxidative stress leads to
excessive generation of oxygen free-radicals and reactive oxygen
species (ROS) [91]. ROS have been implicated in causing vascular
injury and predisposing to autoimmunity in SSc [92]. Interestingly,
there is a cross-talk between oxidative stress and fibrosis, where
oxidative stress stimulates the accumulation of ECM proteins, and

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12. EPIGENETICS AND SYSTEMIC SCLEROSIS

fibrosis generates more oxidative stress [91]. Moreover, in fibroblasts,
TGF-β induces the NADPH oxidase enzyme NOX4, which catalyzes
the reduction of oxygen to ROS. In turn, ROS act as signals to induce
fibroblast activation and myofibroblast differentiation [93].
Interest in the role of oxidative stress in epigenetic regulation is

growing, especially the role of oxidative stress in controlling DNA
methylation. It was demonstrated recently that oxidative stress could
contribute to impaired T-cell extracellular signal-related kinase (ERK)
pathway signaling in SLE, which is another autoimmune disease that
shares several clinical features with SSc that include, but are not limited
to, the presence of Raynaud’s phenomenon and the presence of
circulating autoantibodies. There is evidence that oxidative stress
disrupts ERK signaling in CD41 T cells in vitro, therefore reducing
DNMT1 expression and consequently causing demethylation and
overexpression of methylation-sensitive genes that have been
previously shown to be upregulated in patients with SLE, like CD70
[94]. It remains to be seen whether the oxidative stress effect on ERK
pathway signaling also applies to SSc, and whether antioxidants like
N-acetylcysteine could have therapeutic benefit in treatment of
autoimmune diseases by reversing the oxidative stress state.

12.6 CLINICAL RELEVANCE OF EPIGENETIC
ABERRANCIES IN SSc
We have presented the evidence for epigenetic alterations in different
programs involving several cell types in SSc. In this section, we will
look at these aberrancies from a clinical perspective and discuss the
value of the epigenetic alterations as diagnostic markers, and perhaps
the potential use of the knowledge that we gained from studying epigenetic alterations in SSc as therapeutic strategies.
A potential diagnostic marker, SOX2OT, encodes for one of the long
nonprotein coding RNAs (lncRNAs) that may exert a regulatory role on
stem cell pluripotency [95]. It has been demonstrated that SOX2OT is
hypermethylated across multiple CpG sites in dcSSc fibroblasts, but not
lcSSc fibroblasts, in comparison to control fibroblasts [29]. This observation suggests that the methylation status of SOX2OT might potentially
be a useful marker in differentiating SSc subsets if reproduced and validated in other studies.
There is evidence that expression levels of some miRNAs might correlate with specific features of SSc or with disease activity. For instance,

serum levels of miR-142 correlate with SSc disease severity [67], and
expression of miR-21, miR-29, miR-145, and miR-146 correlates with

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12.7 CONCLUSION

267

disease activity in SSc. Overall, further studies in this field are needed
before miRNAs can be considered useful clinical biomarkers.
There is remarkable interest in finding a disease modifying agent to
treat SSc, based on the unsatisfactory results from most therapeutic
agents that have been used in treatment of SSc up to this date, either
because of nonefficacy (mostly) or because of unacceptable side effect
profile. Therefore, it is not surprising that there is interest in using epigenetic modifying agents in experimental settings in SSc. Trichostatin
(TSA) is one of the HDAC inhibitors that is available for treatment of
myelodysplastic disease. In vitro studies have shown that TSA attenuates expression of collagen I in dermal SSc fibroblasts [96]. Also, TSA
was associated with lower fibrotic end points in an animal model of
skin fibrosis [97]. Despite the fact that these observations suggest a possible role for TSA in the treatment of SSc, the “off-site” effect of TSA, as
an agent with an ability to induce widespread changes in the chromatin,
will perhaps limit TSA usefulness in SSc. Future studies to explore the
use of specific miRNAs as potential treatment strategies in SSc would
be of interest.

12.7 CONCLUSION
In recent years, the field of epigenetics in rheumatic diseases has
grown dramatically and has become one of the paradigms in explaining
the link between environmental exposures and disease susceptibility in

genetically predisposed individuals. The data we have provided in this
chapter suggest new approaches to understand the pathogenesis of a
complex disease like SSc. We have explored several lines of evidence
that confirm substantial epigenetic modifications in SSc, particularly in
fibroblasts, MVECs, and in B and T cells. The evidence extends to
include a role for epigenetic modifications in fundamental pathways
that are involved in the process of fibrosis, such as TGF-β and downstream pathways, and the Wnt/β-catenin signaling pathway. The triggers for the epigenetic alterations in SSc are not clear, but it is
reasonable to suggest a role for occupational exposures, nutritional factors, hypoxia, and oxidative stress as possible triggering mechanisms. It
remains to be determined if epigenetic alterations could be used as biomarkers for disease activity or severity in SSc, or even as therapeutic
strategies. To move the field forward, studies focused on uncovering
the potential pathogenic triggers in SSc, and the mechanisms by which
these triggers induce epigenetic alterations, are warranted. Ultimately,
characterization of the “pathogenic” environment could lead to better
understanding of the disease risk, and probably prevention.

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