Dexamethasone Induces Senescence-Associated Changes in Trabecular Meshwork Cells by Increasing ROS Levels Via the TGFβ/Smad3-NOX4 Axis

Abstract

Glaucoma is a serious complication of glucocorticoid (GC) therapy arising through elevations in intraocular pressure (IOP). Dexamethasone (DEX) is reported to contribute to elevated IOP through different effects on the trabecular meshwork but whether DEX contributes to glaucoma development through the induction of cellular senescence is still unclear. We explored the actions of DEX on transformed human trabecular meshwork cells (HTMCs) using RNA-seq and conducted bioinformatic analyses to determine the affected pathways. Among the 4,103 differentially expressed genes identified in transformed HTMCs treated with 400 nM DEX (2,036 upregulated and 2,067 downregulated genes, respectively), bioinformatic analyses revealed significant enrichment and potential interplay between the transforming growth factor beta (TGFβ)41; signaling and cellular senescence pathways. DEX treatment induced senescence changes in primary and transformed HTMCs as indicated by increases in SA-β-gal positivity, interleukin (IL)-6 secretion, and senescence-associated heterochromatin foci (SAHF) along with selective accumulation of senescence marker p15 and elevations in reactive oxygen species (ROS) levels. Notably, the DEX-induced senescence changes were rescued by treatment with the TGFβ/Smad3 pathway inhibitor SIS3. Furthermore, we show that DEX increases cellular ROS levels via upregulation of NADPH oxidase 4 (NOX4) through activation of Smad3, and that SIS3 decreases ROS levels by downregulating NOX4. Instructively, inhibiting NOX4 with GLX351322 and scavenging ROS with NAC were both effective in preventing DEX-induced senescence changes. Similarly, we found in the mouse model that DEX-ac upregulated p15 and NOX4 expression in the trabecular meshwork, with cotreatment with GLX351322 alleviating elevations in IOP. We establish that DEX induces senescence changes in HTMCs by increasing ROS levels via the TGFβ/Smad3/NOX4 axis, increasing IOP and contributing to glaucoma development.