Activated fibroblasts are considered principal mediators of fibrogenesis. Unlike in physiological wound repair, where fibroblast activation is spontaneously reversible, the fibroblast activation associated with fibrogenesis is perpetuated, and fibroblasts maintain their activated phenotype even when cultured in vitro
. Here we found that the demethylating agent 5′-azacytidine normalizes the phenotype of fibrotic fibroblasts in vitro
and ameliorates experimental kidney fibrosis in mice. Even though we are aware that the effect of 5′-azacytidine is not limited to fibroblasts, this led us to hypothesize that hypermethylation of specific genes in fibroblasts is pivotal in perpetuating fibrogenesis. To gain insights into possible genes that are affected by hypermethylation in fibrotic fibroblasts, we performed a genome-wide methylation screen and identified 12 genes that are selectively hypermethylated in primary human fibrotic fibroblasts. Among these genes, we focused on RASAL1
, an inhibitor of Ras. Our studies highlight commonalities between cancer progression and perpetuated fibrogenesis41
. The capacity of fibrotic fibroblasts to proliferate in serum-free media until they reach senescence resembles the autonomous growth of cancer cells. Because, to our knowledge, mutated oncogenes cannot yet be linked to fibrogenesis, it is possible that commonalities between cancer and fibrosis are based on common epigenetic modifications. We provide evidence that epigenetic RASAL1
silencing causes fibroblast activation and fibrogenesis, similar to cancer cells that are enabled to proliferate in a growth factor–independent manner upon RASAL1
. Furthermore, it remains to be determined whether the activated phenotype observed in fibrotic fibroblasts and in cancer-associated fibroblasts share common underlying mechanisms. Our studies further highlight differences between fibrogenesis and physiological repair. Fibroblast activation is associated with transcriptional RASAL1 repression in both the settings of physiological kidney repair and pathological fibrogenesis. However, whereas reversible fibroblast activation (typical of physiological repair) is associated with reversible RASAL1 suppression without its hypermethylation, sustained fibroblast activation typical of fibrotic kidneys is associated with irreversible RASAL1 expression due to hypermethylation of the RASAL1 promoter. This suggests that hypermethylation perpetuates fibroblast activation and ultimately fibrogenesis by imprinting pathways of fibroblast activation that are engaged during the transient fibroblast activation typical of physiological repair. We further reveal that hypermethylation associated with fibrogenesis is mediated by the methyltransferase Dnmt1 and that pathological hypermethylation by Dnmt1 can be induced by long-term exposure to the profibrotic growth factor TGF-β1. Additional studies are required to gain further insights into the mechanisms that cause Dnmt1 to hypermethylate specific genes.
Although our studies demonstrated that the RASAL1 hypermethylation correlates strongly with fibrogenesis and absence of RASAL1 methylation correlates with physiological repair upon kidney injury, future studies manipulating RASAL1 in vivo are needed to elucidate whether RASAL1 silencing alone is sufficient to induce fibrogenesis in the kidney or whether it modulates the rate at which induced fibrosis progresses.
Our studies emphasize Ras hyperactivity as a potential therapeutic target for kidney fibrosis, confirming previous studies that suggested a prominent role of Ras signaling in renal fibrogenesis31
. Previous studies have shown that increased Ras signaling within the fibrotic microenvironment is due to autocrine and paracrine growth factor stimulation. Our results show a new mechanism of epigenetic RASAL1
silencing causing increased intrinsic Ras-GTPase activity in fibroblasts. These results suggest that the antifibrotic capacity of Ras inhibitors should be further explored.
To our knowledge, this is the first report of hypermethylation as a mechanism of perpetuating fibroblast activation and fibrogenesis. In summary, hypermethylation of specific genes contributes to fibrogenesis. Future studies are needed to elucidate the utility of methylated genes as diagnostic markers to predict fibrosis and the possible therapeutic efficacy of methylation inhibitors in progression of fibrogenesis.