Transforming Growth Factor
1 Rescues Serum
Deprivation-induced Apoptosis via the Mitogen-activated Protein Kinase
(MAPK) Pathway in Macrophages*
Beek Yoke
Chin
§,
Irina
Petrache¶,
Augustine M. K.
Choi
**, and
Mary E.
Choi
§§
From the
Toxicological Sciences, Environmental Health
Sciences, and ¶ Division of Pulmonary and Critical Care Medicine,
The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205 and
the
Section of Pulmonary and Critical Care Medicine and

Section of Nephrology, Department of
Internal Medicine, Yale University School of Medicine and the Veterans
Affairs Connecticut Healthcare Systems, New Haven, Connecticut
06520
 |
ABSTRACT |
Cell death and cell survival are central
components of normal development and pathologic states. Transforming
growth factor
1 (TGF-
1) is a
pleiotropic cytokine that regulates both cell growth and cell death. To
better understand the molecular mechanisms that control cell death or
survival, we investigated the role of TGF-
1 in the
apoptotic process by dominant-negative inhibition of both
TGF-
1 and mitogen-activated protein kinase (MAPK)
signaling pathways. Murine macrophages (RAW 264.7) undergo apoptosis
following serum deprivation, as determined by DNA laddering assay.
However, apoptosis is prevented in serum-deprived macrophages by
the presence of exogenous TGF-
1. Using stably
transfected RAW 264.7 cells with the kinase-deleted dominant-negative
mutant of T
R-II (T
R-IIM) cDNA, we demonstrate
that this protective effect by TGF-
1 is completely
abrogated. To determine the downstream signaling pathways, we examined
TGF-
1 effects on the MAPK pathway. We show that
TGF-
1 induces the extracellular signal-regulated kinase
(ERK) activity in a time-dependent manner up to 4 h
after stimulation. Furthermore, TGF-
1 does not rescue
serum deprivation-induced apoptosis in RAW 264.7 cells transfected with
a dominant-negative mutant MAPK (ERK2) cDNA or in wild type RAW
264.7 cells in the presence of the MAPK kinase (MEK1) inhibitor. Taken
together, our data demonstrate for the first time that
TGF-
1 is an inhibitor of apoptosis in cultured
macrophages and may serve as a cell survival factor via T
R-II-mediated signaling and downstream intracellular MAPK signaling pathway.
 |
INTRODUCTION |
Apoptosis, the process of programmed cell death, is an integral
part of normal embryonic development, inflammatory response, and
tumorigenesis (1). It is a highly regulated series of well coordinated
events characterized by distinctive morphologic and biochemical changes
involving nuclear and chromatin condensation, cell membrane blebbing,
and loss of cellular integrity forming distinct apoptotic bodies, as
well as endonuclease activity resulting in DNA fragmentation and
ultimately cell death (2). Regulatory mechanisms controlling cell death
is as fundamental as those regulating cell growth in achieving the
homeostatic balance between cell survival and cell death and involve a
complex interplay of specific regulatory genes in signaling cells to
either live or die.
Transforming growth factor
1
(TGF-
1)1 is a
25-kDa polypeptide, belonging to a superfamily of multifunctional
cytokines, that regulates cellular growth and differentiation and
extracellular matrix production (3). Moreover, TGF-
1 has
been shown to be a potent modulator of apoptosis in a variety of cell
types, including epithelial cells, hepatocytes, hematopoietic cells,
and lymphocytes, which undergo programmed cell death in response to
TGF-
1 (4-7). We have previously reported the induction
of apoptosis by TGF-
1 in endothelial cells (8). However,
more recent studies suggest that TGF-
1 also possesses
the ability to inhibit apoptosis, further affirming the multifunctional
nature of this cytokine (9).
TGF-
1 elicits multiple biological responses by
interaction with two transmembrane receptor serine/threonine kinases
known as TGF-
type I receptor (T
R-I) and TGF-
type II receptor
(T
R-II) (3). T
R-II is a constitutively active kinase, which binds TGF-
1 directly and recruits T
R-I to form a
"heteromeric" complex, and the signaling cascade is initiated upon
transphosphorylation of the GS domain of T
R-I by T
R-II (10).
T
R-I alone does not exhibit significant binding of
TGF-
1 ligand when assessed by cross-linking analysis,
and T
R-II is unable to signal without T
R-I (10). Thus, T
R-II
is required for initial ligand binding and phosphorylation of T
R-I
to initiate the signaling cascade. We have previously reported the
critical role of T
R-II in the TGF-
1 signaling pathway
to induce apoptosis in endothelial cells (8). Interference with
T
R-II-mediated signal transduction by a dominant-negative mutant of
T
R-II blocked TGF-
1-induced endothelial cell
apoptosis and associated capillary morphogenesis in vitro
(8).
Although molecular cloning of the TGF-
receptors have furthered our
understanding of the mechanism of TGF-
1 signaling, the downstream signaling pathways activated after the initial receptor interaction with ligand to mediate multiple TGF-
1
responses remain poorly understood. Recent studies support the
involvement of the mitogen-activated protein kinase (MAPK) pathways in
TGF-
1 signaling (11-14). Moreover, activation of the
MAPK-dependent pathways has been implicated in the process
of apoptosis (15, 16). Members of the MAPK family, like the TGF-
receptors, are structurally related serine/threonine kinases that are
actively involved in cellular events such as growth, differentiation,
and cellular responses to environmental stress (17, 18). There are
three groups of the MAPK family members identified to date: the
extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), also
known as p44 and p42 MAPKs, respectively; the c-Jun N-terminal
kinase/stress-activated protein kinase (JNK/SAPK); and the p38 (18,
19). The signal transduction cascades involved in the activation of
MAPKs require a well coordinated series of three protein kinase
reactions, propagating the phosphorylation and the activation of the
next kinase in their respective pathways. The MAPKs require dual
phosphorylation at the threonine and tyrosine sites by MAPK kinases,
the MEKs and MKKs that are specific for ERK, JNK, and p38, which are in
turn activated by MAPK kinase kinases (MKKKs) via serine/threonine phosphorylation (19, 20). The MAPK cascades display evolutionary conservation and are implicated to play essential roles in the regulation of cell growth, differentiation, and apoptosis.
To better understand the molecular mechanism controlling cell death or
survival, we investigated the role of TGF-
1 in the apoptotic process by dominant-negative inhibition of both
TGF-
1 and MAPK signaling pathways. In this study, we
utilized serum withdrawal or deprivation to induce apoptosis by
decreased availability of cell survival factors. We show that serum
deprivation induces apoptosis in murine macrophages (RAW 264.7) and
that TGF-
1 is able to prevent serum-deprived macrophages
from undergoing apoptosis. This "rescue" is inhibited in cells
transfected with a dominant-negative mutant of T
R-II
(T
R-IIM), suggesting the critical role of T
R-II in TGF-
1 signaling to prevent serum
deprivation-induced apoptosis. Furthermore, we demonstrate that
TGF-
1 rapidly induces ERK1/ERK2 MAPK activity.
TGF-
1 fails to rescue RAW 264.7 cells from serum deprivation-induced apoptosis upon stable transfection with a dominant-negative mutant MAPK (ERK2) cDNA or in the presence of the
MEK1 inhibitor. Taken together, our data suggest that
TGF-
1 rescues macrophages from serum deprivation-induced
apoptosis via T
R-II-mediated signaling and downstream intracellular
MAPK signaling pathway.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Recombinant human TGF-
1 and TGF-
were obtained from Life Technologies, Inc. The p44/42 MAPK,
phosphospecific p44/42 MAPK (Tyr-204), and phosphospecific Elk-1
(Ser-383) rabbit polyclonal antibodies, purified Elk-1 fusion protein,
ERK2, and phosphospecific SAPK/JNK (Thr-183/Tyr-185) and
phosphospecific p38 (Thr-180/Tyr-182) rabbit polyclonal antibodies were
purchased from New England Biolabs, Inc. (Beverly, MA). The MEK1
inhibitor, PD098059, was also obtained from New England Biolabs, Inc.
Constructs--
A truncated T
R-II construct
(T
R-IIM), lacking the serine/threonine kinase domain,
but containing the full transmembrane spanning and extracellular
domains, was generated by polymerase chain reaction (PCR) using a rat
T
R-II cDNA as the template, as described previously (8). Primer
sequences were as follows: sense primer
5'-GTTAAGGCTAGCGACGGGGGCTGCCATG-3'; antisense primer 5'-GGCGGTCGACTAGACACGGTAACAGTAGAAG-3'. These contained the
sequences for the restriction enzymes NheI and
SalI, respectively (underlined), for directional cloning,
and a stop codon in the antisense primer. The PCR-amplified product was
cloned into the pMAMneo (CLONTECH), a
glucocorticoid-inducible mammalian expression vector, containing a
neomycin-resistant gene. Correct directionality and in-frame sequences
of the PCR product ligated in pMAMneo were verified by restriction
mapping with EcoRI, BamHI, and HindIII
and sequencing by the dideoxy chain termination technique using
Sequenase 2.0 (United States Biochemical Corp.). The MAPK-WT (wild type
ERK2) and the MAPK-TA (dominant-negative mutant of ERK2) constructs used in this study were provided by Dr. Andrew Larner (21).
Cell Culture and Transfection--
The murine peritoneal
macrophage cell line, RAW 264.7, was obtained from ATCC (Rockville,
MD). The cells were maintained in Dulbecco's modified Eagle's medium
(DMEM) (Life Technologies, Inc.) supplemented with 10% FBS (HyClone)
and gentamicin (50 µg/ml) in a humidified atmosphere of 5%
CO2 and 95% air at 37 °C. To generate clones that
stably expressed T
R-IIM, RAW 264.7 cells were
transfected using Lipofectin (Life Technologies, Inc.) as follows.
Cells grown to approximately 50% confluency on 100-mm dishes (Falcon)
were incubated with 10 µg of DNA (T
R-IIM) and 50 µl
of Lipofectin suspension in DMEM at 37 °C in 5% CO2.
After a 5-h incubation, medium containing 20% FBS in DMEM was added to
make a final concentration of 10% FBS and incubated further for
48 h. Then the DNA/Lipofectin-containing medium was changed to
10% FBS in DMEM (no antibiotics) and incubated for another 24 h.
To select for transfectants, cells were treated up to 800 µg/ml G418
(Life Technologies, Inc.) in DMEM containing 10% FBS, and the medium
was changed every 2-3 days. G418-resistant colonies emerged at
approximately 10 days after transfection and were subcloned using ring
cylinders, expanded, and maintained in DMEM containing 10% FBS,
Geneticin (200 µg/ml), and gentamicin (50 µg/ml). Two independent,
stably transfected clones expressing the T
R-IIM, named
10-2 and 10-3, were expanded. Confirmation of mRNA expression was
obtained by reverse transcription-polymerase chain reaction using
primer pairs that contain species-specific sequences that recognize
only the transfected T
R-IIM construct and not the
endogenous wild-type T
R-II.
To generate clones that stably expressed the MAPK-WT or the MAPK-TA,
the corresponding constructs were co-transfected with pcDNA3
(Invitrogen), a mammalian expression vector containing a
neomycin-resistant gene, using Lipofectin, as described above. The
stable transfectants were also selected in medium containing 800 µg/ml G418 and then subcloned and maintained in 200 µg/ml G418. Confirmation of mRNA expression was obtained by reverse transcription-PCR.
Induction of Apoptosis/Genomic DNA Isolation and
Analysis--
To induce apoptosis, cells grown on 100-mm dishes
(Falcon) to 90% confluency were placed in DMEM containing 0.5% FBS
for 24 h. In experiments involving treatment with cytokines, the
cells were incubated in the absence or presence of exogenous
TGF-
1 (1 ng/ml-100 ng/ml) or TGF-
(1 ng/ml-100
ng/ml) at 37 °C for 24 h. In experiments with MEK1 inhibitor,
PD098059, cells were incubated in the absence or presence of 30 µM PD098059 for 24 h. The concentration of 30 µM was chosen as it is the optimal concentration
inhibiting ERK without imparting cellular toxicity in these cells. For
experiments involving exposure of RAW 264.7 cells to hyperoxia, the
cells were placed in a tightly sealed modular chamber (Billup-Rothberg,
Del Mar, CA) with 5% CO2 and 95% O2 at
37 °C. Control cells were maintained in 5% CO2 and 95% air at 37 °C.
Genomic DNA isolation was performed using the Puregene kit (Gentra
Systems, Inc.) according to the manufacturer's directions. Briefly,
cells were lysed directly on the plate after medium removal with lysis
buffer followed by a 1-h incubation with RNase A. The cell lysates were
precipitated for proteins and spun at 2000 × g for 15 min. Then, isopropyl alcohol was added to the supernatant to
precipitate the DNA. After an alcohol wash, the DNA was hydrated and
quantified, and 20 µg was analyzed on 1.5% agarose gel
electrophoresis. The T
R-IIM stable transfectants were
preincubated in the presence or absence of 1 µM
dexamethasone for 24 h prior to serum deprivation and treatment
with exogenous TGF-
1. The MAPK-WT and MAPK-TA stable transfectants
were also subjected to serum withdrawal and TGF-
1 as
described above. Each of the experiments was repeated at least three times.
Cell Survival Assay--
Determination of cell viability was
done by trypan blue exclusion assay. Cells grown on 12-well plates to
90% confluency were induced to undergo apoptosis as described above,
and at the indicated time periods, cells in each of the wells were
collected, centrifuged, and resuspended in 0.5 ml of DMEM. Then
aliquots of 0.1 ml were incubated with trypan blue dye (Life
Technologies, Inc.) for 5 min followed by cell counting by
hemocytometer. Both live (unstained) and dead (blue) cells were counted
from the same randomly selected fields. The results were expressed as
percentages of surviving cells that did not take up the trypan blue dye
in the total cell population. The experiments were performed in
triplicate and repeated two times.
Western Blot Analysis--
Total cellular extracts were obtained
for the Western analyses by lysis of cells in buffer containing 1%
Nonidet P-40, 20 mM Tris, pH 8.0, 150 mM NaCl,
1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml aprotinin. Protein
concentrations of the cell lysates were determined by Coomassie Blue
dye-binding assay (Bio-Rad). An equal volume of 2× SDS loading buffer
(0.125 mM Tris-HCl, pH 7.4, 4% SDS, and 20% glycerol) was
added, and the samples were boiled for 5 min. Protein samples (100 µg) were resolved on 12% SDS-polyacrylamide gel electrophoresis,
then electroblotted onto nitrocellulose membranes (Bio-Rad). The
membranes were incubated with p44/42 MAPK, phosphospecific p44/42 MAPK,
phosphospecific SAPK/JNK, or phosphospecific p38 rabbit polyclonal
antibodies (1:1000) for 1.5 h, followed by incubation with
horseradish peroxidase-conjugated anti-rabbit antibody for 1.5 h.
Signal development was carried out using LumiGLO (New England Biolabs)
and exposed to x-ray film.
MAPK (ERK1 and ERK2) Activity Assays--
Kinase assays were
performed as described by Marais et al. (22) with minor
modifications. Briefly, cells were lysed in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM Na3VO4,
1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride
and sonicated. Protein concentrations were determined as described for
Western analysis. Total protein (200 µg) samples were incubated with
phosphospecific p44/42 MAPK rabbit polyclonal antibody (1:50) overnight
on a rocker at 4 °C. For a positive control, 20 ng of active MAPK
(ERK2) was incubated with control cell extract. Protein A-Sepharose
beads (Amersham Pharmacia Biotech) were then added to immunoprecipitate
the activated MAPK complex. The immunoprecipitate pellets were
incubated with 1 µg of Elk-1 fusion protein in the presence of 100 µM ATP and a kinase buffer containing 25 mM
Tris-HCl, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4, and 10 mM
MgCl2. The reaction was terminated with SDS loading buffer
(62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% w/v bromphenol blue). The
samples were analyzed on 12% SDS-PAGE and electroblotted as described
for Western blot. ERK activity was assayed by detection of
phosphorylated Elk-1 using a phosphospecific Elk-1 rabbit polyclonal antibody (1:1000). After overnight incubation with the primary antibody
at 4 °C, the membrane was incubated for 1 h with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000) at room
temperature with gentle rocking. The proteins were subsequently detected using LumiGLO (New England Biolabs) and exposed to x-ray film.
All of the assays were repeated three times.
Statistical Analysis--
Statistical significance of the
experimental data for the cell survival assays by trypan blue exclusion
was determined by analysis of variance or the Student's t
test for paired data, as appropriate. p values < 0.05 were considered significant. Data are presented as means ± S.E.
of triplicate determinations.
 |
RESULTS |
Serum Deprivation Induces Apoptosis in RAW 264.7 Cells--
We
first determined whether RAW 264.7 cells underwent apoptosis following
withdrawal of serum. Genomic DNA isolated from RAW 264.7 cells were
assessed for the presence of DNA fragmentation by a "ladder"
pattern on agarose gel electrophoresis, indicative of internucleosomal
cleavage, a hallmark of apoptosis. The induction of genomic DNA
fragmentation was observed in RAW 264.7 cells after 24 h of serum
deprivation (Fig. 1).

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Fig. 1.
Serum deprivation-induced apoptosis in RAW
264.7 cells. Genomic DNA was isolated from RAW 264.7 cells
incubated in the presence of 10% FBS (lane 3) or in 0.5%
FBS (lane 4) for 24 h and fractionated on 1.5% agarose
gel electrophoresis as described under "Experimental Procedures."
Molecular weight markers: 100 bp, lane 1; 1-kb ladder,
lane 2.
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TGF-
1 Rescues Serum-deprived RAW 264.7 Cells from
Apoptosis--
Given that previous studies have implicated the
role of TGF-
1 as a modulator of apoptosis, we examined
the effects of TGF-
1 on serum deprivation-induced
apoptosis in RAW 264.7 cells. As shown in Fig.
2A, genomic DNA fragmentation
was not observed in RAW 264.7 cells upon serum deprivation in the
presence of exogenous TGF-
1 (lanes 3-5, 1, 10, and 100 ng/ml, respectively). This inhibition of DNA fragmentation
by TGF-
1 was associated with increased cell survival, as
shown in Fig. 3. Treatment with exogenous
TGF-
1 (10 ng/ml) resulted in increased cell survival of
93 ± 2% compared with 78 ± 3% after serum deprivation for
24 h (p < 0.01, Student's t test,
n = 3) and 88 ± 4% compared with 49 ± 2%
cell survival after serum deprivation for 48 h (p < 0.005, Student's t test, n = 3). The
increased cell survival with TGF-
1 treatment remained significant up to 96 h following serum deprivation. TGF-
, an analog of epidermal growth factor, chemically distinct from
TGF-
1 and acting through a tyrosine kinase receptor
system, failed to prevent DNA fragmentation in serum-deprived RAW 264.7 cells (data not shown). Furthermore, TGF-
1 did not
rescue the apoptotic process elicited by other stimuli such as
oxidative stress (Fig. 2B), indicating specificity of
TGF-
1-mediated rescue from serum deprivation-induced apoptosis in RAW 264.7 cells.

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Fig. 2.
Effect of
TGF- 1 on serum deprivation-induced
apoptosis in RAW 264.7 cells. A, genomic DNA was
isolated from RAW 264.7 cells incubated for 24 h in the presence
of 10% FBS (lane 1) or in 0.5% FBS with increasing doses
of exogenous TGF- 1 (0, 1, 10, and 100 ng/ml, lanes
2-5) and fractionated on 1.5% agarose gel electrophoresis as
described under "Experimental Procedures." Molecular weight markers
(MW), 1 kb; 100-bp ladder. B, genomic DNA was
isolated from RAW 264.7 cells exposed to normoxia (lane 1)
or to hyperoxia (95% oxygen) for 24 h in the presence of
increasing doses of TGF- 1 (0, 1, 10, and 100 ng/ml,
lanes 2-5) and fractionated on 1.5% agarose gel
electrophoresis as described under "Experimental Procedures."
Molecular weight marker (MW), 1-kb ladder.
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Fig. 3.
Time course of
TGF- 1 effect on cell survival in
serum-deprived RAW 264.7 cells. Cells incubated in the presence of
10% FBS or in 0.5% FBS with and without exogenous
TGF- 1 (10 ng/ml) for the various time periods, as
indicated, were assessed for cell viability by trypan blue exclusion
assay as described under "Experimental Procedures." The results
were expressed as percentages of surviving cells that did not take up
the trypan blue dye in the total cell population. Each data point is
the mean of triplicate determinations ± S.E. *, there was a
significant difference in survival of cells treated with
TGF- 1 compared with the respective control in 0.5% FBS
without TGF- 1 for each time period (p < 0.01, Student's t test, n = 3). **, cell
survival was significantly decreased upon serum deprivation in the
absence of exogenous TGF- 1 treatment (p < 0.05, analysis of variance).
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TGF-
1 Rescues Serum Deprivation-induced Apoptosis
via T
R-II Signaling Pathway--
To determine whether the ability
of TGF-
1 to rescue RAW 264.7 cells from serum
deprivation-induced apoptosis was mediated by T
R-II, we first
generated stably transfected cells overexpressing a dominant-negative
mutant of T
R-II (T
R-IIM). Given that
TGF-
1 signal transduction requires heterodimerization of
T
R-II and T
R-I and transphosphorylation of T
R-I by T
R-II,
the truncated receptor T
R-IIM, which is
membrane-anchored but lacks the cytoplasmic serine/threonine kinase
domain, competes for binding to T
R-I, hence acting in a
dominant-negative fashion to inhibit TGF-
1 signaling (8,
23). As predicted, complete inhibition of TGF-
1 rescue
from serum deprivation-induced apoptosis was observed in cells from two
independent clones (10-2 and 10-3) expressing the truncated receptors,
T
R-IIM (Fig. 4). This
occurred both with and without dexamethasone pretreatment. Although the
T
R-IIM construct was under a glucocorticoid-regulated
promoter, "leakage" of promoter activity occurs during uninduced
conditions and has been previously observed by us and other
investigators (8, 24).

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Fig. 4.
Inhibition of
TGF- 1 effect on serum
deprivation-induced apoptosis by dominant-negative mutant
TGF- type II receptor
(T R-IIM). Genomic DNA was
isolated from RAW 264.7 cells transfected with dominant-negative
T R-IIM and fractionated on 1.5% agarose gel
electrophoresis as described under "Experimental Procedures."
Mutant 10-2 and 10-3 represent two independent clones of cells stably
transfected with T R-IIM. Lane 1, 10% FBS;
lane 2, 10% FBS and 1 µM dexamethasone;
lane 3, 0.5% FBS; lane 4, 0.5% FBS and 10 ng/ml
TGF- 1; lane 5, 0.5% FBS, 10 ng/ml
TGF- 1, and 1 µM dexamethasone.
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TGF-
1 Activates MAPK (ERK1 and ERK2) in RAW 264.7 Cells--
Previous studies have suggested that TGF-
1
exerts its biological effects via the MAPK signaling pathway in several
cell culture systems (11-14). We first determined the levels of ERK1
and ERK2 protein expression in RAW 264.7 cells treated with exogenous
TGF-
1 (10 ng/ml) by Western analyses, using
phosphospecific p44/42 MAPK and p44/42 MAPK antibodies. The
phosphospecific p44/42 MAPK antibodies detect specifically the
phosphorylated forms of ERK1/ERK2, whereas the p44/42 MAPK antibodies
detect total (phosphorylation-state independent) ERK1/ERK2 proteins. As
shown in Fig. 5A, increases in
phosphorylation of ERK1 and ERK2 proteins were observed in cells, as
early as 15 min after stimulation with exogenous TGF-
1. There were no appreciable increases in the activation of JNK or p38
within the same time periods of TGF-
1 treatment (Fig.
5B).

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Fig. 5.
Effect of
TGF- 1 on MAPK (ERK1/ERK2) activity
in RAW 264.7 cells. A, lysates from RAW 264.7 cells
incubated in the absence (Ctl) or in the presence of
exogenous TGF- 1 (10 ng/ml) for the indicated times were
subjected to Western analysis using phosphospecific p44/42 MAPK
(top panel) or p44/42 MAPK (bottom panel)
antibodies as described under "Experimental Procedures." Two bands
are detected corresponding to phosphorylated forms of ERK1/ERK2
proteins (top panel) and two bands corresponding to total
ERK1/ERK2 proteins (bottom panel). B, lysates
from RAW 264.7 cells incubated in the absence (Ctl) or in
the presence of exogenous TGF- 1 (10 ng/ml) for the
indicated times were subjected to Western analysis using
phosphospecific SAPK/JNK (top panel) or phosphospecific p38
(bottom panel) antibodies as described under "Experimental
Procedures." C, immunocomplex kinase assay. Lysates from
RAW 264.7 cells incubated in the absence (Ctl) or in the
presence of exogenous TGF- 1 (10 ng/ml) for the indicated
times were analyzed for MAPK activity as described under
"Experimental Procedures." ERK activity was assayed by
immunoprecipitation with phosphospecific antibody to MAPK (Tyr-204)
followed by detection of phosphorylation of Elk-1 fusion protein at
Ser-383 by Western blotting using a phosphospecific Elk-1 (Ser-383)
antibody. For a positive control (+Ctl), 20 ng of active
MAPK (ERK2) was incubated with a control extract.
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We next examined whether this induction of ERK1 and ERK2 by
TGF-
1 was associated with an increase in MAPK activity
using an immunocomplex kinase assay. Lysates from RAW 264.7 cells
incubated in the presence or absence or exogenous TGF-
1
(10 ng/ml) were subjected to immunoprecipitation using phosphospecific
p44/42 MAPK antibodies. The resulting active ERK1/ERK2
immunoprecipitate was then allowed to phosphorylate Elk-1 fusion
protein, and ERK activity was assayed by the detection of
phosphorylated Elk-1 by Western blot analysis. Exogenous
TGF-
1 (10 ng/ml) induced the increase of the
phosphorylated form of Elk-1 (Fig. 5C). Although there was
some endogenous activity in the control untreated cells, TGF-
1 induced ERK activity within 15 min of
TGF-
1 treatment, with marked ERK activity up to 4 h
of TGF-
1 treatment.
Inhibition of Serum Deprivation-induced Apoptosis by
TGF-
1 Involves the MAPK Pathway--
Given that
TGF-
1 activates the MAPK (ERK) pathway in RAW 264.7 cells, we next examined whether the MAPK signaling pathway mediates the
TGF-
1 rescue of RAW 264.7 cells from serum
deprivation-induced apoptosis. Our first strategy was to inhibit the
MAPK pathway by genetic blockade utilizing a dominant-negative mutant
of ERK MAPK (MAPK-TA). Fig. 6A
demonstrates that TGF-
1 rescues serum deprivation-induced apoptosis in cells transfected with MAPK-WT, as was
previously observed in wild-type RAW 264.7 cells. However, in RAW 264.7 cells that have been transfected with a dominant-negative mutant
MAPK-TA, genomic DNA fragmentation was observed both in the presence of
serum and upon serum deprivation, and treatment with exogenous TGF-
1
failed to prevent apoptosis (Fig. 6B). This suggests that
the rescue effect of TGF-
1 is in part mediated by the
MAPK signaling pathway.

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Fig. 6.
Inhibition of
TGF- 1 effect on serum
deprivation-induced apoptosis by dominant-negative mutant
MAPK. A, genomic DNA was isolated from RAW 264.7 cells
transfected with MAPK-WT incubated for 24 h in the presence of
10% FBS (lane 1) or in 0.5% FBS serum (lane 2),
or in 0.5% FBS and 10 ng/ml TGF- 1 (lane 3),
and fractionated on 1.5% agarose gel electrophoresis as described
under "Experimental Procedures." B, genomic DNA was
isolated from RAW 264.7 cells transfected with dominant-negative mutant
MAPK (MAPK-TA) incubated for 24 h under the following conditions
and fractionated on 1.5% agarose gel electrophoresis as described
under "Experimental Procedures." Lane 1, 10% FBS;
lane 2, 0.5% FBS; lane 3, 10% FBS and 10 ng/ml
TGF- 1; lane 4, 0.5% FBS and 10 ng/ml
TGF- 1.
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Inhibition of MEK1 Prevents the Anti-apoptotic Effects of
TGF-
1 in Serum-deprived RAW 264.7 Cells--
To further
confirm the role of the ERK MAPK pathway in TGF-
1 rescue
of serum deprivation-induced apoptosis, we next utilized a selective
inhibitor of MEK1 (upstream of ERK). In wild-type RAW 264.7 cells
treated with 30 µM of the MEK1 inhibitor PD098059 for
24 h, DNA fragmentation was observed both in the presence of serum
(Fig. 7A, lanes
3 and 4) and upon serum deprivation (Fig. 7B, lanes 3 and 4), and
treatment with exogenous TGF-
1 failed to prevent
apoptosis. Taken together, these results suggest that inhibiting MEK1
disrupts the signaling process of exogenous TGF-
1 by
preventing the activation of the MAPK pathway under conditions of serum
deprivation, thus disrupting the initiation of apoptotic rescue.

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|
Fig. 7.
Effect of MEK1 inhibitor, PD098059, on serum
deprivation-induced apoptosis in RAW 264.7 cells. A,
genomic DNA was isolated from wild-type RAW 264.7 cells incubated for
24 h in the presence of 10% FBS and the following conditions and
fractionated on 1.5% agarose gel electrophoresis as described under
"Experimental Procedures." Lane 1, 10% FBS; lane
2, 10% FBS and 10 ng/ml TGF- 1; lane 3,
10% FBS and 30 µM PD098059; lane 4, 10% FBS,
10 ng/ml TGF- 1, and 30 µM PD098059.
B, genomic DNA was isolated from wild-type RAW 264.7 cells
in the presence of 0.5% FBS and the following conditions and
fractionated on 1.5% agarose gel electrophoresis as described under
"Experimental Procedures." Lane 1, 0.5% FBS; lane
2, 0.5% FBS and 10 ng/ml TGF- 1; lane 3,
0.5% FBS and 30 µM PD098059; lane 4, 0.5%
FBS, 10 ng/ml TGF- 1, and 30 µM PD098059.
|
|
 |
DISCUSSION |
It is now well accepted that the process of programmed cell death,
or apoptosis, serves as a critical force in the development and
homeostasis of multicellular organisms and is carefully regulated by
diverse signals that influence the decision of a cell between life and
death. These signals may act to either promote or inhibit apoptosis,
and the same signal may potentially have opposing effects on different
cell types. One such signaling molecule that may possess both
pro-apoptotic and anti-apoptotic activities is the multifunctional
cytokine TGF-
1, both a potent stimulator and an
inhibitor of cell proliferation. In the present study, we examined the
role of TGF-
1 in modulating apoptosis in cultured
macrophages (RAW 264.7 cells). In order to induce apoptosis, serum was
withdrawn. Serum deprivation has been shown to provoke apoptosis in a
variety of cells including fibroblasts and endothelial cells, as a
result of decreased availability of cell survival factors (8, 25). We
observed that macrophages also undergo apoptosis upon serum withdrawal
or deprivation, as determined by detection of the characteristic genomic DNA laddering (Fig. 1). Remarkably, the presence of exogenous TGF-
1 prevented the macrophages from undergoing
apoptosis upon serum withdrawal (Fig. 2A). This inhibition
of DNA laddering by TGF-
1 was also associated with
increased cell survival (Fig. 3). The anti-apoptotic activity of
TGF-
1 was further confirmed by its inability to rescue
cells from serum deprivation-induced apoptosis when its signaling
receptors are blocked by a kinase-deleted dominant-negative mutant of
T
R-II (T
R-IIM). Since signal transduction requires
heterodimerization of T
R-II and T
R-I, the mutant receptor competes for binding to wild-type T
R-I, hence acting in a
dominant-negative fashion (3, 8, 10). In stably transfected RAW 264.7 cells expressing the T
R-IIM, apoptosis occurred with
serum deprivation, both in the presence or absence of exogenous
TGF-
1 (Fig. 4), indicating that the anti-apoptotic
effect by TGF-
1 is mediated by T
R-II kinase.
Although TGF-
1 has been shown in a number of systems to
be a potent inducer of apoptosis, anti-apoptotic actions of
TGF-
1 are less well known. Sachsenmeier et
al. reported "protective" effects of TGF-
1 by
inhibiting suspension-induced apoptosis in human keratinocytes
following loss of adhesion (9). Treatment of keratinocytes with
TGF-
1 attenuated suspension-induced DNA fragmentation.
Moreover, inhibition of endogenous TGF-
1 by neutralizing antibody to TGF-
1 increased DNA fragmentation following
suspension. Thus, TGF-
1 clearly possesses the ability to
exert both pro-apoptotic and anti-apoptotic effects in different cell
systems, and the differential cellular responses likely are necessary
for proper homeostasis of multicellular organisms. With the findings
that TGF-
1 can promote cell survival in certain cell
types, we were interested in exploring the potential downstream
intracellular pathways responsible for these protective effects of
TGF-
1 in macrophages.
Evidences that TGF-
1 is capable of activating
MAPK-dependent pathways in mammalian cells have been
reported. For instance, rapid activation of ERK1 by
TGF-
1 has been demonstrated in intestinal epithelial
cells and is associated with growth inhibitory effects of
TGF-
1 (12). In other cell types, including HepG2, CHO,
and MDCK cell lines, TGF-
1 has been shown to activate
JNK/SAPK, and dominant-negative forms of various components of the
JNK/SAPK pathway abolished TGF-
signaling (13). We examined whether TGF-
1 is capable of activating MAPK in macrophages and
whether TGF-
1 signals rescue from serum
deprivation-induced apoptosis via the MAPK-dependent
pathway. ERK activity was assayed by two methods. First, increased
phosphorylation of ERK1/ERK2 was determined by Western analyses using
phosphospecific p44/42 MAPK antibodies that detect only the tyrosine
204-phosphorylated forms of ERK1/ERK2. Next, ERK activity was
determined by in vitro kinase assay. Following immunoprecipitation with p44/42 MAPK antibodies to select for the
activated (phosphorylated) MAPK, detection of in vitro
phosphorylation of a known substrate, Elk-1, was determined using
phosphospecific antibodies that detect only the serine
383-phosphorylated Elk-1. Our results show that ERK1/ERK2 was activated
within 15 min of stimulation with exogenous TGF-
1 in
cultured RAW 264.7 cells, and this activation was sustained up to
4 h (Fig. 5C). Accordingly, the sharp increase in
phosphorylation of Elk-1 is observed parallel with increased
phosphorylated forms of ERK1/ERK2 (Fig. 5A). In contrast, we
observed that TGF-
1 failed to activate JNK/SAPK or p38
within this same time period, indicating that TGF-
1 is capable of rapidly activating only the ERK pathway, but not the JNK/SAPK or p38 pathways, in RAW 264.7 macrophages (Fig.
5B).
The ERK pathway is the prototypical MAPK pathway induced by epidermal
growth factor stimulation and implicated in the regulation processes of
cellular proliferation and differentiation (18, 26). Evidence for its
potential importance in the modulation of apoptosis has been provided
by studies in cardiac myocytes. Cardiotrophin 1 (CT-1), a member of the
interleukin 6 family of cytokines, is a potent cardiac survival factor
capable of inhibiting apoptosis in cardiac myocytes via the
activation of an anti-apoptotic signaling pathway that requires
MEKs (MAPK/ERK kinases) (27). We determined whether the ERK pathway was
involved in the apoptotic rescue of macrophages by
TGF-
1, using two independent approaches to block the ERK
signaling pathway. Our transfection studies with the dominant-negative
mutant MAPK (ERK2) in RAW 264.7 cells resulted in the blockade of
TGF-
1 anti-apoptotic effects (Fig. 6B). To further support these findings, we utilized an MEK1-specific inhibitor, PD098059, which blocks MEK1 activation by Raf, thus preventing downstream activation of ERK1/ERK2, but does not inhibit JNK/SAPK or
p38 protein kinase activation. In addition, the PD098059 has been shown
to have little effect on other kinases, including
cAMP-dependent kinase, protein kinase C, and other serine
and threonine kinases (28-30). In our studies, PD098059 effectively
prevented the anti-apoptotic effects of TGF-
1 in
serum-deprived macrophages and provides further evidence for the
requirement of the ERK pathway in the survival function of
TGF-
1 (Fig. 7B).
Interestingly, studies supporting our current findings that
MAPK-dependent pathways are responsible for promoting the
survival effects of TGF-
1 have been documented for other
cytokines in neuronal cells and cardiac myocytes. Nerve growth factor
promotes the survival of neuronal (PC-12) cells via activation of the
ERK pathway to mediate and initiate rescue from apoptosis induced by
serum deprivation, and JNK/SAPK activation along with inhibition of
MAPK (ERK) are required for modulating and inducing apoptosis (31). The
MAPK pathways have also been found to be necessary for CT-1 effects on
promoting survival of serum-deprived cardiac myocytes and blocking MAPK
activation by transfection of a dominant-negative mutant MEK or by
treatment with PD098059 inhibited the survival effect of CT-1 (27).
Furthermore, in the present report, we observed, even in the presence
of serum, induction of apoptosis in RAW 264.7 cells upon blockade
of the MAPK signaling pathways either by a dominant-negative mutant of
MAPK (ERK) (Fig. 6B) or by MEK1 inhibitor, PD098059 (Fig.
7A). Thus, based on our studies and those previous studies,
it is plausible that the MAPKs (ERK) represent a common pathway
targeted by anti-apoptotic cytokines to promote cell survival.
It will be of great interest to determine whether similar
cytokine-induced MAPK-dependent signaling pathways operate
in vivo to promote cell survival, and the present findings
may potentially have important clinical implications. Apoptosis
in vivo is followed almost inevitably by rapid uptake into
adjacent phagocytic cells and represents a critical process in tissue
remodeling, regulation of immune response, or resolution of
inflammatory reactions (32). The importance of TGF-
1 in
the regulation of inflammation is well demonstrated by the observations
that TGF-
knock-out mice have severe and generalized inflammatory
disorders (33, 34). We have identified TGF-
1 as an
inhibitor of apoptosis in cultured macrophages and may serve as a cell
survival factor via the MAPK-dependent pathway. This would
provide macrophages with a cellular defense mechanism to be selectively
spared from toxicity and cell death, a process that would be critical
in the resolution of inflammation. Survival of macrophages is required
to perform their duties of phagocytosis and elimination of adjacent
harmful or injured cells, including lymphocytes, that have undergone
apoptosis, and disorders that compromise macrophage survival could
contribute to chronic inflammatory diseases.
 |
FOOTNOTES |
*
This work was supported in part by Physician Scientist Award
5-K12-DK0129809 from the NIDDK, National Institutes of Health, Grant-in-aid 96015510 from the American Heart Association, and a
Veterans Affairs Career Development Award (to M. E. C.).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.
§
Supported by a Laboratory Scholarship from Toxicological Sciences
at The Johns Hopkins University School of Hygiene and Public Health.
**
Supported by National Institutes of Health Grant R29 HL-55330,
American Heart Association EIA, and National Institutes of Health Grant
R01 AI-42365.
§§
To whom correspondence should be addressed: Section of
Nephrology, Yale University School of Medicine, 333 Cedar St., 2073 LMP, New Haven, CT 06520-8029. Tel.: 203-932-5711 (Ext. 2221); Fax:
203-937-3455; E-mail: mary.choi{at}yale.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
1, transforming growth factor
1;
T
R, TGF-
receptor;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase;
PCR, polymerase chain reaction;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum;
JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein
kinase.
 |
REFERENCES |
-
Ashkenazi, A.,
and Dixit, V. M.
(1998)
Science
281,
1305-1308[Abstract/Free Full Text]
-
Steller, H.
(1995)
Science
267,
1445-1449[Medline]
[Order article via Infotrieve]
-
Attisano, L.,
and Wrana, J. L.
(1996)
Cytokine Growth Factor Rev.
7,
327-339[CrossRef][Medline]
[Order article via Infotrieve]
-
Rotello, R. J.,
Lieberman, R. C.,
Purchio, A. F.,
and Gerschenson, L. E.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
3412-3415[Abstract]
-
Oberhammer, F. A.,
Pavelka, M.,
Sharma, S.,
Riefenbacher, R.,
Purchio, A. F.,
Bursch, W.,
and Schulte-Hermann, R.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
5408-5412[Abstract]
-
Lotem, J.,
and Sachs, L.
(1992)
Blood
80,
1750-1757[Abstract]
-
Andjelic, S.,
Khanna, A.,
Suthanthiran, M.,
and Nikolic-Zugic, J.
(1997)
J. Immunol.
158,
2527-2534[Abstract]
-
Choi, M. E.,
and Ballermann, B. J.
(1995)
J. Biol. Chem.
270,
21144-21150[Abstract/Free Full Text]
-
Sachsenmeier, K. F.,
Sheibani, N.,
Schlosser, S. J.,
and Allen-Hoffmann, B. L.
(1996)
J. Biol. Chem.
271,
5-8[Abstract/Free Full Text]
-
Wrana, J. L.,
Attisano, L.,
Wieser, R.,
Ventura, F.,
and Massagué, J.
(1994)
Nature
370,
341-347[CrossRef][Medline]
[Order article via Infotrieve]
-
Yamaguchi, K.,
Shirakabe, K.,
Shibya, H.,
Irie, K.,
Oishi, I.,
Ueno, N.,
Taniguchi, T.,
Nishida, E.,
and Matsumoto, K.
(1995)
Science
270,
2008-2011[Abstract]
-
Hartsough, M. T.,
Frey, R. S.,
Zipfel, P. A.,
Buard, A.,
Cook, S. J.,
McCormick, F.,
and Mulder, K. M.
(1996)
J. Biol. Chem.
271,
22368-22375[Abstract/Free Full Text]
-
Atfi, A.,
Djelloul, S.,
Chastre, E.,
Davis, R.,
and Gespach, C.
(1997)
J. Biol. Chem.
272,
1429-1432[Abstract/Free Full Text]
-
Frey, R. S.,
and Mulder, K. M.
(1997)
Cancer Res.
57,
628-633[Abstract]
-
Adams, J. M.,
and Cory, S.
(1998)
Science
281,
1322-1326[Abstract/Free Full Text]
-
Ip, Y. T.,
and Davis, R. J.
(1998)
Curr. Opin. Cell Biol.
10,
205-219[CrossRef][Medline]
[Order article via Infotrieve]
-
Davis, R. J.
(1993)
J. Biol. Chem.
268,
14553-14556[Free Full Text]
-
Davis, R. J.
(1994)
Trends Biochem. Sci.
19,
470-473[CrossRef][Medline]
[Order article via Infotrieve]
-
Nishida, E.,
and Gotoh, Y.
(1993)
Trends Biochem. Sci.
18,
128-131[CrossRef][Medline]
[Order article via Infotrieve]
-
Whitmarsh, A. J.,
Cavanagh, J.,
Tournier, C.,
Yasuda, J.,
and Davis, R. J.
(1998)
Science
281,
1671-1674[Abstract/Free Full Text]
-
David, M.,
Petricoin, E.,
Benjamin, C.,
Pine, R.,
Weber, M. J.,
and Larner, A. C.
(1995)
Science
269,
1721-1723[Medline]
[Order article via Infotrieve]
-
Marais, R.,
Wynne, J.,
and Treisman, R.
(1993)
Cell
73,
381-393[Medline]
[Order article via Infotrieve]
-
Wieser, R.,
Attisano, L.,
Wrana, J. L.,
and Massagué, J.
(1993)
Mol. Cell. Biol.
13,
7239-7247[Abstract]
-
Mader, S.,
and White, J. H.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
5603-5607[Abstract]
-
Duttaroy, A.,
Qian, J.-F.,
Smith, J. S.,
and Wang, E.
(1997)
J. Cell. Biochem.
64,
434-446[CrossRef][Medline]
[Order article via Infotrieve]
-
Kyriakis, J. M.,
Banerjee, P.,
Nicolakaki, E,
Dai, T.,
Rubie, E. A.,
Ahmad, M. F.,
Avruch, J.,
and Woodgett, J. R.
(1994)
Nature
369,
156-160[CrossRef][Medline]
[Order article via Infotrieve]
-
Sheng, Z.,
Knowlton, K.,
Chen, J.,
Hoshijima, M.,
Brown, J. H.,
and Chien, K. R.
(1997)
J. Biol. Chem.
272,
5783-5791[Abstract/Free Full Text]
-
Dudley, D. T.,
Pang, L.,
Decker, S.-J.,
Bridges, A. J.,
and Saltiel, A. R.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
7686-7689[Abstract]
-
Pang, L.,
Sawada, T.,
Decker, S. J.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
13585-13588[Abstract/Free Full Text]
-
Alessi, D. R.,
Cuenda, A,
Cohen, P.,
Dudley, D. T.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
27489-27494[Abstract/Free Full Text]
-
Xia, Z.,
Dickens, M.,
Raingeaud, J.,
Davis, R. J.,
and Greenberg, M. E.
(1995)
Science
270,
1326-1331[Abstract]
-
Fadok, V. A.,
Bratton, D. L.,
Konowal, A.,
Freed, P. W.,
Westcott, J. Y.,
and Henson, P. M.
(1998)
J. Clin. Invest.
101,
890-898[Abstract/Free Full Text]
-
Shull, M. M.,
Ormsby, I.,
Kier, A. B.,
Pawlowski, S.,
Diebold, R. J.,
Yin, M,
Allen, R.,
Sidman, C.,
Proetzel, G.,
Calvin, D.,
Annunziata, N.,
and Doetschman, T.
(1992)
Nature
359,
693-699[CrossRef][Medline]
[Order article via Infotrieve]
-
Kulkarni, A. B.,
Huh, C.-G.,
Becker, D.,
Geiser, A.,
Lyght, M.,
Flanders, K. C.,
Roberts, A. B.,
Sporn, M. B.,
Ward, J. M.,
and Karlsson, S.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
770-774[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.