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Mitochondrial Cholesterol: A Connection BetweenCaveolin, Metabolism, and Disease

Marta Bosch1, Montserrat Marı2,

Steven P. Gross3, Jose C. Fernandez-Checa1,4

and Albert Pol1,5,∗

1Equip de Proliferacio i Senyalitzacio Cel·lular, Institutd’Investigacions Biomediques August Pi i Sunyer(IDIBAPS), 08036 Barcelona, Spain2Equip de Proliferacio i Senyalitzacio Cel·lular, IDIBAPS,Consejo Superior de Investigaciones Científicas (CSIC) iUnitat d’Hepatologia, Hospital Clínic i Provincial deBarcelona, Centre d’Investigacions Biomediques EstherKoplowitz, 08036 Barcelona, Spain3Department of Developmental and Cell Biology,University of California, Irvine, CA 92697, USA4Research Center for Alcoholic Liver and PancreaticDiseases, Keck School of Medicine, University ofSouthern California, Los Angeles, CA 90033, USA5Institucio Catalana de Recerca i Estudis Avancats, 08010Barcelona, Spain*Corresponding author: Albert Pol,apols@ub.edu

Caveolin (CAV) is an essential component of caveolae,

cholesterol-enriched invaginations of the plasma mem-

brane of most mammalian cells. However, CAV is not

restricted to plasma membrane caveolae, and pools of

CAV are present in myriad intracellular membranes. CAV

proteins tightly bind cholesterol and contribute to regu-

lation of cholesterol fluxes and distributions within cells.

In this context, we recently showed that CAV1 regulates

the poorly understood process controlling mitochondrial

cholesterol levels. Cholesterol accumulates in mitochon-

drial membranes in the absence of CAV1, promoting

the organelle’s dysfunction with important metabolic

consequences for cells and animals. In this article, we

suggest a working hypothesis that addresses the role of

CAV1 within the homeostatic network that regulates the

influx/efflux of mitochondrial cholesterol.

Key words: caveolae, caveolin, cholesterol, glutathione,

mitochondria, oxidative stress

Received 16 May 2011, revised and accepted for publi-

cation 27 July 2011, uncorrected manuscript published

online 29 July 2011, published online 24 August 2011

Caveolin Regulates Intracellular CholesterolFluxes

Caveolae are distinctive invaginations of the plasma mem-brane of most mammalian cells (1). Organized as highly

condensed domains, they have significant accumulationsof both glycosphingolipids and cholesterol. Caveolin (CAV)is an essential component of caveolae: a clear phenotypeof CAV1- and CAV3-deficient mice (CAV−/−) is the com-plete loss of caveolae (2–5). Conversely, CAV expressionin cells results in the assembly of caveolae at the cellsurface (6,7). Lipids are also indispensable caveolar com-ponents; cholesterol depletion causes flattening of cave-olae (8). Further, there is an important interplay betweenlipids and proteins: cholesterol addition accelerates CAVtransport to the plasma membrane (9), whereas this trans-port is inhibited by depletion of glycosphingolipids (10).

While critical for caveolae, CAV proteins likely have addi-tional functions as they are not entirely restricted to caveo-lae in the plasma membrane. Intracellular CAV is present inmyriad locations, including the Golgi complex (11,12), theendoplasmic reticulum (ER)/Golgi network (13), cis-Golgicisternae (14), in trans Golgi network (TGN)-derived vesi-cles of epithelial cells (15,16), exocytic and endocyticcarriers (17,18), endosomes (19–22), cytosolic complexeswith chaperones (23), cytosolic lipid droplets (24–27),secretory vesicles (28), mitochondria (28) and peroxi-somes (29). Whether these non-caveolar pools of CAVare connected under physiological conditions by specifictrafficking pathways and the biological significance of theprotein in each location also remain unclear (30).

Functionally, CAV1 binds cholesterol with high affin-ity (31), and CAV’s ability to move between these com-partments might contribute to regulation of cholesterolfluxes and distributions within cells (1,9,25). In support ofthis general statement, an ectopically expressed CAVdominant-negative mutant (CAVDGV) promotes a com-plex intracellular lipid imbalance. Expression of CAVDGV

increases the level of free cholesterol in late endosomes,but depletes cholesterol in the Golgi complex and theplasma membrane. Interestingly, although CAVDGV irre-versibly accumulates in the membranes of the ER and onlipid droplets (32), deregulation of cholesterol fluxes mod-ifies signaling pathways at the cell surface, such as theH-Ras-mediated activation of Raf-1. The inhibitory effectof CAVDGV on signaling is completely reversed by replen-ishing the cell membrane with cholesterol and reproducedby depletion of membrane cholesterol (33). Therefore, bymodulation of intracellular cholesterol fluxes CAV mightfacilitate multiple cellular processes potentially occurringin virtually all the organelles of cells.

In this context, we recently showed that CAV1 par-ticipates in the regulation of mitochondrial cholesterol

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levels (34). In the absence of CAV1, cholesterol accu-mulates in mitochondrial membranes. This accumulationhas a high biological cost. Combination of in vivo andin vitro analysis revealed that cholesterol accumulationpromotes the organelle’s dysfunction at four connectedlevels: (i) it reduces the fluidity of the mitochondrial mem-brane, (ii) it reduces the efficiency of the respiratory chain,(iii) it increases the generation of reactive oxygen species(ROS) and (iv) it impairs the uptake of mitochondrial glu-tathione (mGSH), a key mitochondrial antioxidant. Clearlypinpointing causality of cholesterol levels in the organellemalfunction, cholesterol depletion of CAV1−/− mitochon-dria to the wild-type (wt) levels restored mitochondrialfunction. Conversely, cholesterol loading of wt type mito-chondria to the CAV1−/− levels reproduced mitochondrialfailure. Importantly, re-expression by retroviral infection ofCAV1 in CAV1−/− cells rescued mitochondrial function-ality, showing direct causality of CAV1 in mitochondrialdysfunction but also temporality in the accumulation ofcholesterol in mitochondria. Thus, cells can potentiallymodulate mitochondrial cholesterol levels by regulation ofCAV1 expression. Indeed, CAV1 expression is upregulatedat the transcriptional level by cholesterol (35).

At the cellular level, the combination of these four factorshas three decisive consequences: (i) it causes reducedcellular proliferation when the availability of glucose isreduced, (ii) it causes mitochondrial failure that pro-motes apoptosis in a situation when the function ofthe organelle is required and (iii) it results in an ampli-fied susceptibility to apoptotic inducers such as cytokinesand mitochondrial toxins. Indeed, CAV1−/− cells undergoan ROS-mediated apoptosis during nutrient limitation[induced by 2-deoxyglucose (2-DG); Figure 1], during theshift from aerobic glycolysis to mitochondrial oxidativephosphorylation (OXPHOS, promoted by dichloroacetate)or when cells are challenged with pro-apoptotic factors[such as tumor necrosis factor (TNF)-α or the agonisticanti-Fas antibody Jo2]. Supplementation with antioxi-dants highly reduces the apoptosis that CAV1−/− cellsshow in the above situations, clearly pinpointing ROSas the apoptogenic triggers. At the animal level, sucha mitochondrial dysfunction also predisposes CAV1−/−

animals to mitochondrial-related diseases such as steato-hepatitis or the progressive neurodegeneration occurringduring Huntington’s and Alzheimer’s diseases [see alsoRef. (36)]. Further, during liver regeneration after partialhepatectomy – in which glucose levels in serum rapidlyfall – CAV1−/− mice showed reduced cellular proliferationand low survival (37). Supplementation of CAV1−/− micewith glucose restores liver regeneration and mice survivalby reactivation of hepatocyte proliferation.

How Does Caveolin Regulate MitochondrialCholesterol?

The evidence that caveolae are organelles enriched incholesterol with respect to the rest of the plasma

membrane was already provided in pioneering stud-ies (47). In contrast, mitochondria are cholesterol-poororganelles and very little is known about the homeostaticnetwork that regulates the influx/efflux of mitochon-drial cholesterol (48). Elucidation of these pathways hasbecome essential since mitochondrial cholesterol playsa key role not only in organelle’s function but also indisease progression (49). It may acquire epidemic dimen-sions, for instance, because cholesterol accumulates inhepatic mitochondria of obese ob/ob mice (46). Excel-lent reviews on the mechanisms that regulate cholesteroltrafficking in cells and mitochondria can be found in theliterature (48). Thus, here we will specifically center ouranalysis on the emerging role of CAV1 in the regulation ofmitochondrial cholesterol. In this article, we will proposea working hypothesis – based on the new experimen-tal data, combined with previous published results – toaddress a possible mechanism for how cholesterol accu-mulates in the mitochondria of CAV1-deficient cells.

In principle, CAV1 could directly modulate cholesterolin the mitochondria by trafficking through the organelle.Although this has been proposed (28), it does not appearlikely here. In our experimental conditions, we could notdetect CAV1 in mitochondria purified from wt livers (34).However, it is increasingly accepted that cholesterol par-tially reaches mitochondria through specialized domainsof the ER called mitochondrial-associated membranes(MAMs) (40). Interestingly, CAV1 was recently describedas a MAM-resident protein (41), and thus is in principle inan excellent position to regulate cholesterol fluxes fromand into MAM (Figure 1). In support of this possibility, wedetected CAV1 in a crude fraction containing mitochondriaand associated ER (34). Further, CAV1 is established tobind cholesterol in the ER (38), and transports this newlysynthesized cholesterol from the ER to the plasma mem-brane (23,50). Accordingly, cholesterol efflux to the cellsurface is more rapid in cells expressing CAV1 (50), andCAV1 expression enhances cholesterol efflux in hepaticcells (51). Similarly, cholesterol addition rapidly acceler-ates the exocytic trafficking of newly synthesized CAV1from the ER to the plasma membrane (9). Taken together,we believe that this data supports the hypothesis thatCAV1 promotes cholesterol efflux out of the ER, reducingthe availability of cholesterol in MAM, and thus ultimatelythe entry of cholesterol into mitochondria (Figure 1).

At the molecular level, CAV is an integral membrane pro-tein, synthesized in the ER in a signal recognition particle(SRP)-dependent manner (52). Then, the newly synthe-sized protein rapidly goes through a first stage of oligomer-ization into low molecular weight oligomers in a processstabilized by binding with cholesterol (38,39,52). Imme-diately after synthesis, CAV is recruited to ER exit sites(ERES) and the coat protein II (COPII) machinery mediatesits transport to the Golgi complex (39). This immediateforward transport of CAV into the exocytic pathway willdirectly reduce the levels of newly synthesized cholesterolin the membranes of the ER, and ultimately the availability

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A

B C

Figure 1: CAV regulates the influx of mitochondrial cholesterol: metabolic implications. A) CAV (black C) is synthesized in theER and rapidly goes through a first stage of oligomerization into low molecular weight oligomers in a process stabilized by cholesterol(red slashes) (38). Immediately after synthesis, CAV is recruited to ERES (39) and the COPII machinery mediates its transport to theGolgi complex. Exit from the Golgi complex is associated with the formation of high molecular weight oligomers and incorporation intocholesterol-enriched lipid-raft structures. Finally, the mature caveola-like exocytic carrier is incorporated into the plasma membrane (1,17)(detail in B). The physical association between the ER and mitochondria, known as MAM, participates in the influx of cholesterol intothe organelle (40). CAV1 is a MAM-resident protein (41) and thus it is in a position to modulate cholesterol fluxes from/to MAM. Wepostulate that newly synthesized CAV binds cholesterol in the ER and the CAV–cholesterol complexes are rapidly cleared into ERESand transported to the Golgi complex. This immediate forward transport of CAV (active transport) reduces overall levels of newlysynthesized cholesterol in the membranes of the ER, and thus cholesterol levels in MAM and ultimately the influx of cholesterolinto the mitochondria. In contrast, in the absence of CAV (right panel situation in C) cholesterol accumulates in the bulk of the ER,including MAM, and thus increases its transport into mitochondria. Glucose is internalized by cells and rapidly phosphorylated by the keyrate-limiting enzyme hexokinase (42): this process can be inhibited by 2-DG, which is a glucose analog that acts as a competitive inhibitorof glucose phosphorylation. Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK) (43). Inhibition of PDK resultsin the activation of pyruvate dehydrogenase (PDH), a gate-keeping enzyme that shifts glucose metabolism from lactate production tomitochondrial OXPHOS. Mitochondrial OXPHOS represents a major source of ROS, especially in a low energy context (44), and ROS areeffective apoptogenic molecules (45). mGSH is a key antioxidant imported from the cytosol to modulate the levels of ROS, the oxidativestate of the cell, and ultimately apoptosis (46).

of mitochondrial cholesterol via MAM (Figure 1). Next,CAV transport through the Golgi complex is relativelyslower and Golgi pools of newly synthesized CAV areobserved in many cell types (9). The exit of CAV from theGolgi complex is associated with the formation of highmolecular weight oligomers (39) and masking of specificCAV epitopes by incorporation into cholesterol-enrichedlipid-raft structures (9). This most probably occurs in lateGolgi compartments, which are especially enriched inglycosphingolipids and in cholesterol (48). Finally, themature caveola-exocytic carrier is incorporated into theplasma membrane (1,17). At the cell surface, auxiliary pro-teins such as polymerase I and transcript release factor(PTRF)/cavins stabilize the organelle (19,39,53) and – in

contrast to the intracellular pools of the protein – CAVbecomes relatively immobile (18). Because cholesterolhomeostasis is so important, there are clearly multipleregulatory mechanisms. In baby hamster kidney (BHK)cells, the exocytic pathway through the Golgi complex con-tributes only partially to the transport of newly synthesizedcholesterol to the cell surface, and it was estimated as con-trolling approximately 20% of the total (54). Alternatively,it was proposed that a soluble form of CAV associated withchaperone complexes also co-operates in the transport ofnewly synthesized cholesterol from the ER through thecytoplasm to caveolae (23). Although questions remainabout the relative importance of this cytosolic pool, CAValone cannot control all cholesterol transport and in many

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cases – especially in optimal culture conditions – a deficiton this pathway will only have a mild impact on cells.

The hypothesis that CAV1 promotes cholesterol efflux outof the ER and thus ultimately reduces the entry of choles-terol into mitochondria predicted that steroid biosynthesisshould be affected by the absence of CAV1. In steroido-genic cells, after synthesis at the ER, cholesterol istransported into mitochondria to become pregnenolone,the precursor of steroids, by the P450 side chain cleav-age enzyme (CYP11A1). Then, pregnenolone leaves themitochondrion to undergo enzymatic transformation intofinal steroid products. Mitochondrial cholesterol availabilityis the rate-determining step in steroid biosynthesis (55),so pregnenolone levels indicate the rate of mitochon-drial cholesterol influx. In support to our hypothesis, weshowed that reduction of CAV1 protein levels in steroido-genic F2-CHO proportionally increased pregnenolonebiosynthesis. We confirmed that the same mechanismoccurs in animals: concentrations of serum steroids (preg-nenolone, corticosterone and testosterone) were found tobe significantly higher in CAV1−/− mice (34).

Moreover, the model proposed in Figure 1 makes fouradditional predictions that we have experimentally testedin this article (Figure 2). First, in CAV1-deficient cells, thereshould be an intracellular accumulation of newly synthe-sized cholesterol caused by the reduced transport of thenew cholesterol into the plasma membrane. To test this,cells were incubated with the radio-labeled cholesterolprecursor 14[C]-acetate, and intracellular accumulationof newly synthesized cholesterol was determined afterjust 3 h by thin-layer chromatography (TLC). As shownin Figure 2A,B, CAV1-deficient cells accumulate radio-labeled cholesterol and early arrival of this cholesterolto the cell surface, using a non-specific acceptor suchas cyclodextrin, is reduced in CAV1-deficient hepatocytes

and mouse embryonic fibroblasts (MEFs). Third, as a resultof the reduced efflux, cholesterol levels throughout theexocytic pathway followed by CAV1 (39) should be higherin CAV1−/− cells. Indeed, as predicted by others (56), herewe have measured by high-performance liquid chromatog-raphy (HPLC) a significant enrichment in free cholesterollevels in ER purified from the liver of CAV1-deficient mice(Figure 2C; 32.79 ± 10.33%). Finally, our last predictionis that cholesterol levels in the Golgi complex shouldbe also higher in CAV-deficient cells, because CAV exitfrom the Golgi complex is associated with insertion intolipid-raft domains (1,17). After acquisition of raft proper-ties, a single CAV protein seems to be able to organizean estimated number of approximately 140 molecules ofcholesterol (57). Indeed, as shown in Figure 2D, there isa significant enrichment (53.86 ± 5.01%) in the levels offree cholesterol present in a Golgi fraction purified fromCAV1−/− mice liver. Therefore, these results support theworking model proposed in Figure 1 and strongly implicateCAV in the crucial lipid transport occurring throughout theexocytic pathway.

Concluding Remarks

In conclusion, the cholesterol-binding capacity of CAV1,in combination with complex intracellular trafficking,converts CAV1 into an excellent candidate to functionas a sensitive lipid-organizing molecule not only at the cellsurface but also on intracellular membranes. In supportof this general idea, we have particularly shown thatin the absence of CAV1, cholesterol accumulates inmitochondrial membranes and the exocytic pathway. Withthe available data we favor a model in which the increasedinflux of cholesterol into mitochondria is a consequenceof inefficient cholesterol efflux out of the ER. Cholesterolaccumulation in the ER could account for the cholesterol

A B C D

Figure 2: Inefficient exocytic transport of newly synthesized cholesterol in CAV1−/− cells. A) Primary hepatocytes and MEFs wereincubated for 3 h with 14[C]-acetate and its incorporation into free cholesterol analyzed by TLC (example in bottom). Band intensity wascalculated by densiometry and is shown as relative to the intensity of the band corresponding to wt cells in each experiment. B) Efflux ofradio-labeled cholesterol to cyclodextrin was measured during the last hour. The results are shown as relative to the efflux calculated inwt cells in each experiment. C and D) Free cholesterol content of the ER and Golgi complex isolated from wt (white bars) and CAV1−/−

mice liver (black bars) and measured by HPLC. Statistical significances were determined in at least five independent experiments usingthe Student’s t-test, ∗p < 0.05, ∗∗p < 0.01.

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increase in mitochondria, as the magnitudes of theincreases are similar: 32.79 ± 10.33% for ER (Figure 2C)and 39.16 ± 6.38% for mitochondria (34).

This hypothesis of CAV as a cholesterol transporterproposes an alternative view of caveolae. It has beensuggested that mammalian cells use caveolae and lipiddroplets to ensure a supply of lipid-raft componentsto the cell when needed (1) and indeed, regulatory cir-cuits that co-ordinate their functions are followed byCAV (9,32,58,59). However, as cholesterol levels in dif-ferent subcellular membranes can change the organellesfunction (as we saw for mitochondria), it is likely impor-tant to have ‘storehouse’ locations, where it is possible torapidly sequester as well as release extra cholesterol untilneeded, but where such changes will not impair cellu-lar function. We hypothesize that caveolae may serve thisrole. If so, it would explain the dynamic nature of caveolae,especially in response to changes in cholesterol levels, andthe fact that the precise contribution of caveolae and CAVto cell biology and animal physiology has remained difficultto elucidate, in spite of many investigations searching forthe fundamental function of caveolae (1).

As might be expected from subtle impairment ofcholesterol homeostasis, the CAV1 null animal lacks aclear fundamental phenotype. Nonetheless, there are avariety of mild phenotypes that link the loss of CAVfunction with unrelated diseases (3,60,61), which are likelyexplained by metabolic failure and oxidative stress (62). Bydiscovery that CAV1 loss alters mitochondrial function,we provide a single underlying mechanism. In aphysiological context, cells continuously switch betweenaerobic glycolysis and OXPHOS, and thus a lack ofCAV function would progressively result in sustainedoxidative stress and apoptosis, contributing to diseasepathogenesis.

Methods

Animals, cells and primary hepatocytesThey were cultured, treated or isolated exactly as previously described (34).Cholesterol in subcellular fractions was measured by HPLC asdescribed (34,46). Purified ER and Golgi were obtained from mice liveras described elsewhere (63). Statistical analysis of differences were deter-mined using the Student’s t-test, ∗p < 0.05, ∗∗p < 0.01. All data shown inthe graphs are the mean ± SD.

Cholesterol synthesisIntracellular accumulation of newly synthesized cholesterol was deter-mined by TLC by using 14[C]-acetate as the precursor. Cells were platedin 6-well culture plates the day before and finally labeled by incubationwith 1 μCi/mL of 14[C]-acetate for 3 h at 37◦C. After washing with PBS,cells were trypsinized, centrifuged and resuspended in 100 μL of PBS. Analiquot of the homogenate was taken for protein determination and lipidswere extracted according to Bligh and Dyer (64), and separated by TLCby using petroleum ether/diethyl ether/acetic acid (60:40:1) as the solvent.Plates were exposed during 7 days for autoradiography. The band corre-sponding to cholesterol was identified by comigration with a cholesterolstandard. Band intensity was calculated by densitometry and is shown

as relative to the intensity of the band corresponding to wt cells in eachindependent experiment.

Cholesterol effluxCells were plated in 6-well culture plates, growth overnight and labeled byincubation with 1 μCi/mL of 14[C]-acetate for 2 h at 37◦C. After labeling,cells were rinsed 3× with DMEM and incubated for 1 h at 37◦C withserum-free DMEM or DMEM containing 10 mM β-cyclodextrin; 100 μLaliquots were taken out for the liquid scintillation counter and cells wereharvested to determine protein concentrations. Cholesterol incorporatedon the plasma membrane was calculated as the cpm obtained in thepresence of β-cyclodextrin after subtracting the cpm obtained in freeDMEM. Results are shown as relative to the efflux calculated for wt cellsin each independent experiment.

Acknowledgments

A. P. was supported by grants (BFU2011-23745, CSD2009-00016 andMarato de TV3 PI041315), M. M. by PI10/02114, S. P. G. by GM64624/NIHand J. C. F.-C. by SAF2009-11417 and HI2007-0244/MCI, P50-AA-11999/NIAAA/NIH and Marato de TV3 PI041315. We thank Dr CarlosEnrich, Dr Francesc Tebar, Dr Anna Colell, Dr Elina Ikonen and the membersof A. P.’s and J. C. F. C.’s labs for scientific discussion and Dr AmericaGimenez and Josep Ma Marimon from the Animal Facility (UB), and AlbaFajardo, Anna Colell, Adam Kassan, Albert Herms and Susana Nunez fortechnical assistance.

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