The anti-fibrotic factor KLF4 is repressed by the miR-10/TFAP2A/TBX5 axis in dermal fibroblasts: insights from twins discordant for systemic sclerosis

Abstract

Objectives:

Systemic sclerosis (SSc) is a complex disease of unknown etiology in which inflammation and fibrosis lead to multiple organ damage. There is currently no effective therapy that can halt the progression of fibrosis or reverse it, thus studies that provide novel insights into disease pathogenesis and identify novel potential therapeutic targets are critically needed.

Methods:

We used global gene expression and genome-wide DNA methylation analyses of dermal fibroblasts (dFBS) from a unique cohort of twins discordant for SSc to identify molecular features of this pathology. We validated the findings using in vitro, ex vivo, and in vivo models.

Results:

Our results revealed distinct differentially expressed and methylated genes, including several transcription factors involved in stem cell differentiation and developmental programs (KLF4, TBX5, TFAP2A and homeobox genes) and the microRNAs miR-10a and miR-10b which target several of these deregulated genes. We show that KLF4 expression is reduced in SSc dFBs and its expression is repressed by TBX5 and TFAP2A. We also show that KLF4 is anti-fibrotic, and its conditional knockout in fibroblasts promotes a fibrotic phenotype.

Conclusions:

Our data support a role for epigenetic dysregulation in mediating SSc susceptibility in dFBs, illustrating the intricate interplay between CpG methylation, miRNAs and transcription factors in SSc pathogenesis, and highlighting the potential for future use of epigenetic modifiers as therapies.

INTRODUCTION

Systemic sclerosis (SSc) is a complex connective tissue disease whose hallmarks include autoimmunity, inflammation, fibrosis and vasculopathy. SSc predominantly affects women and is the connective tissue disease with the worst survival.¹ Diffuse cutaneous SSc (dcSSc) is characterized by rapid development of skin, lung and other organ fibrosis within 3–5 years, after which skin fibrosis regresses but damages to internal organs persist. Limited cutaneous SSc (lcSSc) shows gradual skin progression with delayed internal organ involvement. Most research studies and clinical trials have focused on dcSSc,² although dermal fibrosis is a hallmark of both dcSSc and lcSSc. The etiology and molecular mechanisms underlying SSc remain elusive and no effective therapeutic treatment exists.

In monozygotic (MZ) twins, the rate of disease concordance is low (4.2%),³ suggesting an important role for epigenetic and environmental factors in SSc susceptibility in addition to a genetic predisposition.⁴ Environmental factors can influence a trait through epigenetic regulation, and epigenetic marks can impact gene expression, thus governing cell function and response to environmental stimuli.⁵ Additionally, early mutations that occurred before the primordial germ cell specification have recently been shown to contribute to phenotypic discordance observed in MZ twins,⁶ a contribution previously underestimated. Due to replication errors, mutations that are specific to one twin happened in 15% of MZ twin pairs, revealing the importance of early cell lineages and mutations that are dependent on DNA methylation. Epigenetic mechanisms play a role in the pathogenesis of autoimmune diseases, and epigenome-wide association studies revealed the existence of differentially methylated (DM) regions associated with systemic lupus erythematosus⁷ and psoriasis,⁸ two other autoimmune rheumatic diseases. Inhibition of DNA methyltransferases has shown promising anti-fibrotic properties in SSc fibroblasts and SSc patients.^9–11 Epigenetic changes have been reported in SSc fibroblasts.^{12, 13} Epigenetic changes in SSc are emerging as important mediators of disease processes including fibrosis and angiogenesis¹¹. As a result, epigenetic modifying drugs may provide viable therapies in SSc.⁹

To our knowledge, our study is the first to combine RNA sequencing (RNAseq) and genome-wide DNA methylation analyses in dFBs, the effector cells in fibrosis¹⁴, of twins discordant for SSc. This unique cohort is the ideal design to assess the role of epigenetic variations in disease etiology.¹⁵

Our study identified several transcription factors (TFs) involved in stem cell differentiation that were DM and differentially expressed (DE) in SSc, including homeobox (HOX) genes, TBX5 and TFAP2A, suggesting that SSc is mediated by an aberrant resumption of developmental pathways. MicroRNAs (miRNAs) of the miR-10 family encoded within HOX gene clusters have a direct regulatory role on these TFs, and are also differentially expressed, highlighting the deregulation of complex mRNA-miRNA regulatory networks in SSc. We also identified KLF4 as a major regulator of fibrosis, cell differentiation and extracellular matrix (ECM) accumulation, and determined that TBX5 and TFAP2A regulate KLF4, which in turn regulates WNT and HOX genes. Mice with conditional fibroblast-targeted loss of KLF4 showed increased dermal hydroxyproline levels and dFBs from these mice had increased expression levels of fibrotic genes. Overexpression of KLF4 in dFBs and human skin tissues in organ culture prevented the TGFβ-induced fibrotic response. Our data suggest that epigenetic events shape the fibrosis in dFBs that leads to SSc.

METHODS

See Supp. File 1 for details of the methods.

RESULTS

Gene expression profiling of twins discordant for SSc

The DE analysis “lcSSc twins vs. healthy twins” (n=8) returned 897 DE genes in dFBs of lcSSc twins (Supp. Fig. 1A; Supp. Table 1); 511 upregulated (q < 0.1, log2FC > 0.6) and 386 downregulated (q < 0.1, log2FC < −0.6) genes. The heatmap showed two clear clusters for healthy and lcSSc samples (Supp. Fig. 1B). To validate these results, another DE analysis was computed for “lcSSc twins vs. unrelated controls” (n=8) and revealed 2164 DE genes in lcSSc twins (Supp. Fig. 2A–B; Supp. Table 2) out of which 308 DE genes are in common with DE genes from the “lcSSc twins vs. healthy twins” comparison (Supp. Fig. 2C; Supp. Table 3). One striking feature of the lcSSc twins’ dataset is that 12 homeobox genes are present, 5 of which are significantly upregulated (HOXB3, HOXB7, HOXB8, HOXC10, PITX1) and 7 are significantly downregulated (HOXB13, HOXD3, HOXD9, HOXD10, HOXD11, HOXD13, HOXD-AS2) (Figure 1A; Supp. Fig. 2D). Of note, WNT5A was significantly upregulated in dFBs of lcSSc twins (Supp. Fig. 2E).

Figure 1: RNAseq and DNA methylation results.

(A) RNAseq revealed that several homeobox genes are differentially expressed in dFBs of lcSSc twins compared to healthy twins and validated with unrelated controls. Significance criteria in RNAseq analysis: q-value < 0.1, log2FC > 0.6 for upregulation, log2FC < −0.6 for downregulation. (B) Validation of several genes of interest by qPCR in lcSSc twins (lcSSc) and unrelated controls (Ctrl). (C) Homeobox genes differentially expressed in dcSSc twins compared to healthy twins. Significance criteria in RNAseq analysis: q-value < 0.1, log2FC > 0.6 for upregulation, log2FC < −0.6 for downregulation. (D) Venn diagram for the comparison of DE genes in dcSSc and lcSSc twins. (E) Genes with increased and reduced methylation status identified in the DNA methylation analysis of dFBs. Significance criteria in DNA methylation analysis: P-value < 10–04, b > 0.20 for increased methylation, b < −0.20 for reduced methylation. (F) Genes that are simultaneously differentially expressed and differentially methylated in dFBs of lcSSc (validated dataset) and dcSSc twins.

The systems level analysis showed enrichment of pathways and ontologies pertaining to ECM components, organ morphogenesis, regulatory region involved in DNA binding, and integral component of the plasma membrane (Table 1; Supp. Table 4). Note that the HOX genes are hit in the query list for “animal organ morphogenesis” and “transcription regulatory region sequence-specific DNA binding” along with several T-box genes (TBX2, TBX3, TBX15) and transcription factors that bind the consensus sequence 5’-GCCNNNGGC-3’ (TFAP2A, TFAP2B) (Supp. Fig. 2E). We used qPCR to validate several genes of interest (Figure 1B). The expression levels of TFAP2A were increased in lcSSc samples compared to unrelated controls while HOXD10 and HOXD11 levels were reduced, in agreement with the RNAseq data. A significant decrease in expression levels of KLF4 was observed, consistent with DE analysis “lcSSc twins vs unrelated controls” (Supp. Table 2). We also detected an increase in HOXA13 levels and a decrease in HOXC5 levels in lcSSc samples (Figure 1B).

Table 1:

Functional enrichment in lcSSc twins.

Enrichment	Hit in Query List
PW: Ensemble of genes encoding ECM and ECM-associated proteins (7.04E-11)	ADAMTSL1, ADAMTS5, ANGPTL1, BMP6, CHRDL1, CILP2, CLEC14A, COL7A1, COL9A3, COL14A1, COL23A1, CRISPLD2, CTSB, CXCL1, CXCL6, EDA, ELFN2, EREG, F7, FBLN7, FBN2, FGF5, FGL2, FRAS1, HAPLN3, IL6, IL11, KAL1, LIF, LOXL3, LOXL4, MMP10, OGN, PCOLCE2, PCSK5, PDGFB, PRG4, RSPO1, SEMA3C, SEMA3E, SRPX2, TGFB3, THBS4, USH2A, WISP1, WNT5A
BP: Animal organ morphogenesis (4.14E-19)	ACTC1, ADAMTS5, ALDH1A2, ALPL, AR, BMP6, CELSR1, CELSR2, COL7A1, COL14A1, DIO3, DLL4, EDA, EMX2, EPHB2, EREG, ETV4, EYA4, FBN2, FOXF1, FRAS1, HEYL, HHEX, HAND2, HMGA2, HOTTIP, HOXB3, HOXB7, HOXB8, HOXB13, HOXD3, HOXD9, HOXD10, HOXD11, HOXD13, ID3, IL6, LIF, MAN2A1, NFIB, NKX3-2, NTRK2, PDGFB, PLAG1, RPS6KA1, SEMA3C, SHANK1, SHROOM2, SOBP, SOX8, TACSTD2, TBX2, TBX3, TBX15, TFAP2A, TFAP2B, TGFB3, TMEM100,TSHR, TSPAN12, USH2A, WNT5A, ZFPM2
MF: Transcription regulatory region sequence-specific DNA binding (5.82E-10)	AR, ATF5, EBF1, EGR1, EGR3, EMX2, EN1, ETV4, FOS, FOXF1, FOXQ1, GLIS1, HAND2, HEYL, HHEX, HMGA2, HOXB3, HOXB7, HOXC10, HOXD3, HOXD9, HOXD10, HOXD13, KLF5, KLF8, LIF, NFIB, NKX3-2, NFE2L3, PITX1, PLAG1, PRDM6, RUNX3, SOX8, SOX10, TBX2, TBX3, TBX15, TFAP2A, TFAP2B
CC: Extracellular matrix (1.53E-10)	ADAMTSL1, ADAMTS5, ALPL, CLEC14A, COL7A1, COL9A3, COL14A1, COL23A1, CRISPLD2, CTSB, ELFN2, F7, FBN2, FGL2, FRAS1, HAPLN3, KAL1, L1CAM, MMP10, OGN, PCOLCE2, PDGFB, PRG4, PTPRZ1, SEMA3C, SRPX2, TGFB3, THBS4, TLR3, USH2A, WISP1, WNT5A
CC: Integral component of plasma membrane (2.38E-08)	ADORA2B, ADRA1D, CADM1, CD9, CD70, CD74, CELSR1, CHRM3, CMKLR1, DLL4, DRD1, EDA, EPHB2, EREG, F2RL1, GPR17, GPR133, HLA-DQA1, HRH2, IL6, ITGB7, KCNN4, LAT, LGR5, LPHN2, LRFN5, NOTCH3, MSR1, MST1R, NTRK2, NTSR1, PPAP2A, PPAP2B, PODXL, PROCR, PTGER2, PTPRD, PTPRZ1, REEP2, ROS1, SCARA5, SCN1A, SEMA3C, SEMA3E, SHANK1, SLC14A1, SLC16A2, SLC16A5, SYT7, TLR3, TRHDE, TSHR, TSPAN12, TSPAN18

This systems level analysis was performed in ToppFun using the 308 DE genes identified in lcSSc twins and validated with unrelated controls. PW: pathway; BP; biological process; MF: molecular function; CC: cellular component. The p-value for each pathway and ontology is shown in parenthesis. Genes in red are upregulated in lcSSc twins, and in blue downregulated.

The comparison of “dcSSc twins vs. healthy twins” (n=7) returned 76 DE genes (Supp. Fig. 3A; Supp. Table 5). The heatmap revealed that the transcriptomic signatures of dcSSc and healthy samples were not markedly different, therefore no clear clustering of the samples was observed; dcSSc and healthy samples appear to be randomly intertwined (Supp. Fig. 3B), suggesting that the transcriptomic signature of dFBs in dcSSc patients is not as deep and well-defined as in their healthy twins. Three HOX genes are present in this dataset (Figure 1C: HOXB-AS3 is downregulated and HOXD10/HOXD11 are upregulated in dFBs of dcSSc twins) as well as the T-box gene TBX5 (upregulated; Supp. Table 5). The systems level analysis did not identify any pathways, likely due to a limited number of DE genes (Table 2; Supp. Table 6).

Table 2:

Functional enrichment in dcSSc twins.

Enrichment	Hit in Query List
BP: Tube development (1.12E-08)	ALX1, ANGPT4, CD24, COL4A3, COMP, HOXD11, IL6, KLF5, LEP, LIF, MIR27B, MYLK, NRXN3, RAMP1, RGCC, SPINT2, TBX5, TNS3, UNC5B
BP: Cell adhesion (8.28E-07)	CD24, CD200R1, CDH8, COL4A3, COMP, EPDR1, IL6, JAM2, LEP, MIR27B, NEGR1, NRXN3, PCDH19, PYCARD, RGCC, S100A10, SORBS1, SPINT2
MF: Calcium ion binding (3.15E-05)	CDH8, COMP, EPDR1, MATN2, PADI2, PCDH19, PLN, S100A10, SCARA3, SYT12, VLDLR
MF: IL6 receptor binding (2.33E-04)	IL6, PYCARD
MF: ECM structural constituent	COL4A3, COL11A1, COMP, MATN2, SCARA3

This systems level analysis was performed in ToppFun using the 76 DE genes identified in dcSSc twins. BP; biological process; MF: molecular function. The p-value for each ontology is shown in parenthesis. Genes in red are upregulated in dcSSc twins, and in blue downregulated.

By comparing the DE genes in lcSSc and dcSSc twins, we identified 9 genes in the intersect (Figure 1D): CDK18, IL6, KLF5, LIF, MST1R and RGCC were commonly upregulated in both disease subtype while HOXD10, HOXD11 and PRDM6 were upregulated in dcSSc twins but downregulated in lcSSc twins.

Methylation profiling of twins discordant for SSc

Methylation profiling of 10 twin pairs discordant for SSc identified 174 DM CpG sites (P < 10⁻⁰⁴) between healthy and SSc twins, 67% of which mapped to gene bodies (116 genes), while the remainder mapped to intergenic regions, and these 116 DM CpG sites in dFBs mapped to 83 distinct genes (Supp. Table 7). A total of 55 CpG sites showed a large reduction (W Beta < −0.20) and 16 showed a large increase (W Beta > 0.20) in DNA methylation status in the SSc twins. At the gene level, 35 DM genes were identified (P < 10⁻⁰⁴) in dFBs of SSc twins, out of which 13 had increased methylation and 22 had decreased methylation (Figure 1E).

Next, we sought to identify genes that are both DM and DE (Figure 1F). In lcSSc twins, HOXB3 and TFAP2A showed an increase in both gene expression and DNA methylation while HOXB8 and HOXC10 had an increase in gene expression but a reduction in DNA methylation status as compared to healthy twins (Supp. Fig. 4A). In dcSSc twins, TBX5 and UNC5B were upregulated and less methylated compared to their healthy twins (Supp. Fig. 4B).

miR-10a and miR-10b mediate HOXD10, TFAP2A, TBX5 and COL1A1 dysregulation

The HOX gene clusters have co-evolved with and contain many miRNAs,¹⁶ including miR-10a and miR-10b (Figure 2A). miR-10a is located upstream of HOXB4 on chromosome 17, while miR10-b is located upstream of HOXD4 on chromosome 2. Both miR-10a-5p and miR-10b-5p expression levels were significantly reduced in lcSSc dFBs compared to healthy twins (Figure 2B–C). In the dcSSc subtype, both miRNAs were also decreased, albeit not significantly for miR-10b-5p. Since miR-10a-5p and miR-10b-5p are downregulated, we expect a negative correlation with their target genes. Accordingly, TFAP2A and TBX5 levels were upregulated in lcSSc and dcSSc dFBs respectively (Figure 1B; Supp. Table 5). This negative correlation was also observed with HOXD10 in dcSSc dFBs (Figure 1C) but not in the lcSSc dFBs in which HOXD10 is downregulated (Figure 1A–B), suggesting that other regulatory mechanisms govern HOXD10 in lcSSc dFBs.

Figure 2: Both miR-10a and miR-10b mediate the dysregulation of TFAP2A, TBX5 and COL1A1 in SSc dFBs.

(A) miR-10a and miR-10b are encoded from the HOX clusters, upstream of the HOXB4 (chromosome 17) and HOXD4 (chromosome 2) respectively. (B) Levels of expression of miR-10a-5p in lcSSc and dcSSc dFBs compared to healthy dFBs. (C) Levels of expression of miR-10b-5p in lcSSc and dcSSc dFBs compared to healthy dFBs. (D) Comparison of predicted miR-10a/miR-10b target genes (obtained from TargetScan database) and validated DE genes in lcSSc twins. Genes in red font are upregulated, and genes in blue are downregulated. Note that HOXD10 is downregulated in lcSSc twins while upregulated in dcSSc twins. (E) Effect of antagomiR-mediated miR-10a-5p (inhib miR10a) and miR-10b-5p (inhib miR10b) silencing on HOXD10 (n=7), TFAP2A (n=10) and TBX5 (n=7) expression levels in normal dFBs transfected for 48hrs. Combined inhibition (inhib combined) was induced by simultaneous delivery of both miR-10a-5p and miR-10b-5p antagomiRs. Scr: scramble antagomiR. (F) Effect of miR-10a and miR-10b mimics in SSc dFBs on HOXD10, TFAP2A, TBX5 and COL1A1 protein abundance by immunoblotting (n=5). The concentration of mimic transfection was 50mM for individual mimic and 25mM for each mimic when transfected together (mimic combined). Scr: scramble mimic control. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars = SEM.

These two miRNAs share many target genes because they have seed sequences that only differ by one base. By comparing the list of predicted gene targets obtained from TargetScan for miR-10a-5p and miR-10b-5p to the DE genes lists in lcSSc (validated) and dcSSc twins, we determined that 10 predicted targets were present as DE genes in our RNAseq dataset: in lcSSc twins ALPL, ANK1, CADM1, HOXB3, HOXD10, SOBP, TFAP2A and VASH1, and in dcSSc twins HOXD10, TBX5 and UNC5B (Figure 2D). Silencing of both miR-10a-5p and miR-10b-5p in normal dFBs (single or combined) significantly increased TFAP2A and TBX5 mRNA levels (Figure 2E), while only the silencing of miR-10b-5p induced a significant increase in HOXD10 expression levels. This is consistent with previous reports stating that miR-10b targets HOXD10 for silencing^17–19. In a complementary approach, transfection of SSc dFBs with miR-10a-5p and miR-10b-5p mimics (individually or combined) significantly reduced HOXD10 protein abundance (Figure 2F), confirming the efficacy of the mimics and showing that miR-10a is also able to silence HOXD10 in SSc dFBs. The combination of both mimics also reduced the translation of TFAP2A, TBX5 and COL1A1 protein levels, and the miR-10a-5p and miR-10b-5p mimics individually exerted a significant translational silencing of TBX5. These results suggest that miR-10a-5p and miR-10b-5p, whether individually or in combination, modulate HOXD10, TFAP2A, TBX5 and COL1A1 levels in SSc.

In early dcSSc, TBX5 and TFAP2A regulate HOX genes via KLF4, NANOG and POU5F1 pathway

Given that HOX genes encode TFs involved in cellular differentiation during embryogenesis,²⁰ and that myofibroblast differentiation is central to SSc and fibrosis pathogenesis,¹⁴ a review of TGFβ and HOX gene regulation led us to examine the embryonic stem cell (ESC) TFs SOX2, KLF4, NANOG and POU5F1 (aka OCT4).²¹ In lcSSc twins, SOX2 was significantly upregulated while KLF4 was downregulated compared to unrelated controls (Figure 1B; Supp. Fig. 5A; Supp. Table 2). Additionally, POU5F1 was downregulated in several samples, albeit not significantly, and NANOG was not detected. In dcSSc and healthy twins, the expression levels of these 4 ESC TFs were different by samples, not by experimental groups (Supp. Fig. 5B; Supp. Table 5). Since the lcSSc and dcSSc patients in the twin cohort had variable disease duration (Supp. File 1), we quantified these ESC TFs in non-twin early dcSSc patients with disease duration of 8–24 months. Upregulation of SOX2 was observed in early dcSSc dFBs along with downregulation of KLF4, NANOG and POU5F1 (Figure 3A). TFAP2A and TBX5 were also upregulated in these cells.

Figure 3: Validation in early disease and regulation of KLF4 function.

(A) Expression levels of ESC pluripotency TFs and other genes of interest in early dcSSc dFBs (n=8, disease duration 8–24 months) compared to levels in normal dFBs from healthy twins (n=7, aged-matched to early dcSSc patients). (B) KLF4 expression levels in dcSSc discordant twins (n=7). (C) Disease duration correlation in dcSSc for NANOG and POU5F1 expression levels. All dcSSc twins (n=7) and non-twin dcSSc patients selected for the early disease study (n=5) were used in this linear regression analysis. (D) Effect of TFAP2A and TBX5 silencing (siRNA) on KLF4 expression levels in early dcSSc dFBs (n=7) transfected for 24hrs. (E) Effect of KLF4 silencing (siRNA) on ACTA2, CTGF, HOXB5, WNT2, WNT4 and WNT16 expression levels in normal dFBs (n=5–6) transfected for 72hrs. Scr: scramble siRNA. (F) Effect of adenoviral overexpression of KLF4 (ad-KLF4) in normal dFBs infected for 24 hrs and stimulated with TGFβ (TGF) or vehicle control (VC) for 72hrs on protein abundance of fibrotic genes (n=5). ad-NULL: null adenovirus. *P<0.05, **P<0.01, ***P<0.001. Error bars = SEM.

In discordant dcSSc twins, KLF4 expression levels were noticeably reduced as compared to healthy twins, albeit not significantly (Figure 3B). We performed a disease duration correlation analysis (Figure 3C and Supp. Fig. 5C) using data from all dcSSc twins (representing intermediate and late disease stages) and early dcSSc patients and identified a strong positive correlation between disease duration and relative mRNA levels of NANOG (P= 0.0129, R²= 0.4769) and POU5F1 (P= 0.0236, R²= 0.4159).

KLF4 is protective against fibrosis

KLF4 is a master regulator of skin and stem cell biology.²² Silencing of TFAP2A and TBX5 in early dcSSc dFBs increased KLF4 expression levels (Figure 3D). In turn, KLF4 silencing in normal dFBs (Figure 3E) significantly increased smooth muscle actin alpha 2 (ACTA2), an indicator of myofibroblast differentiation,¹⁴ and connective tissue growth factor (CTGF), a central pro-fibrotic factor,²³ while decreasing HOXB5 levels, an expression profile mirroring that in SSc. We also examined the effect of KLF4 silencing on the expression levels of several key molecules in the WNT pathway and determined that KLF4 positively regulates WNT2 and WNT16 and negatively regulates WNT4 (Figure 3E). Normal dFBs overexpressing KLF4 and stimulated with TGFβ showed a significant decrease in COL1A1, FN, αSMA and CTGF protein abundance as compared to TGFβ alone, confirming that KLF4 protects against TGFβ-induced fibrogenesis (Figure 3F). These findings were further validated in human skin in organ culture as it is the relevant tissue for SSc and lends direct relevance to the human disease. Similarly to its effect in dFBs, KLF4 reduced the TGFβ1 response in human skin (Supp. Fig. 6). To extend our findings in vivo, we induced fibroblast-specific knock-down of KLF4 in mice (KLF4-KO). In KLF4-KO dFBs, fibrotic genes were highly expressed (Figure 4A). Furthermore, hydroxyproline content was significantly higher in the skin of KLF4-KO mice as compared to control mice (Figure 4B). Since KLF4 regulated WNT gene expression (Figure 3E), we examined its effect on β-catenin. Loss of KLF4 resulted in a significant increase in nuclear chromatin levels of β-catenin, a key downstream component of the canonical Wnt signaling pathway, in dFBs of KLF4-KO mice (Figure 4C). This suggests that the imbalance of WNT gene expression resulted in an activation status in mouse dFBs.

Figure 4: Anti-fibrotic properties of KLF4.

(A) Effect of KLF4 conditional knock-out (KLF4-KO) on the expression levels of fibrotic genes in murine dFBs. Control KLF4-flox mice (Ctrl) received corn oil. (B) Hydroxyproline levels in 3-mm skin punches of control and inducible KLF4-KO mice. Representative images of skin sections stained with Masson’s trichrome showing collagen in blue. The scale bar indicates 100 μm. (C) Protein abundance of β-catenin in the nuclear chromatin fraction of control and KLF4-KO dFBs relative to histone H3. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars = SEM.

Together our data highlight KLF4 as a key anti-fibrotic factor that is downregulated by TFAP2A and TBX5 early in disease progression, leading to fibroblast activation and fibrosis. KLF4 could be a candidate for the development of targeted therapies against fibrosis that would aim to maintain increased levels of this antifibrotic gene.

DISCUSSION

This study is the first to examine genome-wide DNA methylation profiling in combination with RNAseq in dFBs from a unique cohort of twins discordant for SSc categorized by disease subtypes. HOX genes belong to a family of evolutionarily highly conserved genes that encode TFs involved in stem cell differentiation²⁰. In addition to driving stem cells towards their corresponding lineages, the HOX code is also important during adulthood to repair and maintain proper tissue and organ function, and provide cellular positional memory that is crucial to inform the location and cell lineage of new epidermal cells during turnover, a process that is mediated in adult dFBs via HOXA13-WNT5A pathway.²⁴ Our data show that both HOXA13 and WNT5A are upregulated in lcSSc dFBs, implicating this pathway in SSc. Even though HOXA13 function is required for WNT5A expression, it does not directly target WNT5A, which led Rinn et al. to speculate that HOXA13 activates WNT5A directly via distant enhancer sequences, or indirectly via HOXB6.²⁴ Rinn et al.²⁵ examined the HOX signature in primary human dFBs that delineates distal and proximal sections of limbs and showed that HOXA13 is exclusively expressed in adult dFBs isolated from distal sites such as hands, feet and foreskin. Our qRT-PCR data is consistent with Rinn et al.²⁵ in that HOXA13 was upregulated in the lcSSc dFBs as compared to unrelated controls. Our data suggest that deregulation of the HOX zip code, including HOXA, HOXB, HOXC and HOXD genes, is dependent on SSc skin involvement and independent from biopsy location since the expression of HOXB genes is limited to the trunk and non-dermal samples while HOXD4 and HOXD8 are expressed exclusively in the trunk and proximal samples in healthy donors.²⁵

Decreased promoter DNA methylation often leads to increased gene expression as is the case with HOXB8 and HOXC10 in lcSSc twins, and TBX5 and UNC5B in dcSSc twins. In the same reasoning, gene silencing is expected with increased methylation of CpG islands, however, this classic paradigm does not always apply, as illustrated by HOXB3 and TFAP2A which were more methylated yet upregulated in lcSSc twins. Mucientes et al. also observed differential expression of HOX genes independent of their methylation status in stem cells of osteoarthritic patients.²⁶ This suggests that other regulatory and epigenetic mechanisms are implicated in the regulation of these HOX genes, including sequential activation of HOX genes with 3’ to 5’ polarity.^27–29 Our data highlight that perturbation of homeotic genes drives developmental reprogramming in SSc dermal fibroblasts. Interestingly, TBX5 is located on chromosome 12, the same chromosome as the HOXC cluster (Supp. Fig. 4), and is a direct target of HOX genes.³⁰ TBX5 showed reduced methylation status and upregulated gene expression in dcSSc dFBs in our study (Supp. Fig. 4B), a state also observed in the activation of rheumatoid arthritis synovial fibroblasts.³¹

Our data showed that TFAP2A and TBX5 negatively regulate KLF4, an upstream regulator of the ESC factors NANOG, SOX2 and POU5F1³² that control cellular differentiation into extra-embryonic, endodermal, mesodermal, and ectodermal lineages, and also regulate HOX genes³³ (Figure 5). We determined that KLF4 is a negative regulator of ACTA2, CTGF and WNT4 in dFBs, indicative of active fibrotic cascade, and a positive regulator of HOXB5, WNT2 and WNT16. Interestingly, binding motifs for KLF4, POU5F1, and SOX2 exist in the WNT2 promoter region, and WNT2 has been shown to play a role in the nuclear accumulation of β-catenin and cell proliferation in early stages of embryonic fibroblast reprogramming.³⁴ Beazley et al.³⁵ showed that TGFβ inhibits WNT16 expression, leading to the deactivation of Notch signaling in vascular smooth muscle cells (VSMCs). Considering that inhibition of Notch signaling limits both fibrosis and autoimmune activation³⁶ and even reversed established fibrosis in murine model of SSc,³⁷ the role of downregulation of the KLF4-WNT16-Notch signaling axis in dFBs in the development of SSc remains unclear. Due to functional redundancy among HOX5 paralogs, it is important to consider HOXA5 and HOXC5 when assessing the role of HOXB5. Loss of HOX5 leads to downregulation of WNT2,³⁸ consistent with our KLF4 silencing assay in which both WNT2 and HOXB5 were downregulated. Together this suggests that both KLF4 and HOXB5 regulate WNT/β-catenin signaling. Additionally, HOX5 paralogs modulate Th2 inflammation during chronic allergic reaction,³⁹ a role that is unexplored in SSc.

Figure 5: Summary model of the regulatory network.

In SSc dFBs, miR-10a and miR-10b expression levels are low, allowing for the upregulation of two silencing targets TFAP2A and TBX5. KLF4 levels are repressed by TFAP2A and TBX5, which leads to high expression of ACTA2, COL1A1, CTGF and FN, enhancing myofibroblasts differentiation and fibrosis. Low levels of KLF4 also correlate with low levels of HOXB5, WNT2 and WNT16, and high levels of WNT4, suggesting that KLF4 regulate the Wnt signaling pathway. HOXB5 is positively correlated with KLF4 levels, and has been shown to play a role in myofibroblast adhesion to FN.

The fact that TFAP2A overexpression suppressed the expression of the matrix genes collagen types II and X, and aggrecan (ACAN),⁴⁰ and KLF4 negatively regulated the expression of mesenchymal markers ACTA2 and fibroblast specific protein 1 (FSP1),⁴¹ suggests that the TFAP2A-KLF4 axis likely regulates myofibroblast differentiation and fibrosis. Furthermore, the decreased expression levels of POU5F1 and NANOG in early dcSSc parallel those of KLF4 and may reflect a state of reduced plasticity that points to cell fate reprogramming in dFBs.⁴²

MiRNAs of the miR-10 family target TFAP2A,⁴³ TBX5,⁴⁴ KLF4 and several HOX genes for silencing.¹⁶ Both miR-10a-5p and miR-10b-5p are active elements in mediating cancer metastasis^{45, 46} and the fibrogenic response.⁴⁷ Our silencing assay showed that they mediate TFAP2A, TBX5 and HOXD10 dysregulation in dFBs. Our data suggest that it is a combination of decreased DNA methylation and reduced miRNA silencing that maintain high levels of TBX5 in dcSSc dFBs. However, for the epigenetic regulation of TFAP2A, which was upregulated with increased methylation status, may be more driven by miRNAs. Even though miR-10a/b target several DE genes in our dataset, not all of these targets exhibited increased expression levels when miR-10a and miR-10b were downregulated, as is the case for HOXD10 and SOBP in lcSSc twins, which could be due to the complex way these genes are epigenetically or post-transcriptionally regulated.

Our findings suggest that developmental programs deregulated in lcSSc are similarly observed in the early phase of dcSSc. This may be due to skin involvement in lcSSc, although minimal, remains active as the disease progresses, whereas skin involvement regresses in dcSSc approximately 3 years after disease onset. This highlights the importance of including lcSSc patients in studies involving gene expression profiling whether in response to potential therapies or to monitor disease progression.

Some genes linked to differentially methylated CpG sites reported in individual SSc studies were not observed to show evidence of differential methylation in this discordant twin design. This may be due to simple random sampling variation in combination with the power to detect more modest effects. Some may reflect differences in tissues assayed (type, physical location). But some of these genes may not replicate because of the robustness of a discordant twin study design where the test of association is between the twins, eliminating the confounding effects of genetic heterogeneity across samples that cause inflation of the test statistics (i.e., lead to false positive).

This study provides insights into the interplay between different epigenetic mechanisms in SSc-associated dermal fibrosis and implicate TFs and miRNAs in disease pathogenesis. Given that epigenetic regulation is reversible, this suggests that epigenetic modifying agents or CRISPR-based epigenome editing to ameliorate fibrosis may be of relevance in SSc. ⁴⁸

Key messages:

What is already known about this subject?

• SSc is a complex connective tissue disease of unknown etiology.

• Disease concordance in twins with SSc is low, implicating a potential role for epigenetics in disease manifestation.

What does this study add?

• Using global DNA methylation and gene expression, we identified novel programs deregulated in dermal fibroblasts of a unique cohort of twins discordant for SSc.

• Using gain and loss of function studies, we confirmed the functional impact of differentially expressed and methylated transcription factors and miRNAs in mediating dermal fibrosis in SSc.

How might this impact on clinical practice or future developments?

• Since no effective therapy that can halt the progression of fibrosis exists, novel insights into disease pathogenesis and identification of novel potential therapeutic targets are critically needed.