Caveolin-1 negatively regulates SR-BI mediated selective uptake of high-density lipoprotein-derived cholesteryl ester.

The class B, type I scavenger receptor (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. SR-BI is predominantly associated with caveolae in Chinese hamster ovary cells. The caveola protein, caveolin-1, binds to cholesterol and is involved in intracellular cholesterol trafficking. We previously demonstrated a correlative increase in caveolin-1 expression and the selective uptake of HDL cholesteryl esters in phorbol ester-induced differentiated THP-1 cells. The goal of the present study was to determine if the expression of caveolin-1 is the causative factor in increasing selective cholesteryl ester uptake in macrophages. To test this, we established RAW and J-774 cell lines that stably expressed caveolin-1. Transfection with caveolin-1 cDNA did not alter the amount of 125I-labeled HDL that associated with the cells, although selective uptake of HDL [3H]cholesteryl ether was decreased by approximately 50%. The amount of [3H]cholesterol effluxed to HDL was not affected by caveolin-1. To directly address whether caveolin-1 inhibits SR-BI-dependent selective cholesteryl ester uptake, we overexpressed caveolin-1 by adenoviral vector gene transfer in Chinese hamster ovary cells stably transfected with SR-BI. Caveolin-1 inhibited the selective uptake of HDL [3H]cholesteryl ether by 50-60% of control values without altering the extent of cell associated HDL. We next used blocking antibodies to CD36 and SR-BI to demonstrate that the increase in selective [3H]cholesteryl ether uptake previously seen in differentiated THP-1 cells was independent of SR-BI. Finally, we used beta-cyclodextrin and caveolin overexpression to demonstrate that caveolae depleted of cholesterol facilitate SR-BI-dependent selective cholesteryl ester uptake and caveolae containing excess cholesterol inhibit uptake. We conclude that caveolin-1 is a novel negative regulator of SR-BI-dependent selective cholesteryl ester uptake.

Palmitoylation of caveolin-1 is required for cholesterol binding, chaperone complex formation, and rapid transport of cholesterol to caveolae.

We previously demonstrated that a caveolin-chaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae. Caveolin-1 has a 33-amino acid hydrophobic domain and three sites of palmitoylation in proximity to the hydrophobic domain. In the present study, we hypothesized that palmitoylation of caveolin-1 is necessary for binding of cholesterol, formation of a caveolin-chaperone transport complex, and rapid, direct transport of cholesterol to caveolae. To test this hypothesis, four caveolin-1 constructs were generated that substituted an alanine for a cysteine at position 133, 143, or 156 or all three sites (triple mutant). These mutated caveolins and wild type caveolin-1 were stably expressed in the lymphoid cell line, L1210-JF, which does not express caveolin-1, does not form a caveolin-chaperone complex, and does not transport newly synthesized cholesterol to caveolae. All of the caveolins were expressed and the proteins localized to plasma membrane caveolae. Wild type caveolin-1 and mutant 133 assembled into complete transport complexes and rapidly (10-20 min) transported cholesterol to caveolae. Caveolin mutants 143 and 156 did not assemble into complete transport complexes, weakly associated with cholesterol, and transported small amounts of cholesterol to caveolae. The triple mutant did not assemble into complete transport complexes and did not associate with cholesterol. We conclude that palmitoylation of caveolin-1 at positions 143 and 156 is required for cholesterol binding and transport complex formation.

Activated cardiac adenosine A(1) receptors translocate out of caveolae.

The cardiac affects of the purine nucleoside, adenosine, are well known. Adenosine increases coronary blood flow, exerts direct negative chronotropic and dromotropic effects, and exerts indirect anti-adrenergic effects. These effects of adenosine are mediated via the activation of specific G protein-coupled receptors. There is increasing evidence that caveolae play a role in the compartmentalization of receptors and second messengers in the vicinity of the plasma membrane. Several reports demonstrate that G protein-coupled receptors redistribute to caveolae in response to receptor occupation. In this study, we tested the hypothesis that adenosine A(1) receptors would translocate to caveolae in the presence of agonists. Surprisingly, in unstimulated rat cardiac ventricular myocytes, 67 +/- 5% of adenosine A(1) receptors were isolated with caveolae. However, incubation with the adenosine A(1) receptor agonist 2-chlorocyclopentyladenosine induced the rapid translocation of the A(1) receptors from caveolae into non-caveolae plasma membrane, an effect that was blocked by the adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. An adenosine A(2a) receptor agonist did not alter the localization of A(1) receptors to caveolae. These data suggest that the translocation of A(1) receptors out of caveolae and away from compartmentalized signaling molecules may explain why activation of ventricular myocyte A(1) receptors are associated with few direct effects.

High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae.

Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation. In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function. We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immunoprecipitate with caveolin-1. Co-incubation of cells with 10 microgram/ml high density lipoprotein (HDL) prevented oxLDL-induced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 microgram/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL-induced redistribution of eNOS. SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL-induced depletion of caveola cholesterol. We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation. Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.

Characterization of a cytosolic heat-shock protein-caveolin chaperone complex. Involvement in cholesterol trafficking.

Caveolin is a 22-kDa protein that appears to play a critical role in regulating the cholesterol concentration of caveolae. Even though caveolin is thought to be a membrane protein, several reports suggest that this peculiar protein can traffic independently of membrane vesicles. We now present evidence that a cytosolic pool of caveolin is part of a heat-shock protein-immunophilin chaperone complex consisting of caveolin, heat-shock protein 56, cyclophilin 40, cyclophilin A, and cholesterol. Treatment of NIH 3T3 cells with 1 microM cyclosporin A or 100 nM rapamycin disrupted the putative transport complex and prevented rapid (10-20 min) transport of cholesterol to caveolae. The lymphoid cell line, L1210-JF, does not express caveolin, does not form an immunophilin-caveolin complex, and does not transport newly synthesized cholesterol to caveolae. Transfection of caveolin cDNA into L1210-JF cells allowed the assembly of a transport complex identical to that found in NIH 3T3 cells. In addition, newly synthesized cholesterol in transfected cells was rapidly (10-20 min) and specifically transported to caveolae. These data strongly suggest that a caveolin-chaperone complex is a mechanism by which newly synthesized cholesterol is transported from the endoplasmic reticulum through the cytoplasm to caveolae.