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J Biol Chem, Vol. 275, Issue 18, 13802-13811, May 5, 2000
-Adrenoreceptor/cAMP/Protein Kinase A Pathway Involving Src but
Independently of Erk1/2*
,From The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden
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ABSTRACT |
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To identify the signaling pathway that mediates
the adrenergic stimulation of the expression of the gene for vascular
endothelial growth factor (VEGF) during physiologically induced
angiogenesis, we examined mouse brown adipocytes in primary culture.
The endogenous adrenergic neurotransmitter norepinephrine (NE) induced
VEGF expression 3-fold, in a dose- and time-dependent
manner (EC50 For the study of angiogenesis, brown adipose tissue provides a
physiologically interesting model system. In this tissue,
norepinephrine (NE),1 the
adrenergic mediator in sympathetic nerves, is the acute inducer of
thermogenesis (1). During the thermogenic process, the rate of lipid
oxidation in this tissue, and thus the demand for oxygen delivery to
the tissue, is immense. Heat production in brown adipose tissue
accounts for nearly half of the total energy expenditure in a
cold-acclimated animal in the cold, even though the tissue only
constitutes 1-2% of the total body weight (2, 3). The blood supply
needed to support the oxygen requirement under such conditions is
necessarily exceedingly high (2, 3), demanding a rich vasculature,
which is indeed increased during brown adipose tissue recruitment (2,
4).
The regulation of this angiogenesis in brown adipose tissue has so far
not been extensively investigated. Expression of the angiogenic factor
basic fibroblast growth factor, is induced in the tissue by cold
exposure (5) and by NE in cultured brown adipocytes (6). The
VEGF-related factor (vascular endothelial growth factor-B) is expressed
already during development (7), and expression of VEGF (vascular
endothelial growth factor (8)) itself is induced by NE administration
and cold exposure in brown adipose tissue (9), and VEGF expression and
secretion is stimulated by NE in cultured brown adipocytes (10, 11).
Thus, NE appears to mediate cold-induced angiogenesis in brown adipose
tissue by stimulating the expression of angiogenic factors, such as
VEGF. In this respect, the effect of NE may be connected to the general recruitment effect of NE in this tissue, i.e. increased DNA
synthesis (6, 12-14) and increased expression of the brown
adipocyte-specific mitochondrial uncoupling protein UCP1 (15-18).
The aim of the present investigation was to clarify through which
intracellular signaling mechanisms NE stimulates expression of the VEGF
gene. We conclude that NE-induced VEGF expression was exclusively
dependent on a Cell Isolation--
Brown adipocyte precursors were isolated
from 3-4-week-old mice of the NMRI strain, obtained from a local
supplier (Eklunds), principally as described by Néchad et
al. (19) and modified by Bronnikov et al. (14).
Briefly, the cervical, interscapular, and axillary brown adipose tissue
depots were dissected out from each mouse. The depots were pooled and
incubated in the Hepes-buffered Ringer solution detailed by
Néchad et al. (19), containing 0.2% (w/v) crude
collagenase type II (Sigma). The tissue was digested for 30 min at
37 °C, with vortexing every 5 min, after which the digest was
filtered through a 250-µm nylon screen. To allow lipid droplets and
mature adipocytes to float, the digest was put on ice for 20 min, and
the infranatant was then collected and filtered through a 25-µm nylon
screen. The cells in the filtrate were pelleted by centrifugation,
suspended in Dulbecco's modified Eagle's medium and repelleted, after
which they were suspended in culture medium and used for cell culture.
Cell Culture--
The cells were cultured either in Costar
6-well plates (growth area 9.4 cm2 per well, with cells
obtained from 2.5 animals inoculated per plate in 2 ml of culture
medium per well) or in Costar 12-well plates (growth area 3.83 cm2 per well, with cells obtained from 2 animals inoculated
per plate in 1 ml of culture medium per well). The culture medium
consisted of Dulbecco's modified Eagle's medium (routinely Life
Technologies, Inc., without L-glutamine with 4 nM L-glutamine (Life Technologies, Inc.)
added), supplemented with 10% newborn calf serum (PAN Systems), 4 nM insulin (Actrapid Human, Novo), 10 mM Hepes
(Life Technologies, Inc.), and with 50 IU penicillin, 50 µg of
streptomycin (Life Technologies, Inc.), and 25 µg of sodium ascorbate
(Kebo) per ml, at 37 °C in a water-saturated atmosphere of 8%
CO2 in air in a Heraeus CO2 auto-zero B5061
incubator. On day 1, the cultures were washed with prewarmed
Dulbecco's modified Eagle's medium, and fresh prewarmed medium was
then added. Medium was then changed every other day until the day of
experimentation, which was routinely day 6.
Cultures under serum-free conditions were cultured for 5-6 days in the
above medium, after which they were washed and the same culture medium
as described above, without newborn calf serum, but with 0.5% (w/v)
albumin (fraction V, fatty acid free; Roche Molecular Biochemicals)
added. The experiments were performed after approximately 20 h in
this medium.
RNA Isolation and Determination of mRNA Levels for VEGF and
UCP1--
On the indicated day of culture (day 6 if not otherwise
indicated) and after stimulation, the culture medium was discarded and
the cells were dissolved in 0.8 ml of an Ultraspec solution (Biotecx),
and the manufacturer's procedure for RNA isolation was followed. The
RNA concentration was measured on a Beckman DU 50 spectrophotometer
with readings at 260 and 280 nm; the 260/280 nm ratio was routinely
1.7-1.8. 10 µg of total RNA was separated by electrophoresis in an
ethidium bromide-containing agarose-formaldehyde gel, as described by
Bronnikov et al. (20). The intensity of the 18 S and 28 S
rRNA bands under UV light was routinely checked to verify that all
samples were equally loaded and that no RNA degradation had occurred.
The RNA was blotted overnight from the gel to a Hybond-N membrane,
cross-linked, and hybridized as described by Bronnikov et
al. (20). The Hybond-N membrane was then washed three times in
0.1 × SSC, 0.2% SDS at 50 °C for 20 min. The membrane was
sealed in a plastic envelope and exposed to a PhosphorImager screen for 1 day. The screen was analyzed on a Molecular Dynamics PhosphorImager and the overall VEGF mRNA signal was quantified with the ImageQuant program. When the same Hybond-N membrane was analyzed for both VEGF and
UCP1 mRNA, the previous probe was removed by putting the membrane
in 0.2% SDS solution at 100 °C and allowing this solution to cool
down to room temperature.
cDNA Probes--
The VEGF cDNA was a gift from Dr. Georg
Breier, Max-Planck Institute, Bad Nauheim, Germany. It contained the
580-base pair mouse VEGF164 cDNA, which encodes the
VEGF164 isoform (21). This cDNA had been cloned into
the EcoRI-BamHI cloning sites of a pBluescript KS
vector. The vector was amplified by transformation of a TG-1
Escherichia coli strain and the cDNA was isolated. The UCP1 cDNA was that earlier used (18). The probes were labeled with
[ cAMP Analysis--
On day 6 in culture and after stimulation,
the culture medium was aspirated after the indicated time and the cells
were treated principally as described by Bronnikov et al.
(20). Briefly, 0.8 ml of 75% ethanol with 1 mM EDTA was
added to each well, and after 10 min, cells were harvested by scraping.
Ethanol was removed by SpeedVac centrifugation and pellets were
suspended in 0.5 ml of 4 × TE buffer and sonicated 5 s.
After centrifugation, a 25-µl supernatant aliquot from each sample
was used for determination of cAMP levels with the Cyclic AMP Assay
System (Amersham Pharmacia Biotech), according to the manufacturer's instructions.
Erk1/2 Phosphorylation Analysis--
On day 6 in culture and
after stimulation, the culture medium was discarded and the cells were
harvested and analyzed for Erk1/2 phosphorylation as described earlier
(22). Briefly, proteins were separated on 12% polyacrylamide gels.
Electrotransfer to Hybond-C Extra nitrocellulose membranes (pore size
0.45 µm; Amersham Pharmacia Biotech) was carried out in a semidry
electroblotter. After transfer, the membranes were allowed to soak in
Tris-buffered saline for 5 min followed by quenching (5% non-fat dry
milk, 0.1% Tween 20 in Tris-buffered saline) of nonspecific binding
for 1 h at room temperature. The membranes were incubated with
primary antibodies, an Erk1/2 phosphospecific antibody (human Thr(P)
202/Tyr(P) 204; New England Biolabs) or an Erk1/2 antibody (New England
Biolabs) overnight at 4 °C. The primary antibody was detected with a
secondary horseradish peroxidase-conjugated goat anti-rabbit antibody
(New England Biolabs) and enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). The blots were exposed to Kodak X-Omat RP films and
quantified on a Molecular Dynamics densitometer. Erk1/2 phosphorylation
was routinely expressed as the ratio of phosphorylated Erk1/2 to total
Erk1/2.
Chemicals--
The following chemicals were used: norepinephrine
(( Mouse Brown Adipocytes Constitutively Express the VEGF Gene
To examine the regulation of VEGF expression in mouse brown
adipocytes, we first investigated the ability of these cells to express
the VEGF gene constitutively. Total RNA isolated from differentiated
mouse brown adipocytes (on day 6 in culture) was analyzed by Northern
blot analysis. The 580-base pair mouse VEGF164 cDNA
probe (21) was used. On the Northern blot (Fig.
1A), a major band at
approximately 4 kilobases was observed (top arrow), and 4 further bands at lower molecular weight were distinguishable on
densitometric analysis. This Northern blot pattern was similar to that
observed in other cells, tissues, and species (9, 21, 23, 24),
including rat brown adipose tissue (9). During the different treatments
used in this study, the intensity of these bands varied in parallel,
and the total signal was therefore that used for analysis.
90 nM). Also, the
hypoxia-mimicking agent cobalt, as well as serum and phorbol ester,
induced VEGF expression, but the effect of NE was additive to each of
these factors, implying that a separate signaling mechanism for the
NE-mediated induction was activated. The NE effect was abolished by
propranolol and mimicked by isoprenaline or BRL-37344 and was thus
mediated via
-adrenoreceptors. The NE-induced VEGF expression was
fully cAMP mediated, an effect which was inhibited by H-89 and thus was
dependent on protein kinase A activity. Involvement of other adrenergic
signaling pathways (
1-adrenoreceptors, Ca2+,
protein kinase C,
2-adrenoreceptors, and pertussis
toxin-sensitive Gi-proteins) was excluded. The specific
inhibitor of Src tyrosine kinases, PP2, markedly reduced the
stimulation by NE, which demonstrates that a cAMP-dependent
Src-mediated pathway is positively connected to VEGF expression.
However, inhibition of Erk1/2 MAP kinases by PD98059 was without
effect. NE did not prolong VEGF mRNA half-life and its effect was
thus transcriptional, and was independent of protein synthesis. These
results demonstrate that adrenergic stimulation, through
-adrenoreceptor/cAMP/protein kinase A signaling, recruits a pathway
that branches off from the NE-activated Src-Erk1/2 cascade to enhance
transcription of the VEGF gene.
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INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenoreceptor-mediated increase in cAMP levels and
PKA activity. The mediation involved Src tyrosine kinases, unexpectedly
and completely independently of downstream mediation via Erk1/2 MAP
kinases. We thus demonstrate a positive connection between VEGF
expression and a signaling pathway that branches off from the
NE-activated Src-Erk1/2 cascade.
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EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP with a Random Primed DNA Labeling Kit
(Roche Molecular Biochemicals), according to the manufacturer's instructions.
)-arterenol bitartrate), DL-propranolol, prazosin,
isoprenaline, clonidine, A23187,
12-O-tetradecanoylphorbol-13-acetate (TPA), genistein, wortmannin, pertussis toxin, actinomycin D, cycloheximide,
8-bromo-cAMP, and forskolin (all from Sigma), CGP-12177 (CGP-12177A;
Ciba-Geigy), BRL-37344 (SmithKline Beecham), cirazoline, and
1,9-dideoxy-forskolin (RBI), UK 14304 (Tocris Cookson), PD98059 (New
England BioLabs), H-89 and PP2 (Calbiochem). Dimethyl sulfoxide
(Me2SO) was used to dissolve A23187, TPA, forskolin,
PD98059, genistein, and PP2; final concentration per assay was
maximally 0.5%. Ethanol (50%) was used to dissolve prazosin and
actinomycin D; final concentration per assay was maximally 0.5%.
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RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Fig. 1.
Effect of norepinephrine on VEGF gene
expression in brown adipocytes at different stages of
differentiation. Brown adipocytes were isolated and cultured for
3-7 days as described under "Experimental Procedures" and treated
as indicated. Total RNA was isolated and used for Northern blotting.
A, upper panel is a Northern blot of VEGF mRNA in day 6 cultures. Two left lanes, control levels (C);
two right lanes, stimulated with 10 µM NE for
1 h. The sizes of 28 S and 18 S rRNA are indicated to the
left. Right side arrows indicate bands as identified on
densitometric analysis. The lower panel shows ethidium
bromide (EtBr) staining of 28 S rRNA, as visualized under UV light.
B, UCP1 mRNA levels in brown adipocytes on day 3-7 in
culture, with or without 10 µM NE for 1 h. The
values are mean ± S.E. of three experiments with single wells. In
each experiment, the mRNA level in NE-treated day 5 cultures was
set to 1. (The relative increase was lower than that seen earlier (18)
as only 1 h of NE treatment was used.) C, the same
experiments as in B analyzed for VEGF mRNA levels.
To investigate whether the expression of VEGF is a differentiation characteristic, we followed VEGF expression during cell differentiation. Brown adipocytes in primary culture proliferate until confluence is reached, which occurs at approximately day 5-6 after inoculation (14, 19, 20, 25, 26). At this time, differentiation spontaneously occurs. This is verified in Fig. 1B where the ability of NE to induce expression of the brown adipocyte-specific differentiation marker, mitochondrial uncoupling protein-1 (UCP1), is analyzed. As seen, rather high levels of UCP1 mRNA were induced by NE from day 5 (Fig. 1B, black bars), indicating that brown adipocyte differentiation had occurred. As seen in Fig. 1C (gray bars), VEGF was constitutively expressed already in undifferentiated cells, and basal VEGF mRNA levels were similar in all stages of differentiation. This is in contrast to VEGF expression observed in 3T3-F442A adipocytes, which was fully differentiation-dependent (27). Thus, the state of differentiation is not crucial for basal VEGF expression in brown adipocytes.
Norepinephrine Induces VEGF Gene Expression
To investigate the effect of NE on VEGF expression, we treated the cultures with NE for 1 h. VEGF mRNA levels increased approximately 3-fold after NE stimulation (Fig. 1A, two rightmost lanes), which is in accordance with observations in cultured rat brown adipocytes (11). The stimulation by NE was observed at all stages of brown adipocyte differentiation, from proliferating to fully differentiated cells, and was of similar relative magnitude (Fig. 1C, black bars).
When brown adipocytes were stimulated with increasing concentrations of
NE, VEGF mRNA levels increased monophasically, following simple
Michaelis-Menten kinetics, to 4-fold control levels, obtained with 10 µM NE; the EC50 was 70 ± 22 nM (Fig. 2A).
Analysis of mean points from four similar experiments yielded a 3-fold
increase, with similar kinetics; EC50 93 ± 27 nM (not shown).
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In the maintained presence of NE, the VEGF mRNA levels were elevated only during the first few hours and had returned to control levels after 6 h (Fig. 2B). An independent experiment with 30 min NE stimulation of brown adipocytes resulted in only a 30% elevation of VEGF mRNA levels (not shown); thus maximum levels occurred at about 1 h of stimulation. The rapid disappearance of the mRNA signal in these experiments implies a short half-life of VEGF mRNA (see also below). This transient increase in expression is in principal agreement with the time course of VEGF expression induced in rat brown adipose tissue in situ by cold exposure: the expression peaked at 4 h, and had decreased down to control levels within 24 h (9). Based on these experiments, further experiments were performed with 10 µM NE and analyzed after 1 h of stimulation.
Interaction between Norepinephrine and Other Inducers of VEGF Gene Expression
As demonstrated above, NE is a potent inducer of VEGF expression in mouse brown adipocytes. In various other cell types, other factors such as hypoxia, cobalt (as a hypoxia-mimicking agent), serum, growth factors, and phorbol esters have been demonstrated to induce VEGF expression (27-31). We have therefore investigated whether NE is an exclusive inducer of VEGF expression in brown adipocytes, or whether the classical factors mentioned above could induce expression also in these cells. These experiments may also reveal initial information on the intracellular pathway mediating the NE signal, as non-additivity between the NE-induced VEGF expression and that induced by other factors would imply distinct signaling pathways.
Hypoxia (Cobalt)--
The hypoxia-mimicking agent cobalt was not
able to elevate VEGF expression after 1 h of treatment (not
shown), but after 6 h, a 3-fold increase could be seen (Fig.
3A). The elevated VEGF mRNA levels persisted for at least 24 h (not shown). Thus,
hypoxia is probably an inducer of VEGF expression also in brown
adipocytes. The effect of NE was additive to that of cobalt (Fig.
3A). Thus, NE and hypoxia (cobalt treatment) utilize
separate pathways for the induction of VEGF expression.
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Serum-- To investigate the effect of serum factors, serum was removed from the brown adipocyte cultures. When readded the next day, serum induced VEGF expression 4-fold (Fig. 3B). The effect of serum lasted longer than that of NE (>6 h; not shown). NE was able to induce VEGF expression, also in the absence of serum factors (Fig. 3B). The effect of NE was additive to that of serum (Fig. 3B), and thus the signaling pathway mediating the NE effect would also seem to be different from pathways utilized by serum factors.
Phorbol Esters-- The phorbol ester TPA was also a potent inducer of VEGF expression (4-fold) (Fig. 3C), implying that protein kinase C (PKC)-activated pathways can stimulate VEGF expression in brown adipocytes. NE gave a marked increase of VEGF mRNA in addition to that induced by TPA alone (Fig. 3C). Thus, the pathway mediating the NE effect on VEGF expression appears not to involve PKC activation (see also below). Thus, also the classical inducers of VEGF expression, hypoxia (cobalt), serum, and phorbol ester, induced VEGF expression in brown adipocytes, but NE utilized a distinct signaling pathway.
Mediation of Norepinephrine-induced VEGF Gene Expression
To investigate which adrenergic receptors mediate the NE effect on
VEGF expression in brown adipocytes, adrenoreceptor agonists and
antagonists were used (Fig.
4A). The
1-adrenoreceptor antagonist prazosin had no significant
effect on NE-induced VEGF expression, but the
-adrenoreceptor
antagonist propranolol completely abolished it. In accordance with
this, the
1-adrenoreceptor agonist cirazoline had no
inducing effect (Fig. 4A), nor had the
2-adrenoreceptor agonists clonidine (10 µM) or UK14304 (10 µM) (not shown), whereas the
-adrenoreceptor agonist isoprenaline was an equally potent inducer as was NE (Fig. 4C). Thus, only
-adrenoreceptors
mediated the NE-induced VEGF expression.
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Proliferating brown preadipocytes only express the
1-adrenoreceptor subtype whereas fully differentiated
brown adipocytes also express the
3-adrenoreceptor
subtype which, at this stage of differentiation, is the only
-adrenoreceptor subtype coupled to adenylyl cyclase (20). Thus, the
NE-induced VEGF expression observed in proliferating undifferentiated
brown preadipocytes (days 3-4, Fig. 1B) is most likely
mediated by
1-adrenoreceptors. In differentiated brown
adipocytes, the
3-adrenoreceptor agonist BRL-37344
induced VEGF expression to the same extent as did NE (not shown). The
3-adrenoreceptor agonist CGP-12177 (32) (which is a
1- and
2-adrenoreceptor antagonist) also
induced VEGF expression in these cells (not shown). Thus, both
1- and
3-adrenoreceptors were able to
mediate NE-induced VEGF expression in these cells, the coupling
depending on the differentiation stage. To identify which second
messengers had the ability to induce VEGF expression, we examined the
intracellular second messenger systems involved in
adrenoreceptor-mediated signaling: Ca2+ and PKC (from
1-adrenoreceptors), the proposed
3-adrenoreceptor/Gi-protein signaling
pathway (33), and cAMP (from
-adrenoreceptors).
The Ca2+ ionophore A23187, which increases intracellular Ca2+ levels and through this, e.g. induces c-fos expression (34), had no effect on VEGF mRNA levels (Fig. 4A). Concerning PKC activation, it was shown above that the phorbol ester TPA induced a marked elevation of VEGF expression (Fig. 3C), but it was concluded that the NE effect appeared to be independent of PKC, because of the additivity of the effects. To substantiate this, PKC was inactivated prior to NE stimulation, by prolonged exposure to TPA, a treatment that inactivates TPA-sensitive PKC isoforms (35, 36). After TPA pretreatment, no remaining elevation of VEGF mRNA levels was observed (Fig. 4B), and the acute effect of TPA on VEGF mRNA levels was abolished (Fig. 4B). Thus, TPA pretreatment effectively inactivated PKC. However, the NE-induced VEGF expression was not altered by TPA pretreatment (Fig. 4B) and was thus not mediated through a TPA-sensitive PKC pathway.
To investigate the possible involvement of the suggested
cAMP-independent
3-adrenoreceptor signaling through
pertussis toxin (PTX)-sensitive G-proteins, i.e.
Gi-proteins (33, 37, 38), we pretreated brown adipocytes
with PTX. PTX augmented
3-adrenoreceptor-stimulated cAMP
production,2 confirming that
Gi proteins were active. However, NE- or
isoprenaline-induced VEGF expression was not affected by PTX, and PTX
had no effect in itself (Fig. 4C). Thus, no adrenergic
stimulation of VEGF expression was mediated through PTX-sensitive
Gi proteins in a cAMP-independent process.
To investigate the ability of cAMP to induce VEGF expression, brown adipocytes were treated with forskolin to activate adenylyl cyclases. Forskolin induced VEGF expression to the same extent as did NE (Fig. 4A). This effect was not altered by increased levels of intracellular Ca2+ (Fig. 4A). The forskolin analogue 1,9-dideoxy-forskolin, which does not activate adenylyl cyclases, had no effect (not shown). The cAMP analogue 8-bromo-cAMP (1 mM for 1 h) also induced VEGF expression 3-fold (not shown). Thus, increased levels of cAMP are potent stimulators of VEGF expression in brown adipocytes and may mediate the NE effect.
cAMP Is the Mediator of the Norepinephrine-induced VEGF Expression
To establish that cAMP not only is able to induce VEGF expression,
but also is the actual mediator of the NE effect, we first investigated
the correlation between cAMP levels and VEGF expression in brown
adipocytes treated with different doses of forskolin. The elevation of
cAMP levels was dose-dependent (Fig.
5A), as was the elevation of
VEGF mRNA levels (Fig. 5B). However, nearly maximal
induction of VEGF expression occurred already at approximately 50 pmol
of cAMP/well (Fig. 5C). Thus, the ability of cAMP to induce VEGF expression was saturated at this cAMP level.
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To compare the ability of NE- and forskolin-derived cAMP to induce VEGF expression, we measured the cAMP levels induced by NE (Fig. 5D). The NE-induced cAMP levels reached the saturating levels for VEGF expression (approximately 50 pmol of cAMP/well), and the dose-response kinetics were very similar to those for NE-induced VEGF expression (Fig. 2A). Indeed, when NE-derived results were plotted into the forskolin curve (Fig. 5E), they did not deviate from the forskolin results. These results suggest that NE-induced VEGF expression is exclusively dependent on cAMP. To confirm this, we treated brown adipocytes with NE at forskolin concentrations sufficient to saturate cAMP-induced VEGF expression. Under these conditions, NE did not affect forskolin-induced cAMP levels (not shown), nor did it affect forskolin-induced VEGF expression (Fig. 5F). This lack of additivity implies that cAMP is the common mediator of NE- and forskolin-induced VEGF expression (contrast the additivities in Fig. 3).
The Signaling Pathway Downstream of cAMP Is Mediated via PKA and Src but Not via Erk1/2
To identify the further signaling pathway involved in mediating the cAMP-dependent stimulation of VEGF expression, we used inhibitors of intracellular signaling factors.
The classical immediate downstream effector of cAMP, the
cAMP-dependent protein kinase (PKA), could be expected to
mediate the cAMP-dependent signaling. Therefore, we
pretreated brown adipocytes with the PKA inhibitor H-89, which had no
effect in itself, but which abolished both NE- and forskolin-induced
VEGF expression (Fig. 6). That also the
forskolin effect was inhibited demonstrated that H-89 acted by
inhibiting PKA, and not by competing with NE for
3-adrenoreceptor binding (such an effect has been
demonstrated for
1- and
2-adrenoreceptors
(39), but does not seem to apply to
3-adrenoreceptors).3 Thus, the
cAMP-dependent stimulation was mediated through a
PKA-dependent signaling pathway.
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In brown adipocytes, adrenergic
activation may proceed via Src tyrosine kinases, which are activated
via
3-adrenoreceptors in a PKA-dependent
manner in these cells.4
Therefore, to investigate whether Src mediates NE-induced VEGF expression, we pretreated brown adipocytes with the specific inhibitor of Src, PP2. PP2 was fully effective as a Src inhibitor, since NE-induced Erk1/2 phosphorylation was abolished (Fig.
7A). To ensure that PP2 had no
unspecific effect on adrenergic pathways and gene expression, we first
examined whether PP2 inhibited NE-induced UCP1 expression. Clearly PP2
did not have a general inhibitory effect; rather, surprisingly, PP2
markedly potentiated the effect of NE (Fig. 7B), an effect
for which we have no explanation. Concerning the NE-induced VEGF
expression, PP2 had a marked inhibitory effect (Fig. 7C).
Thus, Src was involved in the mediation of the response, although a
Src-independent pathway also existed.
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The MAP kinases Erk1/2 could be expected to mediate the
Src-dependent stimulation of VEGF expression. This cascade
is activated by NE in brown adipocytes in a cAMP-dependent
manner (22, 40). In other cell types, VEGF expression (induced by other
factors) has been implied to be induced via this signaling cascade (24, 30, 41-43). Therefore, we pretreated brown adipocytes with the MAP
kinase/Erk1/2 kinase (MEK1) inhibitor PD98059. In agreement with
earlier observations (22), PD98059 totally abolished NE-induced Erk1/2
phosphorylation (Fig. 8A).
However, PD98059 did not alter the NE-induced VEGF expression (Fig.
8B). Thus, despite the involvement of Src demonstrated
above, Erk1/2 did not mediate NE-induced VEGF expression in brown
adipocytes. In an attempt to characterize factors acting downstream of
Src tyrosine kinases on the stimulation of VEGF expression, we used the
general tyrosine kinase inhibitor, genistein (100 µM,
1 h pretreatment), but it decreased NE-induced VEGF expression to
a lesser extent than did PP2 (not shown).
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Phosphatidylinositol 3-kinase is a substrate for Src (44) and is a factor implicated in the activation of VEGF expression (45). However, wortmannin (100 nM, 1 h pretreatment), an inhibitor of phosphatidylinositol 3-kinase, had no effect on NE-induced VEGF expression (not shown). Thus, phosphatidylinositol 3- kinase-dependent signaling pathways did not appear to be involved.
Norepinephrine Does Not Require Induced Protein Synthesis to Induce VEGF Gene Expression
To investigate whether synthesis of new transcription factors, or
other protein factors, were required for NE-induced VEGF expression, we
examined the effect of the translational inhibitor cycloheximide.
Cycloheximide in itself led to an induction of VEGF mRNA (Fig.
9). A similar effect has been described
in other systems (46, 47). These experiments imply either that a
short-lived protein factor may constitutively inhibit VEGF
transcription or, perhaps more likely, that such a factor may promote
VEGF mRNA degradation (in other systems, cycloheximide has been
demonstrated to stabilize VEGF mRNA (48)).
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The effect of NE was additive to that of cycloheximide (Fig. 9) and
thus not dependent on protein synthesis. Thus, although NE may induce
the gene expression of certain transcription factors (such as
hypoxia-inducible
factor-1
),5 such induction
cannot be involved in the mediation of the NE effect. The lack of need
for protein synthesis is also in accordance with observations in other
systems, with other inducers of VEGF expression (46, 49). Thus, all
factors required for regulation of VEGF expression appeared to be
constitutively produced and present in brown adipocytes.
The Effect of Norepinephrine on VEGF Gene Expression Is Transcriptional
The augmenting effect of NE on VEGF mRNA levels could be due
to an increase in the rate of transcription of the VEGF gene or to an
increase of VEGF mRNA half-life. Indeed, in several cell types, the
half-life of VEGF mRNA is positively affected by other agents, such
as hypoxia (50, 51), PKC activation (48), and nitric oxide (52). To
investigate whether NE functions by prolonging VEGF mRNA half-life,
we treated brown adipocytes with the transcriptional inhibitor
actinomycin D. The VEGF mRNA half-life, as measured after
actinomycin D treatment, was approximately 1.5 h (Fig.
10) (compare also the rate of
spontaneous decrease seen in Fig. 2B), which is similar to
earlier observations (51, 53). NE did not affect the half-life of VEGF
mRNA (Fig. 10). Thus, the effect of NE was probably due to a direct
increase in the rate of transcription.
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DISCUSSION |
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In the present investigation, we have demonstrated that the
adrenergic agent NE induces the expression of the gene for the potent
angiogenic factor VEGF in primary cultures of mouse brown adipocytes.
The stimulatory effect by NE occurred independently of that induced by
other factors known to induce VEGF expression in other cell systems.
Signaling was via a
-adrenoreceptor-induced increase in cAMP levels,
further mediated by PKA. Downstream of cAMP/PKA, the signal was
mediated by Src tyrosine kinases and also through a Src-independent
pathway. However, the Src pathway did not involve Erk1/2 MAP kinases
(Fig. 11).
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Induced synthesis of protein factors was not required for mediation of the adrenergic stimulation. The mechanism of action was not prolongation of VEGF mRNA half-life, and was thus apparently an increase in the rate of transcription.
Norepinephrine Stimulation of VEGF Gene Expression-- That adrenergic stimulation of cultured rat (11) and mouse brown adipocytes stimulated VEGF expression, indicates parenchymal localization of the NE-induced expression observed in intact animals by Asano et al. (9). Although NE is not generally recognized as an inducer of VEGF expression, the effect is not exclusive for brown adipocytes, since also in rat aortic smooth muscle cells, adrenergic stimulation induces VEGF expression (54).
Adrenergic stimulation of VEGF expression in brown adipose tissue is physiologically meaningful in that also the oxygen-demanding thermogenic processes are adrenergically stimulated. However, the transient induction of VEGF expression, demonstrated here in cultured brown adipocytes, where elevated VEGF mRNA levels were observed only for the first few hours during chronic stimulation, may be thought to imply that adrenergic stimulation of VEGF expression may not be of major significance in regulating physiological angiogenesis. VEGF expression induced by the hypoxia-mimicking agent cobalt was maintained for a longer period of time, and thus could be considered more physiologically relevant, as hypoxic conditions may be generated in brown adipose tissue in the cold-exposed animal, due to the extensive oxygen consumption by the adrenergic thermogenic processes (2, 3). However, in brown adipose tissue in cold-exposed rats (9) and mice,6 cold-induced VEGF expression is also transient and thus, in this respect, very similar to our observations in NE-stimulated cultured brown adipocytes. Thus, it is likely that NE is the physiological inducer of VEGF expression and thereby of cold-induced angiogenesis. Apparently, an initial short elevation of VEGF expression is sufficient to support angiogenesis during brown adipose tissue recruitment.
-Adrenoreceptors, cAMP, PKA, and Src but Not Erk1/2 Mediate the
Norepinephrine-induced VEGF Gene Expression--
The classical
-adrenoreceptor pathway, cAMP and PKA, was fully responsible for the
mediation of the NE effect. In several cell types, forskolin and/or
cAMP-analogues have earlier been demonstrated to induce VEGF expression
(27, 46, 54-62). In most of these cell types, as yet only an ability
of cAMP to induce VEGF expression has been demonstrated, but it has
been implied that the relevant physiological activator also in these
cells induces VEGF expression via cAMP. Further mediation has, however, not been clarified.
It is in this context especially noteworthy that in brown adipocytes,
Src tyrosine kinases are activated via a
3-adrenoreceptor/cAMP/PKA pathway.4 This is
of relevance because Src earlier has been demonstrated to mediate
hypoxia-induced VEGF expression (41) and has been suggested to be
involved in tumor-induced angiogenesis by mediating enhanced VEGF
expression (63, 64). Indeed, in brown adipocytes we found that specific
inhibition of Src tyrosine kinases, by PP2 treatment, markedly reduced
VEGF expression in brown adipocytes stimulated by NE; in agreement with
observations in hypoxia-treated cell lines (41), this inhibition was
only partial. Thus, in brown adipocytes, Src tyrosine kinases appear to
be involved in the mediation of NE-induced VEGF expression.
In glioblastoma U87 and fibrosarcoma HT1080 cells, Src-dependent VEGF expression is further mediated via Erk1/2 MAP kinases (41, 43), and the Src-Erk1/2 signaling cascade appears to be important for the enhanced VEGF expression in other tumor cell types (42). However, the Erk1/2 MAP kinases were not involved in mediating the NE-induced VEGF expression in brown adipocytes. The unexpected lack of involvement of Erk1/2 demonstrates the existence of a cAMP-induced Src-mediated signaling pathway, positively connected to VEGF expression, which is not dependent on downstream mediation via Erk1/2.
Src has numerous substrates involved in various cellular processes
(44), but the factors mediating the adrenergic stimulation of VEGF
expression downstream of Src are presently unknown. As the effect of NE
does not involve prolongation of VEGF mRNA half-life, an increase
in the rate of transcription of the VEGF gene is to be expected.
Furthermore, as the NE induction of VEGF expression is not dependent of
protein synthesis, the transcription factor involved must be
constitutively expressed. The Src-dependent NE-induced VEGF
expression may involve the transcription factor Sp1, which can mediate
Src-dependent gene expression (65) and PDGF-induced VEGF
expression (66). Also NF
B may mediate Src-dependent gene expression (44) and thus may be involved. For these transcription factors, putative binding sites (consensus sequences) exist in the VEGF
promoter (67, 68).
In conclusion, we have here demonstrated that adrenergic stimulation of
VEGF expression in brown adipocytes is exclusively mediated by a
-adrenoreceptor/cAMP/PKA signaling pathway. This pathway can utilize
a Src-dependent pathway that branches off from the
Src-Erk1/2 signaling cascade to stimulate expression of the VEGF gene.
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ACKNOWLEDGEMENTS |
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We thank Professor Barbara Cannon for valuable discussions and Dr. Georg Breier for kindly providing the VEGF cDNA clone.
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FOOTNOTES |
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* This work was supported by a grant from the Swedish Cancer Foundation and by the Swedish Natural Science Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 46-8-164130;
Fax: 46-8-156756; E-mail: mf@zoofys.su.se.
§ On leave from the Institute of Cell Biophysics, Russian Academy of Sciences, 142 292 Pushchino, Russia.
2 J. M. Fredriksson, J. M. Lundquist, and J. Nedergaard, unpublished observations.
4 J. M. Lindquist, unpublished observations.
5 J. M. Fredriksson, H. Nikami, and J. Nedergaard, unpublished observations.
6 H. Nikami J. M. Fredriksson, and J. Neergaard, unpublished observations.
3 J. M. Fredriksson, H. Thonberg, K. B. E. Ohlson, B. Cannon, and J. Nedergaard, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: NE, norepinephrine; Erk1/2, extracellular-signal regulated kinase 1/2; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKC, protein kinase C; PTX, pertussis toxin; TPA, 12-O-tetradecanoylphorbol-13-acetate; VEGF, vascular endothelial growth factor; UCP1, uncoupling protein-1.
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