The etiology and pathogenesis of scleroderma (systemic sclerosis; SSc) remain unknown. However, the dominant clinical and pathologic features of SSc include progressive tissue fibrosis and widespread vascular disorder. It is generally believed that interstitial fibroblasts mediate tissue fibrosis in SSc, since these cells synthesize excessive amounts of collagen and other components of the extracellular matrix together with reduced expression of matrix metalloproteinases, leading to excessive matrix accumulation (1, 2). The basis for the altered cellular phenotype in SSc has not been determined, but primary metabolic abnormalities, responses to abnormal environmental signals, and clonal selection have all been hypothesized to play a role (2). Scleroderma-associated cellular abnormalities have been shown to persist in multiple generations of SSc fibroblasts in vitro and the persistence of the profibrotic phenotype outside the disease environment suggests the possible in vivo imprinting of a profibrotic phenotype that is inherited and is transmitted from one generation of fibroblasts to the next. This inherited phenotype can ultimately lead to the development of clinical tissue fibrosis. In the current report, we present data indicating that an epigenetic alteration in SSc fibroblasts controls cellular events characteristic of these fibroblasts.
The term “epigenetics” describes all inherited changes in gene expression that are not coded in the DNA sequence itself. The 2 major mechanisms that are known to mediate epigenetic changes are DNA methylation and histone modification. Methylation of the CpG dinucleotides has long been recognized as a major epigenetic modification of the mammalian genome, and is implicated in imprinting, X chromosome inactivation, embryonic development, defense against retroviral sequences, transcriptional repression of certain genes, and in carcinogenesis (3, 4). CpG islands are stretches of DNA located within the promoter regions of ∼40% of mammalian genes. When methylated, they cause a stable, heritable repression of transcription for the affected gene. Methylation is controlled by a number of DNA methyltransferases (Dnmts).
Histone modification has also been identified as an important epigenetic mechanism (5). Posttranslational modifications of histones, including acetylation and methylation of conserved lysine residues on the amino-terminal tail domains, have been studied closely over the last few years. Generally, the acetylation of histones marks active, transcriptionally competent regions, whereas deacetylated histones are found in transcriptionally inactive regions.
There is a direct causal relationship between methylation-dependent transcriptional repression and histone modifications. Thus, sites of DNA methylation are recognized by a family of proteins that contain a highly conserved methyl-CpG DNA binding domain (MBD). MBD-containing proteins recruit chromatin-modifying enzymes, such as histone deacetylase 1 (HDA-1) and HDA-6, which condense chromatin and repress transcription (6).
Given the stable cell dysregulation associated with SSc, we explored potential epigenetic influence on the expression of type I collagen in SSc fibroblasts, using the Dnmt inhibitor 2-deoxy-5-azacytidine (2-deoxy-5-azaC) and the HDA inhibitor trichostatin A (TSA). Examining the nuclear extracts of fibroblasts from SSc and healthy controls, we assessed the levels of Dnmt, MBD, and HDA. Levels of deacetylated histones in the FLI1 promoter region in fibroblasts were measured by chromatin immunoprecipitation (ChIP) assay. Methylation of promoter region CpG islands was assessed by methylation-specific polymerase chain reaction (PCR) and by sequence analysis of DNA after bisulfite modification. Our results suggest that there are ongoing epigenetic modifications in SSc fibroblasts, which may explain the abnormal cell behavior and offer a new target for therapy.
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- MATERIALS AND METHODS
SSc is a systemic autoimmune disease of unknown etiology and pathogenesis. It is characterized by progressive vasculopathy and widespread tissue fibrosis. Activated fibroblasts are believed to play a key role in the development of tissue fibrosis and disease progression. Numerous in vivo and in vitro studies have helped to characterize the profibrotic phenotype of SSc fibroblasts and have shown that fibroblasts derived from involved SSc skin produce increased amounts of extracellular matrix proteins, including types I, III, VI, and VII collagen and fibronectin, and profibrotic cytokines, including TGFβ and connective tissue growth factor, in association with reduced synthesis of matrix metalloproteinases 1 and 3 (18–20).
This profibrotic phenotype persists in vitro for multiple generations, suggesting an inherited cellular trait that is transmitted from one generation to the next. One possible explanation for the persistence of the phenotype is in vivo clonal selection of a profibrotic fibroblast from a heterogeneous population (21). The clonal selection hypothesis has generated intense interest; however it has not been validated by experimental data. An alternative possibility is that of an acquired epigenetic alteration that is maintained during cell divisions. Epigenetics is defined as the heritable but potentially reversible changes in genetic material, including the DNA and chromatin, that lead to alterations in gene expression (22, 23). Over the last decade, several disease conditions have been linked to epigenetic processes; diseases that have been suggested to be under epigenetic influence include several cancers (24), type 1 diabetes mellitus (25), and inflammatory bowel disease (26).
Our data suggest that an epigenetic mechanism may indeed lead to augmented collagen expression by SSc fibroblasts. First, the addition of inhibitors of DNA methyltransferases and histone deacetylases led to normalization of type I collagen expression levels in SSc fibroblasts, providing evidence of epigenetic influence on collagen gene expression. Second, the levels of factors involved in the maintenance of epigenetic mechanisms are clearly elevated in the nuclear extract from SSc fibroblasts, demonstrating the ability of the cells to sustain an epigenetic process.
We tested the possibility that the augmentation of collagen expression is related to epigenetic repression of a collagen suppressor gene. It is known that DNA methylation inhibitors boost the expression of repressed genes and reduce that of genes that are activated due to repression of a suppressor gene. For example, 2-deoxy-5-azaC has been shown to reduce aberrant p16INK4A RNA transcripts and restore the functional retinoblastoma protein pathway in hepatocellular carcinoma (27), and it can also reduce the expression of an up-regulated vascular endothelial growth factor gene in several leukemias and lymphomas by augmenting the expression of a suppressor gene (28). We examined 3 collagen suppressor genes, smad7, p53, and FLI1, using quantitative real-time PCR along with type I collagen before and after addition of epigenetic modifiers. Another collagen suppressor factor is HDA-1 (29), and it was overexpressed in SSc fibroblasts.
Of the 3 genes investigated, only FLI1 was significantly reduced in SSc fibroblasts and substantially increased after the addition of epigenetic modifiers. FLI1 is a member of the Ets family of transcription factors that has been shown to have roles in hematopoiesis, embryonic development, and vasculogenesis (30). It is also a transcription factor that inhibits collagen gene via an Sp-1–dependent pathway (15). The role of FLI1 as a negative regulator of extracellular matrix genes is supported by the observations that FLI1 protein levels are inversely correlated with production of type I collagen in fibroblast cultures generated from FLI1−/−, FLI1+/−, and FLI1+/+ mouse embryos (14). In the present report we describe an epigenetic regulation of FLI1 gene expression in SSc that results in enhanced type I collagen gene expression. The transfection data in the current study support the notion of a link between levels of FLI1 gene expression and type I collagen expression. Thus, the introduction of FLI1 antisense constructs into normal cells led to a significant increase in type I collagen expression, while the transient transfection of SSc fibroblasts with FLI1 gene led to a decrease in collagen gene expression.
Epigenetic regulation of a gene requires the presence of CpG islands in the promoter region. The promoter of FLI1 contains CpG islands that can be methylated, as shown by binding of a genomic DNA fragment containing 206 nucleotides from positions −161 to 46 of the FLI1 promoter with an MeCP-2 affinity column (31), indicating that the FLI1 proximal promoter region can be methylated and bind to methylated nucleotide binding protein. In the present study we demonstrated epigenetic regulation of FLI1 gene expression in SSc cells, which is related not only to CpG island promoter hypermethylation as shown by the methylation-specific PCR and promoter sequencing results, but also to chromatin deacetylation as shown in the chromatin immunoprecipitation experiments. Moreover, results of the methylation-specific PCR studies of skin biopsy samples demonstrated that almost half of FLI1 promoters are methylated, suggesting that epigenetic repression of the FLI1 gene occurs in vivo. The reported underexpression of FLI1 in SSc skin supports our findings (15). The proportion of methylated FLI1 promoters was clearly higher in SSc fibroblasts than in SSc skin specimens, possibly because of the mixed cellularity of the biopsy samples. Nonetheless, no significant methylation was noted in control cells or biopsy samples.
It is unclear whether FLI1 expression is linked to the development of the full fibrotic phenotype of SSc fibroblasts, including the myofibroblast phenotype, or is related only to the level of type I collagen synthesis. Of interest, other collagen suppressor genes may be involved in different forms of fibrosis; for example, in liver cirrhosis, cytokine signaling 1 gene methylation is associated with hepatic fibrosis (32).
Finally, our observations may have significant implications with regard to therapy, because neither methylation of a gene nor alteration in chromatin structure is irreversible since the gene itself is not mutated in any way by methylation, and the chromatin is not irreversibly changed. Because of this reversibility, epigenetic gene regulation is theoretically amenable to intervention (33). Thus, a better understanding of the role of epigenetics in SSc tissue fibrosis may lead to the development of a novel therapy.