Molecular mechanisms for the emergence and/or persistence of myofibroblasts within the fibroblastic foci of IPF remain unclear. Our previous findings in vitro
and in the bleomycin model and within IPF tissues indicate that the absence of Thy-1 expression in lung fibroblasts correlates with a more profibrotic myofibroblast phenotype, as shown here in (9
). The mechanisms by which Thy-1 expression is regulated in fibroblasts are unclear. We previously demonstrated reversible shedding of Thy-1 associated with exposure of fibroblasts to inflammatory cytokines. However, inflammation is not a consistent feature of fibrotic disorders such as IPF (2
). Loss of expression can result from genetic mutations, such as deletions or insertions, or from epigenetic modifications including promoter hypermethylation.
Hypermethylation of DNA at cytosine residues in CpG islands led to heritable gene silencing via the formation of a repressive chromatin structure (25
). Epigenetic events affect gene expression without affecting the gene sequence itself and are believed to be a primary mechanism causing tumor suppressor genes to be inactive and causing certain cancers (26
). Aberrant promoter methylation is a common mechanism for the transcriptional inactivation of certain genes in many types of cancer, including tumor suppressor genes, cell regulatory genes, and apoptosis-related genes. For example, the tumor suppressor gene p16 is hypermethylated in many tumor types, including colorectal, lung. and breast cancers (27
The absence of Thy-1 expression in a rat T-cell lymphoma cell line has been shown to be associated with DNA hypermethylation in the promoter region and is reversible upon exposure to 5-azadeoxycytidine (28
). Thy-1 has been proposed as a tumor suppressor gene in nasopharyngeal carcinoma (15
) and is down-regulated through promoter region methylation, associated with a more invasive/metastatic clinical phenotype. In this study, we have demonstrated that the absence of Thy-1 expression in Thy-1(−) primary lung fibroblasts and in IPF samples can be attributed in part to epigenetic regulation through Thy-1 promoter hypermethylation (). Demethylation in the Thy-1(−) cells restores Thy-1 gene expression (). Human and rat lung fibroblasts demonstrate similar results.
DNA methylation is regulated in part by DNMT enzymes. This family includes DNMT1, which has maintenance DNA methylase activity; and DNMT3a and DNMT3b, which are de novo
). Over-expression of DNMTs is one possible mechanism of gene hypermethylation (29
). Methyl-CpG-binding proteins are a group of proteins thought to inhibit the binding of transcription factors to gene promoters and thus are proposed as another mechanism of transcription inhibition by hypermethylation (30
). Although there are reports that DNMTs are overexpressed (31
), other studies demonstrate no association between the mRNA expression levels of DNMTs and methyl-CpG–binding proteins and the methylation status of tumor-suppressed genes (33
). In this study, we found neither DNMT1, -3a, -3b, nor MBD1 or Mecp2 methyl-binding proteins to be overexpressed in Thy-1(−) cells. In fact, Thy-1(+) had higher levels of mRNA for the methyltransferases. This was unexpected because DNMT expression, especially DNMT3a and -3b, is usually associated with hypermethylation (29
). However, the regulation of methylation by DNMTs is complex and involves multiple molecular interactions, such that expression of a particular DNMT at the mRNA level may not correlate with hypermethylating activity for a particular gene promoter (35
). Previous studies in colorectal cancer (33
) and hepatocellular carcinoma (36
) failed to show any significant correlation between methylation status and the DNMT mRNA expression levels. Also, because we analyzed serially passaged cell populations from established cultures, it is possible that transiently up-regulated DNMTs and/or MBD in a previous generation determined the aberrant gene methylation status, which is heritable in subsequent cell generations, and that to maintain the pattern, relatively low enzyme expression would be sufficient. Nevertheless, the demethylation of all but one CpG site in the Thy-1 promoter () and the reversibility of Thy-1 expression with AZA strongly suggest that methyltransferase activity is involved in maintaining hypermethylation in Thy-1(−) fibroblasts.
Other well described epigenetic mechanisms involving chromatin modifications, such as histone deacetylation, can result in gene silencing (37
). Recent studies have shown that methylation and histone modifications are related (38
). More and more studies have shown that methylation of gene promoters is only one of many layers of repressive mechanisms (39
). We also investigated whether histone deacetylation contributes to the silencing of Thy-1 gene expression: We treated the Thy-1(−) cells with trichostatin A for 72 hours, which also resulted in re-expression of Thy-1 (data not shown; studies ongoing), suggesting that promoter region methylation and histone deacetylation contribute to the silencing of Thy-1 in Thy-1(−) lung fibroblasts. Other factors, such as transcription factors, may also be affected by AZA or trichostatin A and could promote re-expression of Thy-1. The direct bisulfite sequencing of the promoter region of Thy-1 after treatment with AZA indicates that in Thy-1(−) cells, promoter region demethylation is associated with the re-expression of Thy-1 ( and ). By removal of methyl groups, certain genes can undergo significant changes in structure characterized by partial re-acetylation of histone H3 and H4 and re-methylation of H3(K4), but in other genes, the removal of DNA methylation does not result in appreciable histone reacetylation (40
). Further investigation of the role of histone acetylation in Thy-1(−) cells is ongoing.
The FF within the IPF specimens showed down-regulation or loss of Thy-1 expression by IHC and ISH, whereas the normal samples showed normal expression of Thy-1 ( and ). This is consistent with our pervious findings (9
). The in situ
MSP findings indicate that the loss of Thy-1 expression within the FF in patients with IPF may be caused by promoter region hypermethylation of Thy-1 ( and ). It is possible to speculate that patients in whom Thy-1 expression in fibroblasts is lost by epigenetic regulation may be more prone to develop IPF. Alternately (or in addition), acute inflammation could result in Thy-1 protein shedding associated with additional migration/accumulation of Thy-1(−) myofibroblasts, with persistence of latent TGF-β activation, excessive matrix accumulation, and resistance to apoptosis (9
). The demonstration of Thy-1 expression in epithelial cells overlying fibroblastic foci (, , and ) has not been previously reported. Although the significance of epithelial Thy-1 expression is unclear, it could be suggestive of epithelial-mesenchymal transition because Thy-1 is expressed on normal lung fibroblasts or could indicate emergence of a progenitor cell phenotype because Thy-1 is expressed in most stem cells (7
Taken together with our prior studies, the findings reported here suggest that Thy-1 may function as a “fibrosis suppressor” gene. The reversibility of epigenetic suppression of Thy-1 suggests that it may be possible to restore Thy-1 expression in fibroblasts, altering the profibrotic phenotype of the cells and possibly the clinical outcome in patients with IPF. This is the first report of an epigenetic mechanism causing the silencing of a specific gene in IPF. Transcriptional silencing by CpG island methylation is a common mechanism of silencing of tumor suppressors in cancer (26
). There are reports that in liver fibrosis, loss of expression of certain genes is associated with promoter region hypermethylation (42
). More experiments are needed to unravel how DNA methylation together with other repression mechanisms, such as histone deacetylation, may generate distinct patterns of gene silencing in IPF. These studies could give us novel insights into the causes of pulmonary fibrosis and open the possibility of novel therapeutic approaches to this fatal disease.