Abstract
Despite the association of cholesterol with debilitating pressure-related diseases such as glaucoma, heart disease, and diabetes, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and the stretch-activated transient receptor potential vanilloid isoform 4 (TRPV4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time-dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers. Depleting free membrane cholesterol with m-β-cyclodextrin (MβCD) augmented TRPV4 activation by the agonist GSK1016790A, swelling and strain, with the effects reversed by cholesterol supplementation. MβCD increased membrane expression of TRPV4, caveolin-1, and flotillin. TRPV4 did not colocalize or interact with caveolae or lipid rafts, apart from a truncated ∼75 kDa variant partially precipitated by a caveolin-1 antibody. MβCD induced currents in TRPV4-expressing Xenopus laevis oocytes. Thus, membrane cholesterol regulates trabecular transduction of mechanical information, with TRPV4 channels mainly located outside the cholesterol-enriched membrane domains. Moreover, the biomechanical milieu itself shapes the lipid content of TM membranes. Diet, cholesterol metabolism, and mechanical stress might modulate the conventional outflow pathway and intraocular pressure in glaucoma and diabetes in part by modulating TM mechanosensing.
Highlights
Conversion of sensory information into electrical and chemical signals in eukaryotic cells is modulated by unesterified cholesterol, a planar 27-carbon polycyclic molecule that constitutes ~20% of the total mass of membrane lipids [1, 2]
A subset of experiments was conducted in immortalized juxtacanalicular human trabecular meshwork cells obtained from ScienCell (Catalog#6590) and used up to the 7th passage. Primary cultures of TM (pTM) and hTM cells
The mechanical properties of the lipid bilayer can change under tension [31], but it is not known whether the biomechanical milieu shapes the membrane lipid content
Summary
Conversion of sensory information into electrical and chemical signals in eukaryotic cells is modulated by unesterified cholesterol, a planar 27-carbon polycyclic molecule that constitutes ~20% of the total mass of membrane lipids [1, 2]. In response to mechanical stress and glaucoma, TM cells upregulate the actomyosin cytoskeleton, ECM secretion, and the size/number of focal cell-ECM contacts, thereby increasing cell contractility and tissue resistance to the outflow of aqueous humor [22, 23, 36, 37]. It is not known how TM membrane lipid composition is affected by the biomechanical milieu and whether changes in membrane stiffness impelled by free membrane cholesterol affect TM mechanosensing, intracellular signaling, and cytoskeletal organization
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