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J Lipid Res
2021 Jan 01;62:100145. doi: 10.1016/j.jlr.2021.100145.
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Membrane cholesterol regulates TRPV4 function, cytoskeletal expression, and the cellular response to tension.
Lakk M
,
Hoffmann GF
,
Gorusupudi A
,
Enyong E
,
Lin A
,
Bernstein PS
,
Toft-Bertelsen T
,
MacAulay N
,
Elliott MH
,
Križaj D
.
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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.
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Figure 1The C/P ratio in TM membranes is stretch-dependent. (A) Representative chromatograms and (B) Normalized and averaged cholesterol GS/MS data in control, 1 h and 3 hours stretched primary TM cells; (C) Normalized and averaged phosphatidylcholine (PC) fluorometric data in control, 1 h and 3 hour stretched primary TM cells. Stretch induced a time-dependent decrease in membrane cholesterol level, which was concomitant with increasing content of membrane P.C. N = 3; **, P < 0.01; ****, P < 0.0001.
Figure 2Cholesterol depletion promotes formation of actin stress fibers. Double-labeling for F-actin (phalloidin-Alexa 488 nm) and filipin (405 nm). (A) Untreated preparations show typical stress fiber organization dotted by lipid rafts. (B) 1-hour incubation with MβCD results in dissolution of filipin+ puncta and upregulation of phalloidin-actin fluorescence. (C) Saturated 1:10 admixture of cholesterol (1 mM) and MβCD (10 mM) increased the number of filipin puncta. (D-E) Averaged data for experiments shown in A-C (N = 3-4). *, P < 0.05, **; P < 0.01, ***, p < 0.001 and ****, p < 0.0001. Scale bar = 20 μm.
Figure 3Cholesterol depletion increases the amplitude of TRPV4 agonist-induced Ca2+ signals. (A-B) Ratiometric signals in Fura-2 AM-loaded cells. (A) GSK101-induced elevations are increased in MβCD -treated cells (n = 8-10) (B) Averaged data. MβCD (blue bar) augmented, whereas MβCD:cholesterol (green bar) reduced the amplitude of agonist-induced fluorescence. (C) Fluorimetry, cell populations in 96 wells. 5 nM GSK101 increased Fluo-4 fluorescence. Its effect was facilitated (∼ 12%) by MβCD (N = 4). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; N.S., non-significant.
Figure 4Cholesterol depletion facilitates HTS-induced Ca2+ signals. (A-B) Cytosolic Ca2+ responses in Fura-2 AM-loaded cells. (A) HTS induced [Ca2+]i elevations in representative control, MβCD and MβCD:cholesterol -treated samples (n = 8-10) (B) Averaged data from A. MβCD (blue bar) augmented, whereas MβCD:cholesterol (green bar) reduced HTS-induced [Ca2+]i increases. (C) Fluorimetry. 55% HTS (140 mOsm) increased Fluo-4 signals. This effect was facilitated by MβCD (N = 3). ***, P < 0.001; ****, P < 0.0001; N.S., non-significant.
Figure 4Cholesterol depletion facilitates HTS-induced Ca2+ signals. (A-B) Cytosolic Ca2+ responses in Fura-2 AM-loaded cells. (A) HTS induced [Ca2+]i elevations in representative control, MβCD and MβCD:cholesterol -treated samples (n = 8-10) (B) Averaged data from A. MβCD (blue bar) augmented, whereas MβCD:cholesterol (green bar) reduced HTS-induced [Ca2+]i increases. (C) Fluorimetry. 55% HTS (140 mOsm) increased Fluo-4 signals. This effect was facilitated by MβCD (N = 3). ***, P < 0.001; ****, P < 0.0001; N.S., non-significant.
Figure 6Membrane cholesterol content regulates stretch-dependence of cytoskeletal remodeling. (A) TM cells were stretched in the presence/absence of MßCD and MßCD:cholesterol, and labeled for filipin (405 nm) and phalloidin-Alexa 488 nm in control, 6% stretch (0.5 Hz, 6%, 1 hour), MßCD and MßCD + stretch samples. (B) Averaged and normalized puncta number. Filipin is significantly reduced by stretch and MßCD alone. (C) Stretch and MßCD significantly facilitate F-actin fluorescence. Combined stimulation results in an additional ∼30% increase in stress fiber signal. (N = 3-4) *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Scale bar: 20 μm.
Figure 7Cholesterol depletion increases the number of TRPV4-ir puncta. TRPV4 immunolabeling of primary TM cells. Representative examples of (A) Control, (B) 1-hour treatment with MßCD and (C) 1-hour treatment with MβCD:cholesterol. Inset: zoomed-in region with TRPV4-ir puncta (arrow); (D) Summary of 3 independent experiments, normalized for control cells. The number of TRPV4 puncta is upregulated after incubation with MßCD. *, P < 0.05. Scale bar: 10 μm.
Figure 8Cholesterol depletion enhances TRPV4-mediated membrane currents in Xenopus oocytes (A) Representative current traces from TRPV4-expressing oocytes and uninjected control oocytes in control solution or after 45 min exposure to 50 μM MβCD. (B) I/V curves of TRPV4-expressing oocytes exposed to control solution or MβCD, with uninjected oocytes in inset. Summarized currents obtained at -85 mV shown in the lower inset. The magnitude of TRPV4-mediated currents (at Vm = -85 mV) were compared using Student’s t-test. **, P < 0.01; NS: not significant. N = 9.
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