EGR1/NOX4 pathway regulates oxidative stress and further facilitates fibrosis progression in keloids responses to TGF-β1

Abstract

Background: As classic benign fibroproliferative tumors, keloids remain a major therapeutic challenge due to their complex pathological mechanisms.

Objective: To determine the functional role of transforming growth factor β1 (TGF-β1)/early growth response factor-1 (EGR1)/NADPH oxidases 4 (NOX4) axis in the pathogenesis of keloid fibrosis.

Methods: Differentially expressed genes in keloid tissues and normal skins were analyzed by RNA sequencing. Then, the human skin fibroblast cell line was treated with TGF-β1 at a dose of 10 ng/mL in order to stimulate the TGF-β1/SMAD pathway and the pathway was blocked using the SB431542. Furthermore, EGR1/NOX4 was over-expressed and inhibited by transfecting overexpression plasmids and small interfering RNAs, respectively. The levels of intracellular reactive oxygen species were measured using the DCFH-DA assay, and the expression levels of fibrosis-related genes were assessed by Western blot analysis. Alternately, dual-luciferase reporter analysis verified the targeting relationship between EGR1 and NOX4.

Results: The TGF-β1/SMAD signaling pathway was significantly activated in keloid tissues to promote dermal fibrosis. The level of ROS was increased in keloid fibroblasts. Moreover, TGF-β1 could facilitate the expression of EGR1 through regulating the SMAD pathway in keloids and promoting the fibrotic phenotype of keloid fibroblasts. EGR1 could regulate the production of ROS by targeting NOX4. Furthermore, NOX4-derived ROS could promote fibrotic-like phenotype of keloid fibroblasts and play an important role in keloid fibrosis.

Conclusion: Our findings provide new insights into the mechanisms of the TGF-β1/EGR1/NOX4 pathway in keloid fibrosis, and the TGF-β1/EGR1/NOX4 axis may serve as a potential therapeutic target for keloids.

Keywords: EGR1; Fibrosis; Keloid; NOX4; Oxidative stress.