Role of Epigenetics in the Pathogenesis of Systemic Sclerosis

Chao-Fan Liu, Jia-Xuan Tang, Lu-Bing Zhu*, Ming Li

Department of Dermatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

Abstract Systemic sclerosis(SSc)is characterized by immune dysfunction,vasculopathy,chronic fibrosis of skin and internal organs with complex etiology. With the rapid development and the application in biomedicine of epigenetics,accumulating evidence has shown that epigenetics plays an important role in the pathogenesis of SSc.Environmental factors via epigenetics are needed to trigger and maintain for the disease in the subjects with genetic predisposition to SSc. The role of epigenetics in the pathogenesis of SSc includes hypermethylation of the promoter region of nitric oxide synthase and bone morphogenetic protein receptors II, up-regulation of histone deacetylases 4 and 5 expression,and down-regulation of miR-193b and miR-152 in endothelial cells inducing vascular dysfunction;DNA hypermethylation and hypoacetylation of histone H3 and H4 in Friend leukemia virus integration 1 and Kruppel-like factor 5 genes,and the abnormal expression of miR-29,miR-129-5p and miR-135b in fibroblasts causing excessive fibrosis;DNA hypomethylation in the promoter regions of CD11a and CD70 genes in CD4+T cells resulting in immune dysfunction.Studies on the role of epigenetics in SSc are of great significance for better understanding the pathogenic machanism of SSc, which is helpful to find new molecular targets for treating SSc, and consequently, improve the prognosis of SSc.

Keywords:DNA methylation,epigenetics,fibrosis,histone acetylation,immune dysfunction,micro RNA,systemic sclerosis, vasculopathy

Introduction

Systemic sclerosis (SSc) is an autoimmune connective tissue disease of unknown etiology characterized by immune disorders, vasculopathy, and fibrosis of the skin and internal organs.1Genetic factors were initially considered to be the key factors in the onset of SSc,and a family history was considered the highest risk factor for the occurrence of SSc.2However,it was subsequently discovered that SSc has a low concordance in monozygotic twins with the same genetic background.3The concordance rate for SSc in twins is only 4.2%, which is lower than the concordance rate for other autoimmune diseases,such as systemic lupus erythematosus (11.1%), rheumatoid arthritis (12.3%), and primary biliary cirrhosis(77%).2,4Hence, genetic predisposition seems to play a relatively small role in the pathogenesis of SSc.1

Epigenetics refers to the process of heritable changes in gene expression level without alteration of the DNA sequence per se, which ultimately mediates phenotypic changes.5The three main epigenetic mechanisms are DNA methylation, histone modification, and microRNA(miRNA) regulation. An increasing number of studies have shown that epigenetics plays a critical role in the pathogenesis of SSc. For individuals with a genetic susceptibility to SSc,external environmental factors,such as silica dust, chemicals, drugs, and viral infections, may trigger SSc through epigenetic regulation.5Therefore,we performed the literature search on PubMed using the following strategy ((((epigenetics[MeSH Terms]) OR(DNA methylation[MeSH Terms])) OR (histone acetylation[MeSH Terms])) OR (micro RNA[MeSH Terms]))AND (systemic sclerosis, scleroderma[MeSH Terms])from 2010 to 2020, and discussed the recent findings regarding the contribution of epigenetic regulation to vasculopathy, excessive fibrosis, and immune dysregulation in SSc based on the literature searching results(Fig. 1).

Figure 1. Schematic diagram of the epigenetic regulation involved in systemic sclerosis. BMPRII: bone morphogenetic protein receptor II;COL1A2:collagen type I alpha 2 chain;DKK1:dickkopf related protein 1;DNMT:DNA methyltransferase;ECM:extracellular matrix;EndoMT:endothelial-to-mesenchymal transition; EZH2: enhancer of zeste homolog 2; FLI1: Friend leukemia virus integration 1; H3K27me3:Trimethylation at lysine 27 of histone H3;H3K4me3:Tri-methylation at lysine 4 of histone H3;HDAC5:histone deacetylase 5;ITGA9:Integrin alpha 9;JHDM2A:JmjC-domain-containing histone demethylase 2A;KLF5:Kruppel-like factor 5;NOS3:nitric oxide synthase 3;PARP-1:poly ADP-ribosepolymerase-1;SFRP1:secreted Frizzled-related protein 1;SIRT1:Sirtuin 1;SUV39H2:suppressor of variegation 3-9 homolog 2;TET1: ten-eleven translocation 1.

DNA methylation

DNA methylation is the addition of a methyl group from S-adenosylmethionine to the cytosine nucleotides, which is catalyzed by a family of DNA methyltransferases(DNMTs).1As this process often occurs at the cytosinephosphate-guanine (CpG) islands of the gene promoter region, the DNA configuration becomes denser, which hinders the binding of transcription factors to the target gene promoter region, and thus leads to an inhibition of gene transcription. DNA methylation can be reversible,and ten-eleven translocation(TET)enzymes participate in the process of DNA demethylation, which increases transcription activity.1

Vasculopathy

It is currently believed that microvasculopathy is the initial event in the development of SScthat frequently precedes the onset of fibrosis.6Fish et al.7showed that there is a defect in the production of nitric oxide (NO) by microvascular endothelial cells (MVECs) in patients with SSc. It is well knownthatNO,apotentvasodilator,isproducedunderthe action of NO synthase 3 (NOS3) and is essential for maintaining the function of endothelial cells (ECs). Fish et al.7further reported that the promoter region of NOS3 is hypermethylated in SSc MVECs compared with controls,resulting in an insufficient expression of NOS3,and thus a decreased expression of NO, which may be one of the mechanisms of MVEC dysfunction in SSc.

Increased apoptosis of MVECs is also involved in the vasculopathy of SSc. Wang et al.8reported that the apoptosis level of SSc MVECs is closely related to the expression level of bone morphogenetic protein receptor II(BMPRII), which is involved in the classic signaling pathway of bone morphogenetic protein and is beneficial to the survival, proliferation, and apoptosis resistance of MVECs. The decreased expression of BMPRII in SSc MVECs is due to the CpG hypermethylation of its promoter region.8Furthermore, the DNMT1 inhibitor 5-azacytidine suppresses methylation in the promoter region of BMPRII and subsequently restores the expression level of BMPRII,therefore reducing the apoptosis level of SSc MVECs.8

Excessive fibrosis

Excessive collagen deposition is a pivotal factor in the occurrence and development of SSc. It has recently been demonstrated that Friend leukemia virus integration 1(FLI1), a member of the E-twenty-six transcription factor family,plays a critical role in the antifibrotic process.The main mechanism is inhibiting the transcription of the collagen type I alpha 2 chain (COL1A2) gene in dermal fibroblasts (Fb) and weakening of the synthesis of connective tissue growth factor synergistically with Kruppel-like factor 5(KLF5).9Moreover,the CpG hypermethylation of the promoter region of the FLI1 and KLF5 genes results in a lower expression of the antifibrotic factors FLI1 and KLF5 in SSc Fb than in controls,9indicating that DNA hypermethylation plays a crucial role in the overproduction of collagen in SSc.

The prototypical DNMT inhibitor drug 5-azacytidine has been approved by the FDA for treating myelodysplastic syndrome; this drug has recently been found to suppress the collagen content in SSc dermal Fb, possibly by normalizing the hypermethylated promoter region of FLI1,thereby increasing the expression level of FLI1.10In addition to typical DNMT inhibitors, the non-epigenetic modifier ciprofloxacin(a broad-spectrum antibiotic)also plays an antifibrotic role in SSc-associated interstitial lung disease, possibly by downregulating DNMT1 and upregulating FLI1.11

Altorok et al.12revealed that hypomethylation of the promoter regions of integrin alpha 9(ITGA9)results in an elevated expression of ITGA9 in SSc Fb,which promotes the differentiation of myofibroblasts and activation of the transforming growth factor (TGF)-β signaling pathway.In turn,the activated TGF-β pathway further induces the expression of ITGA9,forming a positive feedback loop to promote the activation, proliferation, and differentiation of SSc Fb, hence aggravating fibrosis. In addition to ITGA9, the decreased expression of poly ADP-ribose polymerase-1 (PARP-1) that results from DNA hypermethylation of its promoter region also results in the abnormal activation of the TGF-β signaling pathway in SSc.13Under normal conditions, TGF-β inactivates Smad3 and Smad-dependent signaling pathways by recruiting PARP-1; however, this negative feedback mechanism is dysregulated due to the decreased expression of PARP-1 in SSc,leading to abnormal activation of TGF-β signaling pathways.13

The Wnt/β-catenin signaling pathway is another key pathway involved in the aberrant fibrogenesis of SSc.Dees et al.14reported that the endogenous Wnt inhibitor Dickkopf-related protein 1 and secreted frizzled-related protein 1 are downregulated in SSc,resulting in excessive activation of the Wnt/β-catenin pathway. Notably, this process is strongly associated with the hypermethylation of its related gene promoter.In a bleomycin-induced skin fibrosis model, DNMT inhibitors reactivate Dickkopfrelated protein 1 and secreted frizzled-related protein 1 to inhibit the Wnt pathway,thereby alleviating skin fibrosis.Together, these findings indicate that DNA hypermethylation is responsible for the continuous activation of Wnt/β-catenin signaling in SSc.

In addition to DNMT,DNA demethylases,such as TET family proteins,also participate in the pathogenesis of SSc.Hattori et al.15found that the mRNA level of TET1 in SSc Fb is increased by 1.68-fold compared with that of control Fb. Interestingly, the expression of TET1 is upregulated through the hypoxia-inducible factor-1a-dependent pathway under hypoxic conditions in SSc Fb.Previous studies have shown that hypoxia-inducible factor-1a is a key regulator of fibrogenesis, and contributes to various pathogenic processes in SSc.16Therefore, it seems that epigenetic changes catalyzed by TET1 in hypoxia may play a role in the aberrant fibrosis of SSc.

Immune disorders

Abnormal activation of the immune system, such as the aberrant activation of T-and B-lymphocytes,and excessive autoantibody production by plasma cells are critical pathogenic factors for SSc. The lymphocyte functionassociated antigen 1a chain (CD11a) encoded by ITGAL is one of the costimulatory molecules expressed by CD4+T- and B-lymphocytes. Wang et al.17reported that the expression of CD11a in peripheral blood cells is higher in patients with SSc than in healthy controls due to hypomethylation of the promoter region of ITGAL,which eventually leads to the proliferation and recruitment of inflammatory cells.Moreover,Jiang et al.18observed that hypomethylation of the promoter region of the CD70 gene in SSc CD4+ T cells results in the upregulation of CD70 expression, generating more CD70/CD27 costimulatory molecules between T- and B-lymphocytes; this further stimulates abnormal proliferation of plasma cells and secretion of autoantibodies, which may finally lead to a break in immune tolerance of SSc.

In addition, regulatory T lymphocytes (Tregs) with immunosuppressive effects are essential for the immune regulation of SSc. Wang et al.19reported that hypermethylation of the promoter region of forkhead/winged helix transcription factor 3(FOXP3)in SSc CD4+T cells leads to reduced expression of the pivotal transcription factor FOXP3 during the generation of Tregs, which in turn causes a decrease in the number of Tregs; treatment of SSc CD4+T cells with DNMT inhibitors reverses these changes.Furthermore,the FOXP3 gene is located on the X chromosome,and the hypermethylation level leads to a reduction in FOXP3 expression,which may contribute to the predominance of females among patients with SSc.

Collectively,the increased apoptosis and dysfunction of MVECs,abnormal proliferation and activation of Fb,and immune dysregulation in SSc are closely related to the aberrant level of DNA methylation of related genes,suggesting that regulation of DNA methylation might be a promising target for improving the prognosis of SSc.

Histone modification

Histones include core histones(H2A,H2B,H3,and H4)and linker histones (H1 and H5). Generally, histone modification regulates transcription by changing the chromatin structure, mainly via histone acetylation,methylation, phosphorylation, and ubiquitination.Among these, histone acetylation and methylation are the commonest modifications.1Histone acetylation refers to the addition of acetyl groups to the lysine residues of H3 or H4 under the control of histone acetyltransferases(HATs) to open the gene promoter region and thereby promote gene transcription. The process of histone deacetylation is catalyzed by class I-IV histone deacetylases (HDACs). Class III HDACs are also known as sirtuins(SIRTs)1-7.The methylation status of histone is controlled by histone methyltransferases and histone demethylases.1

Impaired angiogenesis

Tsou et al.20reported that HDAC5 has an anti-angiogenic effect in SSc ECs, and that the expression of HDAC5 is higher in SSc ECs than in control ECs. The tube-forming ability of SSc ECs is restored by knocking down HDAC5,which indicates that the effect of the regulation of HDACs on acetylation status may be involved in the development of an aberrant number and function of blood vessels in SSc.Tsou et al.21revealed that the expressions of histone methyltransferases EZH2 and histone H3 at lysine 27(H3K27me3) are significantly increased in SSc ECs, and that EZH2 inhibits gene transcription by catalyzing H3K27me3. The tube-forming ability of ECs is restored after the knockdown of EZH2 in SSc ECs,demonstrating that an abnormal level of histone methylation is also one of the main causes of impaired angiogenesis in SSc.21

Excessive fibrosis

Noda et al.9found that hypoacetylation of the histones H3 and H4 of the collagen-inhibiting FLI1 and KLF5 genes in SSc Fb represses gene transcription, thereby reducing the expression of FLI1 and KLF5,and promoting the collagen production of SSc Fb.Furthermore,treatment with the HDAC inhibitor trichostatin A suppresses the level of collagen synthesis in Fb,which may be related to the downregulated TGF-β signaling pathway.9These data collectively suggest that histone acetylation modification regulates the level of fibrosis in SSc.

Imatinib mesylate is a selective protein tyrosine kinase inhibitor that has been widely used to treat blood diseases,such as chronic myeloid leukemia. Asano et al.22further found thatimatinibincreases the binding activityof FLI1 to the promoter of collagen type I alpha 1 chain (COL1A1)and improves the protein stability of FLI1 by affecting the phosphorylation-acetylation cascade, which consequently ameliorates skin fibrosis in patients with SSc. Moreover,numerous clinical and experimental studies have proved that the dual endothelin receptor antagonist bosentan not only improves blood vessel function,but also reduces the phosphorylation levels of FLI1 and increases its protein stability, thus hindering the development of dermal fibrosis.23Inconclusion,reversibleepigeneticmodifications such as demethylation and hyperacetylation of FLI1 might be a promising SSc treatment strategy.

Although some studies have shown that class III HDACs,especially SIRT1,SIRT3,and SIRT7,may exert an inhibitory effect on the fibrogenesis of the skin and pulmonary system in SSc,the role of SIRT1 in SSc fibrosis remains controversial.4Wei et al.24reported that SIRT1 has an antifibrotic effect by blocking the transcription of the TGF-β/Smad pathway; thus, resveratrol, which is an activator of SIRT1, ameliorates the fibrosis induced by TGF-β. However,Zerr et al.25suggested that resveratrol enhances the profibrotic effect of TGF-β by activating SIRT1, and that TGF-β/Smad signal transduction is inhibited in Fb by knocking down SIRT1. Thus, the usefulness of resveratrol as a possible antifibrotic drug for patients with SSc is still debatable.Although inconclusive,the findings of the abovementioned studies at least confirm the importance of histone acetylation in the fibrosis of SSc.

In addition to HDACs, HATs are also involved in the activation of the TGF-β pathway.Ghosh et al.26showed that the expression of HAT p300 is elevated in the Fb and skin of patients with SSc compared with controls,and that HAT p300 participates in the process of promoting fibrogenesis mediated by the TGF-β signaling pathway.Furthermore,TGF-β increases the expression of p300 and promotes its recruitment at the COL1A2 locus, thus elevatingthe level ofhistoneH4 acetylationinthe promoter region of COL1A2, which ultimately stimulates collagen synthesis. Accordingly, p300 has become an ideal therapeutic target for the TGF-β-mediated fibrosis pathway.

Histone methylation also participates in SSc fibrosis.The effect of H3K27me3,which is jointly regulated by histone methyltransferase EZH2 and demethylase JMJD3, in relation to gene repression in SSc fibrosis has been widely studied.21,27Bergmann et al.27found that the profibrotic transcription factor FRA2, epigenetically regulated by EZH2, is enhanced in SSc Fb, along with a reduction in H3K27me3 at the promoter region. When there is promotionof EZH2 or inhibition of JMJD3,the expression level of the promoter region H3K27me3 is upregulated while FRA2 is downregulated,and the collagen content is subsequently reduced. These results suggest that an abnormal level of histone methylation is also involved in the excessive fibrosis of SSc.

Immune dysregulation

Wang et al.28reported that B cells present global H4 hyperacetylation and H3K9 hypomethylation in SSc,which is related to changes in the expression of HDAC2/7,JHDM2A, and SUV39H2. Additionally, the level of overall H4 acetylation in B cells is positively correlated with the disease activity of SSc,and HDAC2 expression is negatively correlated with skin thickness, demonstrating that SSc immune dysfunction is associated with dysregulation of histone acetylation.28

In addition to B cells, Van et al.29found that patients with SSc, including early-stage patients, have changes in H3K4me3 and acetylation of lysine 27 of H3(H3K27ac)markers in the whole genome distribution of monocytes related to immune activation;this suggests that epigenetic changes may already exist in the early stage of the disease and are essential for triggering and maintaining SSc.

The abovementioned evidence demonstrates that histone modification plays a vital role in the occurrence and development of SSc. Thus, drugs or molecules related to the regulation of histone modification might be potential novel targets for ameliorating SSc.

Non-coding RNA

Non-coding RNA (ncRNA) is unable to encode proteins, but affects gene expression through a varietyof mechanisms. Among them, miRNA is about 18 to 25 nucleotides in length,and complementary binds to the 3′-untranslated region of the target mRNA to inhibit translation or induce degradation of the target mRNA,resulting in the repression of target gene expression.1NcRNA also includes long ncRNA(lncRNA)that is more than 200 nucleotides long and is mainly involved in RNA silencing,chromatin remodeling,and post-transcriptional regulation.1Table 1 shows the ncRNA involved in the pathogenesis of SSc.

Table 1 Non-coding RNA involved in systemic sclerosis.

Vasculopathy

It is widely known that an impaired urokinase-type plasminogen activator (uPA)-uPA receptor pathway also participates in the vasculopathy of SSc. Iwamoto et al.30observed that miR-193b is significantly downregulated in the Fb and skin of patients with SSc, accompanied by an increase in uPA. When miR-193b is overexpressed or knocked down, the uPA expression is reversely altered,confirmingthatuPAisthedownstreamtargetofmiR-193b.Overall, the downregulation of miR-193b is responsible for the elevation of uPA and aberrant angiogenesis in SSc.Huang et al.31showed that the expression of miR-152,whose target gene is DNMT1, is downregulated in SSc MVECs in comparison to normal controls. Overexpression of miR-152 decreases the expression of DNMT1 in normal MVECs. In contrast, inhibition of miR-152 expression increases DNMT1 expression and reduces NOS3 expression, similarly to SSc MVECs. Taken together, these data show that impaired angiogenesis is associated with the downregulated expression of miR-152 by enhancing the level of DNA methylation of NOS3 in SSc MVECs.

Aberrant fibrogenesis

Studies have confirmed that miR-29 has a strong antifibrotic effect in a variety of organs, such as the heart, kidney, and lung, and its effect may be mediated through the TGF-β pathway. The level of miR-29 is decreased in both SSc Fb and a bleomycin-induced mouse model of SSc. Overexpression of miR-29 results in a reduced expression of type I and III collagen in SSc Fb.32Furthermore, miR-29a alleviates the expression of Bcl-2 family proteins that govern cell survival or death.Hence,decreased expression of miR-29 is conducive to collagen production and proliferation of SSc Fb.33

Honda et al.34showed that the expression of miR-150 is significantly lower in SSc Fb than in normal Fb.

The expressions of phosphorylated Smad3 and COL1A1 are reduced due to the overexpression of miR-150.Additionally, the expression of miR-150 is higher after treatment with a DNMT inhibitor.Thus,downregulation of miR-150 expression may be associated with hypermethylation of its promoter region in SSc.34

Nakashima et al.35confirmed that IL-17A inhibits the expression of COL1A1 by downregulating miR-129-5p,but this process is markedly inhibited by the TGF-β pathway in SSc Fb.In contrast to the antifibrotic effect of IL-17A, IL-13 promotes fibrogenesis by upregulating signal transducer and activator of transcription 6 expression; miR-135b, which is downregulated in SSc Fb, also promotes signal transducer and activator of transcription 6 expression, thereby enhancing the profibrotic effect of IL-13 in SSc.36

Wang et al.37showed that SSc Fb have an elevated expression of lncRNA TSIX, which acts on the downstream target genes COL1A1 and COL1A2 to promote fibrosis. Another transcriptomics study identified 676 lncRNAs with differential expression in the skin tissues of healthy controls versus patients with SSc. In particular,three antisense lncRNAs (CTBP1-AS2, OTUD6B-AS1,and AGAP2-AS1) are significantly downregulated in SSc38; the role of these lncRNAs in SSc warrants further investigation.

Immune disorders

Rossato et al.39were the first to demonstrate the effect of miRNA on plasmacytoid dendritic cells(pDC)in SSc.The expression of miR-618 is upregulated in SSc pDC,accompanied by the downregulation of interferon regulatory factor 8 in pDC. More importantly, overexpression of miR-618 inhibits the differentiation and activation of pDC while enhancing the secretion of interferon-α.Thus,dysregulation of miR-618 is considered a vital reason for the increased expression of interferon-α in patients with SSc.

In summary, ncRNA, including miRNA and lncRNA,plays a crucial role in the progression of SSc,and there is an interactive relationship between DNA methylation and histone modification. Accordingly, ncRNA is an important focus in the study of the molecular mechanism of SSc.

Conclusions

The study presents several limitations. First of all, when conducting literature retrieval, we tend to select more classic and general aspects of the epigenetic mechanism of SSc,which may not fully reproduce the complexity of SSc pathogenesis. Secondarily, we seldom discuss the role of lncRNA in the epigenetics of SSc.This may be due to the fact that there are relatively few studies in related fields.Finally,we pay more attention to the basic research of the epigenetics in SSc,and there is less discussion on its clinical application. In future study, we will have a more comprehensive and in-depth understanding of the epigenetic mechanism of SSc.

In conclusion, the exact etiology of SSc has not been completely elucidated. However, recent epigenetics research provides new insights into the pathogenesis of SSc,mainly involving the DNA methylation, histone modification, and ncRNA regulation that contribute to the vasculopathy,collagen deposition,and immune disorders of SSc. The greatest advantage of epigenetics is the dynamicity and reversibility of epigenetic modification compared with genetic mutation. Therefore, epigenetics research is expected to offer novel ideas or methods for alleviating or even curing SSc by modifying the expressions of genes.