Hypoxia Negatively Regulates Antimetastatic PEDF in
Melanoma Cells by a Hypoxia Inducible Factor-
Independent, Autophagy Dependent Mechanism
Asuncio ´n Ferna ´ndez-Barral1,2, Jose ´ Luis Orgaz1,2¤a, Valentı ´ Gomez1,2¤b, Luis del Peso1,2, Marı ´a
Jose ´ Calzada2,3, Benilde Jime ´nez1,2*
1Department of Biochemistry, Universidad Auto ´noma de Madrid (UAM) Madrid, Spain, 2Instituto de Investigaciones Biome ´dicas Alberto Sols, CSIC-UAM, Madrid, Spain,
3ServiciodeInmunologia,Hospitalde laPrincesa, Instituto deInvestigacio ´n Sanitaria Princesa and Departamentode Medicina,UniversidadAuto ´nomadeMadrid, Madrid,Spain
Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent
antiangiogenic and antimetastatic activity in a broad range of tumor types. Melanocytes and low aggressive melanoma cells
secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas. PEDF efficiently abrogates a number
of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization,
proliferation, migration, invasiveness and extravasation. In this study, we identify hypoxia as a relevant negative regulator of
PEDF in melanocytes and low aggressive melanoma cells. PEDF was regulated at the protein level. Importantly, although
downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the
hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia. Decreased PEDF protein was not mediated by
inhibition of translation through untranslated regions (UTRs) in melanoma cells. Degradation by metalloproteinases,
implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory
mechanism in melanoma cells. Instead, we found that degradation by autophagy was critical for PEDF downregulation
under hypoxia in human melanoma cells. Our findings show that hypoxic conditions encountered during primary
melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving
degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of
aggressive melanoma cells.
Citation: Ferna ´ndez-Barral A, Orgaz JL, Gomez V, del Peso L, Calzada MJ, et al. (2012) Hypoxia Negatively Regulates Antimetastatic PEDF in Melanoma Cells by a
Hypoxia Inducible Factor-Independent, Autophagy Dependent Mechanism. PLoS ONE 7(3): e32989. doi:10.1371/journal.pone.0032989
Editor: Irina V. Lebedeva, Enzo Life Sciences, Inc., United States of America
Received August 17, 2011; Accepted February 7, 2012; Published March 23, 2012
Copyright: ? 2012 Ferna ´ndez-Barral et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants Ministerio de Educacion y Ciencia SAF2007-62292 and SAF2010-19256 to BJ, SAF2009-11113 to MJC and SAF2008-
03147 to LP. AFB was supported by a CSIC-JAE fellowship, JLO by a Ministerio de Educacion y Ciencia SAF2007-62292 contract and VG by S-SAL-0311_2006 grant.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤a Current address: Randall Division of Cell and Molecular Biophysics, King’s College London, London, United Kingdom
¤b Current address: UCL Cancer Institute, University College London, London, United Kingdom
Serine protease inhibitor (SERPIN) is a large superfamily of
genes that codes for serine protease inhibitors in mammals .
However, there is a small number of SERPIN family members
with non-inhibitory protease activity, among which is included
pigment epithelium-derived factor (PEDF, gene symbol SER-
PEDF was originally described as the most potent angiostatic
factor in the eye . PEDF is produced at high levels by retinal
pigment epithelial (RPE) cells, and counteracts a number of potent
angiogenic growth factors in the retina; ensuring the right balance
of angiogenic regulators that leads to an optimum physiological
pattern of blood vessels for a correct retinal function. A number of
eye pathologies like diabetic retinopathy and eye-related macular
degeneration are associated with loss of PEDF expression, leading
to excessive and aberrant vascularization patterns associated with
loss of vision .
Later studies showed that PEDF is also produced by a wide
variety of epithelial cell types and its role in controlling primary
tumor growth, angiogenesis and metastatic spread has been
explored in a wide range of tumor types [8–10]. Levels of
angiostatic PEDF decrease during the progression of a number of
cancers, such as hepatocellular carcinoma , prostate carcino-
ma [12,13], breast adenocarcinoma , glioblastoma  and
Wilm’s tumors .
We have recently shown that melanocytes are also among the
cell types in our body that produce and secrete high levels of
PEDF , which are comparable to the levels produced by RPE
cells, neural cells or retinoblastoma cells. We [8,17–19] and others
[4,20,21] have described a complex mechanism underlying the
potent inhibition of melanoma metastasis by PEDF. PEDF-
mediated antitumor activity in melanoma and other tumors is
based on its dual action on the tumor microenvironment and on
the tumor cells themselves . PEDF inhibits tumor angiogenesis
by means of induction of apoptosis on endothelial cells and
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modulation of the angiogenic profile of melanoma cells.
Additionally, PEDF exerts a potent inhibitory action on melanoma
cells, inducing apoptosis under stress conditions (such as absence of
growth factors or detachment from the extracellular matrix) and
abrogating migration and invasion. More recently we have
demonstrated that loss of PEDF expression enables melanoma
cells to acquire migratory and invasive properties, as well as
vasculogenic mimicry capability, which altogether is translated
into an increased in vivo metastatic potential [17,19]. Therefore,
regulation of PEDF expression could be critical for the malignant
progression of human melanoma.
The mechanisms responsible of PEDF reprogramming during
the malignant progression of human melanoma are still elusive,
and their identification could be of critical importance to better
understand the biological significance of PEDF in melanoma. The
skin is a mildly hypoxic microenvironment (pO2in the dermal/
epidermal junctions ranging from 0.5% to 10%) that significantly
contributes to melanocyte transformation, as the result of hypoxia
promoting both proliferation and survival, and avoiding senes-
cence . Thus, hypoxia has emerged as a relevant tumor-
promoting environmental factor in skin melanocytes that cooper-
ates with oncogenic BRAF (BRAFV600E) and activation of AKT
pathway for malignant transformation . Furthermore, hypoxia
has been identified as a critical regulator of invasiveness and
epithelial-mesenchymal transition (EMT)  thus promoting
metastasis. Additionally, hypoxia is one of the main regulators of
angiogenic growth factors and inhibitors, which contributes to tilt
the balance toward inducers of angiogenesis and to impose the loss
of relevant angiostatic factors during tumor progression .
Given the central role of hypoxia in tumor progression and
angiogenesis, here we explored whether PEDF expression in
human melanocytes and melanoma cell lines is regulated by
variations in oxygen tension.
Cells respond to hypoxia through a combination of regulatory
mechanisms that results in reduced oxygen consumption and
restoration of oxygen supply. A central regulatory mechanism is
hypoxia-inducible factors (HIFs). HIF is a heterodimer comprising
an oxygen-regulated alpha subunit (HIFa) and a constitutively
expressed beta subunit (HIFb). HIFa family comprises three
members: HIF1a, HIF2a and HIF3a [25–27], which display
differential expression and regulate the expression of a subset of
non-overlapping target genes. Central to the hypoxia response is a
family of 2-oxoglutarate dependent dioxygenases (EGL nine
homolog, EGLNs; also called prolyl-hydroxylases, PHDs) that
require oxygenascosubstrateandconstitutethe mainoxygensensor
mechanism so far characterized [28,29]. PHDs hydroxylate HIFa
in two proline residues [30,31] and this posttranslational modifica-
tion labels HIFa for proteasomal degradation. Reduced oxygen
concentration in hypoxia comprises hydroxylation by PHDs and
consequently HIFa subunits are stabilized. The stabilization of
HIFa allows for the formation of the HIF1a/b heterodimer and
lead to HIF-mediated transcription.
Transcriptional reprogramming through HIFs acts in concert
with inhibition of translation through inactivation of the
mammalian target of rapamycin (mTOR) and activation of the
unfolded protein response (UPR); to effectively achieve hypoxia
adaptation based on changes in metabolism, angiogenesis,
endoplasmic reticulum (ER) homeostasis and autophagy [32,33].
Hypoxia also regulates translation through miRNAs [34,35] and
regulation of RNA-binding proteins (RBPs) . Additionally,
selective degradation of certain target proteins under hypoxia by
diverse degradation routes significantly contributes to hypoxia
tolerance mechanisms [37,38].
Here, we study the general characteristics of the mechanism
responsible for regulation of PEDF expression by hypoxia in
human melanocytes and melanoma cells. Our results show that
reduction of PEDF production by hypoxia has common general
characteristics with previously described regulation of PEDF in
other cell types, and distinct characteristics that specifically involve
degradation by autophagy in neural crest derived cells.
Hypoxia Downregulates PEDF at the Protein Level in
Melanocytes and Melanoma Cell Lines
Seeking for regulators of PEDF relevant in the context of
melanoma progression we explored whether hypoxia could be a
candidate mechanism. In primary cultures of human skin
melanocytes we found that extracellular levels of PEDF protein
(PEDFe) detected by western blot analysis of conditioned medium
gradually decreased under hypoxic (1% O2) (Fig. 1A) and anoxic
(0% O2) conditions (Fig. S1). Downregulation of PEDFe by
hypoxia was detected at 8–12 h and secreted protein levels
remained low after 24–48 h of hypoxia (Fig. 1A and data not
shown). Establishment of hypoxia response in primary melano-
cytes was monitored by detection of hypoxia-inducible factor 2a
(HIF2a) and 1a HIF1a stabilization by western-blot analysis of
whole-cell extracts (Fig. 1B and data not shown). We next
analyzed mRNA levels of PEDF in normoxic versus hypoxic
conditions. Interestingly, we found that PEDF mRNA levels
remained constant over the time course in which we detected
downregulation of extracellular protein levels (Fig. 1C). VEGF
mRNA levels were evaluated under the same experimental
conditions as a well characterized HIF transcriptional target. As
expected, hypoxia induced a large increase in VEGF mRNA levels
in melanocytes (Fig. 1D). These results demonstrate that hypoxia
downregulates secreted levels of PEDF at the protein level in
melanocytes by posttranscriptional mechanisms.
Downregulation of extracellular PEDF by hypoxia was detected
in serum-free conditioned medium and growth factor supple-
mented conditioned medium (Fig. S2A). Although PEDF is very
efficiently secreted and consequently we detected low intracellular
PEDF (PEDFi) levels in melanoma cell lines, additionally, we
checked whether intracellular PEDF levels were modulated by
hypoxia. Our results indicated that PEDFiwas downregulated by
hypoxia and this downregulation was also independent of the
presence or absence of growth factors (Fig. S2B). Rate of DNA
synthesis was not affected by hypoxic conditions used in NHEM
primary melanocytes and SBcl2 melanoma cell line (Fig. S3).
We also examined whether hypoxia downregulated PEDF in
poorly aggressive melanoma cell lines that produce endogenous
PEDF levels similar to primary melanocytes . Hypoxia
downregulated PEDFein SBcl2 and WM164 melanoma cell lines
with a similar kinetics and extent to those found in primary
melanocytes (the melanocyte primary culture M438 was used as a
reference) (Fig. 1E). Also, in agreement with our results in primary
melanocytes, PEDF mRNA levels were not modulated by hypoxia
in the melanoma cell lines tested (Fig. 1F). Regulation of VEGF
mRNA levels by hypoxia was used as positive control (Fig. 1G).
VEGF mRNA induction by hypoxia was confirmed in primary
melanocytes, SBcl2 and WM164 melanoma cell lines.
Hypoxia Inducible Factor Does Not Mediate
Downregulation of PEDF by Hypoxia in Melanocytes and
Melanoma Cell Lines
PHDs are the best characterized cellular oxygen sensors and
they trigger many of the responses to hypoxia. Thus, we next
Hypoxia Downregulates PEDF in Melanoma
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Figure 1. Hypoxia downregulates PEDF at the protein level in melanocytes and human melanoma cell lines. Western blot analysis of
(A) extracellular PEDF (PEDFe) protein levels in conditioned medium (CM) and (B) HIF2a protein levels in whole-cell extracts (B) from M330 primary
melanocytes incubated under normoxia (21% O2) or hypoxia (1% O2) for 12 h or 24 h. b-tubulin was used as loading control. Quantitative RT-PCR
Hypoxia Downregulates PEDF in Melanoma
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decided to assess the effect of PHD inhibition on PEDF regulation.
As PHDs require oxygen, iron ascorbate and 2-oxoglutarate as
cosubstrates, we used the synthetic 2-oxoglutarate antagonist
DMOG (N-(Methoxyoxoacetyl)-glycine methyl ester) to inhibit
their activity. Treatment of primary melanocytes (M330) and
SBcl2 melanoma cells with 1 mM DMOG led to a significant and
time-dependent decrease in secreted PEDF levels detected by
western blot of conditioned medium (Fig. 2A). When we analyzed
the effect of DMOG in PEDFiprotein levels, we also found a
similar dose response and kinetics as for the downregulation of
extracellular PEDF by DMOG (Fig. 2B). As expected, we
observed HIF1a stabilization after DMOG treatment (Fig. 2B),
although a lower dose was required. The effects of DMOG were
further confirmed analyzing the dose response and kinetics of
VEGF mRNA levels (Fig. 2C). These results showed a significant
difference between the DMOG dose required for maximum
downregulation of PEDF protein levels and HIF stabilization;
pointing that decreased PEDF protein was PHD-dependent but
To further explore whether HIF was involved in the decrease in
PEDF protein levels imposed by hypoxia in primary melanocytes
(M13) and SBcl2 melanoma cells, we silenced HIF1a expression
using shRNAmirto HIF1a (shHIF1a delivered by lentiviral
transduction (Fig. 3). Non-silencing (NS) shRNAmir(shNS) was
used as control. Lentiviral transduction of primary melanocytes
(M13) and SBcl2 cells was highly efficient as demonstrated by the
high percentage of GFP positive cells (Fig. 3A). Efficiency of
HIF1a silencing was determined analyzing HIF1a mRNA levels
by quantitative RT-PCR, being this higher than 80% in primary
melanocytes and SBcl2 melanoma cells (Fig. 3B). Despite the
efficient silencing of HIF1a in primary melanocytes, hypoxic
conditions reduced secreted and intracellular PEDF to a similar
extent in shNS and shHIF1a cells (Fig. 3C). To further confirm
HIF1a silencing in melanocytes we checked mRNA levels of the
known HIF direct genes VEGF and BNIP3. Induction by hypoxia
of both HIF genes in melanocytes was efficiently abrogated by
shHIF1a (Fig. 3D). In agreement with our results in melanocytes,
HIF1a silencing in SBcl2 melanoma cells did not interfere with
downregulation of secreted PEDF protein levels by hypoxia
(Fig. 3E). Interestingly, knock-down of HIF1a in SBcl2 melanoma
cells diminished VEGF and BNIP3 mRNA induction by hypoxia,
although to a lesser extent than observed in melanocytes (Fig. 3F).
These results could be explained by differences in the mechanisms
that mediate regulation of expression of HIF target genes in
primary melanocytes versus melanoma cells.
UTRs Are Not Required for PEDF Downregulation by
Hypoxia in Melanoma Cell Lines
To further confirm that PEDF is regulated by hypoxia at the
protein level in melanoma cells, we analyzed the effect of hypoxia
on exogenous PEDF expressed from a heterologous promoter
(CMV promoter in pCEP4-PEDF vector). SBcl2 melanoma cells
were stably transfected with pCEP4 (SBcl2-pCEP4) or pCEP4-
PEDF (SBcl2-pCEP4-PEDF). Downregulation of endogenous
secreted PEDF by hypoxia and DMOG was confirmed in control
analysis of (C) PEDF mRNA levels and (D) VEGF mRNA levels in M330 primary melanocytes incubated in normoxia or hypoxia for 12 h or 24 h. PEDF
and VEGF mRNA levels are shown relative to cells in normoxia after normalization to b-actin. Bars represent average 6 standard deviation (SD)
(**P,0.01). (E) Western blot analysis of PEDFeprotein levels in CM and HIF1a in whole-cell extracts from M438 primary melanocytes, SBcl2 and
WM164 melanoma cell lines incubated in normoxia or hypoxia for 12 h or 24 h. b-actin was used as loading control. Quantitative RT-PCR analysis of
(F) PEDF mRNA levels and (G) VEGF mRNA levels in M438 primary melanocytes, SBcl2 and WM164 melanoma cell lines incubated under normoxia or
hypoxia for 12 h or 24 h. PEDF and VEGF mRNA levels are shown relative to normoxia after normalization to b-actin. Bars represent average 6 SD
Figure 2. Inhibition of PHDs leads to decreased PEDF protein in
normoxia in melanocytes and SBcl2 melanoma. (A) Western blot
analysis of extracellular PEDF (PEDFe) protein levels in conditioned
medium (CM) from M330 primary melanocytes (upper blot) and SBcl2
melanoma cell line (lower blot) treated with different concentrations of
DMOG for 8 h, 16 h or 24 h. (B) Western blot analysis of PEDFeprotein
levels in CM, intracellular PEDF (PEDFi) and HIF1a protein levels in
whole-cell extracts from SBcl2 melanoma cells treated with DMOG for
12 h and 24 h. b-tubulin was used as loading control. (C) Quantitative
RT-PCR analysis of VEGF mRNA levels in SBcl2 melanoma cell line
treated with DMOG for the indicated times. VEGF levels are shown
relative to controls without DMOG in time points, after normalization to
18s rRNA. Bars represent average 6 standard deviation (SD) (**P,0.01;
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SBcl2-pCEP4 cells (Fig. 4A). Regulation of exogenous PEDF
protein was monitored by means of a histidine tag (5-HIS) fused to
PEDF in pCEP4-PEDF vector. Figure 4A shows that hypoxia and
DMOG efficiently reduced exogenous secreted PEDF protein
levels in SBcl2-pCEP4-PEDF cells. Additionally, we confirmed
that intracellular PEDF protein levels decreased in SBcl2-pCEP4
and SBcl2-pCEP4-PEDF cells under hypoxia or DMOG treat-
ment (Fig. 4B). Stabilization of HIF1a protein as a control of
hypoxia response is shown in all the experimental conditions used
(Fig. 4B). Given that exogenous PEDF expressed from pCEP4-
PEDF vector lacked the 59 and 39 untranslated regions (UTRs),
downregulation of PEDF by hypoxia was most likely not mediated
through UTR inhibition of translation. In order to directly asses
the role of SERPINF1 UTRs in the regulation of PEDF by
hypoxia in melanoma cells we cloned the 39UTR of PEDF in a
Renilla reporter construct psiCHECK2 (psiCHECK2-39PEDF)
and analyzed the 39UTR of PEDF using UTR reporter assays in
normoxic versus hypoxic conditions in melanoma cells. We used
the 39UTR of GAPDH (psiCHECK2-39GAPDH) as a control,
together with the empty psiCHECK2 plasmid, since it was
previously shown that hypoxia did not modify the translation of
GAPDH mRNA . We first confirmed that hypoxia downreg-
ulated extracellular and intracellular PEDF protein levels (Fig. 4C)
in SBcl2 cells. As in previous experiments hypoxia response in
SBcl2 cells was confirmed by stabilization of HIF1a detected by
western blot analysis of whole-cell extracts (Fig. 4C). Afterward, in
transient transfection experiments, we found no significant
differences in reporter activity when we compared psiCHECK2-
39UTR PEDF with empty vector or psiCHECK2-39UTR
GAPDH (Fig. 4D), indicating that the 39UTR of PEDF does not
mediate inhibition of translation under hypoxic conditions. These
results were also confirmed in M000921 human melanoma cell
line in which PEDF (intracellular and extracellular) is highly
regulated by hypoxia and presents a higher efficiency of
transfection (Fig. S4).
In summary, these results support that PEDF downregulation is
not mediated by regulation of translation through UTRs.
Degradation by Metalloproteinases or the Proteasome
Does Not Mediate Downregulation of PEDF by Hypoxia
in Melanoma Cell Lines
As no difference in PEDF mRNA level was detected in hypoxia
compared to normoxia in melanocytes or melanoma cells, and
inhibition of translation through UTRs was not involved in the
downregulation of PEDF, it stands to reason that hypoxic
regulation of PEDF could likely occur at the posttranslational
Matrix metalloproteinases type 2 (MMP-2) and type 9 (MMP-9)
belong to the large MMP family of Zn2+- and Ca2+-dependent
extracellular proteinases that are critically involved in the
regulation of migration, invasion and angiogenesis [40,41]. It
has been shown that PEDF produced by RPE cells is degraded
extracellularly by MMP-2 and MMP-9 activated in hypoxic
conditions . Therefore, we studied whether this posttransla-
tional regulatory mechanism could be responsible of decreased the
PEDF protein in neural crest-derived pigment cells (melanocytes
and melanoma cells) under hypoxia. Conditioned medium from
control or DMOG-treated melanocytes and SBcl2 cells were tested
for protein degradation activity against exogenously added
purified recombinant human PEDF (rhuPEDF). In vitro incubation
of rhuPEDF with either direct or concentrated conditioned
medium from DMOG-treated melanocytes and SBcl2 cells did
not produce any significant degradation of the exogenous PEDF
protein (Fig. 5A). Incubation with EDTA was used to inhibit
expected induction of metalloproteinase activity by hypoxia.
Downregulation of endogenous secreted PEDF protein by hypoxia
was confirmed in concentrated conditioned medium from control
versus DMOG-treated melanocytes and SBcl2 cells (Fig. 5A).
Furthermore and in agreement with previous results, cells treated
with the metalloproteinase inhibitor GM6001 did not block
downregulation of secreted PEDF protein levels by hypoxia in
SBcl2 melanoma cells (Fig. 5B).
We next studied whether decreased PEDF protein levels under
hypoxia were a consequence of degradation by the proteasome,
and found that the diminished PEDFeprotein levels in hypoxia
were not recovered when we treated SBcl2 and WM164 cells with
the proteasome inhibitor MG132 (Fig. 5C). As expected, MG132
stabilized HIF1a in normoxic conditions (Fig. 5C).
Hence, these results indicated that neither extracellular
degradation by metalloproteinases nor proteasomal degradation
was implicated in the downregulation of PEDF protein levels by
hypoxia in melanocytes and melanoma cells.
Downregulation of PEDF by Hypoxia in Melanoma Cell
Lines Involves Degradation by Autophagy
Autophagy is a tightly controlled degradation pathway that has
been recently shown to be activated in response to hypoxia [42–
44]. Among the identified autophagy-related proteins, microtu-
bule-associated protein light chain 3 (LC3) has been widely used to
monitor the autophagic response [45,46]. LC3 exists in two forms:
LC3-I (18 kDa) localized in the cytosol and its proteolytic
derivative LC3-II (16 kDa) which is modified by conjugation with
phosphatidylethanolamine and bound to autophagosomal mem-
(SQSTM1/p62) were used to monitor induction of autophagy
by hypoxia . Hypoxia induced accumulation of LC3-II
positive autophagic vacuoles in SBcl2 cells with a rapid and
sustained kinetics (Fig. S5). Induction of autophagy by hypoxia was
Figure 3. Hypoxia-induced downregulation of PEDF is HIF-independent in melanocytes and SBcl2 melanoma. (A) Transduction
efficiency of M13 primary melanocytes (left panels) and SBcl2 melanoma cell line (right panels) after infection with non-silencing (shNS) or shRNAmir
to HIF1a (shHIF1a) lentivirus at multiplicity of infection of 40 (M13) or 60 (SBcl2). Fluorescence images (406magnification) show more than 90% GFP-
positive cells. (B) Quantitative RT-PCR analysis of HIF1a mRNA levels in M13-shNS, M13-shHIF1a primary melanocytes and SBcl2-shNS, SBcl2-shHIF1a
melanoma cell lines. HIF1a mRNA levels are shown relative to control shNS cells after normalization to 18s rRNA. Bars represent average 6 standard
deviation (SD) (**P,0.01; ***P,0.001). (C) Western blot analysis of extracellular PEDF (PEDFe) protein levels in conditioned medium (CM), intracellular
PEDF (PEDFi) and HIF1a protein levels in whole-cell extracts from M13-shNS and M13-shHIF1a primary melanocytes incubated under normoxia (21%
O2) or hypoxia (1% O2) for 16 h and 24 h. b-tubulin was used as loading control. (D) Quantitative RT-PCR analysis of VEGF (left panel) and BNIP3 (right
panel) mRNA levels in M13-shNS (filled bars) and M13-shHIF1a (empty bars) primary melanocytes. VEGF and BNIP3 mRNA levels are shown relative to
M13-shNS under normoxia after normalization to 18s rRNA. Bars represent average 6 SD (*P,0.05; ***P,0.001). (E) Western blot analysis of PEDFe
protein levels in CM, PEDFiand HIF1a protein levels in whole-cell extracts from SBcl2-shNS and SBcl2-shHIF1a melanoma cell lines incubated under
normoxia or hypoxia for 16 h and 24 h. b-actin was used as loading control. (F) Quantitative RT-PCR analysis of VEGF (left panel) and BNIP3 (right
panel) mRNA levels in SBcl2-shNS (filled bars) and SBcl2-shHIF1a (empty bars) melanoma cell lines. VEGF and BNIP3 mRNA levels are shown relative to
SBcl2-shNS under normoxia after normalization to 18s rRNA. Bars represent average 6 SD (**P,0.01; ***P,0.001).
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further demonstrated by decreased the levels of LC3-II and p62 in
SBcl2 and M000921 melanoma cells (Fig. 6A). Quantification of
SBcl2 cells with autophagic vacuoles showed a significant increase
(***P,0.001) in hypoxia versus normoxia (Fig. 6C). As an
additional control we show that ischemic conditions mimicked
by glucose starvation induced a similar pattern of accumulation
and distribution of GFP-LC3 signal than hypoxia in SBcl2 cells
To investigate whether autophagy was implicated on the
degradation of PEDF by hypoxia in SBcl2 melanoma cells we
used bafilomycin A1 (Baf. A1), which inhibits the vacuolar ATPase
and blocks the fusion of autophagosomes with lysosomes . As
expected, Baf. A1 treatment of SBcl2 and M000921 cells induced
accumulation of LC3-II and p62 detected by western-blot (Fig. 6A)
and redistribution of GFP-LC3 fusion construct into punctuate
cytoplasmic structures indicative of accumulation of autophagic
vacuoles (Fig. 6B, C). Figure 6A shows that Baf. A1 treatment
efficiently blocked downregulation of PEDFeand PEDFi(Fig. 6A)
Implication of autophagy in the degradation of PEDF under
hypoxia was further confirmed by silencing of LC3 in SBcl2
melanoma cells. LC3 was efficiently interfered in SBcl2 melanoma
cells using shRNAmirto LC3 (Fig. 7A–B). Silencing of LC3
prevented PEDF donwregulation by hypoxia (Fig. 7C).
These results implied that autophagy-mediated degradation of
PEDF is induced in response to hypoxia in melanoma cells.
PEDF was first identified as an endogenous inhibitor of
angiogenesis in the eye . RPE cells secrete high levels of
PEDF to ensure a proper balance of neovascularization in the
retina. Avascular eye compartments like the cornea and the
vitreous are rich in PEDF. Therefore, PEDF plays a pivotal role
on maintaining the eye vasculature in a quiescent state and loss of
its expression is associated with pathological neovascularization
leading to compromised vision and blindness .
We have recently shown that PEDF is also produced at high
levels by neural crest-derived pigment-producing cells, the skin
melanocytes . The role of PEDF in the control of physiological
skin vascularization remains to be characterized. Given PEDF’s
role as an antiangiogenic factor, we have recently described the
regulation of its expression during the malignant progression of
human melanomas and the functional consequences of loss of
PEDF expression . PEDF expression is high in melanoctyes,
but it is lost during malignization of human melanoma. In vitro and
in vivo functional analysis combined with interference strategies to
silence PEDF, led us to demonstrate that PEDF has a broad
Figure 4. UTRs are not required for PEDF downregulation by hypoxia in SBcl2 melanoma. Western blot analysis from SBcl2-pCEP4 and
SBcl2-pCEP4-PEDF melanoma cell lines incubated with 1 mM DMOG or under hypoxia (1% O2): (A) extracellular PEDF (PEDFe) and Penta-HIS (5-HIS)
protein levels in 24 h conditioned medium (CM) and (B) intracellular PEDF (PEDFi) and HIF1a protein levels in whole-cells extract. b-tubulin was used
as loading control. (C) Western blot analysis of PEDFeprotein levels in 24 h CM, PEDFiand HIF1a protein levels in whole-cell extracts from SBcl2
melanoma cell line incubated under normoxia (21% O2) or hypoxia (1% O2). b-tubulin was used as loading control. (D) UTR-reporter assay in SBcl2
melanoma cell line transfected with a Renilla promoter reporter containing the 39 UTR of PEDF (psiCHECK2-39PEDF), the 39UTR of GAPDH (psiCHECK2-
39GAPDH) or an empty reporter (psiCHECK2) and incubated under normoxia or hypoxia for 24 h. Renilla activity was normalized to luciferase activity,
which is used as an internal control of transfection efficiency. psiCHECK2-39GAPDH was used as a negative control. Bars represent average 6 standard
Hypoxia Downregulates PEDF in Melanoma
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function in melanoma that allows it to dually impinge on the
vascular component of the tumor microenvironment and on
directly counteracting a set of capabilities that enable the
metastatic spread of melanoma cells [17,19].
The functional relevance and multifunctionality of PEDF in
melanoma prompted us to identify an important regulatory
mechanism during melanoma progression. Our results suggest
that PEDF expression could be modulated by two general types of
mechanisms, reprogramming events and loss of expression .
Hypoxia is a hallmark of tumors that results from an imbalance
between oxygen supply and consumption in continuously
proliferating cancer cells in a tumor mass devoid of an adequate
vascular network to cope with imposed oxygen demand.
Consequently, hypoxia is one of the main triggers of tumor
neovascularization and has been shown to contribute to tumor cell
invasion, migration and metastasis .
Signals from the microenvironment such as hypoxia and
inflammation are thought to reprogram and switch melanoma
cells toward an invasive phenotype , and therefore, could be
responsible for the reprogramming of PEDF during melanoma
Previous reports described that PEDF is decreased by hypoxia
in retinoblastoma  and RPE cells , although none of them
studied the role of HIF in PEDF downregulation. Here, we
describe the general characteristics of the mechanism responsible
of decreased PEDF protein levels under hypoxia in human
melanocytes and melanoma cells. We show that secreted PEDF, as
well as intracellular PEDF protein levels, decrease under low
oxygen conditions in primary melanocytes and several human
melanoma cell lines. However, we found no significant differences
in PEDF mRNA levels when we compared hypoxic versus
normoxic conditions, suggesting that regulation of PEDF by
hypoxia was posttranscriptional. This result is in agreement with
previous studies in retinoblastoma cells and RPE cells in which
diminished PEDF protein levels under hypoxia did not correlate
with changes in PEDF mRNA levels [37,48].
Moreover, in support to our results, the meta-analysis of gene
profiling data sets from 16 independent experiments by Ortiz-
Barahona and collaborators  confirmed there was no variation
in PEDF mRNA levels in normoxic versus hypoxic conditions
(data not shown).
HIF plays a central role in the regulation of the cell responses
that allows adaptation to reduced oxygen tension. Although PEDF
was not regulated at the transcriptional level, molecules down-
stream HIF could be involved in PEDF downregulation. We
therefore analyzed whether HIF was implicated in the observed
effects on PEDF in primary melanocytes and melanoma cell lines.
Using lentiviral transduction of shRNA specific to HIF1a, we
demonstrated that decreased PEDF protein levels in melanocytes
and melanoma cells were not mediated by HIF1a.
Alternative mechanisms that participate in the downregulation
of specific targets under hypoxia are the following: (i) regulation of
translation by miRNAs and RBPs , (ii) selective degradation of
hypoxia targets by the proteasome  or metalloproteinases 
(iii) inhibition of translation through mammalian target of
rapamycin (mTOR) kinase, (iv) activation of the unfolded protein
response [33,53] and (v) degradation by autophagy [42–44].
Inhibition of overall protein synthesis has long been accepted as
a general trait of adaptation to hypoxia . Notwithstanding,
translation of specific mRNAs is favored by low oxygen tension.
This is the case of the mRNAs of HIF1a  and VEGFA 
which harbor regulatory elements that promote their preferential
translation under overall translation inhibition imposed by
hypoxia. We have directly addressed whether downregulation of
PEDF by hypoxia occurs at the translational or posttranslational
level. Two independent approaches allowed us to conclude that
translation of PEDF mRNA is unlikely to be regulated by low
oxygen tension. First, exogenous PEDF produced using vector
constructs lacking the 59UTR and 39UTR was effectively
decreased under hypoxic conditions. Furthermore a reporter
construct of PEDF 39UTR was not affected by hypoxic conditions
in several melanoma cell lines. However, the UTRs are highly
conserved in SERPINF1 among different species, which points to
their putative regulatory role by an as yet unidentified mechanism.
Secondly, we used a bioinformatic approach to predict putative
targets sequences for the RBPs HuR and TIA-1 [56,57]. This
program identifies motifs that share two common characteristics:
(i) a primary sequence over 20 bp rich in AU and (ii) a specific
secondary structure named stem-loop. With this approach we
identified a small region rich in AU in the 39 UTR of SERPINF1
Figure 5. Hypoxia-induced downregulation of PEDF in mela-
nocytes and SBcl2 melanoma cells is not mediated by
metalloproteinases or proteasomal degradation. (A) Western
blot analysis of extracellular PEDF (PEDFe) protein levels in 24 h
conditioned medium (CM) from M330 primary melanocytes (upper blot)
and SBcl2 melanoma cell line (lower blot). Cells were treated with 1 mM
DMOG for 24 h and the CM were incubated with 100 ng human
recombinant PEDF (rhuPEDF) and 20 mM EDTA at 37uC for 2 h. (B)
Western blot analysis of PEDFe protein levels in CM from SBcl2
melanoma cell line treated with metalloproteinase inhibitor GM6001
(10 mM) and incubated under normoxia (21% O2) or hypoxia (1% O2) for
24 h. (C) Western blot analysis of PEDFeprotein levels in 16 h CM and
HIF1a protein levels in whole-cell extracts from SBcl2 and WM164
melanoma cell lines after treatment with the proteasome inhibitor
MG132 (5 mM and 1 mM respectively) under normoxia or hypoxia. b-
tubulin was used as loading control.
Hypoxia Downregulates PEDF in Melanoma
PLoS ONE | www.plosone.org8March 2012 | Volume 7 | Issue 3 | e32989
Figure 6. Autophagy is involved in downregulation of PEDF by hypoxia in melanoma cells. (A) Western blot analysis of extracellular PEDF
(PEDFe) protein levels in 24 h conditioned medium (CM), intracellular PEDF (PEDFi), HIF1a LC3 and p62 protein levels in whole-cell extracts from SBcl2
(left) and M000921 (right) melanoma cell lines treated with different concentrations of the autophagy inhibitor bafilomycin A1 (Baf. A1, 50 nM and
200 nM) or DMSO vehicle under normoxic (21% O2) or hypoxic (1% O2) conditions. b-tubulin was used as loading control. (B) Fluorescence images
(636magnification) of GFP-LC3 protein redistribution in SBcl2 melanoma cell line (transduced with pLV-EGFP-LC3 plasmid) treated with 50 nM Baf.
A1 in normoxia or hypoxia for 24 h. (C) Quantification of SBcl2 cells with autophagic vacuoles after Baf. A1 treatment for 24 h under normoxia (filled
Hypoxia Downregulates PEDF in Melanoma
PLoS ONE | www.plosone.org9 March 2012 | Volume 7 | Issue 3 | e32989
(data not shown). However, this sequence was in a non-conserved
region and it was not identified by the in silico analysis as a putative
RBP binding site; most likely due to lack of the secondary structure
necessary for the binding of HuR or TIA-1.
These results prompted us to directly address the implication of
degradation pathways relevant in the context of hypoxia response
as the main mechanism underlying decreased PEDF protein levels
under low oxygen tension conditions in melanocytes and
The first candidate that we explored was the proteasome. The
proteasome has been recently implicated on the selective
degradation of specific targets under low oxygen conditions. This
mechanism is responsible of downregulating the ternary complex
factor net under hypoxia . However, inhibition of the
proteasome using MG132 did not block downregulation of PEDF
levels by hypoxic conditions in melanoma cell lines. Also, the fact
that decreased PEDF protein levels under low oxygen tension were
not mediated by the proteasome makes it unlikely that activation
of the UPR could be involved.
Taking into account a previously reported implication of
metalloproteinases in the downregulation of PEDF protein levels
under hypoxia in RPE cells  we checked whether this
mechanism could be also operating in neural crest-derived
pigment-producing cells. Our results demonstrate that induction
of metalloproteinases by hypoxia was not responsible of decreased
PEDF protein levels in melanoma cell lines.
An alternative degradation pathway that has been recently
demonstrated to be relevant to the hypoxia adaptation response is
degradation by autophagy [33,42–44]. Specific targets downreg-
ulated by hypoxia have been shown to be degraded by autophagy.
We show that hypoxic conditions significantly induced autophagy
in melanoma cells as revealed by the punctuated phenotype of
GFP-LC3 labeling and downregulation of LC3-II and p62 levels.
The autophagy inhibitor Bafilomycin A1 (Baf. A1), which blocks
the fusion of autophagosomes with lysosomes, abrogated down-
regulation of PEDF protein levels under low oxygen tension in
melanoma cells. Silencing of LC3 also prevented PEDF down-
regulation by hypoxia in melanoma cells. These results support
that decreased extracellular PEDF levels by hypoxia are a
consequence of degradation by autophagy of intracellular PEDF,
resulting in loss of its biological activities in pigment-producing
melanocytes and melanoma cells.
Altogether our results point to hypoxia as a permissive
environment associated with decreased production of PEDF by
melanocytes and melanoma cells that in turn impacts on the
acquisition of a more malignant phenotype. Furthermore,
downregulation of PEDF at low oxygen tension is HIF-
independent and occurs at the level of protein degradation
involving the participation of autophagy as the most likely
candidate mechanism. Both hypoxia and autophagy play a
significant role in the context of melanoma progression [22,58–
60], therefore we have identified a relevant mechanism that may
underlie reprogramming of PEDF expression during the malig-
nant progression of melanoma. Loss of PEDF expression during
melanoma malignization enables acquisition of angiogenic,
invasive and metastatic capabilities to melanoma cells [17,19].
Hypoxic conditions at the invasive front could be responsible of
required decreased PEDF levels to enable low proliferation and
increased migration and invasiveness characteristic of invasive
phenotype melanoma cells. Both heterogeneity in tumor oxygen-
ation, as well as colonization of new tissue environments
characterized by higher oxygen tensions than the skin may lead
to PEDF regulation in melanoma lesions  and subsequent
reprogramming back to high PEDF to allow melanoma cells to
gain the proliferative potential required to successfully colonize
Materials and Methods
The present study was approved by the institutional Review
Board of Children’s Hospital Universitario Nin ˜o Jesu ´s (Madrid,
bars) or hypoxia (empty bars). Ten fields from each condition were counted for quantification. Bars represent average 6 standard deviation (SD)
(***P,0.001). (D) Fluorescence images (636magnification) of GFP-LC3 redistribution in SBcl2 melanoma cell line grown in the absence of growth
factors (0% FBS) or ischemic conditions (0% glucose) under normoxia or hypoxia.
Figure 7. LC3 knock-down prevents downregulation of PEDF
by hypoxia in melanoma cells. (A) Transduction efficiency of SBcl2
melanoma cell line after infection with non-silencing (shNS) or shRNAmir
to LC3 (shLC3) lentivirus at multiplicity of infection of 60. Fluorescence
images (206magnification) show more than 90% GFP-positive cells. (B)
Quantitative RT-PCR analysis of LC3 mRNA levels in SBcl2-shNS and
SBcl2-shLC3 melanoma cell lines. LC3 mRNA levels are shown relative to
SBcl2-shNS after normalization to 18s rRNA. Bars represent average 6
standard deviation (SD) (***P,0.001). (C) Western blot analysis of
extracellular PEDF (PEDFe) protein levels in conditioned medium (CM),
intracellular PEDF (PEDFi), HIF1a and LC3 protein levels in whole-cell
extracts from SBcl2-shNS and SBcl2-shLC3 melanoma cells incubated
under normoxia (21% O2) or hypoxia (1% O2) for 24 h. b-tubulin was
used as loading control.
Hypoxia Downregulates PEDF in Melanoma
PLoS ONE | www.plosone.org10 March 2012 | Volume 7 | Issue 3 | e32989
Spain) in accordance with the Helsinki Declaration. Informed
written consent was obtained from all donors of foreskins.
Human melanoma cell lines SBcl2 (radial growth phase) and
WM164 (established from a metastasis) were provided by Dr
Herlyn (The Wistar Institute, Philadelphia, PA, USA) and cultured
as described previously . M000921 melanoma cell line
(established from a metastasis) was provided by Dr Hoek
(University Hospital of Zu ¨rich, Zu ¨rich, Switzerland) and cultured
as reported earlier . Primary human melanocytes were isolated
from foreskins from independent donors and grown as described
previously . Primary cultures of melanocytes obtained from
different single donors were used in this study (M330, M438,
M13). For experiment in Figure S3, primary human melanocytes
from Lonza (Basel, Switzerland) obtained from single donors were
also used (NHEM). Primary cultures of melanocytes were used
between passages 4–6, thus, a limited number of experiments
could be carried out for each particular primary culture and
therefore three different cultures were used.
Reagents and Antibodies
The prolyl-4-hydroxylase inhibitor DMOG (N-(Methoxyoxoa-
cetyl)-glycine methyl ester, 250 mM and 1 mM) was purchased
from Enzo Life Sciences (Farmingdale, NY, USA); EDTA
(20 mM) and autophagy inhibitor Bafilomicyn A1 (Baf.A1)
(50 nM and 200 nM) were purchased from Sigma (St Louis,
MO, USA). The metalloproteinase inhibitor GM6001 (10 mM)
and the proteasome inhibitor MG132 (1 mM and 5 mM) were
from Calbiochem (Darmstadt, Germany). Antibodies used were:
extracellular PEDF (polyclonal; Bioproducts, West Palm Beach,
FL, USA), intracellular PEDF (monoclonal; Chemicon, Billerica,
MA, USA), HIF1a (monoclonal; BD Transduction Laboratories,
Franklin Lakes, NJ, USA), HIF2a polyclonal; Abcam, Cambridge,
UK), b-tubulin (monoclonal; Sigma), b-actin (polyclonal; Santa
Cruz Biotechnologies, Santa Cruz, CA, USA), p62 (polyclonal;
Cell Signaling Technology, Danvers, MA, USA), LC3 (polyclonal;
Cell Signaling Technology) and Penta-His (monoclonal; Qiagen,
Hypoxia conditions were achieved by incubating cells in a
Hypoxystation H35 (Don Whitley Scientific Limited, Shipley,
UK). Hypoxia was generated using a gas mixture of 1% O2, 5%
CO2and 94% N2. In Figure S4 anoxia was generated using a gas
mixture of 0% O2, 5% CO2and 95% N2. Alternatively, hypoxic
like responses were mimicked by incubation with 250 mM and
1 mM DMOG (Enzo Life Sciences).
Conditioned Medium Preparation
For extracellular PEDF (PEDFe) detection, cells were cultured
in basal medium. Later, conditioned medium (CM) was collected,
centrifuged to eliminate cellular debris and treated with protease
inhibitor PMSF (phenylmethylsulfonyl fluoride; Sigma). CM was
concentrated 50 times using Amicon Ultra (Millipore, Billerica,
MA, USA) devices with 10 kDa cut-off in a refrigerated centrifuge.
Afterward, 15–30 ml of CM was loaded per lane and used for
western blot analysis.
A total of 100 ng recombinant human PEDF (rhuPEDF) was
mixed in 10 ml of SBcl2 direct or concentrated CM and when
indicated was treated with EDTA (Sigma) protease inhibitor at
37uC for 1 h. Reaction mixture was subjected to western blot for
Whole-cell extracts were prepared by lysing the cells in Laemmli
buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.1%
bromophenol blue and 100 mM DTT) containing protease and
phosphatase inhibitors (10 mg/ml leupeptin; 10 mg/ml aprotinin;
10 mg/ml sodium orthovanadate; 1 mM PMSF (all from Sigma)).
Whole-cell extracts or CM were separated by SDS-PAGE and
subsequently transferred to PVDF membranes, and afterward,
incubated with appropriate primary and horseradish-conjugated
secondary antibodies and developed with ECL (GE Healthcare,
Buckinghamshire, UK). Shown data are from a representative
experiment that was confirmed on at least two independent
DNA synthesis in primary human melanocytes (NHEM) and
SBcl2 melanoma cell lines in normoxic vs hypoxic conditions was
analyzed by the incorporation of 5-ethynyl-2-deoxyuridine (EdU)
using Click-iT EdU Imaging Kit (Invitrogen, Paisley, UK) as
indicated by the manufacturer. EdU-positive nuclei were counted
in six independent fields using a TCS SP5 DM16000 spectral
confocal microscope (Leica Microsystems, Heidelberg, Germany).
Nuclei were visualized using DAPI (49,6-diamidino-2-phenylin-
RNA Extraction and Quantitative RT-PCR
Total RNA was extracted and purified with the RNeasy Mini Kit
(Qiagen). Total RNA (1 mg/sample) was reverse transcribed to
cDNA (Improm-II reverse transcriptase; Promega, Madison, WI,
USA) and 1 ml of cDNA samples were used as template for
amplification reactions carried out with the LC Fast Start DNA
master SYBR Green I Kit (Roche Applied Science, Basel,
Switzerland) following the manufacturer’s instructions. Oligonucle-
otides used were: 28s rRNA sense 59-cagtacgaatacagaccg-39 and
antisense 59-ggcaacaacacatcatcag-39; b-actin sense 59-cccagagcaaga-
gagg-39 and antisense 59-gtccagacgcaggatg-39; HIF1a sense 59-
gtttactaaggacaagtcacc-39 and ansitense 59-ttctgtttgttgaagggag-39;
and BNIP3 sense 59-gtctggacggagtagc-39 and antisense 59-ggccgactt-
gaccaat-39. For detection of mRNA levels of 18s rRNA, PEDF,
VEGF and LC3B, 1 ml of cDNA samples were used as a template
and amplified with ABI Prism 7900 HT (Applied Biosystems,
Carlsbad, CA, USA) using the following TaqMan probes (Applied
Biosystems): 18 s rRNA (Hs99999901_s1), PEDF (Hs00171467_m1),
LC3B (Hs00797944_s1) and VEGF (Hs00173626_m1). Average 6
standard deviation (SD). Values shown are from a representative
experiment that was confirmed on at least three independent
Lentivirus Production and Transduction of Target Cell
For transduction of cell lines, lentiviruses were used at multiplicity of
infection (MOI) of 40–60 in the presence of 8 mg/ml polybrene
(Sigma) for 8 h. Transduction efficiency was higher than 90%, and
after 72 h, gene overexpression or knock-down was assessed.
For LC3 overexpression we used the lentiviral vector pLV-
EGFP-LC3 plasmid, kindly provided by Dr Soengas (Centro
Nacional de Investigaciones Oncologicas, Madrid, Spain).
Hypoxia Downregulates PEDF in Melanoma
PLoS ONE | www.plosone.org 11March 2012 | Volume 7 | Issue 3 | e32989
HIF1a and LC3 silencing was carried out using the lentiviral
vector pGIPz containing shRNAmirsequence V2LHS_132150
and V3LHS_408637 respectively from Open Biosystems (Thermo
Fisher Scientific, Huntsville, AL, USA). Non-silencing shRNAmir
sequence (shNS), with no homology to known mammalian genes
was used as control, cloned in a pGIPz vector (Open Biosystems).
Generation of PEDF-overexpressing Cell Lines
SBcl2 and M000921 melanoma cell lines were seeded on
21 cm2plates (106cells) and 24 h later were transfected using
Lipofectamine 2000 (Invitrogen) with 8 mg of pCEP4-PEDF
plasmid (provided by Dr Bouck, Northwestern University,
Chicago, IL, USA) or control pCEP4 plasmid. Forty-eight hours
later cells were selected with 300 mg/ml hygromicin B (Sigma) for
two weeks, and then characterized for PEDF overexpression and
used for further studies. pCEP4-PEDF plasmid contains PEDF
cDNA without untranslated regions (59UTR and 39UTR) followed
by a histidine tag at 39.
Plasmids psiCHECK-39 PEDF and psiCHECK-39 GAPDH
were generated by cloning the PEDF 39-UTR or GAPDH 39-
UTR into the multiple cloning site of psiCHECK-2 vector
(Promega) after Not I (Roche Applied Science) and Xho I
(Invitrogen) digestion. The PEDF 39-UTR and GAPDH 39-UTR
were amplified by PCR using the following pairs or oligonucle-
otide primers: for PEDF, primer sense 59-cgctcgagtatcccagtttaa-
tattcc-39 (Xho I site underlined) and antisense 59-cagcggccgctaa-
cagaagttagggataa-39 (Not I site underlined); for GAPDH, primer
sense 59-cgctcgaggaccctggaccaccagc-39 (Xho I site underlined) and
antisense 59-cagcggccgcggttgagcacagggtac-39 (Not I site under-
UTR Reporter Assay
Reporter assays were performed using the SBcl2 and M000921
melanoma cell lines. Cells were seeded on 24-well plates (105cells/
well), and 16 h later were transfected using Lipofectamine 2000
(Invitrogen) with 300 ng of empty plasmid or the indicated
reported construct. Four hours after transfection, medium was
changed and cells cultured for 24 h. Afterward cells were cultured
under normoxic (21% O2) or hypoxic (1% O2) conditions in
serum-free medium for additional 24 h. After the treatment, plates
were kept frozen at 280uC until used. Analysis of Luciferase and
Renilla was performed using the Dual Luciferase Reporter System
(Promega) and a Lumat LB9507 luminometer (Berthold Technol-
ogies, Bad Wildbad, Germany). The Renilla activity was then
normalized to Luciferase activity (constitutive expression). The
results are average and standard deviation (SD) of the values
obtained from two independent experiments in each melanoma
Statistical significance was assessed by two-tailed unpaired
Student’s t-test using GraphPad Instat (GraphPad Software, San
Diego, CA, USA). P-values,0.05 were considered as significant.
concentrations in melanoma cells. Western blot analysis of
extracellular PEDF (PEDFe) protein levels in conditioned medium
(CM) and HIF1a protein levels in whole-cell extracts from
M000921 melanoma cell line incubated in normoxia (21% O2),
PEDF downregulation by different oxygen
hypoxia (1% O2) and anoxia (0% O2) for 16 h and 24 h. b-actin
was used as loading control.
cells in the absence or presence of growth factor. (A)
Western blot analysis of extracellular PEDF (PEDFe) protein levels
in conditioned medium (CM) from WM164 melanoma cell line. (B)
Western blot analysis of intracellular PEDF (PEDFi) and HIF1a
protein levels in whole-cell extracts from WM164 melanoma cell
line. Cells were grown in basal medium with or without fetal bovine
serum (2% FBS or 0% FBS, respectively); or without FBS in the
presence of basic fibroblast growth factor (bFGF) and epidermal
growth factor (EGF) under normoxia (21% O2) or hypoxia (1% O2)
for 24 h. b-tubulin was used as loading control.
Hypoxia downregulates PEDF in melanoma
of primary human melanocytes and SBcl2 melanoma
cells. 5-ethynyl-2-deoxyuridine (EdU) incorporation of NHEM
primary human melanocytes and SBcl2 melanoma cell grown in
normoxic (21% O2) and hypoxic (1% O2) conditions. Cells were
grown in normoxic or hypoxic conditions for 16 h and 24 h
incubated in the presence of 20 mM EdU during the last 4 h. Bars
represent average 6 standard deviation (SD).
Hypoxia does not change DNA synthesis rate
PEDF levels under hypoxia in M000921 melanoma. (A)
Western blot analysis of extracellular PEDF (PEDFe) protein levels
in conditioned medium (CM), intracellular PEDF (PEDFi) and
HIF1a protein levels in whole-cell extracts from M000921-pCEP4
and M000921-pCEP4-PEDF melanoma cell line incubated under
normoxia (21% O2) or hypoxia (1% O2) for 24 h. b-tubulin was
used as loading control. (B) Western blot analysis of PEDFeprotein
levels in 24 h CM and HIF1a protein levels in whole-cell extracts
from M000921 melanoma cell line incubated under hypoxia. b-
tubulin was used as loading control. (C) UTR-reporter assay in
M000921 melanoma cell line transfected with psiCHECK2-
39PEDF, psiCHECK2-39GAPDH or empty vector psiCHECK2.
After transfection, cells were incubated in hypoxia for 24 h. Renilla
activity was normalized to luciferase activity expressed from internal
control. psiCHECK2-39GAPDH was used as a negative control.
Bars represent average 6 standard deviation (SD).
UTRs are not required for downregulation of
SBcl2 melanoma cells. Quantification of SBcl2-GFP-LC3 with
autophagic vacuoles after treatment of different times of hypoxia
(1% O2) (4 h, 8 h, 16 h, 24 h and 48 h). Ten fields from each
condition were assessed. Bars represent average 6 standard
deviation (SD) (*P,0.05; **P,0.01).
Hypoxia induces the autophagic phenotype in
The authors acknowledge with gratitude Dr. M. Gorospe and Dr. I. Lopez
de Silanes for their advice in UTR reporter assay and bioinformatic search
of RBP binding sites on PEDF’s UTRs and Dr. P. Boya and Dr. M.
Soengas for their advice on autophagy.
Conceived and designed the experiments: AFB JLO BJ. Performed the
experiments: AFB JLO. Analyzed the data: AFB JLO BJ. Contributed
reagents/materials/analysis tools: LdP MJC. Wrote the paper: BJ AFB.
Cloned the UTR reporters and performed UTR-reporter assays: VG.
Contributed with the in silico search of HRE sites in SERPINF1: LP LdP.
Hypoxia Downregulates PEDF in Melanoma
PLoS ONE | www.plosone.org12March 2012 | Volume 7 | Issue 3 | e32989
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