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Antagonistic Functions of Tetradecanoyl Phorbol
Acetate-Inducible-Sequence 11b and HuR in the
Hormonal Regulation of Vascular Endothelial
Growth Factor Messenger Ribonucleic Acid Stability
by Adrenocorticotropin
Nadia Cherradi, Cyrille Lejczak, Agnes Desroches-Castan, and Jean-Jacques Feige
Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM), Equipe Mixte 01-05; and
Commissariat a` l’Energie Atomique (CEA), Department of Cellular Responses and Dynamics,
F-38054 Grenoble, France
Expression of vascular endothelial growth factor
(VEGF), an endothelial cell-specific mitogen and a
potent angiogenic factor, is up-regulated by a va-
riety of factors including hypoxia, growth factors,
and hormones. In the adrenal cortex, regulation of
VEGF expression by the pituitary hormone ACTH
ensures the maintenance of the organ vasculature.
We have previously shown that ACTH evokes a
rapid and transient increase in VEGF mRNA levels
in primary adrenocortical cells through transcrip-
tion-independent mechanisms. We further demon-
strated that the zinc finger RNA-binding protein
Tis11b (tetradecanoyl phorbol acetate-inducible-
sequence 11b) destabilizes VEGF mRNA through
its 3ⴕ-untranslated region (3ⴕ-UTR) and that Tis11b
is involved in the decay phase of ACTH-induced
VEGF mRNA expression. In the present study, we
attempted to determine the mechanisms underly-
ing ACTH-elicited increase in VEGF mRNA levels in
adrenocortical cells. We show that ACTH triggers
an increase in the levels of the mRNA-stabilizing
protein HuR in the cytoplasm and a concomitant
decrease in the levels of HuR in the nucleus. This
process is accompanied by an increased associa-
tion of HuR with the nucleocytoplasmic shuttling
protein pp32, indicating that ACTH induces HuR
translocation from the nuclear to the cytoplasmic
compartment. Leptomycin B, a specific inhibitor of
CRM1-dependent nuclear export of pp32, signifi-
cantly reduced ACTH-induced VEGF mRNA levels.
Furthermore, RNA interference-mediated deple-
tion of HuR in adrenocortical cells abrogated
ACTH-induced VEGF mRNA expression. Finally, we
show that Tis11b and HuR exert antagonistic ef-
fects on VEGF 3ⴕ-UTR in vitro. Although both pro-
teins could bind simultaneously on VEGF 3ⴕ-UTR,
Tis11b markedly decreases HuR-binding to this
RNA sequence. Altogether, these results suggest
that the RNA-stabilizing protein HuR is instrumen-
tal to ACTH-induced expression of VEGF mRNA
and that the nuclear export of HuR is a rate-limiting
step in this process. HuR appears to transiently
stabilize VEGF transcripts after ACTH stimulation
of adrenocortical cells, and Tis11b appears to sub-
sequently trigger their degradation. (Molecular En-
docrinology 20: 916–930, 2006)
V
ASCULAR ENDOTHELIAL GROWTH factor
(VEGF) is the major angiogenic cytokine contrib-
uting to the regulation of physiological and patholog-
ical angiogenesis (1, 2). Its expression levels in mam-
malian cells are very tightly regulated. In fact, a 50%
reduction of VEGF expression during embryogenesis,
as observed in VEGF ⫹/⫺ heterozygous mice, results
in lethality (3, 4). Reciprocally, overexpression of VEGF
in adult tissues leads to the formation of angiomas and
perturbs organ development (5). VEGF expression is
regulated by a number of environmental factors in-
cluding hypoxia, growth factors, and several hor-
mones (1, 6). With most of these factors, different
levels of regulation are observed. First, VEGF tran-
scription is activated by the binding of a number of
transcription factors onto specific response elements
located in the promoter of the VEGF gene. These
include HIF-1
␣
and AP-1 for the hypoxic response (7,
8) and Sp1 and AP-2 for the growth factor response (9,
10). Second, VEGF mRNA stability is regulated in re-
sponse to some of these effectors through the binding
First Published Online November 23, 2005
Abbreviations: ARE, AU-rich element; BAC cell, bovine
adrenocortical cell; CRM1, chromosomal region mainte-
nance 1; ELAV, embryonic-lethal abnormal visual; GST, glu-
tathione-S-transferase; HPRT, hypoxanthine phosphoribosyl
transferase; HRP, horseradish peroxidase; LMB, leptomycin
B; NES, nuclear export signal; PVDF, polyvinylidine difluoride;
RNAi, RNA interference; RRM, RNA recognition motif; SDS,
sodium dodecyl sulfate; siRNA, short interfering RNA; SSC,
saline sodium citrate; TBE, Tis11b-binding element; Tis11b,
tetradecanoyl phorbol acetate-inducible-sequence 11b; TK,
thymidine kinase; 3⬘-UTR, 3⬘-untranslated region; VEGF, vas-
cular endothelial growth factor.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
0888-8809/06/$15.00/0 Molecular Endocrinology 20(4):916–930
Printed in U.S.A. Copyright © 2006 by The Endocrine Society
doi: 10.1210/me.2005-0121
916
of stabilizing and destabilizing proteins to AU-rich el-
ements (AREs) located in the 3⬘-untranslated region
(3⬘-UTR) of VEGF mRNA (11–14). Third, translation of
VEGF mRNA into protein is also a controlled mecha-
nism implying several alternative initiation codons (15).
The choice of the preferential initiation codon is mod-
ified under hypoxic conditions (16) or under oncogenic
transformation (17). These different levels of regulation
may cooperate to amplify the effects of such or such
regulator. For example, hypoxia-induced stimulation
of VEGF expression results from both HIF-1
␣
-medi-
ated stimulation of transcription (7) and HuR-mediated
stabilization of VEGF mRNA (11).
HuR is a ubiquitously expressed member of the
ELAV (embryonic-lethal abnormal visual in Drosophila
melanogaster) family of RNA binding proteins, which
also comprises the neuron-specific proteins HuD,
HuC, and Hel-N1 (18, 19). The protein products of all
four genes bind with high affinity and specificity to
AU-rich elements (AREs) in a variety of mRNAs, such
as those encoding VEGF, c-fos,c-jun, IL-3, TNF
␣
, and
granulocyte macrophage colony-stimulating factor,
and are believed to increase mRNA stability, mRNA
translation, or both (20, 21). Although the precise
mechanisms regulating HuR function in mRNA stabi-
lization are poorly understood, increasing evidence
indicates that HuR function is intimately linked to its
subcellular localization (22–24). Indeed, HuR contains
three classical RNA-binding domains [RNA recogni-
tion motifs (RRM)]: the first two (RRM1 and RRM2)
have been implicated in ARE recognition whereas the
third (RRM3) has been suggested to bind the poly A
tail of target mRNAs (25). The region located between
RRM2 and RRM3, known as the hinge region, is es-
sential for HuR subcellular localization. This region
contains a shuttling domain, HNS (for HuR nucleocy-
toplasmic sequence) that is involved in the interaction
of HuR with the acidic phosphoproteins ligands, pp32,
SET
␣
/

, and APRIL (26). Like HuR, these proteins are
primarily nucleoplasmic but shuttle between the nu-
cleus and the cytoplasm. pp32 and APRIL contain
domains homologous to nuclear export signals (NES)
known to interact with CRM1 (chromosomal region
maintenance 1), the nuclear export receptor for the
HIV-1 Rev protein. It has been shown recently that
pp32 and APRIL mediate CRM1-dependent export of
HuR in response to heat shock (27).
We have previously reported that ACTH rapidly and
transiently stimulates VEGF expression by primary cul-
tures of bovine adrenocortical (BAC) cells (28). Inter-
estingly, we observed that this increase in VEGF
mRNA was transcription independent because it was
still observed in the presence of 5,6-dichloro-1-b-
ribofuranosylbenzimidazole (DRB), a potent transcrip-
tion inhibitor (28). We then characterized Tis11b (tet-
radecanoyl phorbol acetate-inducible-sequence 11b)
as a zinc finger RNA-destabilizing protein the synthe-
sis of which is also induced by ACTH, although slightly
later than that of VEGF (29). We hypothesized that
Tis11b might play a role in the control of VEGF mRNA
stability. Indeed, we recently characterized the inter-
action between Tis11b and VEGF mRNA 3⬘-UTR and
identified a 75-bp sequence in this 3⬘-UTR, containing
two AU-rich elements, as the Tis11b binding-element
(TBE) (30). We could show that Tis11b binding to TBE
induces destabilization of VEGF mRNA, resulting in a
reduction of VEGF mRNA half-life from 130 min down
to 60 min. In the present work, we attempted to iden-
tify the mechanism by which ACTH induces VEGF
mRNA. We identified HuR as a potential regulator and
could establish that ACTH induces a rapid increase of
HuR levels in the cytoplasm and a concomitant de-
crease of HuR levels in the nucleus. Moreover, coim-
munoprecipitations experiments revealed that ACTH
induces a molecular complex between the nucleocy-
toplasmic shuttling protein pp32 and HuR in the cyto-
plasm, suggesting that pp32 is involved in HuR accu-
mulation in this compartment. Finally, as the Tis11b-
and HuR-binding sites are quite close to each other on
the VEGF mRNA 3⬘-UTR, we wondered whether these
two proteins might antagonize each other in the con-
trol of VEGF mRNA stability.
RESULTS
Regulation of VEGF, Tis11b, and HuR mRNAs
Expression by ACTH
We have previously shown that ACTH rapidly and
transiently induces the expression of VEGF in primary
cultures of adrenocortical cells (28). Moreover, we re-
cently observed that Tis11b, through its mRNA-desta-
bilizing activity, is involved in the decay phase of
ACTH-elicited VEGF mRNA levels (30). However, the
mechanisms involved in the induction phase of VEGF
mRNA remained to be determined. As this induction
was still observed in the presence of the transcription
inhibitor 5,6-dichloro-1-b-ribofuranosylbenzimidazole
(DRB) (28), we postulated that stabilization of VEGF
mRNA might be involved in this process. Because the
ELAV family members HuC, HuD, and Hel-N1 were
reported as neural-specific RNA-stabilizing proteins,
we focused on HuR, the ubiquitously expressed mem-
ber of ELAV proteins. We first examined the possible
involvement of HuR in ACTH-induced increase of
VEGF mRNA. Figure 1A illustrates an RT-PCR analysis
of VEGF, Tis11b, and HuR mRNA levels in adrenocor-
tical cells stimulated with 10 n
M ACTH for various
periods of time ranging from 0–5 h. As previously
reported (29), VEGF mRNA levels peaked after 2–3 h,
whereas the hormone induced the expression of
Tis11b between 3 and5hoftreatment (Fig. 1, A and
B). In contrast, HuR mRNA levels were unchanged
over the period of ACTH stimulation (Fig. 1, A and B).
Northern blot analysis of HuR mRNA levels in ACTH-
treated cells confirmed that they were very stable for
up to 24 h of hormone treatment (maximal value reach-
ing 137 ⫾ 40% of mRNA levels measured in non-
treated cells, n ⫽ 3, data not shown).
Cherradi et al. • ACTH-Induced VEGF Expression Mol Endocrinol, April 2006, 20 (4):916–930 917
ACTH Does Not Affect Total HuR Protein Content
of BAC Cells
The above results prompted us to examine whether
ACTH could exert a posttranscriptional effect by act-
ing on HuR mRNA translation. As shown in Fig. 2, A
and B, after exposure of BAC cells to 10 n
M ACTH, no
significant changes were observed in HuR protein
content of whole-cell extracts. In contrast, ACTH in-
duced a progressive increase in Tis11b protein con-
tent (Fig. 2, A and B). This increase was apparent as
early as 1 h after stimulation and peaked after 5–6 h.
Tis11b protein expression remained slightly elevated
24 h after ACTH treatment (data not shown).
ACTH Increases Cytoplasmic Levels of HuR
HuR appears predominantly located in the nucleus
within all cell types examined so far (24). Moreover,
HuR-induced stabilization of its mRNA targets is as-
sociated with an increase in cytoplasmic levels of HuR
(24). To assess the subcellular distribution of HuR in
BAC cells as well as the potential effect of ACTH on
HuR localization, BAC cells were incubated with 10 n
M
ACTH for various periods of time ranging from 0–6 h,
and then subfractionated into nuclear and cytoplasmic
fractions. As shown in Fig. 2C, HuR was weakly
present in the cytoplasm (20
g of cytoplasmic ex-
tracts) but was abundant in the nucleus (5
gofnu-
clear extracts) of unstimulated cells. Quantitation of
cytoplasmic and nuclear HuR revealed that ACTH
treatment led to a 2- to 3-fold increase of cytoplasmic
HuR as early as 30 min after stimulation and a con-
comitant decrease in nuclear HuR (Fig. 2D). Cytoplas-
mic HuR levels remained elevated for up to3hand
then decreased by4hofstimulation. In unstimulated
cells, HuR was estimated to be about 5- to 6-fold more
abundant in the nucleus. Hybridization of the same
membranes with antibodies recognizing cytoplasm-
and nucleus-specific proteins (
␣
-tubulin and lamin
A/C, respectively) allowed us to verify that nuclear
proteins did not leak into the cytoplasmic fractions
during cell fractionation (Fig. 2C). To determine the
subcellular localization of Tis11b in BAC cells, the
same blots were probed with affinity-purified poly-
clonal anti-Tis11b antibodies (30). As shown on Fig.
2C, Tis11b was exclusively detected in the cytoplas-
mic fractions. Upon prolonged exposure of the mem-
brane (15 min), a very weak Tis11b signal could be
detected in the nuclear fractions (data not shown).
Exposure of BAC cells to ACTH induced a robust
increase in Tis11b protein levels in the cytoplasm (Fig.
2C), which was correlated to the induction of Tis11b
that we observed in whole-cell extracts (Fig. 2A).
Altogether, these results indicate that ACTH treat-
ment of BAC cells leads to a rapid increase of HuR
protein content in the cytoplasm with a concomitant
decrease of HuR protein content in the nucleus and to
a delayed increase of Tis11b protein in the cytoplasm.
HuR Interacts with the Nucleocytoplasmic
Shuttling Protein pp32 in BAC Cells
In light of the observation that increases in cytoplas-
mic HuR are largely due to the CRM1-dependent ex-
port of nuclear HuR, which involves complexes be-
tween HuR and the nucleocytoplasmic shuttling
proteins pp32 and/or APRIL (26, 27), we further tested
whether these proteins might contribute to ACTH-in-
duced accumulation of HuR in the cytoplasm using
coimmunoprecipitation experiments. Because we could
Fig. 1. Effect of ACTH on Expression of VEGF, Tis11b, and
HuR mRNAs
A, Representative ethidium bromide staining of VEGF,
Tis11b, and HuR mRNAs amplified by RT-PCR. Primary cul-
tures of BAC cells were treated with 10 nM ACTH for the
indicated periods of time. VEGF, Tis11b, and HuR mRNA
levels were then analyzed by RT-PCR as described in Mate-
rials and Methods. B, Quantitation of VEGF, Tis11b, and HuR
mRNA levels of independent experiments (n ⫽ 3–5). mRNA
level values were normalized to HPRT mRNA levels and are
expressed as fold induction over control values at time 0
(unstimulated cells).
918 Mol Endocrinol, April 2006, 20(4):916–930 Cherradi et al. • ACTH-Induced VEGF Expression
not detect APRIL in whole BAC cell extracts (data not
shown), we focused on pp32. Cytoplasmic and nuclear
BAC extracts were exposed to anti-pp32 antibody.
Probing the immunoprecipitates with anti-HuR antibody
revealed that pp32 is associated with HuR in the cyto-
plasm and that ACTH treatment increases this associa-
tion (Fig. 3, A and B). The same blot was probed with
anti-pp32 antibody to check for pp32 immunoprecipita-
tion. In the nucleus, HuR-pp32 complexes are less abun-
dant as compared with the cytoplasm and are not sig-
nificantly altered by hormone treatment (Fig. 3, A and B).
These results suggest that ACTH-induced increase in
cytoplasmic HuR levels involves pp32-mediated HuR
delocalization from the nucleus to the cytoplasm of BAC
cells.
Leptomycin B (LMB) Prevents ACTH-Induced
Expression of VEGF mRNA
CRM1-dependent nucleocytoplasmic shuttling of
NES-containing proteins such as pp32 has been
shown to be impaired by LMB, a compound that co-
valently modifies a critical cystein residue in CRM1
and thereby prevents CRM1-pp32 interaction via NES
(31). Moreover, CRM1 has been shown to be instru-
mental in the nuclear export of HuR (26, 27). Our
observation that ACTH treatment stimulates the for-
mation of HuR-pp32 complexes in the cytoplasm
prompted us to examine the effect of LMB on ACTH-
induced increase in VEGF mRNA levels. BAC cells
were stimulated with ACTH in the absence or in the
Fig. 2. Effect of ACTH on Tis11b and HuR Protein Levels in Total Cell Extracts and Subcellular Fractions
A, BAC cells were treated with 10 n
M ACTH for the indicated periods of time. Tis11b and HuR protein levels of whole-cell
extracts (10
g) were analyzed by Western blot as outlined in Materials and Methods. The Western blot was subsequently probed
with an anti-
␣
-tubulin monoclonal antibody to assess equal loading of samples. B, Quantitation of HuR and Tis11b protein levels
of total cell extracts in three independent experiments. Protein level values were normalized to
␣
-tubulin protein levels. C, BAC
cells were treated with 10 nM ACTH as indicated in panel A. Nuclear (5
g) and cytoplasmic (20
g) fractions were prepared as
described in Materials and Methods and subjected to Western blot analysis to monitor Tis11b and HuR expression. The same
membranes were sequentially probed with antibodies recognizing cytoplasm- and nucleus-specific proteins (
␣
-tubulin and lamin
A/C, respectively) to assess the quality of the fractionation process and to check for equal protein loading. D, Cytoplasmic and
nuclear HuR protein levels were normalized to
␣
-tubulin and lamin protein levels, respectively, and are expressed as a fraction
of total cytoplasmic or total nuclear protein content (n ⫽ 2).
Cherradi et al. • ACTH-Induced VEGF Expression Mol Endocrinol, April 2006, 20 (4):916–930 919
presence of LMB. As shown in Fig. 4A, the hormone-
induced increase of HuR in the cytoplasm was abol-
ished at all time points of stimulation in the presence of
LMB (5 ng/ml).
RT-PCR analysis of VEGF mRNA levels revealed
that cotreatment of BAC cells with ACTH and LMB
prevented the increase in VEGF mRNA expression
elicited by ACTH (Fig. 4B). Quantitation of two inde-
pendent experiments showed that after2hofACTH
treatment, VEGF mRNA levels were induced up to
290 ⫾ 60% of control levels and that, in the presence
of LMB, maximal induction reached only 147 ⫾ 60% of
control levels. LMB also had a discrete effect by itself
on the basal expression of VEGF mRNA.
HuR Knockdown Inhibits ACTH-Induced Increase
in VEGF mRNA Levels
To provide further evidence of the involvement of HuR
in the regulation of VEGF mRNA expression by ACTH,
we used RNA interference (RNAi) to more selectively
target HuR. Using a specific short interfering RNA
(siRNA) targeting exon 2 in HuR gene, we showed by
both RT-PCR and Western blotting that this siRNA
was effective in decreasing HuR mRNA and protein
levels in BAC cells after 48 h of treatment (Fig. 5, A and
B, time point 0). Quantitation of HuR mRNA levels in
three independent experiments revealed that HuR
gene expression was knocked down by 70–85% (data
not shown). In these experiments, HuR protein levels
were barely detectable upon prolonged exposure of
the Western blot (data not shown). BAC cells pre-
treated either in the presence of a negative control
siRNA or in the presence of HuR siRNA were further
stimulated with 10 n
M ACTH, and VEGF mRNA levels
were analyzed by RT-PCR. In this particular experi-
ment, cells pretreated with negative control siRNA
displayed a 2.5-fold increase in VEGF mRNA ex-
pression after4hofexposure to ACTH (Fig. 5, A and
C). In contrast, HuR siRNA impaired ACTH-elicited
up-regulation of VEGF mRNA levels (Fig. 5, A and C).
Moreover, silencing HuR expression led to a sub-
stantial reduction in basal VEGF mRNA expression
down to 39 ⫾ 19% of controls (n ⫽ 3, data not
shown).
Antagonistic Effects of Tis11b and HuR on
VEGF mRNA
A previous study using cotransfections of Tis11b
(pCMV-Tis11b) and firefly luciferase cDNA cloned up-
stream of VEGF mRNA 3⬘-UTR (Luc-V3⬘ construct)
allowed us to demonstrate that VEGF mRNA 3⬘-UTR
confers a Tis11b-mediated decrease in reporter gene
activity, which was closely related to a decrease in
luciferase transcript stability (30). Furthermore, similar
experiments using cotransfections of HuR and Luc-
V3⬘ construct revealed that HuR-induced increase in
luciferase activity reflects an increase in luciferase
transcript stability (32). Because HuR and Tis11b ap-
pear to have antagonistic activity on VEGF mRNA and
because the Tis11b binding site on VEGF mRNA 3⬘-
UTR is very close to a recently described HuR binding
site (32), we investigated whether these two proteins
could compete with each other to regulate VEGF
mRNA expression. We first determined the effect of
expressing each protein alone on luciferase activity.
COS7 cells were transiently transfected with increas-
ing doses of either pCMV-Tis11b or pCMV-HuR plas-
mids and a fixed concentration of Luc-V3⬘. Figure 6A
shows that luciferase activity was decreased in a
dose-dependent manner by Tis11b, a statistically sig-
nificant decrease being observed with a plasmid
amount as low as 0.5 ng (62.5 ⫾ 7.9% of controls; P ⬍
0.01, n ⫽ 3). In contrast, HuR expression led to an
Fig. 3. Coimmunoprecipitation of HuR from BAC Cytoplas-
mic and Nuclear Extracts Using Anti-pp32 Antibodies
A, BAC cells were treated with 10 nM ACTH for the indi-
cated periods of time. Cytoplasmic (250
g protein) and
nuclear fractions (70
g protein) were immunoprecipitated
with antihuman pp32 antibodies as mentioned in Materials
and Methods. Precipitates were electrophoresed on a 12%
denaturing gel, transferred to PVDF membrane, and probed
with HuR or pp32 polyclonal antibodies. B, Quantitation of
HuR levels in cytoplasmic and nuclear extracts. HuR protein
levels were normalized to the IgG light chain bands.
920 Mol Endocrinol, April 2006, 20(4):916–930 Cherradi et al. • ACTH-Induced VEGF Expression
increase in the reporter gene activity over a narrow
window of the cotransfected HuR plasmid (Fig. 6B). A
statistically significant increase of luciferase activity
was observed with 0.1 ng of pCMV-HuR (156.8 ⫾
13.8% of controls; P ⬍ 0.001, n ⫽ 3; Fig. 6A). Exper-
iments were then performed to determine the compet-
itive effects of HuR and Tis11b coexpression on VEGF
mRNA 3⬘-UTR. As shown in Fig. 6C, the luciferase
activity recorded after cotransfection of 1 ng of pCMV-
Tis11b and 0.1 ng of pCMV-HuR was significantly
lower than the luciferase activity obtained with 0.1 ng
of pCMV-HuR alone (81.8 ⫾ 2.3% as compared with
152.2 ⫾ 6.3% of controls, respectively; P ⬍ 0.001, n ⫽
3). This inhibitory effect of Tis11b on HuR-induced
luciferase activity was observed with a concentration
of pCMV-Tis11b as low as 0.2 ng. Conversely, the
luciferase activity measured after cotransfection of 1
ng of pCMV-Tis11b and 0.1 ng of pCMV-HuR was
significantly higher than the one measured with
pCMV-Tis11b alone (81.8 ⫾ 2.3% as compared with
48.6 ⫾ 2.1% of controls, respectively; P ⬍ 0.01, n ⫽
3). Western blot analysis of cell extracts from these
transfection experiments revealed that COS7 cells
constitutively express HuR protein but not Tis11b pro-
tein (Fig. 6C, lower panel). As expected, transfection of
either 0.05 ng or 0.1 ng of pCMV-HuR plasmid led to
an increased expression of HuR. By contrast, Tis11b
expression was barely detectable with 0.2 ng of
pCMV-Tis11b plasmid but clearly detected after trans-
fection of 1 ng of plasmid.
We next investigated the effect of HuR and Tis11b
coexpression on Luc-V3⬘ mRNA levels (Fig. 6D).
Fig. 4. Effect of LMB on ACTH-Induced Increase in VEGF mRNA Levels
A, BAC cells were treated with 10 nM ACTH for the indicated periods of time, in the presence or in the absence of LMB (5 ng/ml).
When used, LMB was added 15 min before ACTH treatment. Cytoplasmic (30
g) and nuclear fractions (5
g) were subjected
to Western blot analysis of HuR. B, Representative ethidium bromide staining of VEGF mRNA levels amplified by RT-PCR in BAC
cells stimulated with ACTH in the presence or in the absence of LMB (5 ng/ml). C, Quantitation of VEGF mRNA levels in BAC cells
stimulated with ACTH in the presence or in the absence of LMB, expressed as fold induction of mRNA levels at time 0
(unstimulated cells). Each point is the mean value from two separate experiments.
Cherradi et al. • ACTH-Induced VEGF Expression Mol Endocrinol, April 2006, 20 (4):916–930 921
Northern blot analysis of COS7 total RNA revealed that
transfection of 0.1 ng of pCMV-HuR increased Luc-
V3⬘ mRNA levels similarly to the reporter gene activity.
Cotransfection of 0.2 and 1 ng of pCMV-Tis11b with
0.1 ng of pCMV-HuR abolished HuR-induced increase
in Luc-V3⬘ mRNA. Finally, Luc-V3⬘ mRNA level was
decreased in a dose-dependent manner by Tis11b.
Altogether, these results suggest that Tis11b and HuR
could antagonize each other to regulate VEGF mRNA
expression through its 3⬘-UTR.
Tis11b and HuR Compete for Binding to the
3ⴕ-UTR of VEGF mRNA
Because Tis11b and HuR appeared to antagonize
each other in the regulation of VEGF mRNA expres-
sion, experiments were performed to determine
whether these proteins could compete with each other
for the binding to VEGF 3⬘-UTR. Tis11b-expressing
bacterial extract was mixed with increasing amounts
of purified glutathione-S-transferase (GST)-HuR fusion
protein and purified GST-HuR was mixed with increas-
ing amounts of Tis11b-expressing bacterial extract.
These mixtures were exposed to UV light in cross-
linking assays with VEGF 3⬘-UTR RNA probe. As
shown in Fig. 7B, a covalent ribonucleoprotein com-
plex with an apparent molecular mass of 38 kDa was
detected when Tis11b-expressing bacterial extract
was incubated with VEGF 3⬘-UTR (lane 2). This com-
plex was not observed in the presence of control bac-
terial extracts (lane 1). On the other hand, a covalent
ribonucleoprotein complex with an apparent molecu-
Fig. 5. Effect of HuR Repression by RNAi on ACTH-Induced Increase in VEGF mRNA Levels
A, BAC cells were transfected either with HuR-specific siRNA or a negative control siRNA as described in Materials and
Methods. Culture medium was changed 48 h later, and cells were treated for the indicated periods of time with or without 10 nM
ACTH. At each time point of stimulation, total RNA was isolated and RT-PCR analysis was performed to determine HuR, VEGF,
or HPRT mRNA expression levels. B, Western blot analysis of HuR protein levels in whole-cell extracts (10
g), showing that HuR
siRNA was effective in knocking down HuR protein levels. In this particular experiment, blots for HuR were exposed for 2 min.
Despite prolonged exposure of the membrane (15 min), HuR was barely detectable in protein extracts derived from HuR
siRNA-treated cells. C, Quantitation of the RT-PCR experiment represented in panel A, in which HuR repression was evaluated
to 85%. Results obtained from three independent experiments revealed that ACTH-induced increase in VEGF mRNA levels was
altered to a lesser extent when HuR repression was about 70% (data not shown).
922 Mol Endocrinol, April 2006, 20(4):916–930 Cherradi et al. • ACTH-Induced VEGF Expression
Fig. 6. Antagonistic Effects of Tis11b and HuR in the Regulation of VEGF mRNA Stability
COS7 cells were transfected as outlined in Materials and Methods. The pLuc-V3⬘ construct contains the full-length 3⬘-UTR of
the rat VEGF mRNA (2201 bp) (30). A, Dose-dependent effect of Tis11b on pLuc-V3⬘ reporter gene activity. Results are expressed
as relative light units of firefly luciferase activity over relative light units of Renilla luciferase activity. B, Dose-dependent effect of
HuR on pLuc-V3⬘ reporter gene activity. C, Effect of Tis11b and HuR coexpression on pLuc-V3⬘ reporter gene activity. The dose
giving the maximal effect of HuR on luciferase activity (0.1 ng) was used with 0.2 or 1 ng of Tis11b to perform competition studies.
Transfections were performed in triplicate, and values are means ⫾ SE from three independent experiments. ⫹, ⫹⫹⫹, significantly
different from control (0 ng of pCMV-Tis11b or pCMV-HuR) with P ⬍ 0.05 and P ⬍ 0.001, respectively. There was a statistically
significant decrease in luciferase activity for (HuR 0.1 ng ⫹ Tis11b 1 ng) compared with HuR 0.1 ng (***, P ⬍ 0.001), as well as
a statistically significant increase in luciferase activity for (HuR 0.1 ng ⫹ Tis11b 1 ng) compared with Tis11b 1 ng (**, P ⬍ 0.01).
There was a statistically significant decrease in luciferase activity for (HuR 0.1 ng ⫹ Tis11b 0.2 ng) compared with HuR 0.1 ng
(**, P ⬍ 0.01), as well as a statistically significant increase in luciferase activity for (HuR 0.1 ng ⫹ Tis11b 0.2 ng) compared with
Tis11b 0.2 ng (**, P ⬍ 0.05). In the lower panel, COS7 cell extracts (10
g) were immunoblotted using anti-HuR or anti-Tis11b
antibodies to check for HuR and Tis11b protein expression in transfected cells. HuR and Tis11b are indicated with arrows. In this
cell line, Tis11b is undetectable at basal levels. D, Effect of Tis11b and HuR coexpression on pLuc-V3⬘ reporter gene mRNA levels.
COS7 cell total RNA (20
g) was analyzed by Northern blot as indicated in Materials and Methods. Luc, Luciferase.
Cherradi et al. • ACTH-Induced VEGF Expression Mol Endocrinol, April 2006, 20 (4):916–930 923
lar mass of 64 kDa was detected when GST-HuR
fusion protein was incubated with VEGF 3⬘-UTR (lane
6). When 1
g of GST-HuR was added to 2
gof
Tis11b, both complexes of 38 and 64 kDa were de-
tected, indicating that Tis11b and HuR could bind
simultaneously to the VEGF 3⬘-UTR RNA probe (lane
3). Increasing the amount of GST-HuR in the presence
of 2
g of Tis11b resulted in a slight decrease in the
intensity of the Tis11b-RNA complex and a corre-
sponding increase in HuR-RNA complex (lanes 4 and
5). Increasing the amount of Tis11b in the presence of
1
g of HuR resulted in a marked decrease in the
intensity of the HuR-RNA complex (lanes 7–9). Unex-
pectedly, no increase in Tis11b-RNA complex was
observed, suggesting that optimal Tis11b binding to
VEGF 3⬘-UTR requires additional factors, which re-
main to be identified. Indeed, we could observe an
increase of Tis11b binding to VEGF 3⬘-UTR over a
narrow window of Tis11b doses ranging from 1–3
g,
followed by a decrease of Tis11b binding to VEGF
Fig. 7. Binding of HuR and Tis11b to VEGF 3⬘-UTR RNA
A, Restriction map of the 2201-bp long 3⬘-UTR of VEGF mRNA. TBE is located between nucleotides 1161 and 1235 (30). The
40-bp functional HuR binding site is located between nucleotides 1285 and 1325 (32). Flags with white circles represent the
nonameric ARE motifs UUAUUUA(A/U)(A/U), and those with black circles represent the pentameric motif AUUUA. B, VEGF
full-length 3⬘-UTR RNA probe was mixed either with bacterial cell extracts containing Tis11b (2
g) and increasing doses of
purified GST-HuR (0, 1, 2, or 5
g) or purified GST-HuR (1
g) and increasing doses of Tis11b (0, 2, 4, or 6
g). The reaction
mixtures were treated with UV radiation and were analyzed by electrophoresis as outlined in Materials and Methods. The positions
of migration of the HuR and Tis11b RNA-protein complexes are indicated with arrows. ns, Nonspecific band observed with the
control bacterial extract. C, Quantitation of Tis11b binding to VEGF 3⬘-UTR in the presence of increasing doses of HuR (squares)
and of HuR binding to VEGF 3⬘-UTR in the presence of increasing doses of Tis11b (circles) in two to four independent
experiments; 100% represents either the binding of Tis11b in the absence of HuR or the binding of HuR in the absence of Tis11b.
FL, Full length.
924 Mol Endocrinol, April 2006, 20(4):916–930 Cherradi et al. • ACTH-Induced VEGF Expression
3⬘-UTR at higher doses (data not shown). Quantitation
of two to four independent experiments shows that
increasing doses of Tis11b markedly decreased HuR
binding to VEGF 3⬘-UTR (to 23.2 ⫾ 9.5% of HuR
binding in the absence of Tis11b; Fig. 7C, n ⫽ 3).
DISCUSSION
VEGF has been shown in mammals to be a key me-
diator of angiogenesis in such diverse physiological
and pathological processes as embryogenesis, female
estrous cycle, diabetic retinopathy, and tumor devel-
opment (1). The expression of the VEGF gene is con-
trolled at many levels including transcription (10),
mRNA stability through the binding of regulatory pro-
teins to the 3⬘-UTR (33), and mRNA translation via
internal ribosome entry site sequences located in the
5⬘-UTR (15). Posttranscriptional regulation of VEGF
mRNA is a key control point in VEGF expression under
hypoxic conditions. Indeed, hypoxia-mediated in-
crease of VEGF mRNA levels is due, in large part, to an
increase in VEGF mRNA half-life after its stabilization
by the RNA-binding protein HuR (11).
Transcriptional regulation of VEGF expression by
hormones in endocrine tissues, including endome-
trium, ovaries, and adrenal cortex, has been exten-
sively studied (34–39). In contrast, the possible in-
volvement of posttranscriptional regulation in
hormonally regulated expression of VEGF has re-
ceived much less attention to date. We have previ-
ously shown that the trophic hormone ACTH triggers a
rapid and transient increase in VEGF mRNA levels in
adrenocortical cells via transcription-independent
mechanisms (28). We could further demonstrate that
the decay phase of ACTH-induced VEGF mRNA levels
involves the RNA-destabilizing protein Tis11b (30). In
the current study, we aimed at investigating the mo-
lecular mechanisms of ACTH-elicited increase in
VEGF mRNA levels. Four major conclusions can be
drawn from the present work: 1) ACTH induces a rapid
delocalization of the RNA-stabilizing protein HuR from
the nucleus to the cytoplasm in adrenocortical cells
without affecting total cellular HuR mRNA and protein
levels; 2) Blocking nuclear export of NES-bearing pro-
teins by LMB impairs ACTH-induced expression of
VEGF mRNA; 3) Silencing the expression of HuR by
siRNA markedly inhibits ACTH-mediated induction of
VEGF mRNA levels; and 4) Tis11b and HuR exert an
antagonistic action on VEGF mRNA in vitro.
First, our finding that the levels of cytoplasmic and
nuclear HuR are potently influenced by the trophic
hormone ACTH provides a new insight into the regu-
lation of HuR in adrenocortical cells, suggesting a
connection between the ability of HuR to stabilize
ACTH-induced labile mRNAs and the subcellular tar-
geting of these mRNAs. Various other stimuli can shift
the nucleo-cytoplasmic distribution of HuR. Cytoplas-
mic localization of HuR is associated with conditions
of cellular stress including heat shock (40), UV irradi-
ation (41), and amino acid starvation (42), as well as
stimulation with lipopolysaccharide in the case of
macrophages (43). HuR can bind to AU-rich regions in
c-fos mRNA, a CU-rich region in c-jun mRNA, and a
U-rich domain in c-myc and VEGF mRNAs (11, 20, 44,
45), all of these genes being early-response genes to
ACTH in the adrenal cortex (28, 46). Our observation
that ACTH induces an increase in cytoplasmic levels of
HuR suggests that HuR may play a role in stabilizing
these labile mRNAs. Our results showing that 1) HuR
coimmunoprecipitates with the nucleocytoplasmic
shuttling protein pp32 in the cytoplasm of ACTH-stim-
ulated cells and 2) ACTH increases transiently the
association of HuR with pp32 in the cytoplasm argue
that the hormone triggers nuclear export of HuR to the
cytoplasm. Interestingly, there is accumulating evi-
dence that the nuclear-cytoplasmic localization of
HuR is modulated by signal transduction pathways
(47–51). ACTH is known to increase intracellular cAMP
levels in cultured adrenocortical cells. We have previ-
ously shown that forskolin, an activator of adenylate
cyclase, was as potent as ACTH in increasing VEGF
mRNA levels (28). In the present study, stimulation of
BAC cells with ACTH in the presence of H89 (10
M),
a potent and specific inhibitor of protein kinase A,
completely abolished the hormone-induced increase
of HuR in the cytoplasm (data not shown). Moreover,
stimulation of BAC cells with forskolin (10
M) led to a
substantial increase of HuR levels in the cytoplasm
[ⱕ165% of HuR protein content of nonstimulated cells
after 1–2 h of stimulation (data not shown)]. These
observations suggest that cAMP and protein kinase A
are involved in the hormone-induced delocalization of
HuR to the cytoplasm. The potential targets of protein
kinase A, however, remain to be determined in future
studies. Because pp32 is a phosphoprotein (26), it is
tempting to speculate that pp32 shuttling or its inter-
action with HuR could be regulated by phosphoryla-
tion. This hypothesis is worth testing because we
failed to identify phosphorylated forms of HuR in re-
sponse to ACTH (data not shown).
Second, we observed that LMB, a specific inhibitor
of the nuclear export receptor CRM1, significantly im-
paired ACTH-induced increase in VEGF mRNA. Al-
though it is possible that LMB inhibits other compo-
nents of the nucleocytoplasmic trafficking machinery,
we hypothesize that ACTH-induced association of
pp32 and HuR most likely confers CRM1-dependent
export on HuR. Therefore, disruption of the nuclear
export of HuR protein partners by LMB might lead to
retention of HuR in the nucleus and thereby might
impair ACTH-induced increase in VEGF mRNA levels.
Indeed, we observed that LMB prevented the ACTH-
mediated increase of HuR in the cytoplasm of BAC
cells as well as the hormone-induced increase in VEGF
mRNA levels, thus indicating that HuR export to the
cytoplasm is instrumental in ACTH-induced VEGF
mRNA.
Cherradi et al. • ACTH-Induced VEGF Expression Mol Endocrinol, April 2006, 20 (4):916–930 925
Because BAC cells already have a high level of HuR,
defining the functions of HuR in the cellular response
to ACTH using the overexpression approach could
lead to effects that might not reflect the true function
of the endogenous protein. To clearly establish the
role of HuR in ACTH-induced VEGF mRNA, we dis-
rupted the expression of endogenous HuR in BAC
cells using RNAi. Interestingly, RNAi-mediated deple-
tion of HuR leads to a complete inhibition of hormone-
induced expression of VEGF, indicating that HuR is a
limiting factor in the induction phase of VEGF mRNAs
by ACTH. HuR expression was significantly reduced
(by 80–85%) 48 h before ACTH treatment. In our RNAi
experiments in which HuR expression was reduced by
only 70%, inhibition of ACTH-elicited VEGF mRNA
levels was not complete (data not shown), suggesting
that low levels of HuR were sufficient to stabilize VEGF
mRNA. These results, together with those we reported
on the involvement of the mRNA-destabilizing protein
Tis11b in the decay phase of ACTH-elicited VEGF
mRNA levels, indicate that ACTH controls VEGF ex-
pression in adrenocortical cells mainly by posttran-
scriptional mechanisms. The pivotal role of HuR in the
stabilization of VEGF mRNA by hypoxia has been ex-
tensively studied (52). To our knowledge, this work is
the first to report a hormonal up-regulation of VEGF
mRNA expression that is mediated by the RNA-stabi-
lizing protein HuR.
Fourth, concomitant overexpression of Tis11b and
HuR revealed that Tis11b completely abrogated HuR-
induced luciferase activity on a heterologous tran-
script consisting of luciferase cDNA cloned upstream
of VEGF mRNA 3⬘-UTR (Luc-V3⬘). Our results showing
that HuR increases luciferase activity derived from
Luc-V3⬘ in vitro are similar to those reported by Gold-
berg-Cohen et al. (32). Using a similar reporter con-
struct, these authors have shown further that the in-
crease in reporter activity was related to HuR binding
to a 40-bp RNA element (nucleotides 1285–1325)
within VEGF mRNA 3⬘-UTR, which confers increased
stability to the heterologous transcript. Tis11b and
HuR bind to distinct but very close RNA elements on
VEGF mRNA 3⬘-UTR [nucleotides 1161–1235 (30) and
1285–1325 (32) for Tis11b and HuR, respectively]. Our
UV cross-linking experiments indicate that Tis11b and
HuR can simultaneously, as well as individually, bind
to VEGF 3⬘-UTR RNA probe. However, Tis11b potently
prevents HuR binding to VEGF 3⬘-UTR, a finding that
is consistent with the inhibitory effect of Tis11b on
HuR-mediated increase in reporter gene activity and
mRNA level (cotransfection experiments; Fig. 6, C and
D). For as yet unidentified reasons, the decrease in
HuR binding to VEGF 3⬘-UTR in the presence of in-
creasing doses of Tis11b was not paralleled by an
increase in Tis11b binding. Similar results showing
that HuR and tristetraprolin (TTP, also named Tis11),
the most studied member of the Tis11 protein family,
can bind simultaneously and competitively to granu-
locyte macrophage colony-stimulating factor 3⬘-UTR
have been reported by Raghavan et al. (53). More
recently, Lal et al. (54) provided evidence that HuR and
the mRNA-destabilizing factor AUF1 can bind target
transcripts on both distinct, nonoverlapping sites, and
on common sites in a competitive fashion. They pro-
pose that the fate of the mRNA target depends on HuR
and AUF1 abundance, the target RNA sequence, and
the subcellular compartment investigated. Experi-
ments aiming at dissecting the molecular mechanisms
governing the binding of Tis11b and HuR to specific or
common sites on VEGF 3⬘-UTR are under way.
At the functional level, after stimulation of adreno-
cortical cells by ACTH, HuR, which predominates in
the nucleus, may shuttle to the cytoplasm where it may
stabilize VEGF mRNA, allowing it to be translated.
Subsequently, Tis11b expression is induced and the
cytoplasmic level of Tis11b increases. The relative lev-
els and binding affinities of HuR and Tis11b for their
specific sequences in VEGF mRNA 3⬘-UTR may de-
termine the fate of VEGF transcripts, with HuR pre-
dominance promoting VEGF mRNA stabilization and
Tis11b predominance promoting VEGF mRNA degra-
dation. At some point (between 4 and6hofstimula-
tion by ACTH, according to our data), Tis11b may
predominate and facilitate VEGF mRNA degradation.
This model provides a mechanism by which VEGF
gene could be transiently expressed in BAC cells.
In conclusion, this work reports for the first time that
ACTH triggers a rapid nuclear export of HuR into the
cytoplasm of adrenocortical cells followed by an in-
duction of Tis11b protein synthesis and cytoplasmic
accumulation, two processes that appear to be re-
sponsible for the transient stimulation of VEGF mRNA
and protein accumulation. In an in vivo model of ad-
renal cortex tissue regression triggered by the sup-
pression of pituitary ACTH secretion, we recently ob-
served that adrenocortical VEGF expression is
dramatically reduced during this process, resulting in
massive disorganization and regression of the capil-
lary network (55). It will therefore be of great interest to
establish the respective contributions of HuR and
Tis11b to the in vivo regulation of adrenal vasculature
by ACTH in future studies.
MATERIALS AND METHODS
Reagents
Synthetic ACTH
1–24
was purchased from Neosystem (Stras
-
bourg, France). Culture media and sera were from Invitrogen
(Cergy Pontoise, France). All chemicals were obtained from
Sigma Chemical Co. (St. Louis, MO) and were of the highest
purity grade available.
BAC Cell Culture and Treatments
Bovine adrenal glands were obtained from a local slaughter-
house. Zona fasciculata-reticularis cells were prepared by
enzymatic dispersion with trypsin, and primary cultures were
established as described in detail elsewhere (56). BAC cells
were kept at 37 C in Ham’s F12 medium supplemented with
926 Mol Endocrinol, April 2006, 20(4):916–930 Cherradi et al. • ACTH-Induced VEGF Expression
10% horse serum, 2.5% fetal calf serum, 100 U/ml penicillin,
100
g/ml streptomycin, 20
g/ml gentamycin, under 5%
CO
2
-95% air atmosphere. On d 4, cells cultured in 10-cm
petri dishes (3 ⫻ 10
6
cells per dish) were stimulated with 10
n
M ACTH for the indicated periods of time before subcellular
fractionation or processing for total RNA isolation as de-
scribed hereafter. In experiments designed to silence HuR
expression (RNAi), BAC cells were transfected ond2of
culture.
Preparation of Subcellular Fractions
Total cell extracts, as well as nuclear and cytoplasmic frac-
tions, were prepared using the Protein and RNA Isolation
System (Ambion, Inc., Austin, TX) according to the manufac-
turer’s instructions. A protease inhibitor cocktail [10
g/ml of
leupeptin, 1
g/ml of aprotinin, 1
g/ml of pepstatin, and 25
g/ml of 4-(2-aminoethyl-benzenesulfonyl fluoride] was
added to the provided buffers. Briefly, 10-cm petri dishes
(3 ⫻ 10
6
BAC cells per dish) were lysed on ice in 400
l of Cell
Disruption buffer and collected with a rubber spatula. A por-
tion of the total cell lysate was used for RNA isolation (300
l),
and the remainder was kept for protein analysis (100
l).
For nuclear and cytoplasmic lysate preparation, BAC cells
(3 ⫻ 10
6
cells per 10-cm petri dish) were trypsinized, washed
in PBS, and then suspended in 400
l of Cell Fractionation
Buffer. After a 15-min incubation on ice, nuclear and cyto-
plasmic fractions were separated by centrifugation (5 min at
500 ⫻ g). The nuclear pellet was lysed in 400
l of Cell
Disruption buffer and kept 10 min on ice to ensure complete
disruption before processing the sample for protein analysis.
Protein concentration was determined using a Micro BCA
Protein Assay Kit (Pierce Chemical Co., Rockford, IL).
RNA Isolation and RT-PCR
BAC cell total RNA was extracted using the RNAgents kit
(Promega Corp., Charbonnie` res, France) according to the
manufacturer’s instructions. This system consistently yields
50–80
g total RNA/3 ⫻ 10
6
cells. For RT-PCR analysis of
Tis11b, VEGF, HuR,orHPRT gene expression, 1
g of total
RNA was reverse transcribed with Superscript II reverse tran-
scriptase (Invitrogen) and PCR amplified using Taq polymer-
ase (QBiogen, Illkirch, France). Amplification of VEGF mRNA
isoforms was performed using the primers and amplification
conditions described by Gaillard et al. (28). The size of the
expected amplified fragments was 462 bp and 332 bp for the
two major VEGF transcripts, VEGF
165
and VEGF
121
, respec
-
tively. The primers for PCR of Tis11b were as follows: 5⬘-
CGAAGAAAACGGTGCCTGTAAG-3⬘ and 5⬘-AGTAGGTG-
AGCCCAAGAGGTCATC-3⬘. This primer pair sequence
amplifies a 354-bp fragment. The amplification conditions
were as follows: 94 C for 5 min followed by 25 amplification
cycles, each consisting of 94 C for 1 min, 55 C for 1 min, 72
C for 1 min, and 72 C for 5 min for final extension. The primers
for hypoxanthine phosphoribosyl transferase (HPRT) amplifi-
cation were as follows: 5⬘-GCCATCACATTGTAGCCCTCT-3⬘
and 5⬘-TGCGACCTTGACCATCTTTGG-3⬘. This primer pair
sequence amplifies a 305-bp fragment. The amplification
conditions were as follows: 94 C for 5 min followed by 25
amplification cycles, each consisting of 94 C for 1 min, 55 C
for 1 min, 72 C for 1 min, and 72 C for 5 min for final
extension. The primers for HuR amplification were as follows:
5⬘-ATGACCCAGGATGAGTTACGAAGC-3⬘ and 5⬘-GTTCA-
CAAAGCCATAGCCCAAG-3⬘. This primer pair sequence am-
plifies a 111-bp fragment. The amplification conditions were
as follows: 94 C for 5 min followed by 25 amplification cycles,
each consisting of 94 C for 1 min, 52 C for 1 min, 72 C for 1
min, and 72 C for 5 min for final extension. PCR products
were analyzed on 2% agarose ethidium bromide-containing
gels, visualized using a Vistra FluorImager (Molecular Dynam-
ics, Sunnyvale, CA) and quantitated using ImageQuant soft-
ware (Molecular Dynamics). Preliminary experiments were
done to select the starting amounts of RNA so that there was
a linear relationship between the amount of input RNA and
the OD of the HPRT band on ethidium bromide-stained gel.
SDS-PAGE
SDS-PAGE was performed according to Laemmli (57). Total
proteins or subcellular fraction extracts (5–20
g/lane) were
solubilized in sample buffer [60 mM Tris-HCl (pH 6.8), 2%
sodium dodecyl sulfate (SDS), 5%

-mercaptoethanol, 10%
glycerol, 0.01% bromophenol blue], boiled for 5 min and
loaded onto a 12% SDS-PAGE minigel (Mini Protean II Sys-
tem; Bio-Rad Laboratories, Hercules, CA). Electrophoresis
was performed at 150 V for 1 h.
Western Blot Analysis
SDS-PAGE-resolved proteins were electrophoretically trans-
ferred onto a polyvinylidine difluoride (PVDF) membrane ac-
cording to Towbin et al. (58). After transfer, the membrane
was incubated in a blocking buffer (PBS buffer containing
0.1% Tween 20 and 5% nonfat dry milk) for1hatroom
temperature. The blots were probed sequentially with anti-
bodies to a peptide fragment (amino acids 49–63) of Tis11b
protein (1:500; CovalAb, Lyon, France), antihuman HuR (1:
1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
monoclonal anti-
␣
-tubulin (1:200,000, a generous gift from
Dr. D. Job, CS-U366 INSERM, CEA-Grenoble, France), and
antilamin A/C (1:15000, a generous gift from Dr. Deloulme,
TS-EMI 01–04 INSERM, CEA-Grenoble, France) for2hin
PBS containing 0.1% Tween. The membrane was thoroughly
washed with the same buffer (3 ⫻ 10 min), and then incu-
bated for 1 h with either horseradish peroxidase (HRP)-la-
beled goat antirabbit IgG (immunodetection of Tis11b and
HuR) or HRP-labeled goat antimouse IgG (immunodetection
of tubulin), or HRP-labeled goat antiguinea pig IgG (immuno-
detection of lamin A/C). The PVDF sheet was washed as
above, and the antigen-antibody complex was revealed by
Enhanced Chemiluminescence, using the Western blotting
detection kit from Amersham Biosciences (Buckinghamshire,
UK) and BioMax Kodak films (Sigma).
Immunoprecipitation Assay
BAC cells cultured in 10-cm petri dishes (3 ⫻ 10
6
cells per
dish) were stimulated with 10 n
M ACTH for the indicated
periods of time. Subcellular fractions (cytoplasm and nuclei)
were prepared as indicated previously. Lysates was cleared
by centrifugation for 5 min at 12,000 ⫻ g at 4 C. The antihu-
man pp32 (I1PP2A) polyclonal antibody (Santa Cruz Biotech-
nology, Santa Cruz, CA) was added at a concentration of 2
g/ml to whole supernatants (⬃250
g protein for cytoplasm
and 70
g protein for nuclei), which were then gently rocked
for2hat4C,before being incubated for 30 min with Protein
A/G Sepharose beads. Immunoprecipitates were pelleted,
washed four times with radioimmunoprecipitation buffer, an-
alyzed by SDS-PAGE, and transferred to a PVDF membrane.
Blots were probed with anti-HuR or anti-pp32 antibodies,
which were used at 1:1000.
RNAi
Expression of HuR (ELAVL1, GenBank accession no.
NM_001419) was inhibited by transfection of a predesigned
siRNA duplex targeted to exon 2 of human HuR gene (Am-
bion). siRNA sense and antisense sequences were 5⬘-GGAU-
GAGUUACGAAGCCUGtt-3⬘ and 5⬘-CAGGCUUCGUAACU-
CAUCCtt-3⬘, respectively. Adrenocortical cells were transfe-
cted 1 d after plating with 10 n
M of either HuR siRNA duplex
Cherradi et al. • ACTH-Induced VEGF Expression Mol Endocrinol, April 2006, 20 (4):916–930 927
or negative control siRNA (Ambion), using siPORT lipid rea-
gent (Ambion). Typically, cells were analyzed for the loss of
HuR mRNA and protein expression 48 h after transfection
using RT-PCR and immunoblotting. At this time point, culture
medium was changed, and cells were treated for the indic-
ated periods of time with or without 10 nM ACTH.
Transient Transfections and Dual Luciferase
Activity Assay
COS7 cells were grown in DMEM supplemented with 10%
fetal calf serum, 100 U/ml penicillin, 100
g/ml streptomycin,
and 25
g/ml gentamycin and transfected in 12-well plates
using lipofectamine (Invitrogen) according to the manufactur-
er’s recommendations. Plasmid pLuc-V3⬘ contains the firefly
luciferase cDNA cloned upstream of the rat VEGF 3⬘-UTR and
downstream of the thymidine kinase (TK) promoter (30). Plas-
mid pCMV-murine HuR was provided by Dr. Jonathan
LaMarre (University of Guelph, Ontario, Canada). pCMV-
Tis11b plasmid was described previously (30). pRL-TK en-
coding Renilla luciferase was obtained from Promega Corp.
Various amounts of either pCMV-Tis11b, pCMV-HuR, or
both (0.05–1 ng) were cotransfected with 500 ng pLuc-V3⬘,
25 ng of pRL-TK, and pUC19 up to a total of 700 ng plasmid
DNA into 1.5 ⫻ 10
5
cells. Renilla and firefly luciferase activ
-
ities were measured sequentially 48 h after transfection with
the Dual-Luciferase reporter assay system (Promega Corp.)
on a LUMAT LB 9507 luminometer (EGG-Berthold, Bad Wild-
bad, Germany). Results are expressed as relative light units
of firefly luciferase activity over relative light units of Renilla
luciferase activity to compensate for variations in transfection
efficiency. Each transfection condition was performed in
triplicate.
Northern Hybridization
Total RNA from COS7 cells was extracted using the
RNAgents kit (Promega) according to the instructions of the
manufacturer. RNA (15⫺20
g) was size fractionated on a
1% formaldehyde agarose gel, vacuum transferred onto Hy-
bond-n ⫹ membranes (Amersham Biosciences) and fixed by
UV cross-linking. Northern blots were pre-hybridized in Rapid
Hybridization Buffer (Amersham Biosciences) at 65 C for 30
min. [
␣
32
P]dCTP-labeled luciferase cDNA probe (2 ⫻ 10
6
cpm/ng DNA; Rediprime random primer labeling kit, Amer-
sham Biosciences) was then added, and the incubation was
continued for2hat65C.Blots were washed for 5 min and
15 min successively at room temperature in 2⫻ saline sodium
citrate (SSC), 0.1% SDS, and then for 15 min in 1⫻ SSC,
0.1% SDS. The final wash was performed at 65 C for 15 min
in 0.5⫻ SSC, 0.1% SDS. RNA-cDNA hybrids were visualized
on phosphor screen (Molecular Dynamics, Inc.) after a 12- to
24-h exposure period. Blots were stripped and reprobed with
18S cDNA probe to assess RNA loading.
Recombinant Protein Expression and Purification
Tis11b recombinant protein was produced as reported else-
where (30). GST-HuR fusion protein was produced using
Escherichia coli BL21 strain transformed with pGEX-5X2-
HuR plasmid (provided by Dr. J. A. Steitz, Yale University
school of Medicine, New Haven, CT). Bacteria were grown at
37CtoanA
600
nm of 0.6 in 2YT medium (16 g/liter tryptone,
10 g/liter yeast extract, 5 g/liter NaCl) containing 100
g/ml of
ampicillin. The GST-HuR fusion protein was induced with 0.1
mM isopropyl-

-D-thiogalactopyranoside for 2 h and purified
using MicroSpin GST Purification Module (Amersham Bio-
sciences) according to the manufacturer’s instructions.
Briefly, cells were harvested by centrifugation (2500 ⫻ g at 4
C for 5 min). The pellet was resuspended with ice-cold PBS
containing 0.1 mg/ml lysozyme and lysed by repeated freeze/
thawing (10 times). Clarified lysate was mixed for 10 min with
Glutathione Sepharose 4B Microspin column. After two
washes with PBS, GST-HuR fusion protein was eluted with
10 mM reduced glutathione. Purity of GST-HuR protein was
examined by Coomassie blue staining after SDS-PAGE
analysis.
RNA-Protein UV Cross-Linking Assay
[
32
P]UTP-labeled and unlabeled riboprobes were synthesized
in vitro using pSp64 plasmid containing the entire VEGF
3⬘-UTR and the Riboprobe SP6 in vitro Transcription System
(Promega). Integrity of RNA transcripts was visualized on 1%
agarose ethidium bromide-containing gel. RNA transcripts
(1 ⫻ 10
6
cpm) were incubated for 20 min at room temperature
with 1–6
g of either Tis11b-containing bacterial extract or
GST-HuR fusion protein, in 10 mM HEPES (pH 7.6), 3 mM
MgCl
2
,40mM KCl, 5% glycerol, 0.5% Nonidet P-40, and 2
m
M dithiothreitol. Yeast tRNA (50 ng/
l) and heparin (2
g/
l)
were then added for 10 min. Mixtures were exposed to UV
light for 30 min on ice. of RNase T1 (100 U) (Invitrogen) were
then added for 20 min, and RNA-protein complexes were
analyzed by 12% SDS-PAGE and autoradiography.
Statistical Analysis
Results are expressed as means ⫾
SE. The mean values were
compared by ANOVA using Fisher’s test. A value of P ⬍ 0.05
was considered as statistically significant. Quantitation of
immunoblots and autoradiograms was performed using a
Molecular Imager FX and Quantity One software (Bio-Rad).
Acknowledgments
We thank Dr. Jonathan LaMarre (Department of Biomedi-
cal Sciences, Ontario Veterinary College, University of
Guelph, Ontario, Canada) and Dr. J. A. Steitz (Yale University
school of Medicine, New Haven, CT) for their generous gift of
pCMV-HuR and pGEX-HuR plasmids, respectively. We also
thank Dr. Didier Job and Dr. Jean-Christophe Deloulme for
providing us the antitubulin and antilamin A/C antibodies,
respectively.
Received March 11, 2005. Accepted November 14, 2005.
Address all correspondence and requests for reprints to:
Dr J. J. Feige, Institut National de la Sante´ et de la Recherche
Me´ dicale (INSERM), Equipe Mixte 01-05, De´ partement Re´-
ponses et Dynamique Cellulaires, laboratoire ANGIO, CEA-
Grenoble, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9,
France. E-mail: jjfeige@cea.fr.
This work was supported by the Institut National de la
Sante´ et de la Recherche Me´ dicale (INSERM, Equipe Mixte
01-05), the Commissariat a` l’Energie Atomique (Direction des
Sciences du Vivant/De´ partement Re´ sponses et Dynamique
Cellulaires), the Cance´ ropole Rhoˆ ne-Alpes, and the Fonda-
tion de France (Grant 2004009572). N.C. was supported by
the Ligue Nationale Contre le Cancer and the Fondation de
France.
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Erratum
In Table 1 of the article titled “Steroid Deficiency Syndromes in Mice with Targeted
Disruption of Cyp11a1”(Mol Endocrinol 16:1943–1950, 2002) by M.-C. Hu et al., the values
of potassium in the serum appear to be higher than normal due to an unknown systematic
problem in the measurement process. The correct values for normal serum potassium
levels should be 3–8 m
M. The conclusions of the paper are not affected. The authors regret
the error.
930 Mol Endocrinol, April 2006, 20(4):916–930 Cherradi et al. • ACTH-Induced VEGF Expression
