Regulatory role of cholesterol in modulating actin dynamics and cell adhesive interactions in the trabecular meshwork

Abstract The trabecular meshwork (TM) tissue plays a crucial role in maintaining intraocular pressure (IOP) homeostasis. Increased TM contractility and stiffness are directly correlated with elevated IOP. Although cholesterol is known to be a determinant of glaucoma occurrence and elevated IOP, the underlying mechanisms remain elusive. In this study, we used human TM (HTM) cells to unravel the effects of cholesterol on TM stiffness. We achieved this by performing acute cholesterol depletion with Methyl-β-cyclodextrin (MβCD) and cholesterol enrichment/replenishment with MβCD cholesterol complex (CHOL). Interestingly, cholesterol depletion triggered notable actin depolymerization and decreased focal adhesion formation, while enrichment/replenishment promoted actin polymerization, requiring the presence of actin monomers. Using a specific reporter of phosphatidylinositol 4,5-bisphosphate (PIP2), we demonstrated that cholesterol depletion decreases PIP2 levels on the cell membrane, whereas enrichment increases them. Given the critical role of PIP2 in actin remodeling and focal adhesion formation, we postulate that cholesterol regulates actin dynamics by modulating PIP2 levels on the membrane. Furthermore, we showed that cholesterol levels regulate integrin α5β1 and αVβ3 distribution and activation, subsequently altering cell-extracellular matrix (ECM) interactions. Notably, the depletion of cholesterol, as a major lipid constituent of the cell membrane, led to a decrease in HTM cell membrane tension, which was reversed upon cholesterol replenishment. Overall, our systematic exploration of cholesterol modulation on TM stiffness highlights the critical importance of maintaining appropriate membrane and cellular cholesterol levels for achieving IOP homeostasis.