Cholesterol trafficking is required for mTOR
activation in endothelial cells
Jing Xua, Yongjun Danga, Yunzhao R. Rena, and Jun O. Liua,b,1
Departments ofaPharmacology andbOncology, The Johns Hopkins School of Medicine, Baltimore, MD 21205
Edited* by Gregg L. Semenza, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved January 28, 2010 (received for review
September 22, 2009)
a signaling network that regulates cell growth and proliferation in
cholesterol homeostasis, however, has remained unknown. We
a newly identified inhibitor of angiogenesis, itraconazole, leads to
inhibition of mTOR activity in endothelial cells. Inhibition of mTOR
by itraconazole but not rapamycin can be partially restored by
well as siRNA knockdown of Niemann–Pick disease type C (NPC) 1
and NPC2also causeinhibition of mTORin endothelialcells. In addi-
tion, both the accumulation of cholesterol in the lysosome and
inhibition of mTOR causedby itraconazolecan be reversedby thap-
sigarin. These observations suggest that mTOR is likely to be
involved in sensing membrane sterol concentrations in endothelial
cells, and the cholesterol trafficking pathway is a promising target
for the discovery of inhibitors of angiogenesis.
to regulate cell growth and proliferation (1, 2). mTOR exists in two
and mTORC2. Although mTORC1 is involved in regulating trans-
lation, ribosomal biogenesis, and autophagy mediated, in part, by
been shown to affect cellular cytoskeleton as well as Akt phosphor-
ylation(7).Amongthe upstreamsignals that are knowntoaffect the
mTOR pathway are growth factors, nutrients such as amino acids,
cellular energy status, and a variety of environmental stresses.
responsible for conferring the fluidity and impermeability to cellular
membranes, and hence the existence of individual cells. It is also
known to play an essential role in signal transduction as an essential
component of lipid rafts (8). There are two sources of cholesterol:
those that are acquired from extracellular space via LDL receptor-
mediated endocytosis (10). Both pools of cholesterol require proper
intracellular transport to reach their final destinations. Given its key
cholesterol homeostasis is under tight control. Cells employ at least
two sensor proteins, Scap and 3-hydroxy-3-methylglutaryl CoA re-
ductase, to monitor the levels of membrane sterols and to regulate
cholesterol biosynthesis (11). How membrane cholesterol levels
regulate cell proliferation, however, has remained unknown.
Hopkins Drug Library, for previously undescribed inhibitors of an-
giogenesis, and one of the most potent hits was identified as the
antifungal drug itraconazole (12). We showed that itraconazole
Although itraconazole was found to inhibit partially the human lan-
he mammalian target of rapamycin (mTOR) pathway plays a
partial, decrease in the proliferation of endothelial cells, the pre-
cise molecular mechanism of action of itraconazole has remained
unknown. In an attempt to deconvolute the mechanism of inhib-
ition of endothelial cells by itraconazole further, we uncovered a
link between intracellular cholesterol trafficking and the mTOR
pathway in endothelial cells. Herein, we report that itraconazole
causes blockade of cholesterol egress from endosomal/lysosomal
compartments to the plasma membrane, which, in turn, leads to
inhibition of both mTORC1 and mTORC2. We provide multiple
lines of evidence that mTOR activity in endothelial cells requires
proper cholesterol trafficking, adding plasma membrane choles-
terol to the list of signal inputs to regulate the mTOR pathway.
Itraconazole Up-Regulates p27 Expression and Down-Regulates p21
Expression in Endothelial Cells. Given that itraconazole causes cell
cycle arrest in the G1 phase, we determined its effects on the ex-
the G1-S transition. No appreciable changes were seen in the levels
of CDK2, Cyclin D, and p53; however, the expression of Cyclin A,
manner (Fig.1). Strikingly,the level ofp27 was up-regulated,rather
than inhibited, by itraconazole (Fig. 1).
Itraconazole Inhibits Both mTORC1 and mTORC2 in Endothelial Cells.
angiogenesis (14). We thus determined the effect of itraconazole on
inhibited the phosphorylation of p70S6K and 4E-BP1 in a dose-
dependent manner (Fig. 2A). In contrast, itraconazole had no effect
on the phosphorylation of either ERK or JNK, demonstrating a high
4E-BP1, which lie downstream of mTORC1, we examined the
phosphorylation state of Akt, which lies downstream of mTORC2
(15). Unlike p70S6K, which suffered from a decrease in phosphor-
remained largely unchanged until 8 h after itraconazole treatment
phosphorylation of Akt at both sites was inhibited by the drug in a
dose-dependent manner (Fig. 2C). These results suggested that itra-
conazole inhibited both mTORC1 and mTORC2, and inhibition of
mTORC2 likely occurred as a secondary consequence of mTORC1
Author contributions: J.X. and J.O.L. designed research; J.X., Y.D., and Y.R.R. performed
research; J.X. and J.O.L. analyzed data; and J.X. and J.O.L. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| March 9, 2010
| vol. 107
| no. 10www.pnas.org/cgi/doi/10.1073/pnas.0910872107
In addition to blocking cell cycle progression in the G1 phase,
rapamycin has been shown to induce autophagy in both yeast and
mammalian cells (17, 18). We thus determined whether itraconazole
was also capable of inducing autophagy in human umbilical vein
endothelial cells (HUVECs). Microtubule-associated protein 1 light
chain 3 (LC3) is a widely used marker of autophagy. During autoph-
agy, the cytosolic form of LC3 (LC3-I) is conjugated to phosphatidy-
lethanolamine to form a conjugate (LC3-II), which can then be
recruited to the autophagosome membranes (19). When HUVECs
were treated with itraconazole, we observed a dose-dependent and
time-dependent induction of LC3-II (Fig. 2 D and E). Using indirect
immunofluorescence, we also found that LC3 exhibited a punctate
of HUVECs with itraconazole (Fig. S1A). Together, these results
suggested that the antiangiogenic activity of itraconazole is attribut-
effects of rapamycin (20).
Itraconazole Inhibits Cholesterol Trafficking in Endothelial Cells. The
inhibitory effect of itraconazole on endothelial cell proliferation has
14α-demethylase, and hence de novo cholesterol biosynthesis (12).
We thus wondered whether the inhibition of the mTOR pathway by
was widely used to directly incorporate cholesterol to cell plasma
membranes (21). When cholesterol/cyclodextrin complex was added
to HUVECs, it partially reversed the inhibition of endothelial cell
suggesting that itraconazole caused a depletion of cholesterol on the
plasma membrane of endothelial cells.
To confirm the effect of itraconazole on the cholesterol level in
plasma membrane further, we applied filipin, a specific fluorescent
marker of unesterized cholesterol (22), to visualize and follow the
distribution of free cholesterol in HUVECs in the absence and the
presence of itraconazole. In control cells, cholesterol was clearly
visible on the plasma membrane as well as on membranes of intra-
cellular organelles such as the nuclear membrane and the ER (Fig.
plasma membrane under itraconazole treatment. Surprisingly, the
pattern was reminiscent of late endosomes and lysosomes. We thus
and lysosome (23), which revealed that free cholesterol was trapped
within the late endosomal and lysosomal compartments by itracona-
zole. This was further confirmed by the lack of colocalization of free
cholesterol and protein disulfide isomerase, an ER marker, or cyto-
chrome C, a mitochondria marker (Fig. S1 B and C). These obser-
vations suggested that itraconazole inhibited cholesterol trafficking
out of the lysosomes and raised the possibility that inhibition of
intracellular cholesterol trafficking can lead to inhibition of the
Proper Cholesterol Trafficking Is Essential for mTOR Activation in
Endothelial Cells. To assess the causal relation between inhibition
of cholesterol traffickingby itraconazole andinhibition of the mTOR
pathway, we treated HUVECs with itraconazole in the presence and
absence of cyclodextran-cholesterol and determined the phosphor-
ITRA [ M] 0.010.1 0.313
1.0 0.84 0.75 1.82.5 3.1
HUVECs. HUVECs were treated with itraconazole (ITRA) at the indicated
concentrations for 24 h, and cell lysates were subjected to SDS/PAGE fol-
lowed by Western blot analysis with the indicated antibodies. The relative
band intensities of p27 normalized against those of tubulin from the cor-
responding lanes were provided.
Down-regulation of p21 and up-regulation of p27 by itraconazole in
0.01 0.1 0.3130
0.01 0.1 0.3130
0.01 0.1 0.3130
Time (h) 0.512480
in Fig. 1. (B and E) HUVECs were treated with ITRA (2 μM) for the indicated time, and cell lysates were subjected to SDS/PAGE followed by Western blot
analysis with the indicated antibodies.
Itraconazole (ITRA) inhibits both mTORC1 and mTORC2 in HUVECs. (A, C, and D) HUVECs were treated with ITRA and processed in the same manner as
Xu et al. PNAS
| March 9, 2010
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ylation state of p70S6K. As shown in Fig. 3C, inhibition of phos-
phorylation of p70S6K by itraconazole was partially but significantly
reversed by cyclodextran-cholesterol adduct but not by either choles-
terol or cyclodextran alone. In addition, the induction of LC3-II and
phosphorylation of 4E-BP1 by itraconazole were partially but signi-
ficantly rescued by the cyclodextran-cholesterol complex (Fig. S2 A
and B). In contrast, the inhibition of mTOR by rapamycin in
HUVECs was unaffected by the cyclodextran-cholesterol complex
(Fig. S2C). These results suggest that disruption of intracellular
cholesterol trafficking from the endosome/lysosome to the plasma
Intracellular cholesterol trafficking has been shown to require two
cholesterol binding proteins within the lumen of late endosome and
lysosome, the Niemann–Pick disease type C (NPC)1 and NPC2 pro-
teins (24–26). As their names imply, genetic mutations in either gene
disease in humans and to cause blockade of intracellular cholesterol
trafficking (27, 28). To determine whether inhibition of intracellular
cholesterol trafficking by alternative means also caused inhibition
of the mTOR pathway in endothelial cells, we knocked down
NPC1 and NPC2, respectively, using siRNA in HUVECs (Fig. 4A)
anddetermined theeffects onthephosphorylationofp70S6K.The
siRNA oligonucleotides were identified that caused appreciable,
albeit incomplete, down-regulation of NPC1 and NPC2 proteins in
HUVECs by 24 h posttransfection. Similar to itraconazole, knock-
down of NPC1 or NPC2 also led to the inhibition of p70S6K
of several other kinases, including PKCα, ERK, and JNK (Fig. 4A
NPC2, two small molecule inhibitors of intracellular cholesterol
trafficking are known, U18666A and imipramine (29). Both
U18666A and imipramine have been shown to cause accumulation
of cholesterol in the late endosome and lysosome. We thus deter-
mined their effects on endothelial cell proliferation and mTOR
activity using phosphorylation of p70S6K as a readout. Both
U18666A and imipramine inhibited HUVEC proliferation in a
dose-dependent manner, with IC50values comparable to those for
inhibition of cholesterol trafficking (Fig. S3 A and B). They also
exhibited a dose-dependent inhibition of p70S6K phosphorylation
(Fig. 4 B and C) with effective concentrations similar to those
1µg/mLcholesterol (0.1% CD)
2µg/mLcholesterol (0.1% CD)
4µg/mLcholesterol (0.1% CD)
Percent of Control
0.84 220.127.116.11 0.14 0.45 0.070.83 1.00.100.08 0.10
HUVEC proliferation and mTOR activity. (A) HUVECs were seeded into 96-well plates and incubated with the indicated drugs in the presence and absence of
20 μg/mL cholesterol, cyclodextrin (CD), or cholesterol/CD complex for 24 h. Cells were then pulsed with [3H]-thymidine for 6 h. Cell proliferation rates were
normalized to those of cells treated with DMSO, cholesterol, CD, or cholesterol/CD complex only. (B) HUVECs were treated with 2 μM itraconazole for 24 h and
then fixed for immunostaining. (C) HUVECs were treated with 2 μM itraconazole in the presence of CD or cholesterol/CD complex for 24 h, and cell lysates
were subjected to SDS/PAGE followed by Western blot analyses. The values provided below the bottom of the gel are the relative intensities of the p-S6K1
normalized against those of the S6K1 bands.
Itraconazole causes accumulation of cholesterol in the late endosome/lysosome, and exogenous cholesterol partially rescues itraconazole inhibition of
| www.pnas.org/cgi/doi/10.1073/pnas.0910872107Xu et al.
required for inhibiting intracellular cholesterol trafficking (Fig.
S3C). Similar to itraconazole, neither U18666A nor imipramine
affected the phosphorylation of PKCα, ERK, or JNK, displaying
high specificity for the mTOR signaling pathway (Fig. 4 B and C).
U18666A, and imipramine on insulin- and amino acid-stimulated
mTOR activation in HUVECs. All three inhibitors effectively in-
hibited the activation of mTOR induced by either insulin or amino
acids (Fig. S4 A and B). Thus, proper cholesterol trafficking is nec-
essary for mTOR activationby multiple upstreamsignalinginputs in
endothelial cells. In contrast to the treatment with small molecule
inhibitors, however, knockdown of either NPC1 or NPC2 only had
marginal effects on mTOR activation by insulin or amino acids (Fig.
S4 C and D). It is possible that the knockdown was incomplete and
that the remaining NPC1 and NPC2 proteins were sufficient to
support mTOR activation by insulin or amino acids.
The mTOR pathway has been shown to play a key role in protein
synthesis by regulating cap-dependent translation (30, 32). We won-
dered whether cholesteroltrafficking alsoregulates proteinsynthesis
in endothelial cells. A luciferase reporter assay was thus performed.
As shown in Fig. S5A, pateamine A (PatA), a known inhibitor
of eukaryotic translation initiation, dramatically inhibited cap-
dependent translation. Somewhat surprisingly, rapamycin had a
much smaller effect on cap-dependent translation than PatA,
which suggested that the dependence of cap-dependent trans-
lation on mTOR is not as pronounced in endothelial cells. The
agreement with that observed previously (33). Nevertheless, itra-
conazole also had a small but significant effect on cap-dependent
translation similar to rapamycin. Furthermore, knockdown of
either NPC1 or NPC2 had a similar effect on cap-dependent
translation (Fig. S5B). The similar effects of itraconazole, rapa-
mycin, and NPC1/2 knockdown on cap-dependent translation in
endothelial cells are consistent with their common inhibition
Reversal of Itraconazole-Induced Accumulation of Cholesterol in Late
Endosomes/Lysosomes and Inhibition of mTOR by Thapsigargin. The
NPC phenotype has recently been shown to be attributable to the
dysregulation of endosomal/lysosomal calcium homeostasis, and th-
apsigargin, which releases ER calcium, was shown to correct the
cellular NPC phenotype (34). We thus examined the effect of thap-
by itraconazole. Thapsigargin restored the transport of cholesterol
from endosomes/lysosomes to the plasma membrane in HUVECs
by itraconazole but conferred resistance to both U18666A and im-
ipramine (Fig. 5B), indicating that restoration of cholesterol traf-
ficking by thapsigargin confers resistance to mTOR inhibition by all
three inhibitors of intracellular cholesterol transport. In contrast,
cholesterol trafficking, could not be reversed by thapsigargin (Fig.
S6A). In addition to its effect on ER calcium release, thapsigargin is
response, to reverse the inhibition of the mTOR pathway by itraco-
nazole, U18666A, and imipramine. Unlike thapsingargin, tunicamy-
by any of the three inhibitors (Fig. S6B). Together, these results
indicated that proper cholesterol trafficking is required for mTOR
signaling in endothelial cells.
administered cholesterol/cyclodextrin complex on the inhibition of
itraconazole, the inhibition of mTOR by either U18666A or imipr-
amine was significantly reduced in the presence of cholesterol/cyclo-
dextrin, consistent with the notion that the inhibitory effects of those
the lysosome to the plasma membrane (Fig. 5C).
Itraconazole is a widely used antifungal drug. We recently repor-
potent antiangiogenic activity both in vitro and in vivo (12). In the
itraconazole specifically inhibited the mTOR pathway in endo-
thelial cells. It is well known that mTOR plays an essential role in
angiogenesis. Rapamycin and its analogue, temsirolimus, both
inhibit tumor angiogenesis in vivo and directly inhibit endothelial
cells: mTORC1 and mTORC2, both of which are required for
or the small molecule inhibitors U18666A and imipramine leads to mTOR
inhibition. (A) HUVECs were transfected with the indicated siRNA oligonu-
cleotides for 24 h before they were lysed for Western blot analysis. (B and C)
HUVECs were treated with the indicated drugs for 24 h before they were
harvested for immunoblot analyses.
Blockade of cholesterol trafficking by siRNA knockdown of NPC1/2
Xu et al.PNAS
| March 9, 2010
| vol. 107
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mediates an earlier and transient response, whereas mTORC2’s
only inhibit mTORC1 but inhibit mTORC2 after long incubation
because of its disruption of mTORC2 assembly (20). The dual
to rapamycin’s antiangiogenic activity (33). In this study, we found
that itraconazole, like rapamycin, also inhibited both mTORC1 and
mTORC2 in endothelial cells on prolonged exposure. The dual
inhibition of both mTORC complexes by itraconazole offers a plau-
sible explanation for the potent cell cycle effect of itraconazole on
Further mechanistic deconvolution revealed that itraconazole
interferes withcholesterol trafficking and causesanapparent NPC
phenotype in endothelial cells. Whether the blockade of choles-
terol trafficking through the late endosome and lysosome is solely
responsible for the depletion of cholesterol in the plasma mem-
inhibit de novo cholesterol biosynthesis, which could also lead to
depletion of cholesterol in the plasma membrane. To distinguish
between thetwoalternative effects of itraconazole as thepotential
cause of mTOR inhibition, we also determined the effects of itra-
by measuring the cellular concentrations of lanosterol. Indeed,
were cultured in thepresenceofmevalonate(Table S1).However,
lanosterol became undetectable when exogenous mevalonate was
left out of the growth medium, suggesting that the de novo cho-
lesterol biosynthetic pathway was inactive. It remains to be deter-
and lysosome, or by a combination of both. It is possible that the
cholesterol depletion in the plasma membrane by itraconazole is
trafficking through the lysosome.
Building on the preliminary observations with itraconazole, we
and imipramine, and demonstrated that inhibition of cholesterol
trafficking alone is sufficient to inhibit mTOR activity. It is worth
to inhibit cholesterol biosynthesis (38). Unlike U18666A and itra-
conazole, however, imipramine has no effect on cholesterol biosyn-
thesis, and its cellular effects can only be attributed to blockade of
cholesterol trafficking. That imipramine also caused inhibition of
mTOR activity indicates that inhibition of intracellular cholesterol
trafficking alone is sufficient to block the mTOR pathway. This
conclusion was further supported by the demonstration that knock-
down of NPC1 or NPC2, known mediators of cholesterol transport
through the late endosome and lysosome, also inhibited mTOR
activity in HUVECs.
input for the mTOR pathway. Thus, in the absence of proper cho-
lesterol distribution to the plasma membrane (and likely other intra-
inhibition of endothelial cell proliferation. It is apparent that the
the plasma membrane. However, it is unclear whether there exists a
whether it is mediated through a specific receptor. It is formally pos-
sible that the depletion of cholesterol from the plasma membrane
could cause a global defect in either nutrient transport and/or dys-
function of certain cell surface receptors required for endothelial
We have shown that activation of mTOR by such upstream sig-
nals as insulin and amino acids is sensitive to inhibition by itraco-
nazole, suggesting that the site of inhibition lies somewhere in the
common pathway downstream of both insulin and amino acid sig-
Rag guanosine triphosphatases (GTPases), which have recently
been reported as regulators of mTORC1 (39). We overexpressed
constitutively active Rag GTPases to activate mTOR in the pres-
ence ofitraconazole.Interestingly, itraconazole also inhibitedRag-
mediated mTOR activation (Fig. S7A). Moreover, we also exam-
ined the effect of itraconazole on the subcellular distribution of
mTOR and found it to be unaffected by itraconazole (Fig. S7B).
in the mTOR pathway that cross-talks with cholesterol trafficking.
We have previously shown that itraconazole is selective for endo-
thelial cells, suggesting that endothelial cells may be particularly
mTOR activity in 293T and HeLa cells. We have previously shown
that HeLa cells are significantly less sensitive to itraconazole than
HUVECs (12). Similarly, 293T cells are also significantly less sensi-
resistant to the highest concentrations of itraconazole applied (Fig.
S8A). In agreement with the cell proliferation data, the mTOR
activity in both 293T and HeLa cell lines, as measured by p70S6K
phosphorylation, is also less sensitive to inhibition by itraconazole
(Fig. S8 B and C) than that in HUVECs (Fig. 2A). Similar to itra-
Im ipra mine
Contro l I TR A
TG TG + I TR A
Im ipra mine
the inhibition of cholesterol trafficking and mTOR activity by itraconazole
(ITRA), U18666A, and imipramine. (A and B) HUVECs were preincubated with
1 μM TG for 1 h before they were treated with 2 μM itraconazole, 30 μM
filipin staining (A) or harvested for immunoblot analysis (B). (C) HUVECs were
incubated with 10 μM U18666A or 30 μM imipramine in the presence or
absence of 4 μg/mL cholesterol/0.1% cyclodextrin complex for 24 h before
being harvested for immunoblot analyses using the indicated antibodies.
Effects of thapsigargin (TG) and cholesterol/cyclodextrin complex on
| www.pnas.org/cgi/doi/10.1073/pnas.0910872107Xu et al.
effect on p70S6K phosphorylation (Fig. S8D), further underscoring
the unique sensitivity of the mTOR pathway in endothelial cells to
perturbation of cholesterol trafficking. Why do endothelial cells ex-
hibit unique high sensitivity to inhibitors of intracellular cholesterol
transport? Itis temptingtospeculate that endothelialcellsmay have
evolveda higher levelof cholesterol intake fromthe plasma they are
naturally exposed to, and thus greater dependence on transport of
cholesterol from endosomes/lysosomes. The essential role of cho-
lesterol homeostasis in the activity of the mTOR pathway in endo-
thelial cells suggests that the intracellular cholesterol trafficking
for developing inhibitors of angiogenesis, as exemplified by the
unique antiangiogenic activity of itraconazole.
Materials and Methods
Materials. HUVECs and medium were purchased from Lonza, Inc. Cells were
cultured in endothelial cell growth medium (EGM)-2 media at 5% CO2. 293T
Cyclin A, Cyclin D, Cyclin E, p21, p27, p53, tubulin, Akt, p-JNK, S6K, NPC2,
protein disulfide isomerase, and cytochrome C were purchased from Santa
(ser473), and p-ERK were purchased from Cell Signaling. Antibody for NPC1
was purchased from Proteintech. Antibody for p-pkcα was purchased from
Millipore. Antibody for LC3b was purchased from Abcam. LAMP-1 antibody
developed by J.T. August and James Hildreth was obtained from the Devel-
opmental Studies Hybridoma Bank under the auspices of the National Insti-
and NPC2 (SI03026632, SI03093951) and control oligos were purchased from
Qiagen. Cholesterol, filipin, and (2-hydroxypropyl)-β-cyclodextrin were pur-
chased from Sigma.
Filipin Staining. Cellswerefixedwith4%(wt/vol)paraformaldehydeinPBSfor
30 min and stained with 50 μg/mL filipin in PBS at room temperature for 2 h.
Cells were then washed with PBS three times and mounted. Images were
captured using a Zeiss LSM510 confocal microscope.
Cholesterol Rescue Assay. The cholesterol/cyclodextrin complexes were pre-
pared as described previously (21). Cells were treated with cholesterol/cyclo-
dextrin complexes along with the indicated drugs for 24 h before they were
used for immunoblotting or cell proliferation assay.
ACKNOWLEDGMENTS. We are grateful to Dr. Peter Espenshade and Ms.
Clara Bien for technical assistance and Drs. Kun-Liang Guan and Ta-Yuan
Chang for provision of Rag plasmids and cell lines. We thank Benjamin Nacev
and Joong Sup Shim for critical comments on the manuscript. The financial
Institute, The Clinical and Translational Science Award, Keck Foundation,
Patrick C. Walsh Prostate Cancer Research Fund, and Commonwealth Foun-
dation is gratefully acknowledged.
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