Background/Purpose:

Tissue fibrosis caused by pathological activation of fibroblasts is a major hallmark of systemic sclerosis (SSc). Epigenetic gene silencing of anti-fibrotic genes such as FLI1 by DNA methylation has been implicated in the pathogenesis of SSc. Also increased activation of the Wnt pathway with decreased expression of endogenous inhibitors has recently been shown to play a prominent role in tissue fibrosis in SSc. However, the molecular mechanisms leading to the decreased expression of Wnt inhibitors is incompletely understood. In the present study, we evaluated whether the expression of endogenous Wnt inhibitors might be silenced through DNA methylation.

Methods:

The methylation status of endogenous Wnt inhibitors in leukocytes and fibroblasts was evaluated by methylation-specific PCR. Gene expression in fibroblasts and human skin was analyzed by real-time PCR and immunohistochemistry. The expression of endogenous Wnt inhibitors was also evaluated in the mouse model of bleomycin-induced dermal fibrosis. 5-aza-2-deoxycytidine (5-aza) was used to inhibit DNA methyltransferases (Dnmts) in cultured fibroblasts and mice.

Results:

Significant hypermethylation of the promoters of DKK1 and SFRP1 was observed in leukocytes isolated from blood samples of SSc patients. Increased methylation in the promoter region of DKK1 and SFRP1 was also found in cultured fibroblasts from SSc patients. This hypermethylation resulted in decreased gene transcription by 56 ± 5 % for DKK1 and by 89 ± 3 % for SFRP1 (p < 0.05 for both) compared to fibroblasts from healthy subjects. Of note, treatment with 5-aza re-activated the transcription of SFRP1 in SSc fibroblasts from 11 ± 3 % to 52 ± 16 % of control levels and the expression of DKK1 was completely reversed to control levels (p < 0.05 each). Consistent with the reduced mRNA levels, the protein levels of both DKK1 and SFRP1 were severely decreased in skin of SSc patients as analyzed by immunohistochemistry. In addition, decreased expression of DKK1 and SFRP1 was also found in experimental fibrosis. In the model of bleomycin-induced dermal fibrosis, the mRNA levels of DKK1 decreased by 73 ± 5 % (p = 0.036) upon bleomycin challenge and those of SFRP1 by 35 ± 3 % (p = 0.004). Both genes were re-activated by treatment of bleomycin-challenged mice with 5-aza. Compared to untreated mice injected with bleomycin, the mRNA levels increased upon treatment with 5-aza by 504 ± 42 % (p = 0.024) for DKK1 and by 131 ± 23 % (p = 0.015) for SFRP1. Similar results were obtained for protein levels. Consistently, 5-aza significantly reduced bleomycin-induced dermal fibrosis with decreased dermal thickening and reductions in hydroxyproline content and myofibroblast counts.

Conclusion:

We demonstrate that the endogenous Wnt inhibitors DKK1 and SFRP1 are downregulated by promoter hypermethylation in SSc. Inhibition of Dnmts by 5-aza re-activated gene expression in SSc fibroblasts and in experimental fibrosis. As different inhibitors of Dnmts are already approved for other diseases and are well tolerated, our findings might have direct translational implications and provide a novel approach to inhibit Wnt signaling in SSc.

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« Back to 2012 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-wnt-inhibitors-dkk1-and-sfrp1-are-downregulated-by-promoter-hypermethylation-in-systemic-sclerosis/