Cholesterol depletion induces anoikis‐like apoptosis via FAK down‐regulation and caveolae internalization

Abstract

Caveolae (lipid rafts), microdomains of the plasma membrane, are known to contain various signalling molecules and consequently are involved in the regulation of many biological functions. To investigate the role of the caveolae in cell survival and adhesion, we disrupted the caveolae by depletion of cholesterol, a major lipid component of the caveolae, with methyl-beta cyclodextrin (MbetaCD) treatment of A431 cells. We found that cholesterol depletion induced an anoikis-like cell death involving actin reorganization, resulting in a decrease in cell spreading and an increase in cell detachment, which was reversed by cholesterol addition. Disruption of caveolae led to the down-regulation of FAK, Src activation, tyrosine phosphorylation of caveolin-1 and mobilization of caveolae markers, GM1 and caveolin-1, from the cell surface to the cytoplasm, which were also recovered by cholesterol addition. The expression of dominant-active FAK was able to delay caveolae internalization and apoptosis and attenuated Akt inactivation by MbetaCD, whereas dominant-negative FAK expression resulted in enhanced apoptosis. Moreover, FAK down-regulation by si-RNA resulted in Akt inactivation and thus increased cell death by MbetaCD treatment. Our results suggest that the cholesterol content and/or surface levels of the caveolae affect the activity of FAK, which in turn regulates caveolae internalization and cell survival.

Antitumoral Alkylphospholipids Induce Cholesterol Efflux from the Plasma Membrane in HepG2 Cells

“…As stated under Results and in accordance with reports by other authors using different cells lines (Li et al, 2006;Park et al, 2009), the exposure of HepG2 cells to 5 mM M␤CD significantly depletes their cholesterol content and at the same time causes them to become round in shape. These changes are reversed by cholesterol replenishment.…”

“…Because cholesterol is an essential lipid component of the rafts involved in Akt activation and, as we have demonstrated here, cholesterol is depleted by exposure to APLs, we looked to see whether changes in plasma-membrane cholesterol levels altered the activation state of Akt. We used M␤CD as a positive control and found that incubation with M␤CD decreased the phosphorylation of Akt and that, after replenishment with cholesterol, the levels of phosphorylated Akt returned to control values, all of which are in accordance with data reported in other cell lines after treatment with cyclodextrins (Park et al, 2009). Likewise, the depletion of cholesterol produced after exposure to APLs led to a reduction of Akt phosphorylation in HepG2 cells, suggesting that APLs may act on Akt via the depletion of plasma-membrane cholesterol.…”

“…Cells exposed to the different APLs for 30 min became abnormally rounded in shape. A similar morphological change has been reported by other authors in cells depleted of cholesterol by exposure to M␤CD (Park et al, 2009). Thus, in our study, we further examined whether the presence of cholesterol might prevent the changes in cell morphology induced by APLs.…”

“…A similar reduction in the cholesterolreleasing efficiency of M␤CD caused by the presence of cholesterol has been reported previously (Li et al, 2006;Park et al, 2009), and it is now accepted, in fact, that M␤CD can act as a cholesterol donor-acceptor system because the ratio between the quantities of cholesterol and cyclodextrin in the complexes determines whether it will act as cholesterol acceptor or donor. At the moment, we cannot offer any unequivocal explanation for the cholesterol buffering effect on the APL-induced cholesterol efflux, but it is probable that the capability of APL micelles to accept cholesterol decreases concomitantly with an increase in the quantity of exogenous cholesterol complexed with them.…”

Nongenomic Steroid- and Ceramide-Induced Maturation in Amphibian Oocytes Involves Functional Caveolae-Like Microdomains Associated with a Cytoskeletal Environment1

“…In this respect, it has been demonstrated that cholera toxin B subunit in association with its receptor, GM1, can be internalized via caveolae/rafts [48] and that MbCD may remove gangliosides, although this is related to high MbCD concentrations [65]. In addition, disruption of caveolae by MbCD led to the mobilization of GM1 and caveolin-1 from the plasma membrane to the cytoplasm in A431 cells [66].…”