2-Hydroxyestradiol regulates extracellular matrix deposition through estrogen receptor beta activation in airway smooth muscle cells

Airway remodeling in asthma is characterized by increased extracellular matrix (ECM) production and deposition by airway smooth muscle (ASM) cells. Existing studies have shown contrasting effects of 17β-estradiol (E₂) in regulating ASM cellular remodeling via differential activation of estrogen receptors (ERs: α and β). Even though downstream metabolites of E₂ (2-hydroxyestradiol: 2-HE and 16-hydroxyestradiol: 16αHE₂) are gaining recognition for their biological roles in various cellular systems, their role in ASM remodeling remains largely unexplored. Here, we explore the effects of 2-HE and 16αHE₂, a highly potent metabolites, on ECM remodeling in ASM. ECM mRNA's/proteins expression and deposition were determined by Western blotting, qRT-PCR, and In-Cell Western analysis. Interaction of metabolites with ERs was performed using a docking study and their impact on regulation of an estrogen response element (ERE) was monitored via a luciferase reporter assay. Further, the ER-specific effect of metabolites was validated using shRNA-mediated ERα and ERβ knockdown ASM cells. 16αHE₂ exposure showed no notable changes in transforming growth factor-β (TGF-β)-induced ECM proteins expression and deposition, whereas 2-HE exposure blunted the TGF-β effects. Molecular docking unveiled the binding of 16αHE₂ with ERα, while 2-HE more strongly bound to ERβ, which was also confirmed by ERE-luciferase assay. In ERβ knockdown ASM cells, 2-HE inhibited the TGF-β-induced phosphorylation of SMAD2/3, AKT, and ERK1/2. However, 16αHE₂ failed to elicit any of these effects. Furthermore, 2-HE significantly decreased the TGF-β-induced transcriptional activities of AP-1 and NF-κB. Overall, our findings suggest 2-HE blunts TGF-β-induced ECM through ERβ; therefore, it may serve as a novel therapeutic target for airway remodeling and asthma.