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INTRODUCTION |
The complex balance between cell proliferation and the normal cell
death in a tissue is crucial for the maintenance of tissue homeostasis.
It is fundamental for the normal physiology of the organism, conferring
functionality and viability. Alterations in this balance, leading to
excessive cell death, cell survival, cell proliferation, or a
combination of some of them, could play a role in a great number of
disease states (1).
The regulation of mitotic cell growth has been extensively studied, and
the dependence of growth factors and their signaling pathways in
different cell types established (2). There is convincing evidence that
cells also depend on hormone and/or growth factor stimulation for their
survival. In the absence of signals from other cells, the cell may
activate an intrinsic suicide program and kill itself with the typical
features of programmed cell death (3).
Several peptide growth factors (e.g.
NGF,1 insulin-like growth
factor-1, bFGF, and platelet-derived growth factor) have been shown to
promote cell survival of specific cell types, and the characterization
of their corresponding signaling pathways has identified proteins that
are critical mediators of cell survival. For example, insulin-like
growth factor-1 was recently shown to promote the survival of
cerebellar neurones, which was mediated via phosphatidylinositol
3-kinase-dependent activation of the protein kinase Akt,
also known as PKB or RAC (4). Basic fibroblast growth factor (bFGF) has
been shown to suppress tumor necrosis factor-
mediated apoptosis in
L929 cells via a Erk1/2 dependent pathway (5). Similarly, it was
recently also shown that insulin-like growth factor-1 attenuated tumor
necrosis factor- induced apoptosis in cultured fetal brown
adipocytes (6).
Norepinephrine (NE) is a neurotransmitter with pleiotropic effects on
brown adipocytes. It has been implicated both in the regulation of
brown adipose tissue (BAT) hyperplasia and in the differentiation of
brown adipocytes. Furthermore, the acute thermogenic response to low
ambient temperature is mediated by NE released from sympathetic
neurones innervating the tissue. Two phases can be distinguished during
physiological activation (i.e. cold exposure of an animal)
of BAT: an initial phase responsible for the acute heat production; a
recruitment phase involving mitochondriogenesis, increased protein, and
DNA synthesis, ultimately resulting in an increase in the number of
cells in the tissue (7). Likewise, when a cold adapted animal is
transferred to higher ambient temperature there is an abrupt cessation
of the thermogenic activity in the tissue and a new adaptation process
starts. This adaptation involves rapid degradation of RNA and proteins,
decreasing mitochondrial content, the amount of nuclear DNA in the
tissue decreases, and eventually the BAT mass is decreased (8).
Although the regulation of the cell number in BAT has long been
considered in terms of mitotic cell growth (9-14), physiological regulation of the cell survival may also play an important part in the
tissue hyperplasia. The concerted action of these two processes, decreased apoptosis accompanied by increased mitotic cell growth, would
be a way to rapidly increase the tissue size to meet the metabolic
demand for increased thermogenesis.
In the present study we demonstrate that low ambient temperature
(4 °C) caused a rapid increase in the phosphorylation of Erk1/2 in
BAT, and promoted the survival of cells in the tissue. Termination of
the stimulus, by transferring cold adapted animals to 28 °C, caused
a marked increase in the rate of apoptosis in the tissue. We further
show that in primary cultures of BAT Erk1/2 has a critical role in
promoting NE- and bFGF-dependent cell survival. NE
stimulated the Erk cascade through both 1- and
-adrenergic receptors mediated by Ca2+ and cAMP via the
Erk kinase MEK. NE also stimulated the expression and secretion of bFGF
from the cells, which further promoted the cell survival via
MEK-dependent activation of Erk1/2. This is, to our
knowledge, the first demonstration of adrenergic regulation of cell
survival mediated via the Erk1/2 cascade.
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MATERIALS AND METHODS |
Animals and Treatments--
3-week-old male mice (NMRI out-bred
strain; Eklunds, Stockholm, Sweden) were kept at 28 °C for at least
7 days with free access to food and water. At the start of the
experiments the animals were either maintained at 28 °C or
transferred to 4 °C for the indicated times. The animals were
sacrificed (CO2 euthanasia) and the interscapular BAT
dissected, frozen in liquid nitrogen, and stored at 
70 °C until
analyzed. For primary cultures of BAT, mice were kept at 22 °C with
free access to food and water for 2-3 days before sacrifice.
Cell Culture--
Brown adipocyte precursor cells were isolated
from the interscapular, the axillary, and the cervical BAT depots of
3-week-old male mice, and grown in culture as earlier described (15,
16). Briefly, pooled tissues was minced in a Hepes-buffered solution containing 0.2% (w/v) collagenase type II (Sigma) and digestion was
allowed for 30 min at 37 °C. The digest was filtered through 250- and 25-µm nylon filters to remove undigested parts and mature cells.
The precursor cells were pelleted by centrifugation (700 × g), washed in Dulbecco's modified Eagle's medium,
pelleted, and resuspended in 0.5 ml of culture medium/mouse. The
precursor cells were inoculated into multiwell culture dishes (Corning, Falcon) at a density of 1-2 × 104
cells/cm2. Cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% newborn calf serum, 4 nM insulin, 25 µg/ml sodium ascorbate, 10 mM
Hepes, 4 mM glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin at 37 °C in an atmosphere of 8% CO2 in
air. The cells were washed with Dulbecco's modified Eagle's medium on
day 1 and the medium was changed on day 1, 3, 5, and 7 of culture.
Hoescht DNA Content Determination--
The DNA amount in cell
cultures and tissue homogenate was measured by a method described by
Downs and Wilfinger (17), and modified by West et al. (18).
Calf thymus DNA (Sigma), the concentration determined
spectrophotometically at 260 nm, was used as standard. Tissue
homogenates and cultured cells were sonicated and 50 µl of the cell
suspension was treated with 1.45 ml of ice-cold 10 mM EDTA
(pH 12.3) in order to dissolve the DNA. Samples were incubated at
37 °C for 30 min and subsequently cooled on ice, neutralized with 75 µl of 1 M KH2PO4, and left on ice
for 10 min. One volume of Hoescht 33258 (Serva) dissolved in Tris
buffer, pH 7.0 (0.2 µg/ml), was added to the samples and the
fluorescence measured in a Perkin-Elmer fluorescence spectrophotometer
(MPF-2A; excitation 
350 nm, emission 455 nm).
Agarose Gel Assay for DNA Fragmentation--
Interscapular BAT
from warm adapted (28 °C, 5 weeks) and cold adapted (4 °C, 5 weeks) mice were homogenized in a lysis buffer containing 5 mM Tris, 20 mM EDTA, and 0.5% Triton X-100 (pH
8.0) and left on ice for 30 min. The tissue homogenates were
centrifuged (14,000 rpm, 30 min) to separate the cytosolic and the
nuclear fraction. The cytosolic fraction from the same amount of cells (determined by Hoescht 33258 DNA measurements) were treated with 200 µg/ml proteinase K (Sigma) overnight at 50 °C. Protein was extracted once with phenol/chloroform followed by one chloroform extraction. DNA was precipitated with 0.1 volume of 2.5 M
NH4Ac and 2 volumes of 100% ethanol. Precipitated DNA was
dissolved in 100 µl of TE (10 mM Tris, 1 mM
EDTA, pH 7.4) and treated with 100 µg/ml RNase (Ambion) for 90 min at
37 °C. DNA was precipitated as above, and the DNA fragments were
separated on a 1.5% SEAPLAQUE GTG (FMC BioProducts) agarose gel and
visualized with ethidium bromide under UV light.
Enzyme-linked Immunosorbent Assay for Internucleosomal DNA
Fragmentation--
DNA fragmentation in the tissues and cell cultures
was quantified by measuring cytosolic histone-associated DNA fragments using an enzyme-linked immunosorbent assay kit (Boehringer Mannheim). Interscapular BAT was homogenized in the lysis buffer and left on ice
for 30 min. The homogenates were centrifuged at 14,000 rpm in an
Eppendorf centrifuge for 30 min at 4 °C to separate the cytosolic
and nuclear fractions.
For the cell cultures, medium was discarded and the cells were lysed
directly in the well with lysis buffer for 30 min on ice. The cells
were harvested by scraping in the lysis buffer and centrifuged 14,000 rpm for 30 min at 4 °C. Histone-associated DNA fragments was
measured in the supernatant (cytosolic fraction) according to the
manufacturer's recommendation. Briefly, microtiter plate wells
(Costar) were coated with anti-histone antibody for 60 min at room
temperature or 4 °C overnight. The samples were added and incubated
in the wells for 90 min at room temperature allowing antibody histone
binding. To ensure equal loading of cells in the assay a volume
corresponding to the same amount of DNA, determined in the cell
homogenates/lysates, from each sample was analyzed. The wells were
washed, and an anti-DNA-antibody conjugated with peroxidase added to
the wells, incubated for 90 min at room temperature and washed. The
immunocomplex was detected by a substrate solution containing ABST®
(2,2'-azino-di-3-ethylbenzthiazoline sulfonate) and the absorbance
measured at 405 nm in a Titrtek Multiscan. The absorbance values were
used to calculate the percentage of DNA fragmentation compared with
control, with the control values set to 100%.
TUNEL Assay--
Primary cultures of BAT were grown in culture
on glass coverslips until day 8. Medium was discarded and the cells
were washed and fixed in 4% paraformaldehyde in phosphate-buffered
saline for 30 min at room temperature. Cells were incubated with 0.3% H2O2 in methanol for 30 min at room temperature
to block endogenous peroxidase. Staining was performed with the
In Situ Cell Death Detection Kit POD from Boehringer
Mannheim according to the manufacturers instructions. The amount of
positive (apoptotic) cells were analyzed by light microscopy.
Erk1/2 Kinase Activity--
Erk1/2 kinase activity was measured
using a Erk1/2-dependent Elk-1 phosphorylation assay (New
England BioLabs), according to the manufacturer's instructions. After
the indicated times, cells were washed once with ice-cold
phosphate-buffered saline and lysed directly in the well with the
provided lysis buffer supplemented with 2 mM Pefabloc®SC
(Boehringer Mannheim) protease inhibitor. Activated Erk1/2 was
immunoprecipitated from 200 µl of cell lysates using 4 µl of a
phospho-specific (human Tyr(P)-204) Erk1/2 antibody (rabbit polyclonal
IgG) and 20 µl of anti-rabbit IgG-agarose (50% beads in
phosphate-buffered saline; Sigma). Immunocomplexes were incubated for
30 min at 30 °C in 50 µl of kinase buffer containing 1 µg of
GST-Elk1 fusion protein (expressed in Escherichia coli) (New
England BioLabs). The phosphorylation of Elk-1 was analyzed by Western
blotting with a phospho-Elk-1 (Ser-383) specific antibody (rabbit
polyclonal IgG) and chemiluminescence.
Western Blot--
Proteins were separated on 12.5%
polyacrylamide gels and electrotransferred to Hybond-C Extra
nitrocellulose membranes (pore size, 0.45 µm; Amersham) with a
semidry electroblotter. After transfer, the membranes were allowed to
soak in Tris-buffered saline (TBS) for 5 min followed by quenching (5%
non-fat dry milk, 0.1% Tween 20 in TBS) of nonspecific binding for
3 h at room temperature or overnight at 4 °C. The membranes
were incubated with primary antibodies, Erk1/2 phosphospecific antibody
(human Tyr(P)-204, New England BioLabs), Erk2 antibody (New England
BioLabs), B-Raf antibody (Santa Cruz Biotechnology), or Rap-1 antibody
(Transduction Laboratories), overnight at 4 °C. The primary antibody
was detected with the Phototope®-horseradish peroxidase Western blot
Detection kit (New England BioLabs) according to the manufacturer's
recommendation, or by using a secondary horseradish
peroxidase-conjugated goat anti-rabbit antibody (Sigma) and enhanced
chemiluminescence (ECL, Amersham). The blots were exposed to Kodak
X-Omat RP films and quantified on a Molecular Dynamics densitometer.
Chemicals--
Basic fibroblast growth factor (heparin
stabilized, human), L-norepinephrine bitartate (arterenol),
isoprenaline (L-isoproterenol D-bitrate),
phorbol 12-myristitate 13-acetate (TPA), A23187, forskolin, and
collagenase (type II) were obtained from Sigma. Cirazoline was from
RBI. CGP-12177 was a gift from Ciba-Geigy. Anti-bFGF neutralizing
antibody was from R&D Systems. Bovine serum albumin (albumin, fraction
V) was from Boehringer Mannheim. PD98059 was from New England BioLabs.
All agents were freshly dissolved in water except TPA, forskolin, and
PD98059 which were dissolved in dimethyl sulfoxide.
Statistical Analysis--
Results are presented as the mean
values ± S.E. The Student's t test (unpaired) was
used to test for differences between the different treatments and/or
controls (*, p 
0.05; **, p 0.01; (***, p 0.001).
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RESULTS |
Decreased Rate of Apoptosis in Brown Adipose Tissue of Cold Exposed
Animals--
In an attempt to investigate the role of programmed cell
death or apoptosis in the physiological regulation of brown adipose tissue hyperplasia cytosolic fragmented DNA, a hallmark of apoptosis, was analyzed in mice cold exposed (4 °C) for 5 weeks. It was found that the amount of cytosolic fragmented DNA was markedly decreased in
BAT of the cold adapted animals (Fig.
1A). We next analyzed the rate
of DNA fragmentation in the tissue of mice exposed to cold for up to 20 days. It was found that after 1 day of cold exposure DNA fragmentation
in the tissue was significantly decreased by more than 50%
(p 0.05), which was sustained for the whole 20-day
experimental period (Fig. 1B). The effect of cold was
specific to the brown fat cell lineage, since epididymal white fat did not show any alteration in the DNA fragmentation under these
physiological conditions (not shown).
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Fig. 1.
Ambient temperature regulation of apoptosis
in mouse brown adipose tissue. A, mice were cold
exposed (4 °C), or kept at near thermoneutral temperature (28 °C)
for 5 weeks, and DNA fragmentation in the interscapular BAT was
analyzed by an agarose gel assay, as described under "Materials and
Methods." Mw, DNA molecular weight markers. B,
time course of DNA fragmentation in the interscapular BAT of mice
exposed to cold for different lengths of time. Mice were pre-acclimated
to 28 °C for at least 7 days, and then cold exposed
(Clod), or remained at 28 °C (Control), for
the indicated times. All animals were sacrificed on the same day,
except for some of the control animals that were also killed at the
start of the experiment. DNA fragmentation was measured using an
enzyme-linked immunosorbent assay, as described under "Materials and
Methods." C, time course of DNA fragmentation in the
interscapular BAT of cold-adapted animals transferred to near
thermoneutral temperature. Mice were acclimated to 4 °C for at least
3 weeks and then transferred to 28 °C for the indicated times
(Readaptation). Control animals were kept at 28 °C
(Control). The animals were sacrificed and DNA fragmentation
was analyzed as described in B. Results are normalized to
the mean value of the controls. Values are mean ± S.E. from two
animals per point. Data are representative of two to four independent
experiments each done in duplicate.
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Increased Rate of Apoptosis in Brown Adipose Tissue of Cold Adapted
Animals Transferred to Thermoneutral Temperature--
We further
investigated if the reversal process (i.e. increased
apoptosis) could be seen in BAT of cold adapted animals
(i.e. 4 °C for at least 3 weeks) transferred to near
thermoneutral temperature (28 °C). It was found that DNA
fragmentation increased significantly in the tissue within 10 days at
28 °C (p 0.01), reaching control levels after 20 days (Fig. 1C). The data show that cessation of the
sympathetic stimulus leads to an increased rate of apoptosis in the tissue.
Taken together, these data indicate that physiological stimulation
(i.e. sympathetic nerve activation) of BAT promotes the survival of cells within the tissue, and thus that the regulation of
cell survival could be a process involved in the cold-induced hyperplasia of BAT.
NE Promoted Cell Survival in Primary Cultures of Brown Adipose
Tissue--
In order to study the signaling pathways and the molecular
mechanisms behind the cold-dependent cell survival we used
the primary culture system of mouse BAT. These cells are able to fully differentiate in vitro, as shown both by electron microscopy
and their ability to express the UCP-1 gene (16, 19, 20). The cells
show two distinct stages associated with time after plating, preconfluent proliferating cells (0-5 days in culture) and confluent differentiating cells (6 days and older). Besides the difference in
cell density, there is also a marked difference in fat accumulation between these two stages, which further indicate the differentiation state of the cells (15, 19). Since NE has been implicated in the
regulation of both proliferation and differentiation of brown
adipocytes (16, 20, 21), we hypothesized that NE also could be involved
in promoting the survival of the cells. In order to test this, primary
cultures of BAT were stimulated on day 4 or day 6 with NE (1 µM) for different lengths of time (i.e. 2 to 4 days of stimulation). In order to ensure sustained hormonal activity,
NE was added to the cultures every 24 h. It was found that NE
significantly decreased (
50%) the DNA fragmentation in cultures
treated with the hormone, compared with control cells (Fig.
2). Furthermore, the effect of NE
stimulation on cell survival showed a clear temporal dependence, since
only preconfluent proliferating cells were found to respond to the NE
treatment with decreased DNA fragmentation (Fig. 2). Once the cells
started the differentiation process NE was without effect. Moreover,
the effect of NE treatment on the cell survival remained for at least 2 days despite removal of the hormone (Fig. 2), indicating some kind of
cellular memory. Alternatively, the expression of an
autocrine/paracrine growth factor may be induced by the NE stimulation,
and secreted from the cells ensuring cell viability.
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Fig. 2.
The effect of chronic NE stimulation on
internucleosomal DNA fragmentation in brown adipocytes. BAT
precursor cells were grown in culture for 4 days. On day 4 the cells
were stimulated, except the controls (C), with 1 µM NE for 48 h. On day 6 the cultures were deprived
of NE, and grown for an additional 48 h; or 4-day cultures were
stimulated with 1 µM NE for 96 h; or 6-day cultures
were stimulated with 1 µM NE for 48 h. Medium was
changed on day 1, 3, 5, and 7. Fresh NE was added every 24 h. All
cells were harvested on day 8, and assayed for DNA fragmentation as
described under "Materials and Methods." Results are normalized to
the mean value of the controls. Values are means ± S.E. of two to
four experiments each done in duplicate, **, p < 0.01 indicate differences compared with control.
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We also analyzed the percentage of apoptotic cells in control and
NE-treated cultures, by counting positive cells in a TUNEL assay. It
was found that 8.9% of the cell in the control cultures were
apoptotic, and that NE stimulation markedly decreased the number of
apoptotic cells to 4.8% of the total cell population (Table
I). Thus, in the growing cultures 10%
of the cell population are dying by apoptosis. NE treatment of the
cultures markedly decreases the number of apoptotic cells (TUNEL
positive) by 50%, which correlates well to the NE-induced decrease in
DNA fragmentation (cf. Fig. 2).
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Table I
TUNEL staining of cultures of brown adipose tissue after chronic NE
treatment
BAT precursor cells were grown in culture for 4 days. The cells were
treated for 4 days, except controls, with 1 µM NE. Cells
were fixed and stained for DNA breaks with the in situ Cell
Death Detection Kit according to the manufacturers instructions. The
number of positive cells was determined by light microscopy.
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Characterization of the Adrenergic Receptors and Intracellular
Signaling Pathways Involved in Promoting Cell Survival--
Since NE
binds to both 1- and -adrenergic receptors, we
treated preconfluent cells (day 4) for 48 h with specific agonists for either 1- or -receptors (i.e.
cirazoline and isoprenaline, respectively). It was found that
cirazoline (1 µM) was able to fully mimic the effect of
NE on cell survival, whereas isoprenaline (1 µM)
partially mimicked the effect of NE (Fig.
3). Similarly, when the cells were
treated with the 3-receptor agonist CGP12177 (1 µM) (22) DNA fragmentation was decreased to the same
level as in isoprenaline-treated cells (not shown).
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Fig. 3.
The effect of adrenergic agonists, TPA, and
forskolin on internucleosomal DNA fragmentation in brown
adipocytes. BAT precursor cells were grown in culture for 4 days.
On day 4 the cells were stimulated for 48 h, except the controls
(C), as indicated: 1 µM NE, 1 µM
cirazoline (Cir), 1 µM isoprenaline
(Iso), 50 nM TPA, 5 µM forskolin
(Forsk). Medium was changed after 24 h and fresh agents
added. Cells were harvested on day 6, and assayed for DNA fragmentation
as described under "Materials and Methods." Results are normalized
to the mean value of the controls. Values are means ± S.E. of
four to eight experiments each done in duplicate; *, p < 0.05; **, p < 0.01; and ***, p < 0.001 indicate differences compared with control.
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Activation of 1-receptors has been shown to cause
increases in the intracellular Ca2+ level and stimulation
of protein kinase C (PKC) activity (23, 24). Agonist stimulation of
-receptors leads to activation of adenylyl cyclase, resulting in
increased intracellular cAMP level and activation of protein kinase A
(PKA). In order to further characterize the adrenergic signals involved
in the regulation of cell survival we analyzed the effect of these
post-receptor signals by treating the cells with the PKC activator TPA
(50 nM), and the adenylyl cyclase activator forskolin (5 µM) for 48 h. As shown in Fig. 3, TPA treatment of
the cells did not significantly alter the cell survival. However, there
was a tendency for increased apoptosis in the TPA-treated cell.
Forskolin, on the other hand, was found to mimic the effect of NE on
cell survival, indicating that PKA-dependent
phosphorylation could be involved in promoting cell survival.
NE-induced bFGF Expression and an Autocrine/Paracrine Effect of
bFGF--
It was recently shown that NE could induce bFGF expression
in primary cultures of rat BAT (25). Similarly, in primary cultures of
mouse BAT we found that NE increased the expression of bFGF mRNA
and that the cells expressed FGF-receptor-1
mRNA.2 In order to
investigate the role of bFGF and a possible autocrine effect of bFGF,
we stimulated the cells with bFGF, or NE together with a bFGF
neutralizing antibody, and analyzed the effects on cell survival. It
was found that bFGF stimulation of the cells promoted cell survival to
the same extent as NE stimulation did, and that the bFGF antibodies
attenuated the NE-dependent survival (Fig.
4). In a control experiment we also
tested whether the bFGF antibody could inhibit the effect of bFGF on
cell survival. It was found that the effect of bFGF was abrogated by
the bFGF antibody (not shown), which show that the antibody indeed
inactivated bFGF. These data indicated that part of the NE effect was
mediated by bFGF, and suggested a possible interaction of the NE and
bFGF signal in promoting the survival. In order to test for such an interaction, NE and bFGF were added together to the cells. As shown in
Fig. 4, simultaneous NE and bFGF stimulation of the cells significantly
decreased the DNA fragmentation by 74 and 50% compared with control
cells and NE- or bFGF-stimulated cells, respectively. These results
suggest an interaction of the NE and bFGF signal in promoting the
survival of brown adipocytes.
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Fig. 4.
The effect of bFGF, NE, and bFGF neutralizing
antibody on internucleosomal DNA fragmentation in brown
adipocytes. BAT precursor cells were grown in culture for 4 days.
On day 4 the cells were treated for 48 h, except the controls
(C), as indicated: 1 µM NE, 10 ng/ml bFGF, and
10 µg/ml bFGF neutralizing antibody, cells were pretreated with
-bFGF for 1 h before NE addition (-bFGF). Medium
was changed after 24 h and fresh agents added. Cells were
harvested on day 6, and DNA fragmentation was assayed as described
under "Materials and Methods." Results are normalized to the mean
value of the controls. Values are mean ± S.E. of three to eight
independent experiments each done in duplicate: **, p < 0.01, and ***, p < 0.001 indicate differences
compared with control. #, p < 0.05; ##,
p < 0.01; and  , p < 0.05 indicate
differences compared with NE and bFGF, respectively.
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NE- and bFGF-induced Activation of Erk1/2 in Primary Cultures of
Brown Adipose Tissue--
Since bFGF, via its receptor, is known to
activate the Erk1/2 signaling pathway, and NE recently also was shown
to be a potent activator of Erk1/2 (26, 27), the data suggested that
Erk1/2 may have a role in promoting NE- and bFGF-dependent
survival of brown adipocytes. In order to investigate the effect of NE
and bFGF on Erk1/2 activation in primary cultures of BAT, we first analyzed the effects of NE and bFGF on Erk1/2 phosphorylation with a
phospho-specific Erk1/2 antibody. As shown in Fig.
5A, stimulation of
preconfluent cells with either NE (1 µM) or bFGF (10 ng/ml) for 10 min caused a 5-fold increase in Erk1/2
phosphorylation. We next analyzed whether NE and bFGF stimulated Erk1/2
phosphorylation corresponded to increased Erk1/2 kinase activity, by
measuring Erk1/2-dependent Elk-1 phosphorylation in an
in vitro kinase assay. As shown in Fig. 5B,
stimulation of the cells with either NE or bFGF for 10 min resulted in
a 5-fold increase in Erk1/2 activity. In order to exclude the
possibility that the effect of NE on Erk1/2 activation was mediated by
NE induced secretion of bFGF and an autocrine effect of bFGF, we
measured the effect of NE stimulation in the presence of the bFGF
antibody. It was found that the NE stimulated Erk1/2 kinase activity
was unaffected by the presence of the bFGF antibody (data not shown).
These results show that acute NE stimulation of brown adipocytes cause
the phosphorylation and activation of Erk1/2. It further shows that the
acute effect of NE is mediated through the adrenergic receptors and not
via an autocrine/paracrine effect of bFGF.
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Fig. 5.
NE- and bFGF-induced phosphorylation and
activation of Erk1/2 in brown adipocytes, and inhibition by
PD98059. A, BAT precursor cells were grown in culture
for 3 days. On day 3 the medium was changed and the cells preincubated
in culture medium containing 0.5% serum for 2 h. The cells were
stimulated for 10 min, as indicated: 1 µM NE, 10 ng/ml
bFGF. Erk1/2 phosphorylation (P-Erk1/2) was measured using
Western blotting with a phospho-specific Erk1/2 antibody, an Erk2
antibody was used as a loading control, as described under "Materials
and Methods." B, BAT precursor cells were grown in culture
for 5 days. On day 5 the cells were treated for 10 min, as indicated: 1 µM NE, 10 ng/ml bFGF, 50 µM PD98059, the
cells were pretreated for 1 h before NE or bFGF addition
(PD98059). The Erk1/2 kinase activity was analyzed in an in
vitro kinase assay, measuring Elk-1 phosphorylation at Ser-383
with a phospho-specific Elk-1 antibody, as described under "Materials
and Methods." The IgG band represents the antibody used for
immunoprecipitation. Data are representative of two independent
experiments each done in duplicate.
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We also tested whether it was possible to inhibit the activation of
Erk1/2 by pretreating the cells with the MAP/Erk kinase (MEK)
inhibitor, PD98059 (50 µM), for 1 h before NE or
bFGF stimulation of the cells. As shown in Fig. 5B, PD98059
abrogated the NE and bFGF induced activation of Erk1/2. These data show
that in brown adipocytes NE and bFGF activates the Erk cascade through
a MEK-dependent signaling pathway.
1- and -Adrenergic Receptor-mediated
Phosphorylation of Erk1/2--
In order to characterize the adrenergic
receptors involved in the activation of Erk1/2, the cells were
stimulated for 10 min with specific 1- and -receptor
agonists (i.e. cirazoline and isoprenaline, respectively).
As shown in Fig. 6, both cirazoline (1 µM) and isoprenaline (1 µM) markedly
increased the phosphorylation of Erk1/2 (2-3-fold) compared with
control cells. Since 1-receptor stimulation cause
increases in the intracellular Ca2+ level and activation of
PKC, the cells were treated with the Ca2+ ionophore A23187
(1 µM) and the PKC activator TPA (50 nM) for 10 min, and the phosphorylation of Erk1/2 assayed. As shown in Fig. 6,
both A23187 and TPA markedly increased the phosphorylation of Erk1/2
(5- and 10-fold, respectively) compared with control cells. Notably,
TPA was the most potent inducer of Erk1/2 phosphorylation tested. In
order to mimic -receptor stimulation, cells were treated with
forskolin (5 µM) (i.e. increasing the
intracellular cAMP level) for 10 min and the phosphorylation Erk1/2
analyzed. As shown in Fig. 6, forskolin caused a 3-fold increase in
the phosphorylation of Erk1/2, which show that in brown adipocytes cAMP
is a potent activator of Erk1/2.
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Fig. 6.
1- and -adrenergic receptor
mediated Erk1/2 phosphorylation in brown adipocytes. BAT precursor
cells were grown in culture for 3 days. On day 3 the medium was changed
and the cells preincubated in culture medium containing 0.5% serum for
2 h. The cells were treated for 10 min, as indicated: 1 µM NE, 1 µM cirazoline (Cir), 1 µM isoprenaline (Iso), 50 nM TPA,
5 µM forskolin (Forsk), or 1 µM
A23187 (A23). Erk1/2 phosphorylation (P-Erk1/2)
was measured using Western blotting with a phospho-specific Erk1/2
antibody, an Erk2 antibody was used as a loading control, as described
under "Materials and Methods." Data are representative of two
independent experiments each done in duplicate.
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Expression of Rap1 and B-Raf in Brown Adipocytes--
It has
recently been shown that cAMP activates the Erk cascade through a PKA-,
Rap1-, and B-Raf-dependent signaling pathway (28). Although
Rap1 and B-Raf are most abundantly expressed in cells of neuronal
origin, expression has been shown in other cell types (29, 30). In
order to investigate whether brown adipocytes expressed B-Raf and Rap1,
4-day-old primary cultures of BAT were analyzed by Western blot with
specific antibodies against B-Raf and Rap1. As shown in Fig.
7A, the 67-kDa short form of
B-Raf was the predominantly expressed isoform in the brown adipocytes.
Similarly, we also found that Rap1 was expressed in the brown
adipocytes (Fig. 7B), indicating that a cAMP > PKA > Rap1 > B-Raf signal cascade is present in brown
adipocytes.
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Fig. 7.
Expression of B-Raf and Rap1 in brown
adipocytes. Western blot analysis of of B-Raf expression
(A): mouse cerebellum (positive control) (lane 1)
and 4-day old primary cultures of BAT (lane 2). Western blot
analysis of of Rap1 expression (B): A431 cells (positive
control) (lane 1) and 4-day old primary cultures of BAT
(lane 2).
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Inhibition of MEK Abrogates the Effect of NE and bFGF on the Cell
Survival--
Our data showed that both NE and bFGF activated Erk1/2
in primary cultures of BAT, and suggested that NE- and
bFGF-dependent cell survival could be mediated by the
Erk1/2 signaling pathway. In order to investigate the role of Erk1/2 on
cell survival we exposed the cells to either NE or bFGF in combination
with the MEK inhibitor PD98059 (50 µM). As shown in Fig.
8, inhibition of MEK abolished the effect
of NE and bFGF on cell survival, indicating a critical role for Erk1/2
in promoting NE- and bFGF-dependent cell survival.
Furthermore, PD98059 in itself significantly increased cell death
compared with untreated control cells, suggesting that an intact Erk1/2
signaling pathway also is important for the "normal" cell
survival.
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Fig. 8.
The effect of PD98059 on NE- and
bFGF-dependent survival of brown adipocytes. BAT
precursor cells were grown in culture for 4 days. On day 4 the cells
were treated for 48 h, as indicated: 1 µM NE, 10 ng/ml bFGF, 50 µM PD98059, the cells were pretreated for
1 h before NE or bFGF addition (PD98059). Medium was changed after
24 h and fresh agents added. Cells were harvested on day 6 and the
DNA fragmentation was assayed, as described under "Materials and
Methods." Values are mean ± S.E. of five to eight experiments
done in duplicate: *, p < 0.05; ***,
p < 0.001 indicate differences compared with control.
###, p < 0.001 and , p < 0.001 indicate differences compared with NE and bFGF,
respectively.
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Cold-induced Phosphorylation of Erk1/2 in Brown Adipose
Tissue--
The results form the cell cultures showed a critical role
for Erk1/2 in promoting NE- and bFGF-dependent cell
survival. In order to investigate whether the
cold-dependent cell survival in BAT could be correlated to
Erk1/2 activation, we analyzed the effects of cold exposure on Erk1/2
phosphorylation in BAT. Mice were cold exposed for different lengths of
time (i.e. 30 min to 20 days) and the phosphorylation of
Erk1/2 was assayed in BAT. It was found that within 30 min of cold
exposure the phosphorylation of Erk1/2 was markedly increased, and that
the phosphorylation level of Erk1/2 was sustained during the whole
20-day experimental period (Fig. 9).
These data show a clear correlation between Erk1/2 activation and
cold-dependent cell survival, and indicate that the Erk1/2
signaling pathway may have a critical role in promoting cold-dependent cell survival in BAT.
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Fig. 9.
Cold-induced Erk1/2 phosphorylation in mouse
brown adipose tissue. Three-week-old mice were pre-acclimated to
28 °C for 7 days and then placed in the cold (4 °C), or remained
at near thermoneutral temperature (28 °C), for the times indicated.
All animals were sacrificed on the same day. Total protein from
interscapular brown adipose tissue was analyzed for Erk1/2
phosphorylation (P-Erk1/2) by Western blotting with a
phospho-specific Erk1/2 antibody, an Erk2 antibody was used as a
loading control, as described under "Materials and Methods." The
gel show one representative series from one experiment done in
duplicate.
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Although the in vivo data could be interpreted as if
sustained activation of the Erk cascade is obligatory to decrease the rate of apoptosis in the tissue, the in vitro data showed
that the cells only had to be stimulated for 48 h with NE to
decrease cell death (cf. Fig. 2). The in vitro
data further showed that the effect of NE treatment on cell survival
remained for at least 2 days despite removal of the hormone
(cf. Fig. 2), indicating that sustained activation of the
Erk cascade may not be necessary to induce a decrease in the rate of apoptosis.
Different Kinetics of the NE- and bFGF-induced Activation of
Erk1/2--
In order to investigate the kinetics of the NE- and
bFGF-induced phosphorylation of Erk1/2, we stimulated 4-day-old BAT
primary cultures for different lengths of time with NE (1 µM) or bFGF (10 ng/ml). As shown in Fig.
10, A and B, NE
caused a rapid increase in the phosphorylation of Erk1/2 (8-fold)
within 10 min of stimulation. The level of phosphorylated Erk1/2
decreased within 30 min of NE stimulation to 3-fold over basal, and
was back to control levels within 4 h of stimulation. In contrast,
bFGF caused sustained phophorylation of Erk1/2; the activation of Erk
was sustained for more than 4 h following bFGF stimulation.
However, the initial kinetics of bFGF- and NE-induced Erk1/2 activation
was similar.
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Fig. 10.
Kinetics of the NE- and bFGF-induced Erk1/2
phosphorylation in primary cultures of brown adipose tissue.
A, BAT precursor cells were grown in culture for 4 days. On
day 4 the medium was changed and the cells preincubated in culture
medium containing 0.5% serum for 2 h and then treated with 1 µM NE or 10 ng/ml bFGF for the times indicated. Erk1/2
phosphorylation (P-Erk1/2) was measured using Western blotting with a
phospho-specific Erk1/2 antibody, an Erk2 antibody was used as a
loading control, as described under "Materials and Methods."
B, computation of the results obtained in A, the
control level was set to 100%. The sum of P-Erk1 and P-Erk2 are
shown.
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Different Kinetics of the NE- and bFGF-promoted Cell
Survival--
Since NE and bFGF showed different kinetics in the
activation of the Erk cascade, it was possible that the effect of NE
and bFGF stimulation also could show different kinetics in promoting the cell survival. In order to test this, the cells were stimulated on
day 5 for 24 h with either NE (1 µM) or bFGF (10 ng/ml). As shown in Fig. 11, 24 h
bFGF stimulation of the cells decreased the rate of apoptosis. In
contrast, 24 h of NE stimulation was not enough to alter the cell
survival, irrespective of when the stimulation was initiated
(i.e. day 4-5 (not shown) or day 5-6).
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Fig. 11.
Kinetics of the NE- and bFGF-induced cell
survival. BAT precursor cells were grown in culture for 5 days. On
day 5 the cells were treated for 24 h, except the controls
(C), as indicated: 1 µM NE, 10 ng/ml bFGF. All
cells were harvested on day 6, and assayed for DNA fragmentation as
described under "Materials and Methods." Results are normalized to
the mean value of the controls.
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DISCUSSION |
In the present study we demonstrate that cold acclimation
(4 °C) of an animal is accompanied by a decreased rate of apoptosis in BAT. Although acute cold exposure (less than 5 h) did not alter the rate of normal cell death (apoptosis) significantly, the rate of
apoptosis in BAT was markedly reduced already within 24 h of cold
exposure. The reduced rate of apoptosis was sustained as long as the
animals were exposed to the cold, that is, at least for up to 5 weeks
of cold exposure. In analogy, when cold-adapted animals were
transferred to near thermoneutral temperature (28 °C), leading to
decreased sympathetic stimulation of the tissue, the rate of apoptosis
increased within 10 days, reaching control level after 20 days. Thus,
the data show that the regulation of cell survival is a process
involved in the cold-induced hyperplasia of BAT, as well as in
eliminating redundant cells during the transition from the thermogenic
to the non-thermogenic state. The data further indicated that some
factors, released during the cold-dependent recruitment of
the tissue, could promote the survival of the cells, and that certain
cells in the tissue apparently are destined to undergo apoptosis unless
rescued by these survival factors.
There are several examples of growth factor-dependent cell
survival in the developing vertebrate. For example, the survival of
developing neurones depend on neurotrophic factors (e.g. NGF for sympathetic neurones) that are secreted by the target cells they
innervate; those that fail to get enough neurotrophic factors (e.g. NGF) die by typical programmed cell death (31, 32). However, there are only a few examples of hormonal or growth
factor-dependent regulation of cell survival (or apoptosis)
in tissues of adult animals. In adult hamsters, for example, the
reproductive activity is determined by the photoperiod; long days
stimulate testis development, while short days induce testis
regression. An increased rate of testis cell apoptosis has been shown
in Djungarian hamsters exposed to short photoperiods, which correlated
with decreases in serum testosterone levels. Similarly, when the
hamsters were transferred to long photoperiods testis cell apoptosis
was decreased, correlating with increased testosterone levels (33).
The immediate response of an animal to a cold environment is the
activation of the sympathetic nervous system; NE is released at the
nerve terminals triggering several physiological responses in BAT (34).
We show here that NE stimulation, agonist stimulation of
1- and -receptors (cirazoline and isoprenaline,
respectively), and bFGF stimulation of primary cultures of BAT promoted
the survival of the cells. NE and bFGF stimulation (48 h) of the cells
caused a similar decrease in the rate of apoptosis (50% decrease in DNA fragmentation). Furthermore, the effect of NE treatment on the cell
survival remained for at least 2 days despite removal of the hormone.
Similarly, cessation of the sympathetic stimulus, in the intact animal,
caused a delayed increase in the DNA fragmentation in BAT. These data
suggest that both the NE and the cold stimulus caused alterations in
the cellular composition of relatively stable character. The
anti-apoptotic effect of NE also showed a clear temporal dependence;
once the cells reached confluence and started differentiating NE was
without effect. It could be speculated that this may also be the case
in physiologically activated BAT, and thus that sympathetic stimulation
of BAT may only promote the survival of the proliferating precursor
cells in the tissue.
Intracellular Signals Promoting Cell Survival--
As the
NE-dependent cell survival apparently was mediated via both
1- and -receptors, it was possible that
Ca2+- and cAMP-dependent signaling pathways
could be involved in promoting the survival. We found that increasing
the intracellular cAMP level by treating the cells with forskolin
mimicked the effect of NE. However, forskolin was found to be more
potent than selective -receptor stimulation (i.e.
isoprenaline), which may be due to different levels of intracellular
cAMP generated by these agents (forskolin being the more potent), and
the duration of the signal. Thus, it is possible that the magnitude and
duration of the cAMP signal may be critical for the physiological
response of the cell.
Increases in the intracellular cAMP level has been shown to either
induce apoptosis or protect cells from apoptosis, depending on the cell
type and the circumstances. For example, in neutrophils and PC12 cells,
cAMP raising agents have been shown to protect the cells from apoptosis
(35-37) whereas it induces apoptosis in a variety of other cell types
(38-41). Clearly, our data show that in brown adipocytes increases in
the intracellular cAMP level promotes the cell survival.
In contrast to the effect of the 1-receptor agonist
cirazoline, TPA activation of PKC did not affect cell survival.
Similarly, in PC12 cells, growth factor withdrawal-induced cell death
cannot be rescued by TPA treatment (37). It has been suggested that Erk
and the stress-activated kinase, c-Jun N-terminal kinase (JNK) and p38
MAPK, cascade may have opposing effects on nerve cells, and that the
dynamic balance between Erk and the JNK/p38-MAPK signaling pathways is
important in determining whether a cell survives or undergoes apoptosis
(42). The induction of apoptosis in PC12 cells is likely dependent on
the balance between anti-apoptotic and apoptotic signals
(i.e. Erk and JNK/p38-MAPK, respectively) (42, 43). It has
recently been shown that TPA activates the p38 MAPK, Erk, and JNK
cascades in A3.01 T cells, and that both p38 MAPK and JNK were
synergistically activated by TPA and ionomycin (Ca2+
ionophore) (44). It is possible that TPA treatment of brown adipocytes
also could lead to activation of stress-activated JNK/p38 MAPK
cascades. Thus, the inability of TPA to rescue brown adipocytes from
apoptosis, despite that it was found to cause the most pronounced phosphorylation of Erk1/2, could be due to simultaneous stimulation of
the Erk and the stress-activated JNK/p38 MAPK cascade by TPA, and this
may alter the balance between anti-apoptotic and apoptotic signals.
Moreover, the observation that TPA stimulation of brown adipocytes
showed a tendency to increase the cell death, rather that the opposite,
gives further support to this notion.
Activation of Erk1/2 in Brown Adipocytes--
In agreement with
previous findings in rat brown adipocytes and rat cardiomyocytes (26,
27, 45), we found that NE, 1-, and -receptor agonists
activated the Erk cascade in mouse brown adipocytes. In cardiomyocytes,
1- and -receptor agonists synergistically activate
Erk through PKA- and PKC-dependent pathways; the activation of Erk was also shown to be dependent on the influx of extracellular Ca2+ (27, 45). Although we have not directly analyzed for a
possible synergism between 1- and -receptor
stimulation on Erk activation, our results show that, with the
concentrations used, NE stimulation of the cells resulted in a
5-fold increase in Erk1/2 phosphorylation, whereas cirazoline or
isoprenaline stimulation of the cells caused a 2-3-fold increase in
Erk1/2 phosphorylation. These data indicate that NE-induced Erk
activation is mediated via both 1- and -adrenergic receptors through increases in intracellular Ca2+ and cAMP
levels. The data further suggest that NE-induced Erk activation could
be mediated via Ca2+- and cAMP-dependent
kinases. Apparently, in brown adipocytes the signals from
1- and -receptors converge at some point in the
activation of Erk.
In PC12 and HEK-293 cells, extracellular signals that increase the
intracellular Ca2+ level activates the Ras > Raf > MEK > Erk pathway through Pyk2 (i.e. the
proline-rich tyrosine kinase, belonging to the family of non-receptor
protein tyrosine kinases) (46, 47). It has been suggested that
Ca2+/calmodulin could be involved in the activation of Pyk2
(47). Since we here show that both 1-receptor
stimulation and Ca2+ ionophore treatment of the cells
induces Erk activation, it could be suggested that
1-receptor activation of the Erk cascade in brown
adipocytes could be mediated by a
Ca2+/calmodulin-dependent Pyk2 pathway.
cAMP elevating agents has been shown to have both stimulatory and
inhibitory effects on Erk activation depending on the cell type (48),
and a number of possible sites for the interaction of cAMP/PKA in the
Erk signaling pathway has been described (28, 48-51). In brown
adipocytes, forskolin (i.e. increased intracellular cAMP
level) caused a 3-fold increase in Erk1/2 phosphorylation, which was
similar to the effect of -receptor stimulation. These results
indicate that -receptor-stimulated Erk activation is mediated by
cAMP/PKA. It has recently been shown that cAMP/PKA activates the Erk
cascade through a B-Raf and Rap1-dependent pathway (28).
Since we here show that brown adipocytes express both Rap1 and B-Raf,
it is possible that cAMP may activate the Erk cascade through the
PKA > Rap1 > B-Raf > MEK signaling pathway.
bFGF stimulation of the cells also results in Erk activation, similar
to the effect of NE. However, the kinetics of NE- and bFGF-induced
Erk1/2 phosphorylation was markedly different. NE caused transient Erk
activation (i.e. the phosphorylation of Erk1/2 peaked within
10 min, was markedly reduced within 30 min, and was back to control
level within 4 h of NE stimulation), while bFGF caused sustained
Erk activation (i.e. the phosphorylation of Erk1/2 was
sustained for more than 4 h following bFGF stimulation). Similarly, in PC12 cells, the activation of Erk is sustained for several hours following NGF stimulation, while epidermal growth factor
stimulation cause transient Erk activation. These differences may in
part be due to the differences in receptor down-regulation of the
epidermal growth factor receptor and the NGF receptor (TrkA) (52).
However, York et al. (53) recently reported that the activation of Erk by NGF, in PC12 cells, involves two distinct pathways: an initial rapid phase of Erk activation mediated by Ras, and
a sustained phase of Erk activation mediated by Rap1. It was further
shown that the cAMP analogue 8-(4-chlorophenylthio)-cAMP was able to
activate Rap1 for prolonged periods. cAMP raising agents has also been
shown to induce sustained Erk activation in PC12 cell (54).
As NE stimulation cause increases in the intracellular Ca2+
and cAMP level, it could be suggested that NE activates both the Ras
pathway and the Rap1 pathway in brown adipocytes. In analogy with NGF
(Ras and Rap1 activator), NE could be expected to cause sustained Erk
activation in brown adipocytes. However, as mentioned above, NE induces
transient Erk activation in brown adipocytes. This could not be
explained by a similar transient increase in the intracellular cAMP
level; the cAMP level in brown adipocytes remains markedly elevated
after 30 min of NE
stimulation.3 These
apparently contradictory results could perhaps be explained by some
form of interaction between different signals activated by NE. One
possibility is that NE also activates inhibitory signals, such as a
protein phosphatase(s), that could limit the activation of Erk.
Pretreating the cells with the MEK inhibitor PD98059 abrogated the NE-
and bFGF-induced activation of the Erk cascade, which show that, in
brown adipocytes, the signaling pathways used by G-protein-coupled
receptors (adrenergic) and tyrosine kinase receptors (FGFR) intersect
at MEK or upstream of MEK.
We further show that cold exposure of the animals caused a rapid
increase in the phosphorylation of Erk1/2 in BAT. Already within 30 min
of cold exposure the phosphorylation of Erk1/2 was markedly increased,
and the phosphorylation level was sustained as long as the animals were
exposed to the cold. Since the cold-induced activation of Erk was both
rapid and sustained in the tissue, it could be suggested that the
initial activation of Erk is due to the acute effect of sympathetic
stimulation mediated via adrenergic receptors, and that bFGF could be
responsible for the sustained activation. Interestingly, the
cold-induced phosphorylation pattern of Erk1 and Erk2 was similar
between the two isoforms, whereas in the cultured cells Erk2 was
the more heavily phosphorylated isoform (cf. Figs. 6
and 9).
Erk1/2-dependent Survival of Brown
Adipocytes--
Since both NE and bFGF were found to activate the Erk
signaling pathway, it was possible that the signal for cell survival could be mediated via the Erk pathway. We show here that both the NE-
and bFGF-dependent cell survival are dependent on Erk activation; inhibition of MEK with PD98059 abrogated the anti-apoptotic effect of both bFGF and NE. We further show that the anti-apoptotic effect of NE was slower than the effect of bFGF; it was necessary to
stimulate the cells for 48 h with NE to reduce the rate of apoptosis, while the effect of bFGF appeared within 24 h of
stimulation. This together with the finding that NE, in contrast to
bFGF, causes transient Erk activation may suggest that additional
NE-dependent signals (i.e. separate from the Erk
pathway) could be involved in promoting cell survival. However, it may
also be explained by down-regulation of the adrenergic receptors
involved in transmitting the signal. It has previously been shown that
NE stimulation of brown adipocytes cause a rapid down-regulation of
3-adrenergic receptor mRNA; within 2 h of NE
stimulation the cells were almost devoid of 3-receptor
mRNA. However, the down-regulation was transient to its nature and
within 18 h of NE stimulation the level of
3-receptor mRNA was back to control level (55).
Similarly, NE has also been reported to cause a transient
down-regulation of 1-adrenergic receptors (56, 57).
Thus, the transient down-regulation of the adrenergic receptors may in
part explain the differences between NE and bFGF in promoting cell
survival (i.e. 48 h NE exposure is required, while
24 h bFGF exposure is sufficient). It could be suggested that the
initial exposure of the cells to NE, causing transient Erk activation,
induces the expression of genes that are anti-apoptotic. However, as
the initial exposure to NE also causes a transient receptor
down-regulation, NE needs to be readded to the cells at a time when the
receptors have reappeared. Since we followed this scheme, the
readdition of NE to the cells may induce a second peak of Erk
activation, which could further enhance the expression of genes that
are anti-apoptotic.
Alternatively, since our data indicate that NE promotes the cell
survival partly by increasing the expression of bFGF and partly by a
direct effect of NE (Fig. 12), the
differences between NE and bFGF in promoting cell survival may in part
be explained by an autocrine/paracrine effect of bFGF. It is possible
that 24 h of NE stimulation is not sufficient for the accumulation of bFGF to a biologically relevant level, but within 48 h of NE stimulation such a level could be reached. However, since the neutralizing bFGF antibody only partially inhibited the
NE-dependent cell survival, additional
NE-dependent signals may be involved. The observation that
cell survival was markedly enhanced by simultaneous NE and bFGF
stimulation, compared with either NE or bFGF alone, may support the
notion that additional NE-dependent signals
(i.e. separate from the Erk pathway) could be involved.
These data also indicate that NE- and bFGF-dependent
signals in some way cooperates in promoting the cell survival. Although
Erk activation is required for the anti-apoptotic effect of both NE and
bFGF, PKA- and Ca2+-dependent kinases may
augment the Erk-dependent cell survival. It is possible
that co-stimulation of these pathways (e.g. Erk and PKA)
could enhance the expression of genes that are anti-apoptotic. Interestingly, it was recently shown that NGF-induced sustained Erk
activation, nuclear translocation of Erk, and maximal activation of
Elk-1, a member of the the Ets family of transcription factors, was
attenuated by PKA inhibition (54). Thus, the cooperation between the
Erk pathway and PKA may dictate the physiological response to growth
factors and neurotransmitters, such as NE.
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Fig. 12.
Tentative model for the
NE-dependent survival of brown adipocytes. NE
stimulates the Erk cascade via both 1- and
-adrenergic receptors through increases in the intracellular
Ca2+ and cAMP level. NE also increases the expression and
secretion of bFGF, which leads to further activation of Erk. Erk has a
critical role in promoting NE- and bFGF-dependent survival
of brown adipocytes.
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PKA and Erk can activate common transcription factors, such as Elk-1
and the cAMP response element-binding protein, CREB (28, 58-61).
Activated CREB binds to specific sites, cAMP responsive elements (CRE),
present in the promoter of cAMP responsive genes. Elk-1 binds together
with a serum response factor to serum response elements present in the
promoter of many genes. As CREB and Elk-1 can be activated both by the
PKA pathway and the Erk pathway, the transcription of genes that are
anti-apoptotic could be regulated by either pathway. Furthermore,
simultaneous stimulation of the Erk pathway and the PKA pathway may
enhance the transcription of genes containing both serum response
element and CRE sites in their promoters. Although the Bcl-2 promoter
containes a functional CRE and Bcl-2 expression in B cells is dependent
on CREB phosphorylation (62), we could not detect increased Bcl-2
expression in cold exposed animals.2 However, the
transcription of other anti-apoptotic genes in brown adipocytes could
perhaps be dependent on CREB and/or Elk-1 activation. Although the
anti-apoptotic effect of NE and bFGF could be dependent on
transcriptional activation of genes that are anti-apoptotic, post-translational modifications may be important as well. The phosphorylation of pro-apoptotic proteins (i.e. the Bcl-2
family member, Bad) can inactivate the cell-intrinsic death machinery (63, 64), while the phosphorylation of anti-apoptotic proteins (i.e. Bcl-2) can promote cell death (65). Since the
serine/threonine kinase Erk, due to its rather broad nature of
substrate recognition (Pro-Leu-(Ser/Thr)-Pro or (Ser/Thr)-Pro), can
phosphorylate a large number of proteins (66), the anti-apoptotic
effect of NE and bFGF could be due to Erk-dependent
phosphorylation of already existing proteins. This does not necessarily
mean that activated Erk is directly involved in the phosphorylation and
regulation of anti-apoptotic or pro-apoptotic proteins, but the
activity of other kinases or phosphatases could be activated via the
Erk pathway. Taken together, Erk may promote the survival of brown adipocytes by regulating the expression of genes that are
anti-apoptotic and pro-apoptotic, and/or via post-translational
modifications inactivate the cell-intrinsic death machinery; the
specific mechanism(s) remains to be elucidated.
Top
Abstract
Introduction
To whom correspondence should be addressed: Dept. of Metabolic
Research, The Wenner-Gren Institute, Arrhenius Laboratories F3,
Stockholm University, S-106 91 Stockholm, Sweden. Tel.: 46-8-164130; Fax: 46-8-156756; E-mail: stefan{at}metabol.su.S.E.
