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: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
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| vol. 107
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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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