TLX controls angiogenesis through interaction with
the von Hippel-Lindau protein
Zhao-jun Zeng1,2, Erik Johansson1, Amiko Hayashi1, Pavithra L. Chavali1, Nina Akrap1, Takeshi Yoshida1,3,
Kimitoshi Kohno3, Hiroto Izumi3and Keiko Funa1,*
1Sahlgrenska Cancer Center, University of Gothenburg, Box 425, SE 405 30 Gothenburg, Sweden
2Molecular Biology Research Center, School of Biological Science and Technology, Central South University, Changsha 410078, China
3Department of Molecular Biology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807-8555, Japan
*Author for correspondence (email@example.com)
Biology Open 1, 527–535
TLX is known as the orphan nuclear receptor indispensable
for maintaining neural stem cells in adult neurogenesis. We
report here that neuroblastoma cell lines express high levels of
TLX, which further increase in hypoxia to enhance the
angiogenic capacity of these cells. The proangiogenetic
activity of TLX appears to be induced by its direct binding
to the von Hippel-Lindau protein (pVHL), which stabilizes
TLX. In turn, TLX competes with hydroxylated hypoxia-
inducible factor (HIF-a) for binding to pVHL, which
contributes to the stabilization of HIF-2a in neuroblastoma
during normoxia. Upon hypoxia, TLX increases in the nucleus
where it binds in close proximity of the HIF-response element
on the VEGF-promoter chromatin, and, together with HIF-2a,
recruits RNA polymerase II to induce VEGF expression.
Conversely, depletion of TLX by shRNA decreases the
expression of HIF-2a and VEGF as well as the growth-
promoting and colony-forming capacity of the neuroblastoma
cell lines IMR-32 and SH-SY5Y. On the contrary, silencing
HIF-2a will slightly increase TLX, suggesting that TLX acts to
maintain a hypoxic environment when HIF-2a is decreasing.
Our results demonstrate TLX to play a key role in controlling
angiogenesis by regulating HIF-2a. TLX and pVHL might
counterbalance each other in important fate decisions such as
self-renewal and differentiation, as well as angiogenesis and
? 2012. Published by The Company of Biologists Ltd. This is
an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial Share Alike
Key words: TLX, VHL, HIF-2a, Angiogenesis, Neuroblastoma
The orphan nuclear receptor TLX (NR2E1) has been recognized
as an important transcription factor for maintenance of neural
stem cells in an undifferentiated state. TLX is expressed in neural
subventricular zone where neurogenesis continues in adulthood
(Shi et al., 2004). These stem cells reside in a so-called ‘‘niche’’,
which is localized in close proximity to blood vessels, supporting
stem cells/progenitors and their expansion and migration (Shen et
al., 2008). TLX-null mice exhibit progressive retinopathies due to
the inability of retinal astrocytes to produce TLX, playing an
important role in the vascular development (Uemura et al., 2006).
Indeed, in TLX-null mutant mice, the retinal astrocyte network is
disorganized along with poorly developed blood vessels
(Miyawaki et al., 2004; Yu et al., 2000). It has been proposed
that a hypoxic environment in the developing retina upregulates
TLX in immature astrocytes to induce angiogenesis. When
astrocytes mature by establishing a close contact with blood
vessels, a normoxic environment prevails and TLX becomes
downregulated in the astrocytes, suppressing angiogenesis.
However, the regulation of TLX expression during hypoxia and
the mechanism behind this angiogenic switching still remains
The progression of tumors depends on the capacity to maintain
its self-renewing cell population, i.e., tumor-initiating cells
(TICs). We therefore examined whether TLX might be
involved in the angiogenesis important for maintaining the
TICs of the nervous system tumors. Neuroblastoma is the most
common extracranial solid childhood tumor of the sympathetic
nervous system. It is thought to arise from the embryonic neural
crest cells during and after their migration from the neural
tube. Aggressive tumor cells typically lack differentiation
markers of the sympathetic nervous system, similar to the
neural crest progenitors (Pietras et al., 2008). The hallmark
of undifferentiated neuroblastoma is its high degree of
angiogenicity via a fine network of capillaries. TICs can be
enriched from neuroblastoma, which constitutively express HIF-
2a to activate angiogenesis in and around the tumors (Holmquist-
Mengelbier et al., 2006). In this regard, it is interesting to
examine whether TLX may be involved in the development and
progression of neuroblastoma, not only by stimulating self-
renewal of TICs but also through its proangiogenic function.
Furthermore, since HIF is a key regulator of the cellular response
to hypoxia, we wanted to elucidate the relationship between TLX
and HIF proteins.
HIF proteins are heterodimers, composed of one of the three a
subunits and the HIF-1b, a constitutive nuclear protein. In the
presence of oxygen, HIF-a is prolyl hydroxylated, followed by
interactions with von Hippel-Lindau protein (pVHL) that
possesses E3 ubiquitin-ligase activity, and becomes degraded
by the proteosome (Kaelin, 2007b). The stabilized HIF
heterodimer activates hypoxia-inducing genes through binding to
their hypoxia-responsive elements (HREs), resulting in induction
of angiogenic factors such as vascular endothelial factor (VEGF)
and erythropoietin (Bunn et al., 1998; Wiesener et al., 2001). The
inactivation by mutations of the VHL gene is associated with an
increased risk of a variety of tumors, such as clear cell renal
carcinoma, pheochromocytoma, and hemangioblastoma, in an
allele-specific manner. However, HIF-a activation is not sufficient
for the development of these tumors (Kaelin, 2007a). Herein, we
report a novel interaction between TLX and VHL, facilitating the
stabilization of HIF-as in normoxia, and binding of TLX to the
VEGF promoter in hypoxia. These results suggest a role of TLX in
the switch mechanism of angiogenesis, in addition to the
maintenance of neural stem cells. This finding might provide an
integral view on the roles of VHL and TLX in oxygen control and
Materials and Methods
Cell cultures and chemicals
The human neuroblastoma cell lines IMR-32, SH-SY5Y, and SK-N-BE2C, as
well as the primate kidney epithelial Cos-1 cell line were cultured as described
previously (Wetterskog et al., 2009). IMR-32 cells were cultured in a defined
medium for neurosphere formation (Chavali et al., 2011). The VHL-deficient
786 cells, stably transfected with VHL or its control vector (786-VHL, 786-0),
were cultured as described (Iliopoulos et al., 1995). Porcine aorta endothelial
cells (PAEC) were cultured in the same manner as described for SH-SY5Y. For
hypoxia studies, cells were grown in 1.7% O2unless indicated otherwise. For
testing the stability of TLX, Cos-1 cells were treated with 25 mg/ml
cyclohexamide. For silencing TLX, shRNA vectors (20 nmol/L; Qiagen) were
microporated into IMR-32 and SH-SY5Y using a microporator (Digital Bio)
according to vender’s instruction. When siRNA oligonucleotides (Qiagen) were
used, FugeneHD (Roche) was employed for transfection. The concentrations of
other chemicals used were 10 mM for MG132 and 100 mM for 2,29-Bipyridyl
(BP), both purchased from Sigma-Aldrich.
Cells were harvested and RNA was isolated using the TRIzol reagent (Invitrogen)
and ethanol precipitation. cDNA was synthesized using oligo-dT primers and M-
MuLV reverse transcriptase (Thermo Scientific). Quantitative real-time PCR
analysis was performed using the StepOne Real-Time PCR system (Applied
Biosystems). Relative mRNA expression was determined by normalizing to
expression of the reference gene HPRT1 or GAPDH. Primer sequences used for
qPCR are as follows: HPRT, Forward primer (FP), 59-TTT GCT TTC CTT GGT
CAG GC-39, Reverse primer (RP), 59-GCT TGC GAC CTT GAC CAT CT-39;
GAPDH, 59-AAA AGC GGG GAG AAA GTA GG-39, RP, 59-CTA GCC TCC
CGG GTT TCT CT-39; TLX, FP, 59-CTG GCT GTA TCT GGC ATG AA-39, RP,
59-TCT AAA TCG AGC CAC CAC CT-39, VHL, FP, 59-ATT AGC ATG GCG
GCA CAC AT-39, RP, 59-TGG AGT GCA GTG GCA TAC TCA T-39.
Cells were seeded in six-well plates, lysed, and proteins were immunoblotted as
previously described (Wetterskog et al., 2009). The following primary antibodies
were used. TLX (LifeSpan Biosciences, Santa Cruz Biotechnology, R&D
Systems), VHL and VEGF-A (BD Biosciences), HA, Flag, and actin (AC-40)
(Sigma-Aldrich), HIF-1a, HIF-2a, b-actin, b-tubulin, Glucose transporter-1
(Glut1) (Santa Cruz Biotechnology), and GAPDH (Millipore). BAF57 was made
as described in (Shiota et al., 2008). Separation of proteins into nuclear and
cytoplasmic fractions (NF, CF) was done as described elsewhere (Kashiwagi et al.,
2011). b-tubulin, BAF57, and b-actin were used as the cytoplasmic, nuclear, and
total protein controls, respectively.
Cells were plated on chamber slides and used for immunostaining (Chavali et al.,
2011) with antibodies described above and KI67 (Chemicon). As specificity
controls, we used transfection of expression plasmid or control vector into cells not
expressing the protein of interest as well as omission of primary antibody. Alexa
488 or 594-conjugated IgG (Invitrogen) was used as a secondary antibody, and
stained cells were observed by epi-illumination fluorescence microscope (Olympus
High Content microscopy). Images were acquired digitally by using the software
Protein binding assays (immunoprecipitation)
To generate TLX deletion constructs, cDNA of full-length mouse TLX (kind gift
of Dr. Uemura) was used as template for PCR using primers as listed below, with
flanking restriction sites for EcoRI and XhoI. The amplified cDNAs were cleaved
and ligated into the EcoRI/XhoI sites of the pcDNA3 vector containing N-terminal
Flag-tag. Primer sequences for TLX deletion constructs are as follows: TLX-FL,
FP, 59-GTT GAA TTC ATG AGC AAG CC GCC-39, RP, 59-GCG CTC GAG
TTA GAT GTC ACT GGA TTT G-39, TLX-DN, FP, 59-TAT GAA TTC GAA
TCA GCT GCC AGG CTT-39, TLX DC, RP, 59-TTT CTC GAG TTA ACA CAC
GGA CTC AGT-39, TLX DC341-385, RP, 59-TTT CTC GAG TTA GAA TCG
GCA GGG TTG G-39.
HA-VHL30and its mutant VHL30D95-123 in pcDNA3 were kind gifts of Dr.
Krek (Hergovich et al., 2003). At 48 h after transfection using Fugene HD (Roche)
with various expression vectors, cells were lysed and lysates were used for
immunoprecipitation and for expression control of transfected plasmids by
immunoblotting as previously described (Izumi et al., 2001). The different
deletion constructs of TLX expressing recombinant glutathione S-transferase
(GST) fusion protein and the full-length VHL30and VHL30D95-123 expressing
thioredoxin (Thio)-His fusion proteins were made from the plasmid (His-Patch
ThioFusion System, Invitrogen) in E. coli and subsequently purified and used for
in vitro binding assay, as described previously (Uramoto et al., 2002). For
precipitation and blotting, antibodies against GST or Thio (Invitrogen) were used.
Promoter reporter assay
Promoter assays were performed as described by using a VEGF-promoter in a
pGL3 vector (Ryuto et al., 1996). Expression vectors used were HA-VHL30and
Flag-TLX as described above. SH-SY5Y cells were incubated with 2,29 BP for the
indicated time periods before being harvested. Standardization was done by
normalization with co-transfected renilla activity.
Supernatants from IMR-32 cells was collected after 0, 4, and 24 h of hypoxia and
VEGF concentrations were measured using the human VEGF colorimetric ELISA
kit (ThermoFisher) according to the manufacturer’s instruction.
Colony formation assay in soft agar
Agar (Chemicon) was dissolved in culture medium to 0.8% and plated in culture
plates (bottom layer). After the bottom layer had coagulated, cells were seeded in
0.4% agar and added on top of the bottom agar layer. Cells were covered with
medium and cultured at 37˚C in a 5% CO2incubator. Colonies were counted
manually after three weeks.
Chromatin immunoprecipitation (ChIP) assay
Protein and DNA were cross-linked by incubating cells with formaldehyde at a
final concentration of 1% for 10 min at RT, and ChIP assay was done as previously
described (Chavali et al., 2011). The fixed chromatin was incubated at 4˚C with
2 mg of antibodies against TLX, HIF-2a, RNA Polymerase II (PolII), VHL and
mouse or rabbit Ig (Santa Cruz) as a negative control. Actin promoter was used to
rule out non-specific binding. The primers for VEGF promoter were 59-TTT TCA
GGC TGT GAA CCT TG-39 and 59-GAT CCT CCC CGC TAC CAG-39 yielding
a 233 bp product.
All quantitative analysis were repeated at least three times and values represent
means 6 SD of one representative experiment, each done in triplicate. Overall
significance was determined by submitting data to one-way analysis of variance.
Significance of between-group differences was determined by various post-hoc
comparisons as noted in Legends. Significance is labeled as * p,0.05, ** p,0.01,
Expression and subcellular localization of TLX and VHL in
neuroblastoma cell lines upon hypoxia
We first examined by immunoblotting whether neuroblastoma
cell lines derived from the developing peripheral nervous system
might express TLX. TLX expression differs among the IMR-32,
SK-N-BE2C, and SH-SY5Y neuroblastoma cell lines (Fig. 1A).
However, it is clearly higher compared with the human normal
fibroblast AG1518 that expresses almost no TLX. It is well
recognized that hypoxia up-regulates the expression of TLX in
neuroprogenitors and retinal astrocytes during development
(Chavali et al., 2011; Uemura et al., 2006). To test whether
TLX induces angiogenesis in neuroblastoma 528
hypoxia induces TLX expression in neuroblastoma, IMR-32 and
SH-SY5Y were cultured in 1.7% O2, which efficiently induces
neuroprogenitor proliferation (Chavali et al., 2011), for 0, 4, 24,
48 h. The porcine aorta endothelial cell (PAEC) line was
included because of its high hypoxia-sensitivity. The TLX
expression increased in neuroblastoma cells and in PAEC
following 4, 24, and 48 h of hypoxic treatment, along with
glucose transporter-1 (Glut-1), which was used as a marker for
hypoxia (Fig. 1B).
Since HIF-1a is the most well-studied hypoxia-induced
transcription factor with multiple target genes, we compared its
expression pattern with that of TLX in IMR-32 following 6 h of
hypoxia (Fig. 2A). Almost all cells stained positive for both TLX
and HIF-1a, whereas none of these cells expressed HIF-1a in
normoxia, even though a few cells expressed TLX quite strongly.
In normal cells, HIF-as are constantly degraded by pVHL.
However, in neuroblastoma HIF-2a is often constitutively
expressed, which is further upregulated upon hypoxia, but the
up-regulation occurs much later than with HIF-1a (Holmquist-
Mengelbier et al., 2006). In order to find out whether TLX has
any relation with VHL we next stained IMR-32 cultured in
spheres for TLX and VHL (Fig. 2B). TLX became up-regulated
when neuroprogenitors were cultured in a defined medium
stimulating neurosphere formation (Chavali et al., 2011). In this
condition, TLX was actually expressed stronger in the cells
located close to the surface of the spheres while cells in the center
are stained more for pVHL that was mostly in the cytoplasm. In
fact, TLX colocalized with KI67, a marker for dividing cells,
indicating that TLX is associated with proliferating cells in the
In order to see the subcellular localization of pVHL and TLX
in normoxia, proteins from IMR-32, SH-SY5Y, and SK-N-BE2C
were fractionated into nuclear and cytoplasmic proteins and
separately immunoblotted (supplementary material Fig. S1). In
this condition, 3% (IMR-32), 14% (SK-N-BE2C), and 17% (SH-
SY5Y) of the total VHL protein was detected in the nucleus. TLX
expression was found in the nuclear fraction in both IMR-32 and
SK-N-BE2C cells, whereas in SH-SY5Y a small fraction of TLX
was found in the cytoplasm. We then examined whether VHL
expression changes upon hypoxia with regard to its level and
localization in relation to those of HIF-1a and -2a. Fractionated
proteins from IMR-32, SH-SY5Y, and SK-N-BE2C were
immunoblotted before and after 12 h hypoxia (Fig. 2C). pVHL
was mostly expressed in the cytoplasm in agreement with the
immunofluorescence (Fig. 2B). The pVHL in the nucleus
decreased upon hypoxia, especially in IMR-32 and SK-N-
BE2C. Adequate separation was confirmed by cytoplasmic b-
tubulin and mostly nuclear BAF57 transcription factor. In
hypoxia both HIF-1a and HIF-2a were accumulated only in the
Fig. 1. Hypoxia increases TLX expression in neuroblastoma. (A) TLX
expression in 3 neuroblastoma cell lines was evaluated by immunoblotting and
compared with the normal fibroblast cell line AG1518 with GAPDH as a
loading control. (B) Immunoblotting of TLX in PAEC, IMR-32, SH-SY5Y at
indicated time points following hypoxia. Glut-1 is used as hypoxia control and
b-actin as input control.
Fig. 2. Subcellular localization of TLX. (A) Immunofluorescence staining of
IMR-32 for TLX (green), HIF-1a (red) and nuclei (DAPI; blue) in normoxia
and after 6 h of hypoxia. The staining was standardized digitally to optimize the
difference (upper panel). Bar, 50 mm. (B) IMR-32 cells cultured in neurosphere
media were stained for TLX (green) and VHL (red, upper panel) or Ki67 (red,
lower panel) and the images were taken as Z-stacks. (C) Nuclear (N) and
cytoplasmic (C) expression of VHL, HIF-1a, HIF-2a, was compared by
immunoblotting in normoxia (N) and after 12h hypoxia (H) in SH-SY5Y, IMR-
32, and SK-N-BE2C cells. b-tubulin, BAF57, and b-actin were used as the
cytoplasmic, nuclear, and total protein controls. (D) Effect of transfected
pVHL30on the stability of TLX protein was examined with cyclohexamide
pulse chase at 48 h after transfection of Flag-TLX and HA-VHL into Cos-1
cells, and cell lysates were analyzed at indicated time points. Changes of TLX
expression during the time course was depicted in a graph. Values were
standardized by GAPDH. Comparing means of 3 repeated experiments yielded
a significant difference.
TLX induces angiogenesis in neuroblastoma529
nucleus in contrast to pVHL that decreased. SK-N-BE2C cells
contained HIF-2a in the nucleus and even slightly in the
cytoplasm. These results suggest that in the nucleus the
expression of VHL and TLX/HIF-a correlate negatively even
though they may exist simultaneously in the nucleus. However,
the mRNA levels of VHL and TLX as examined by qPCR
remained unaltered in hypoxia (supplementary material Fig. S2).
Thus, the protein expression of TLX seems to be stabilized upon
hypoxia as in neuroprogenitors (Fig. 1B) (Chavali et al., 2011).
Since pVHL acts as an ubiquitin ligase, we wanted to confirm
that TLX is not a degradation target of pVHL. We thus performed
a cyclohexamide (CHX) pulse-chase experiment in Cos-1 cells
over-expressing exogenous Flag-TLX together with HA-VHL or
vector (Fig. 2D). Cells were harvested 48 h after transfection
following treatment with CHX for various time periods.
Coexpression of HA-VHL resulted in stabilization of TLX. This
was also tested in VHL-deficient renal carcinoma 786-0 cells,
where levels of pVHL and endogenous TLX were analyzed after
transfection of VHL or its control plasmid in the presence of the
proteasome inhibitor MG132 (supplementary material Fig. S3).
TLX binds VHL and stabilizes HIF-a, enhancing the VEGF-
Next, we examined whether there is any interaction between TLX
and pVHL, which might explain the stabilization of TLX and
HIF-a. In order to determine TLX binding site to pVHL, Cos-1
cells were cotransfected with Flag-TLX and HA-full-length
VHL30, or the deletion mutant VHL30D95-123, which failed to
bind microtubules (Hergovich et al., 2006). Twenty residues on
the b-sheets of pVHL N67 to W117 are direct binding sites to
HIF-as (Min et al., 2002) and among these amino acids a
mutation of Y98, Y111, or Y117 made it incapable to bind HIF-
as (Ohh et al., 2000). TLX was co-precipitated with the full-
length pVHL30but hardly with pVHL30D95-123 (Fig. 3A). This
suggests that the region 95-123 is important for binding to TLX.
We also examined whether TLX and pVHL can bind each
other directly without any involvement of other proteins, and if
this is the case, we wish to determine which region of TLX would
bind pVHL. To this end we used GST-pull down of different
GST-fused TLX deletion mutants and ThioHis-tagged full-length
VHL and its deletion mutant as shown in Fig. 3B. The result
showed that deletion of the C-terminus (D187-385) of TLX
abrogated VHL binding, while the N-terminal deletion construct
(D1-187) was able to bind to pVHL. Even deletion of the C-
terminal histone deacetylase (HDAC)5 interaction site (D341-
385) could bind, indicating that the ligand-binding site is
necessary for pVHL binding. However, pVHL30D95-123 bound
Fig. 3. Physical interaction of TLX and VHL. (A) Cotransfection and
coimmunoprecipitation of Flag-TLX with VHL30, VHL30D95–123, or vector
alone using Cos-1 cells. (B) Illustration of various TLX deletion constructs and
the full-length and deletion mutant of VHL, and the result of binding (upper
panel). In vitro coimmunoprecipitation of various deletion constructs of GST-
TLX (FL, DN, DC, DC341-385) and ThioHis-VHL30or ThioHis-VHL30D95–
123 by GST pull-down. Protein expression was detected by CBB stain (lower
panel). (C) Competition assay between HIF-a and TLX for the binding to
pVHL30in the presence of MG132. Same amounts of Flag-HIF-1a or Flag-
vector and HA-VHL or HA-vector were cotransfected into Cos-1 cells with
increasing amounts of HA-TLX and lysates were precipitated by anti-Flag (left
panel). IMR-32 was transfected with increasing amounts of TLX and protein
lysate was coimmunoprecipitated with endogenous HIF-2a by using a HIF-2a
antibody and immunoblotted with HIF-2a, VHL, TLX, and GAPDH antibodies
TLX induces angiogenesis in neuroblastoma530
very weakly to the full-length TLX. The N-terminal deleted
construct (D1-187) and to a lesser extent the far-most C-terminal
D341-385 construct could bind pVHL30D95-123, suggesting that
exposing the ligand-binding region of TLX might increase its
affinity to pVHL.
We further examined whether HIF-a and TLX could compete
in binding to VHL involving the region containing residues
95-123. We thus treated cells with MG132 and performed
coimmunoprecipitation with overexpressed Flag-HIF-1a in
normoxia (Fig. 3C, left panel), since the pVHL- binding sites
of all HIF-as are conserved. Cos-1 cells were transfected with
Flag-HIF-1a together with HA-VHL30and HA-TLX. When cells
were transfected with increasing amounts of HA-TLX plasmid,
the binding of pVHL to HIF-1a decreased. Furthermore, when
we overexpressed increasing amounts of TLX in IMR-32 cells
and the lysate was immunoprecipitated by endogenous HIF-2a,
we saw that increased amounts of TLX diminished the amount of
VHL to be coprecipitated with HIF-2a. These results suggest that
increased amounts of TLX could prevent pVHL from binding to
prolyl-hydroxylated HIF-a (Fig. 3C, right panel).
Since TLX protein levels increased in the IMR-32 nucleus in
hypoxia, we examined whether TLX could cooperate with HIF-a
through binding to the TLX-binding motif (AAGTCA) close to
the HRE element in the VEGF-promoter (Fig. 4A). In the 59 and
39 of the HRE-sequence, 3 TLX-consensus-like sequences are
present in the VEGF-promoter. We used the VHL-deficient 786
cells stably transfected with VHL and its control vector (786-
VHL, 786-0) for the promoter-reporter assays. We transfected
those cell lines with TLX, VHL, or both TLX and VHL, together
with the VEGF promoter-luciferase vector in these cell lines and
measured their respective promoter activities (Fig. 4A). In 786-0,
TLX activated the promoter-reporter 1.8-fold in normoxia in the
presence of stabilized HIF-a caused by the loss of VHL. Thus,
TLX activates the reporter not only by sequestering pVHL. As
expected, VHL overexpression, which destabilizes HIF-a,
decreased the activity. In 786-VHL, overexpression of TLX did
not render any significant increase in the reporter activity. This
may be due to depletion of HIF-a by abundant pVHL. We made a
similar experiment in SH-SY5Y cells by using 2.29BP that
mimics a hypoxic condition. In normoxia, similarly to 786-0
cells, over-expression of TLX increased the promoter activity 2-
fold, and excessive VHL decreased the activity to 30% of the
control. In hypoxia, the activation due to TLX overexpression
was marginal, although it counteracted the effect of VHL
overexpression. These results suggest that TLX enhances the
effect of HIF-a. Indeed, during hypoxia TLX protein levels
increased, which was accompanied by a decrease of VHL protein
levels and an apparent increase of VEGF protein levels after
approximately 48 h. This supports the hypothesis that TLX
protein might induce expression of VEGF. (Fig. 4B,C). The
effect of TLX is larger in normoxia, since TLX stabilizes HIF-a
by sequestering VHL in accordance with VHL overexpression,
which will remove this effect.
TLX silencing suppresses cell growth, colony formation, and
VEGF production of neuroblastoma cells
In order to investigate the biological roles of endogenous TLX in
neuroblastoma, we silenced TLX in IMR-32 and SH-SY5Y cells.
Several stable cell lines carrying TLX shRNA were made from
both cell lines (supplementary material Fig. S4A). In agreement
with the role of TLX in self-renewal of neural stem cells, the
growth of silenced clones was clearly slower compared with the
sh-controls (Fig. 5A). Concordant with its potent proliferation-
differentiation of both of these neuroblastoma cell lines
(supplementary material Fig. S4B,C). Since IMR-32 has N-
Myc amplification and a more immature phenotype than SH-
SY5Y, we examined in more detail the IMR-32 shTLX clones
and compared them with the wild type and vector controls. When
these clones were spread on agar plates, and the numbers of
formed colonies were counted after 21 days, a significant
of TLXexpression ledto
Fig. 4. TLX activates but VHL represses the VEGF promoter. (A) The
activity of VEGF promoter-luciferase was measured after transfection of TLX,
VHL30, TLX and VHL30, and control in 786-0 compared with 786-VHL (left
panel) or in SH-SY5Y in normoxia compared to 24 h treatment with 200 mM
of hypoxia mimetic 2,29BP (right panel). The proximal VEGF promoter,
indicating one HRE and three TLX-consensus-like sequences, is illustrated in
the upper panel. The relative reporter activity is expressed with control set to
100 6 SD. For the 786 cells, overall ANOVA showed highly significant
between-groups differences with overall F57.55 (p50.0004). For SH-SY5Y
cells, overall F559.22 (p,0.0001). Pairwise differences were analyzed with
Tukey’s post-hoc tests, as shown. (B) Protein expression of TLX, VHL30,
VEGF, and a-actin in IMR-32 and SYSH-5Y at indicated time points in
hypoxia. (C) Concentrations of VEGF in supernatant of IMR-32 were analyzed
by ELISA following hypoxia for 0, 4, and 24 h.
TLX induces angiogenesis in neuroblastoma 531
Fig. 5. Effects of TLX silencing on cell growth and
angiogenetic factors. (A) Cell growth curves of
wildtype, vector controls (V1, V2), and TLX-depleted
SH-SY5Y (S2, S6, S7) and IMR-32 (I2, I3, I4) clones
during 1, 3, 5 days after seeding. Bars indicate means
6 SD. Colony transformation assay of vector (V2) and
wild type (Ctrl) controls, and shTLX (I2, I3, I4) clones
of IMR-32 (right panel). Overall ANOVA showed
highly significant differences with F5125.4
(p,0.0001). Using Dunnett’s post-hoc test, each clone
was compared with the vector (V) as shown (*).
(B) Comparison of the expression of VEGF165in
normoxia between shTLX clones (I3, I4), control
vector clones (V1, V2), and wild type, with GAPDH
as loading control (upper left panel). The expression of
different isoforms of VEGF is shown in sh-TLX and
sh-control (upper right panel). Expression of TLX,
HIF-2a, and VHL is compared by immunoblotting at
indicated time points upon hypoxia. Expression of a-
actin is shown as a loading control (lower panel).
(C) ChIP assay was performed to see proteins bound
to the proximal VEGF-promoter with antibodies
against TLX, HIF-2a, RNA polymerase II (PolII), and
non-specific IgG, using chromatin collected from
IMR-32 wild type, control vector (Cont), and shTLX
(I3) in normoxia (left panel) and at 24 h of hypoxia
(right panel). Amounts of PCR products covering
HRE and its upstream region are shown as relative
values 6 SD with the binding of HIF2-a in normoxia
TLX induces angiogenesis in neuroblastoma532
reduction of colonies was found in shTLX clones as compared
with the sh-control and wild-type cells (Fig. 5A).
Next, we examined whether silencing of TLX affects the
response of neuroblastoma to hypoxia. Silencing of TLX indeed
decreased the expression of VEGF in normoxia (Fig. 5B). In
hypoxia, TLX gradually increased in controls, whereas in shTLX
cells it remained low until 48 h when the silencing effect was
diminished. In this experiment, 3% O2was applied to prevent
detachment of cells at later time points. The expression of HIF-2a
is markedly reduced in shTLX cells. We found that VEGFA121
and VEGFA165isoforms (Koch et al., 2011) were expressed in
shTLX control cells, and both isoforms were indeed diminished
in shTLX. In addition, VHL was increased in shTLX cells,
supporting the notion that expression of TLX correlates
negatively with that of VHL but positively with HIF-2a. The
increase of VHL might contribute to the differentiation of TLX-
silenced IMR-32 cells.
So far, we found that TLX overexpression activated the
VEGF-promoter in normoxia but, in hypoxia, the effect was
somewhat mild, which could be explained by the endogenous
increase of TLX. In order to see whether TLX actually binds the
VEGF-promoter chromatin, ChIP was performed in IMR-32-
derived cell clones, wild type, sh-control, and shTLX, on the
VEGF-promoter containing the HRE-consensus site (Fig. 5C). In
normoxia, no TLX chromatin binding was detected in any of the
cell lines, which agrees with the fact that TLX binds VHL and
becomes sequestered. However, HIF-2a and RNA polymerase II
(PolII) bound in both wild type and sh-control, while binding was
greatly diminished in the shTLX clone. This suggests that in
normoxia, TLX will indirectly activate HRE-regulated promoters
through stabilizing HIF-2a. In hypoxia, both TLX and HIF-2a
bind chromatin with an apparent difference from shTLX cells.
However, the decrease of PolII binding was relatively small in
the shTLX cells. VHL was not detected on the promoter
chromatin at any time (not shown).
TLX and VHL interaction increases in early hypoxia, stabilizing
HIF-a to induce angiogenesis
Having seen the binding of both HIF-2a and TLX in proximity to
the HRE site of the promoter, we asked whether both TLX and
HIF-2a exist in endogenous complexes with pVHL. Thus,
endogenous pVHL was immunoprecipitated in both IMR-32
and SH-SY5Y, and the bound proteins were evaluated at 0, 4, and
24 h in 1.7% hypoxia (Fig. 6A). In SH-SY5Y, the amount of
precipitated pVHL increased to its maximum at 4 h in the
hypoxic condition, whereas its expression in input protein
decreased. In both cell lines, the same transient increase of
TLX coimmunoprecipitated with pVHL at 4 h was seen, which
might facilitate a faster stabilization of the remaining HIF-a.
TLX expression in input protein continued to increase at 24 h,
especially in IMR-32. Interestingly, HIF-2a, but not HIF-1a, was
coimmunoprecipitated with pVHL, reaching maximum after 4 h
in hypoxia and then to decrease at 24 h in SH-SY5Y. However,
much lesser amount of HIF-2a was precipited in IMR-32, where
only faint bands were detected for input of both HIF-as. We also
coimmunoprecipitate VHL in wild type-, shTLX-, and the sh-
control-IMR-32 cells (supplementary material Fig. S5).
We next examined the expression of TLX, VHL, HIF-1a, HIF-
2a in shTLX, sh-control, and wild type cells in normoxia and
after 4 h of hypoxia (Fig. 6B). As expected, shTLX cells
TLX could reciprocally
expressed hardly any HIF-2a but HIF-1a remained almost
unaffected. We then asked whether the decreased expression of
TLX affects the amounts of HIF-as in the VHL complex in
normoxic and hypoxic conditions (Fig. 6C). Binding of HIF-1a
was unaltered in IMR-32 cells in both conditions, but HIF-2a was
clearly decreased in shTLX cells in both normoxia and hypoxia,
when compared with the sh-controls.
Silencing of HIF-2a and VHL affect the expression of TLX, HIF-
2a, and VHL
In order to see whether decreased HIF-2a or VHL expression will
affect TLX expression, we used siRNA oligos for HIF-2a, VHL,
and TLX in normoxic and hypoxic conditions (Fig. 7). Since
hypoxia affected the attachment of transiently siRNA-transfected
IMR-32 cells, we used 2.29BP to mimic hypoxic condition in SH-
SY5Y. HIF-2a depletion led to a slight up-regulation of TLX in
both conditions and an increased VHL level only in hypoxia.
Depletion of VHL resulted in decreased TLX levels, as expected.
In concordance with Fig. 5B, TLX depletion increased VHL in
normoxia and slightly so in hypoxia, but decreased HIF-2a in
both normoxic and hypoxic conditions.
Hypoxia is a well-recognized mechanism contributing to tumor
progression both through stimulation of blood vessels and by
direct effects on tumor growth and invasion. We demonstrate
herein that neuroblastoma cells express enhanced levels of TLX
protein, which further increase in hypoxia to maintain cancer
stem cells. In our search for a mechanism behind the
angiogeneticactivity of TLX, weidentifieda physical
Fig. 6. TLX and VHL complex in vivo. (A) Proteins (160 mg)
coimmunoprecipitated with a VHL antibody from cell lysates of IMR-32 and
SH-SY5Y taken at different time points in hypoxia (0, 4, 24 h) were blotted
with the indicated antibodies. Lysates in normoxia precipitated with non-
specific IgG were used as controls. Expression of each input protein is shown
by immunoblotting, with GAPDH as an input control. (B) Cell lysates of IMR-
32 (shTLX, sh-control) in normoxia and 4 h hypoxia were blotted with
indicated antibodies. (C) Cell lysates as above were coimmunoprecipitated with
VHL antibody and blotted with indicated antibodies in the same manner
TLX induces angiogenesis in neuroblastoma 533
interaction of TLX with pVHL, leading to stabilization of TLX
and HIF-2a. In normoxia, TLX is able to stabilize HIF-a by
sequestering pVHL, which might be a reason for the increased
HIF-2a often seen in stem cells of advanced tumors (Qing and
Simon, 2009), such as glioma (Li et al., 2009) and neuroblastoma
(Holmquist-Mengelbier et al., 2006). HIF-2a has also been
suggested to be more important than HIF-1a for the development
of renal tumors (Covello et al., 2005; Kondo et al., 2002).
Depletion of HIF-2a in VHL-null renal carcinoma was shown
to efficientlysuppress tumor-forming
Furthermore, HIF-1a and HIF-2a possess different roles in
hypoxic gene control (Qing and Simon, 2009). In concordance
with this, the expression kinetics of TLX resembles more that of
HIF-2a than HIF-1a. pVHL binds to a larger amount of TLX in
early hypoxia than in normoxia, which may be a mechanism for a
rapid stabilization of a partially hydroxylated pool of HIF-a.
Although TLX and HIF-2a do not directly bind to each other,
both are stabilized in the nucleus during hypoxia and bind the
VEGF promoter, recruiting RNA polymerase II. Indeed, a similar
mechanism has been proposed for coactivation between the
orphan nuclear receptor HNF4 and HIF-ab recruiting p300 on the
erythropoietin gene (Bunn et al., 1998). TLX would thus employ
somewhat different mechanisms in promoting angiogenesis, i.e.,
in normoxia by sequestering pVHL and in hypoxia by acting as a
transcription factor binding the VEGF-promoter.
Several mechanisms have been proposed to explain how HIF-a
becomes stabilized in normoxia or mild hypoxia in solid
tumors, creating a hypoxic microenvironment. In aggressive
neuroblastoma, rapid proliferation of TICs may render local
hypoxia. Since dividing cells in the IMR-32 tumorspheres express
TLX, HIF-2a can be stabilized. Furthermore, TLX was shown to
lead to HIF-a stabilization in early hypoxia (Mottet et al., 2003).In
normoxia and early hypoxia, TLX occupies the HIF-binding sites
of pVHL, resulting in stabilization of HIF-a, which concords with
the reduction of HIF-a by depletion of TLX. Moreover, the pVHL-
bound TLX appears to be more stable, leading to further
stabilization of HIF-2a. Interestingly, HIF-2a, but not HIF-1a,
was coimmunoprecipitated with pVHL, which agrees with the
finding that HIF-2a may sustain for a longer time than HIF-1a
upon hypoxia in neuroblastoma (Holmquist-Mengelbier et al.,
Recent reports demonstrate that pVHL can be SUMOylated at
K171 and becomes stabilized to shuttles into nuclei, which might
have otherwise been ubiquitinated and degradated (Cai and
Robertson, 2010). Moreover, the SUMOylation of pVHL
increases in early hypoxia (,6 h) and inactivates the ubiquitin
ligase and other tumor suppressor functions of VHL (Cai et al.,
2010). In fact, the binding of TLX to pVHL was slightly
increased at 4 h in SH-SY5Y cells, indicating that pVHL might
be stabilized upon hypoxia. The exact relationship between TLX
and the modified pVHL during the course of hypoxia remains to
be elucidated. Hypoxia might lead inactivation of pVHL
suppressor functions, stabilizing TLX to stimulate proliferation
of neuroblastoma TICs.
Silencing of HIF-2a or VHL by siRNA affects the expression
levels of TLX, HIF-2a, and VHL in normoxic and hypoxic
conditions. We thus confirmed that TLX regulates expression
levels of HIF-2a, and pVHL regulates those of TLX. However,
silencing of HIF-2a slightly upregulated TLX, suggesting that
TLX could prolong a hypoxic environment when HIF-2a
decreased. The exact mechanism behind the effect could not be
completely identified in this study. However, the fact that the
reduction of pVHL or HIF-2a rapidly affected the levels of other
two proteins might suggest that there are intimate relationships
between these three proteins. Furthermore, the observed
(supplementary material Fig. 4B,C) might also be due to an
increase of pVHL, in agreement with a report where VHL
neuroblastoma cells (Murata et al., 2002).
Taken together, the relationship between TLX and pVHL
changes according to the degree of oxygenation. In normoxia,
their effects might rather be directed towards counterbalancing
proliferation and differentiation. In hypoxia, TLX increases to
stimulate angiogenesis, but when blood vessels are fully
developed and the oxygen level increases, TLX becomes
differentiate neural stem cells. In this way, TLX may act as a
switch molecule for angiogenesis, sustaining the homeostasis in
by TLX depletion
We thank Drs. W. Krek (VHL30, VHL30D95-123), A. Uemura
(TLX), and C. Simon (HIF-2a) for plasmids, Drs. A. Harris and W.
Kaelin for 786 cell lines, L. Green for reading the manuscript, and
the Center for Cellular Imaging for assistance. This work was
supported by grants from the Swedish Science Council, the Swedish
Cancer Society, the Swedish Childhood Cancer Foundation, the
IngaBritt and Arne Lundberg Research Foundation, the Va ¨stra
Go ¨taland Region County Council (ALF), and BioCARE, a National
Strategic Research Program at University of Gothenburg, Sweden.
P.L.C. is a postdoc scholar supported by the Swedish Institute and
Fig. 7. Effects of silencing HIF-2a and VHL. Silencing HIF-2a, VHL and
TLX was performed by siRNA oligonucleotides. Effects on the expression
levels of TLX, HIF-2a, or VHL in normoxia and hypoxia were examined by
immunoblotting. Hypoxia was created by addition of 2.29BP for 8 h at 24 h
after transfection, and immunoblotting data was normalized by GAPDH. Si-
controls at normoxia were set to 100, and comparisons were done with values
of si-controls, in normoxia and hypoxia, respectively (upper panel). Schematic
illustration of the stimulatory and inhibitory relationships between TLX, HIF-
2a, and VHL are depicted (lower panel).
TLX induces angiogenesis in neuroblastoma 534
the Assar Gabrielsson Foundation. E.J. is supported by a postdoc
scholarship from Swedish Childhood Cancer Foundation.
The authors declare that there are no competing interests.
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