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Ching Yuan, Megan Twite, Elizabeth Nelson; The anti-fibrotic Halofuginone regulates the expression of Egr-1 in human corneal fibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5246.
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Transcriptional factor early growth response-1 (Egr-1) plays important roles in the pathogenesis of fibrosis. Unlike regulation of other transcription factors, which rely on post-translational modifications (i.e., phosphorylation), regulation of Egr-1 signaling is mediated via its biosynthesis and by interaction with regulatory molecules, such as Nab2. We have previously demonstrated the anti-fibrotic efficacy of halofuginone, a compound derived from herbal alkaloids, in cultured human corneal fibroblasts (Nelson et al., Molecular Vision 2012). In the current study, we identified Egr-1 as the downstream effector of halofuginone.
Human corneal fibroblasts were extracted from donor corneas provided by Minnesota Lions Eye Bank. To delineate the profibrotic roles of Egr-1 in the corneal fibroblasts, Egr-1 transgene as well as an Egr-1-specific DNA enzyme (ED5) were transfected into cells to either constitutively overexpress or suppress Egr-1 expression. Human corneal fibroblast cells were treated with halofuginone and TGFβ-1 to investigate the expression profile of Egr-1, Nab2, and other fibrosis-related genes using qRT-PCR, Western blot, and immunohistochemistry. The transactivation of collagen type I gene by Egr-1 after halofuginone treatment was evaluated using (1) a reporter plasmid containing the luciferase gene driven by the collagen type I promoter, and (2) promoter-transcription factor pull down assay.
Our results showed that overexpression or knockdown of Egr-1 modulated the expression of fibrotic markers, including alpha smooth muscle actin, and fibronectin in human corneal fibroblasts. Egr-1 also bound directly to the collagen type I promoter and facilitated the production of collagen. Halofuginone significantly reduced the expression of Egr-1 in a dose- and time-dependent manner according to the Western blot and qRT-PCR results. Immunostaining experiments revealed that the fast nuclear translocation of Egr-1 induced by TGFβ-1 was also abolished with halofuginone treatment.
The current study confirms the profibrotic role of Egr-1 in human corneal fibroblasts. Egr-1 signaling is among the divergent fibrotic pathways affected by halofuginone, and may serve as a promising target for anti-fibrotic applications in the cornea.
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