Venkatraman Manickam,1Ajit Tiwari,1Jae-Joon Jung,1Resham Bhattacharya,2Apollina Goel,3Debabrata Mukhopadhyay,2
and Amit Choudhury1
1Department ofAnatomy and Cell Biology, University of Iowa, Iowa City, IA;2Department of Biochemistry and Molecular Biology, Mayo Clinic College of
Medicine, Rochester, MN; and3Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, IA
Vascular endothelial growth factor recep-
tor 2 (VEGFR2) plays a key role in physi-
ologic and pathologic angiogenesis.
Plasma membrane (PM) levels of VEGFR2
are regulated by endocytosis and secre-
tory transport through the Golgi appara-
tus. To date, the mechanism whereby the
tus remains incompletely characterized.
We show in human endothelial cells that
binding of VEGF to the cell surface local-
ized VEGFR2 stimulates exit of intracellu-
lar VEGFR2 from the Golgi apparatus.
BrefeldinAtreatment reduced the level of
traffics through the Golgi apparatus en
route to the PM. Mechanistically, we show
that inhibition of syntaxin 6, a Golgi-
localized target membrane-soluble N-
tor (t-SNARE) protein, interferes with
VEGFR2 trafficking to the PM and facili-
tates lysosomal degradation of the
VEGFR2. In cell culture, inhibition of syn-
taxin 6 also reduced VEGF-induced cell
proliferation, cell migration, and vascular
tube formation. Furthermore, in a mouse
ear model of angiogenesis, an inhibitory
form of syntaxin 6 reduced VEGF-
induced neovascularization and perme-
ability. Our data demonstrate the impor-
tance of syntaxin 6 in the maintenance of
cellular VEGFR2 levels, and suggest that
the inhibitory form of syntaxin 6 has good
potential as an antiangiogenic agent.
Members of the vascular endothelial growth factor (VEGF) family
bind to cell-surface receptors to regulate both physiologic and
pathologic angiogenesis.1The activities of the VEGF-A isoform
are mediated primarily through its interactions with 2 high-affinity
receptor tyrosine kinases expressed on the vascular endothelium:
VEGF receptor 2 (VEGFR2, KDR, Flk-1) and VEGFR1 (Flt-1).2
VEGF-mediated angiogenic signaling has been attributed primarily
to the signal transduction processes that are initiated by VEGFR2.3
Cell-surface VEGFR2 internalized by the clathrin-dependent endo-
cytic process is constitutively recycled back to the plasma mem-
brane (PM).4-7Upon VEGF stimulation a fraction of the internal-
ized endocytic pool of VEGFR2 is sorted toward late endosomes
and lysosomes for degradation, whereas the remainder is recycled
to the PM.4,5,8-10Antiangiogenic approaches currently in use work
by blocking VEGF binding to VEGFR2 and/or signaling by the
receptor tyrosine kinases.11These strategies highlight the impor-
tance of intracellular transport mechanisms that coordinate the
expression of VEGFR2 at distinct subcellular locations.
The Golgi apparatus is a central hub for membrane trafficking
across the mammalian cell. It receives newly synthesized proteins
and lipids from the endoplasmic reticulum (ER), modifies many of
these cargoes as they pass through, and finally sorts them to various
destinations as they exit.12-15Thus the Golgi apparatus is a likely
candidate for regulating VEGFR2 trafficking. Unraveling the
precise transport pathway and the molecular players may offer
better understanding of regulation of the VEGFR2 function.
Nevertheless, to date it remains unclear how secretory transport
through the Golgi apparatus coordinates the cell-surface expression
In eukaryotic cells, most membrane fusion steps require soluble
N-ethylmaleimide–sensitive factor attachment protein receptors
(SNAREs).16,17The SNAREs are classified into 2 major classes
based on the presence of a glutamine (Q SNAREs or t-SNAREs) or
an arginine (R SNAREs or v-SNAREs) in the center of the SNARE
motif.16Syntaxin 6, syntaxin 10, and syntaxin 16 are members of
the t-SNARE protein family, and are localized primarily in the
Golgi apparatus but also in endosomes, and are involved in the
transport of molecules to and from the Golgi apparatus.18-23
Previously, we showed that syntaxin 6 regulates the post-Golgi
transport of membrane microdomain components to the PM.18
However, to date the SNARE complex that mediates VEGFR2
trafficking remains unidentified.
In the current study, we investigated the possibility that syntaxin
6 affects the post-Golgi transport and cell-surface levels of
VEGFR2, as well as VEGF-induced angiogenesis. We show that in
quiescent endothelial cells a pool of VEGFR2 is present in the
Golgi apparatus, and that this Golgi pool of VEGFR2 is rapidly
depleted in response to stimulation with VEGF.Among syntaxin 6,
syntaxin 10, and syntaxin 16, syntaxin 6 was found to maintain
cellular levels of the VEGFR2. When syntaxin 6 function was
selectively inhibited, VEGFR2 was targeted to lysosomes for
degradation, and the levels of VEGF-induced proliferation, migra-
tion, and morphogenesis were decreased. These results show for
the first time that syntaxin 6 regulates post-Golgi trafficking of
Submitted June 21, 2010; accepted November 3, 2010. Prepublished online as
Blood First Edition paper, November 9, 2010; DOI 10.1182/blood-2010-06-291690.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by TheAmerican Society of Hematology
1425BLOOD, 27 JANUARY 2011?VOLUME 117, NUMBER 4
For personal use only.on December 14, 2015. by guest
VEGFR2 as well as VEGF-induced angiogenic processes. Further-
more, expression of an inhibitory form of syntaxin 6 was found to
block VEGF-induced angiogenesis, providing the first demonstra-
tion that an inhibitory mutant t-SNARE can act as a potent
The rabbit monoclonal antibody (mAb) against human VEGFR2 (55B11)
was purchased from Cell Signaling Technology. The goat anti–human
VEGFR2 antibody, goat anti–human VEGFR1 antibody, and recombinant
VEGF-A165 (VEGF) were purchased from R&D Systems. The goat
polyclonal antibody (pAb) against syntaxin 6 and the rabbit polyclonal
antibody against VEGFR1 were obtained from Santa Cruz Biotechnology.
The mAb against lysosome-associated membrane protein 2 (Lamp2) was
obtained from the Developmental Studies Hybridoma Bank at the Univer-
sity of Iowa. The mAbs against early endosome-associated antigen
1 (EEA1), syntaxin 6, and trans-Golgi network 46 (TGN46), the rat
anti–mouse CD31 antibody, and growth factor–reduced Matrigel were
obtained from BD Biosciences. Recombinant adenovirus expressing the
cytosolic inhibitory form of syntaxin 16 (syntaxin 16-cyto) was a gift from
Dr Gwyn W. Gould (University of Glasgow). Rabbit pAb against syntaxin
6 and syntaxin 16 and mouse mAb against syntaxin 16 were purchased from
Synaptic Systems. Fugene 6 HD transfection reagent, protease inhibitor,
and phosphatase inhibitor cocktail tablets were obtained from Roche
Diagnostics. Alexa Fluor–conjugated secondary Abs were obtained from
Invitrogen, Molecular Probes. Vectashield mounting medium was pur-
chased from Vector Laboratories. The mAbs against ?-tubulin, cyclohexi-
mide (CHX), chloroquine (CHQ), bafilomycin AI (Baf), and lactacystin
(Lac) were purchased from Sigma-Aldrich. Protein A and protein
G Sepharose beads and Amplify-fluorographic Reagent were purchased
from GE Healthcare. SuperSignal West Femto ECL reagent was obtained
from Thermo Scientific.35S-methionine and cysteine (EasyTag Express
35S Protein labeling mix) were obtained from PerkinElmer.
Primary human umbilical vein endothelial cells (HUVECs) were obtained
from Lonza and cultured on collagen-coated plates in complete medium
(endothelial cell basal medium containing supplements from Lonza).
HUVECs were used only between passages 3 and 7. The 293T human
embryonic kidney cells with simian virus 40 large T antigen (ATCC) were
maintained in Dulbecco modified minimum essential medium containing
10% fetal bovine serum. Treatment with VEGF-A165was carried out at
50 ng/mLconcentrations unless otherwise indicated.
Adenoviral infections, plasmids, and short interfering and
short hairpin RNAs
Recombinant adenovirus expressing the cytosolic domains of syntaxin
6 and syntaxin 16 (designated syntaxin 6–cyto and syntaxin 16–cyto) were
used as described previously.18,24Except as noted, cells were infected at
shown with the indicated adenovirus (titer ? 1.5 ? 107plaque-forming
units/mL) at a multiplicity of infection of 1:75 in serum-free cell-culture
medium.After 12 hours of infection, the medium was replaced with normal
medium supplemented with 10% serum, and the cells were used for the
experiments 12 to 24 hours later. We cloned both full-length human STX10
(residues 1-249) and a cytosolic form (residues 1-228) into the enhanced
green fluorescence protein (EGFP) plasmid vector pEGFP-C1 (Clontech);
the expected sequence (NM_003765) for each construct was confirmed.
EGFP-syntaxin 10 was used for cellular localization studies.
RNA interference–mediated knockdown was performed using Silencer
Select predesigned short interfering RNAs (siRNAs) against STX6
(5?-GCAACUGAAUUGAGUAUAA-3?) and STX16 (5?-CAGCGAUUG-
GUGUGACAAA-3?) in conjunction with the siPORT NeoFX transfection
reagent from Ambion. The specificity and extent of knockdown of both
proteins was determined 24, 48, and 72 hours after transfection of 293T
cells, by immunoblotting. In the case of 293T cells, both syntaxin 6 and
syntaxin 16 were reduced by approximately 90% at the protein level (data
not shown). RNA interference was less successful in HUVECs, which are
less transfectable; the fraction of cells showing at least a 10-fold reduction
in syntaxin 6 or syntaxin 16 levels after transfection was approximately
12% to 15%, as judged by immunofluorescence (supplemental Figure 5,
available on the Blood Web site; see the Supplemental Materials link at the
top of the online article).
Immunoblotting, immunoprecipitation, and35S-methionine
Procedures for labeling protocol are described in supplemental Methods.
Cell-surface biotinylation studies
Measurements of the cell surface–localized pool of biotinylated VEGFR2
and intracellular degradation of this population were performed using
methods previously described.10Details of cell-surface biotinylation
measurements are provided in supplemental Methods.
Internalization and degradation of surface VEGFR2
The internalization of VEGFR2 from the cell surface and its degradation
were analyzed using previously described methods.25We used an E-tagged
single chain fragment variable (ScFv) mAb against the extracellular domain
of human VEGFR2 (ScFvA7), which has been reported to be devoid of
biologic activity.25,26Cells were maintained at 10°C (a temperature at which
endocytosis does not occur)27with 10 ?g/mL of antibody in Hanks
minimum essential medium (HMEM) buffer ([pH 7.4], 13.8mM HEPES
acid, 137mM NaCl, 5.4mM KCl, 5.5mM glucose, 2.0mM glutamine,
0.4mM KH2PO4, 0.18mM Na2HPO4, 1.25mM CaCl2, and 0.08mM MgSO4)
for 30 minutes. Before stimulation with VEGF, cells were washed in
ice-cold HMEM buffer with 1% BSA to remove unbound Ab, and
maintained in fresh buffer. Cells were then incubated with VEGF at 10°C
for an additional 30 minutes to allow VEGF to bind to surface VEGFR2.
Samples were then incubated at 16°C for 30 minutes in HMEM buffer as
previously described, to allow internalized VEGF-VEGFR2 complexes to
accumulate in early endosomes.27Subsequently, samples were moved to a
cold station maintained at 10°C, and the remaining surface-bound antibody
was removed by an acid wash (3 washes with ice-cold 50mM glycine in
HMEM buffer [pH 2.5] and 2 washes with HMEM buffers [pH 7.5]). The
cells were then subjected to a chase at 37°C in HMEM buffer. At each of
several time points, the cells were moved to a 10°C cold station and were
acid washed to remove any antibody that may have recycled to the surface
during the chase. To assess the intracellular distribution of the anti-
VEGFR2 antibody, cells were fixed, permeabilized, and incubated with
fluorescein isothiocyanate (FITC)–labeled rabbit antibody against its E-tag.
Samples were then processed for fluorescence microscopy.
Microscopy and image analysis
For immunofluorescence studies, cells were grown on acid-washed glass
coverslips. Cells were fixed, permeabilized, and then labeled with primary
antibodies. Microscopy and image analysis are described in supplemental
Endothelial cell proliferation, migration, and vascular tube formation
assays are conducted. Detailed protocols for HUVECs proliferation, wound
healing, Boyden chamber migration, and Matrigel-based morphogenesis
assays are provided in supplemental Methods.
1426 MANICKAM et al BLOOD, 27 JANUARY 2011?VOLUME 117, NUMBER 4
For personal use only. on December 14, 2015. by guest
Contribution: V.M. performed in vitro experiments; A.T. per-
formed in vitro and animal experiments; J.-J.J. performed in
vitro experiments; R.B. conducted wound healing and animal
experiments; A.G. conducted Boyden chamber migration assays
and analyzed the results; D.M. designed the mouse ear angiogen-
esis assay and analyzed the data; and A.C. designed the study,
performed in vitro experiments, analyzed the data, and wrote the
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Amit Choudhury, PhD, Department of Anat-
omy & Cell Biology, University of Iowa, 51 Newton Rd, Iowa City,
IA52242; e-mail: firstname.lastname@example.org.
2. Waltenberger J, Claesson-Welsh L, SiegbahnA,
Shibuya M, Heldin CH. Different signal transduc-
tion properties of KDR and Flt1, two receptors for
vascular endothelial growth factor. J Biol Chem.
4. BrunsAF, Herbert SP, OdellAF, et al. Ligand-
stimulated VEGFR2 signaling is regulated by co-
ordinated trafficking and proteolysis. Traffic.
5. Ewan LC, Jopling HM, Jia H, et al. Intrinsic
tyrosine kinase activity is required for vascular
endothelial growth factor receptor 2 ubiquitina-
tion, sorting and degradation in endothelial cells.
6. Lampugnani MG, Orsenigo F, Gagliani MC,
Tacchetti C, Dejana E. Vascular endothelial cad-
herin controls VEGFR-2 internalization and sig-
naling from intracellular compartments. J Cell
7. SalikhovaA, Wang L, LanahanAA, et al. Vascular
endothelial growth factor and semaphorin induce
neuropilin-1 endocytosis via separate pathways.
Circ Res. 2008;103(6):e71-79.
8. Jopling HM, OdellAF, Hooper NM, Zachary IC,
Walker JH, Ponnambalam S. Rab GTPase regu-
lation of VEGFR2 trafficking and signaling in en-
dothelial cells. Arterioscler Thromb Vasc Biol.
9. LanahanAA, Hermans K, Claes F, et al. VEGF
receptor 2 endocytic trafficking regulates arterial
morphogenesis. Dev Cell. 18(5):713-724.
10. GampelA, Moss L, Jones MC, Brunton V,
Norman JC, Mellor H. VEGF regulates the mobili-
zation of VEGFR2/KDR from an intracellular en-
dothelial storage compartment. Blood. 2006;
11. Ferrara N, Kerbel RS.Angiogenesis as a thera-
peutic target. Nature. 2005;438(7070):967-974.
12. Allan BB, Balch WE. Protein sorting by directed
maturation of Golgi compartments. Science.
13. Glick BS, Malhotra V. The curious status of the
Golgi apparatus. Cell. 1998;95(7):883-889.
14. Mellman I, Simons K. The Golgi complex: in vitro
veritas? Cell. 1992;68(5):829-840.
15. Pelham HR, Rothman JE. The debate about
transport in the Golgi–two sides of the same
coin? Cell. 2000;102(6):713-719.
16. Chen YA, Scheller RH. SNARE-mediated mem-
brane fusion. Nat Rev Mol Cell Biol. 2001;2(2):98-
17. Hong W. SNAREs and traffic. Biochim Biophys
18. ChoudhuryA, Marks DL, Proctor KM, Gould GW,
Pagano RE. Regulation of caveolar endocytosis
by syntaxin 6-dependent delivery of membrane
components to the cell surface. Nat Cell Biol.
19. Kuliawat R, Kalinina E, Bock J, et al. Syntaxin-6
SNARE involvement in secretory and endocytic
pathways of cultured pancreatic beta-cells. Mol
Biol Cell. 2004;15(4):1690-1701.
20. Perera HK, Clarke M, Morris NJ, Hong W,
Chamberlain LH, Gould GW. Syntaxin 6 regulates
Glut4 trafficking in 3T3-L1 adipocytes. Mol Biol
21. Wang Y, Tai G, Lu L, Johannes L, Hong W,
Tang BL. Trans-Golgi network syntaxin 10 func-
tions distinctly from syntaxins 6 and 16. Mol
Membr Biol. 2005;22(4):313-325.
22. Wendler F, Page L, Urbe S, Tooze SA. Homotypic
fusion of immature secretory granules during
maturation requires syntaxin 6. Mol Biol Cell.
23. Wendler F, Tooze S. Syntaxin 6: the promiscuous
behaviour of a SNARE protein. Traffic. 2001;2(9):
24. Proctor KM, Miller SC, Bryant NJ, Gould GW.
Syntaxin 16 controls the intracellular sequestra-
tion of GLUT4 in 3T3-L1 adipocytes. Biochem
Biophys Res Commun. 2006;347(2):433-438.
25. Grazia Lampugnani M, ZanettiA, Corada M, et al.
Contact inhibition of VEGF-induced proliferation
requires vascular endothelial cadherin, beta-
catenin, and the phosphatase DEP-1/CD148.
J Cell Biol. 2003;161(4):793-804.
28. Bhattacharya R, Senbanerjee S, Lin Z, et al. Inhi-
bition of vascular permeability factor/vascular en-
dothelial growth factor-mediated angiogenesis by
the Kruppel-like factor KLF2. J Biol Chem. 2005;
29. Nagy JA, Feng D, Vasile E, et al. Permeability
properties of tumor surrogate blood vessels in-
duced by VEGF-A. Lab Invest. 2006;86(8):767-
33. Smith RM, Jarett L. Ultrastructural basis for
chloroquine-induced increase in intracellular insu-
lin in adipocytes: alteration of lysosomal function.
Proc Natl Acad Sci U S A. 1982;79(23):7302-
34. Yoshimori T, YamamotoA, Moriyama Y, Futai M,
Tashiro Y. BafilomycinA1, a specific inhibitor of
vacuolar-type H(?)-ATPase, inhibits acidification
and protein degradation in lysosomes of cultured
cells. J Biol Chem. 1991;266(26):17707-17712.
35. Fenteany G, Standaert RF, Lane WS, Choi S,
Corey EJ, Schreiber SL. Inhibition of proteasome
activities and subunit-specific amino-terminal
threonine modification by lactacystin. Science.
36. PetterssonA, Nagy JA, Brown LF, et al. Hetero-
geneity of the angiogenic response induced in
different normal adult tissues by vascular perme-
ability factor/vascular endothelial growth factor.
Lab Invest. 2000;80(1):99-115.
37. Nagy JA, DvorakAM, Dvorak HF. VEGF-Aand
the induction of pathologic angiogenesis. Annu
Rev Pathol. 2007;2:251-275.
38. ScottA, Mellor H. VEGF receptor trafficking in
angiogenesis. Biochem Soc Trans. 2009;37(Pt
39. Watson RT, Hou JC, Pessin JE. Recycling of
IRAP from the plasma membrane back to the
insulin-responsive compartment requires the
Q-SNARE syntaxin 6 but not the GGAclathrin
adaptors. J Cell Sci. 2008;121(Pt 8):1243-1251.
40. Cheng J, Cebotaru V, Cebotaru L, Guggino WB.
Syntaxin 6 and CAL mediate the degradation of
the cystic fibrosis transmembrane conductance
regulator. Mol Biol Cell. 21(7):1178-1187.
41. Ferrara N. Vascular endothelial growth factor:
basic science and clinical progress. Endocr Rev.
42. Ferrara N, Gerber HP, LeCouter J. The biology of
VEGF and its receptors. Nat Med. 2003;9(6):669-
43. Matsumoto T, Claesson-Welsh L. VEGF receptor
signal transduction. Sci STKE. 2001;2001(112):
44. Pan Q, Chanthery Y, Liang WC, et al. Blocking
neuropilin-1 function has an additive effect with
anti-VEGF to inhibit tumor growth. Cancer Cell.
45. Sawamiphak S, Seidel S, Essmann CL, et al.
Ephrin-B2 regulates VEGFR2 function in devel-
opmental and tumour angiogenesis. Nature.
46. Wang Y, Nakayama M, Pitulescu ME, et al.
Ephrin-B2 controls VEGF-induced angiogenesis
and lymphangiogenesis. Nature. 465(7297):483-
SYNTAXIN 6 REGULATES VEGFR2 LEVELANDANGIOGENESIS1435BLOOD, 27 JANUARY 2011?VOLUME 117, NUMBER 4
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online November 9, 2010
2011 117: 1425-1435
Mukhopadhyay and Amit Choudhury
Venkatraman Manickam, Ajit Tiwari, Jae-Joon Jung, Resham Bhattacharya, Apollina Goel, Debabrata
and angiogenesis by Golgi localized t-SNARE syntaxin 6
Regulation of vascular endothelial growth factor receptor 2 trafficking
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