From the Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Received for publication, January 23, 2003
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ABSTRACT |
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Tubular atrophy resulting from epithelial
cell loss is one of the characteristic features in the development of
chronic renal interstitial fibrosis. Although the trigger(s) and
mechanism for tubular cell loss remain undefined, the hyperactive
transforming growth factor (TGF)- Epithelial cell loss characterized as tubular atrophy is
considered to be a hallmark in the development of chronic renal
interstitial fibrosis (1-3). This pathologic process not only directly
contributes to the progressive loss of renal function but also
exacerbates the accumulation and deposition of extracellular
matrix components leading to tissue fibrosis, probably because
of the collapse and concentration of extracellular matrix surrounding
the lost cells. In this regard, tubular atrophy and interstitial
fibrosis are often inter-dependent, mutually stimulating
events that ultimately lead to end stage renal failure (4). Although
the mechanism of epithelial cell loss remains uncertain, it is
conceivable to assume that tubular atrophy primarily results from
apoptotic cell death under pathologic conditions (5-7), because there
is little or no evidence for the presence of necrosis in chronically
diseased kidneys. However, the trigger(s) and underlying mechanism
responsible for tubular cell apoptosis in vivo are largely unknown.
Transforming growth factor- Numerous studies indicate that TGF- In the present study, we demonstrate that although TGF- Antibodies and Reagents--
The antibodies for cleaved
caspase-9, full-length and cleaved caspase-8, cleaved caspase-3,
phospho-specific p38 MAP kinase, phospho-specific ERK1/2,
phospho-specific JNK, and total JNK were purchased from Cell Signaling
Technology, Inc. (Beverly, MA). The antibody against phospho-specific
Smad-2 was obtained from Upstate Biotechnology (Lake Placid, NY). The
anti-Smad-7 (sc-7004), anti-Smad-2/3 (sc-6032), anti-full-length
caspase-3 (sc-7148), total p38 MAP kinase (sc-535), and anti-actin
(sc-1616) antibodies were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). The antibody against total ERK1/2 was obtained from
Sigma. Affinity-purified secondary antibodies were purchased
from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Recombinant human TGF- Cell Culture and Treatment--
Human proximal tubular
epithelial cells (HKC) were kindly provided by Dr. L. Racusen of The
Johns Hopkins University (26). Cells were cultured in Dulbecco's
modified Eagle's medium-F-12 medium supplemented with 10% fetal
bovine serum (27). HKC cells were seeded at ~ 70% confluence in
complete medium containing 10% fetal bovine serum. Twenty-four h
later, the cells were changed to serum-free medium and incubated for
16 h. Cells were then treated with recombinant TGF- Apoptosis Detection by Fluorescent Dye H-33258
Staining--
Nuclear chromatin morphology was examined by staining
with the fluorescent dye H-33258 (5 µg/ml) as described previously
(28). Briefly, adherent and detached cells were pooled, washed with phosphate-buffered saline, fixed with 3% paraformaldehyde, and followed by staining with H-33258 for 10 min at 37 °C. Cell smears were prepared on glass slides, observed, and photographed on a Nikon
Eclipse E600 Epi-fluorescence microscope equipped with a digital camera
(Melville, NY). Apoptotic cells with characteristic nuclear
condensation and fragmentation were counted in at least ten random
fields and expressed as a percentage of the total cell number
(apoptotic index).
TUNEL Staining--
In situ detection of DNA
fragmentation was performed using terminal
deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)
staining. A fluorescein-based TUNEL staining protocol was employed by
using an apoptosis detection system (29). Cells were washed with
phosphate-buffered saline, fixed with 3% paraformaldehyde, and then
treated with 0.2% Triton X-100 for 10 min. After pre-equilibration in
100 µl of buffer containing 200 mM potassium cacodylate,
25 mM Tris-HCl, pH 6.6, 0.2 mM dithiothreitol,
0.25 mg/ml bovine serum albumin, and 2.5 mM cobalt
chloride, strands of DNA were end-labeled by incubation at 37 °C for
1 h in 50 µM fluorescein-12-dUTP, 100 µM dATP, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, and terminal deoxynucleotidyltransferase. The reaction was stopped by adding 2× sodium chloride/sodium citrate hybridization buffer for 15 min. After washing, the slides were mounted
and observed on a Nikon Eclipse E600 Epi-fluorescence microscope.
DNA Laddering--
DNA laddering was assessed essentially
according to the procedure described previously (28). Briefly, adherent
and detached cells were combined and lysed in 10 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 0.2% Triton X-100 on ice for
20 min. After a spin at 13,000 × g at 4 °C for 20 min, the supernatant (rich in low molecular weight DNA) was incubated
with protease K (200 µg/ml) at 55 °C for 16 h and 20 µg/ml
RNase A at 37 °C for 1 h and extracted with phenol-chloroform.
The DNA was precipitated by ethanol and separated by electrophoresis on
a 1.0% agarose gel containing ethidium bromide.
Western Immunoblot Analysis--
Detection of pro- and cleaved
caspases was performed by immunoblotting using specific antibodies. HKC
cells following different treatments were washed with
phosphate-buffered saline and lysed in Chaps cell extract buffer
containing 50 mM
piperazine-N,N'-bis(2-ethanesulfonic acid), pH
6.5, 2 mM EDTA, 0.1% Chaps, 5 mM
dithiothreitol, 20 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml
aprotinin, and 1 mM phenylmethylsulfonyl fluoride. After
freezing and thawing three times, cell lysates were centrifuged at
13,000 × g at 4 °C for 10 min; the supernatants
were added with SDS sample buffer (62.5 mM Tris-HCl, pH
6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and
0.1% bromphenol blue). Samples were heated at 100 °C for 5 ~10
min before loading and separated on pre-casted 10 or 15%
SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). The proteins were
electrotransferred to a nitrocellulose membrane (Amersham
Biosciences) in transfer buffer containing 48 mM
Tris-HCl, 39 mM glycine, 0.037% SDS, and 20% methanol at
4 °C for 1 h. Nonspecific binding to the membrane was blocked
for 1 h at room temperature with 5% nonfat milk in TBS buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20).
The membranes were incubated for 16 h at 4 °C with various
primary antibodies in TBS buffer containing 5% milk at the dilutions
specified by the manufacturers. The binding of primary antibodies was
followed by incubation for 1 h at room temperature with the
secondary horseradish peroxidase-conjugated IgG in 1% nonfat milk. The
signals were visualized by enhanced chemiluminescence reagent (ECL;
Amersham Biosciences).
Establishment of Stable Cell Line Overexpressing Smad-7--
HKC
cells were transfected with the pSmad-7 expression vector (kindly
provided by Dr. P. ten Dijke of Ludwig Institute for Cancer Research,
Uppsala, Sweden) (30) using the LipofectAMINETM 2000 according to the instructions specified by the manufacturer (Invitrogen). Twenty-four h after transfection and every 3-4 days thereafter, the cells were re-fed with fresh selective medium containing G418 (Invitrogen) at a final concentration of 800 µg/ml. Neomycin-resistant clones were first visible after 7 days and continuously cultured in selective medium for about 14 days. The clones
were then individually transferred into 6-well plates for expansion
using a cloning cylinder (Sigma). After two further passages in
selective medium, expanded independent clones were cultured in standard
medium. As a mock-transfection control, the pcDNA3 empty vector was
used to transfect HKC cells in a manner identical to the pSmad-7
plasmid. Overexpression of Smad-7 was confirmed in the stable cell line
by Western blot analysis and by immunofluorescence staining.
Immunostaining was performed according to the protocol described
previously (31).
Statistical Analysis--
All data examined were expressed as
mean ± S.E. For Western blot analysis, quantitation was carried
out by scanning and analyzing the intensity of the hybridization
signals using the NIH Imagine program. Statistical analysis of the data
was performed using SigmaStat software (Jandel Scientific, San Rafael,
CA). Comparison between groups was made using one-way analysis of
variance followed by the Student's-Newman-Kuels test. A p
value of less than 0.05 was considered to be statistically significant.
TGF-
To further confirm TGF- TGF-
Staurosporine also induced pro-caspase-8 activation in tubular
epithelial cells in a time-dependent manner. However,
pre-incubation with TGF- TGF- TGF-
Besides Smad signaling, we also examined the effect of TGF- Overexpression of Inhibitory Smad-7 Abrogates Smad-2 Activation but
Does Not Block TGF-
We examined and compared the effects of TGF- TGF-
We next examined the effects of blockade of p38 MAP kinase activation
on cell death and caspase-3 activation. As shown in Fig.
9, A and B, SC68376
markedly abolished TGF- Although TGF- The necessity for two hits to induce tubular epithelial cell death may
potentially have its biologic advantages, which could provide an
effective means for preventing renal tubules from unwanted, accidental
cell loss under normal physiologic conditions. It is reasonable to
speculate that such two-hit scenario will retain double checkpoints to
ensure the death/survival of tubular cells under tight control in
vivo. Transient TGF- Although the exact death cues responsible for tubular epithelial cell
apoptosis in the obstructed kidneys in vivo remain to be
identified, there are a plenty of candidates that could serve as the
"second hit" to trigger apoptotic program in the tubular cells that once were primed by hyperactive TGF- The finding that TGF- The present study attempted to dissect the signaling pathways that lead
to TGF- Our results provide a mechanistic link between TGF- In summary, we have shown in this report that TGF-1 signaling has long been suspected
to play an active role. Here we demonstrate that although TGF-
1 did
not induce cell death per se, it dramatically potentiated
renal tubular cell apoptosis initiated by other death cues in
vitro. Pre-incubation of human kidney epithelial cells (HKC) with
TGF-
1 markedly promoted staurosporine-induced cell death in a time-
and dose-dependent manner. TGF-
1 dramatically
accelerated the cleavage and activation of pro-caspase-9, but not
pro-caspase-8, in HKC cells. This event was followed by an accelerated
activation of pro-caspase-3. To elucidate the mechanism underlying
TGF-
1 promotion of tubular cell death, we investigated the signaling
pathways activated by TGF-
1. Both Smad-2 and p38 mitogen-activated
protein (MAP) kinase were rapidly activated by TGF-
1, as
demonstrated by the early induction of phosphorylated Smad-2 and p38
MAP kinase, respectively. We found that overexpression of inhibitory
Smad-7 completely abolished Smad-2 phosphorylation and activation
induced by TGF-
1 but did not inhibit TGF-
1-induced apoptosis.
However, suppression of p38 MAP kinase with chemical inhibitor SC68376
not only abolished p38 MAP kinase phosphorylation but also obliterated
apoptosis induced by TGF-
1. These results suggest that hyperactive
TGF-
1 signaling potentiates renal tubular epithelial cell apoptosis by a Smad-independent, p38 MAP kinase-dependent mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1
(TGF-
1)1 is a pleiotropic
protein that plays a central role in tissue fibrogenesis after injury (8-11). Many lines of evidence implicate hyperactive TGF-
1 as a
major pathologic factor in the initiation and progression of chronic
renal interstitial fibrosis. Overexpression of TGF-
1 axis is found
in virtually every type of chronic renal diseases in experimental
animal models and in patients (12-14). Inhibition of TGF-
1
signaling by various strategies prevents renal fibrotic lesions and
tubular atrophy and attenuates renal dysfunction (15-18). Conversely,
transgenic mice overexpressing TGF-
1 develop chronic renal disease
with increased expression of fibrotic matrix proteins (19, 20).
However, despite a causal relationship between hyperactive TGF-
1
signaling and chronic renal fibrosis in which tubular atrophy is a
characteristic feature, there is little direct, convincing evidence
demonstrating that TGF-
1 per se induces tubular
epithelial cell apoptosis.
1 is an important regulator of
cell survival and apoptosis under diverse circumstances (21-23).
However, the fate of the cells after TGF-
1 treatment is often
determined by cellular context and experimental conditions. For
instance, TGF-
1 acts as death stimulus inducing apoptotic death in
fetal hepatocytes, podocytes, and certain neuronal cells (22, 23),
whereas it also elicits pro-survival activity to protect macrophages
against apoptosis (24, 25). As to renal tubular epithelial cells,
preliminary studies in our laboratory failed to demonstrate that
TGF-
1 by itself significantly affects their survival in cultured
conditions (data not shown). Such disparity between TGF-
1 signaling
and cell apoptosis implies that hyperactive TGF-
1 signaling alone
may not be sufficient for causing tubular epithelial cells to die. This
observation led us to propose a "two-hit model" in which
both TGF-
1 signaling and a second death cue work in concert to lead
to tubular epithelial cells undergoing apoptosis.
1 by itself
did not induce tubular epithelial cells to die, it markedly potentiated
cell apoptosis initiated by other death cues by accelerating the
activation of pro-caspase-9 and -3. This action of TGF-
1 is likely
mediated by a mechanism independent of Smad signaling. Our results
suggest that TGF-
1 promotes tubular epithelial cell death by
increasing their susceptibility to secondary death stimuli.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 was purchased from R & D Systems
(Minneapolis, MN). SC68376 (p38 MAP kinase inhibitor) and fluorescent
dye H-33258 were purchased from Calbiochem (La Jolla, CA). Cell culture
medium, fetal bovine serum, and supplements were obtained from
Invitrogen. The apoptosis detection system was purchased from
Promega (Madison, WI). Staurosporine and all other chemicals were of
analytic grade and were obtained from Sigma unless otherwise indicated.
1 for
various periods of time as indicated. Staurosporine was added to the
cultures at the final concentration of 1 µM, followed by
incubating for additional periods of time ranging from 1 to 24 h.
The cells were then collected at different time points for apoptosis
detection and caspases activation analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 Potentiates Renal Tubular Epithelial Cell
Apoptosis--
Incubation of human kidney proximal tubular epithelial
HKC cells with various concentrations of TGF-
1 for 2 days did not significantly affect cell viability and apoptosis as evidenced by
nuclear condensation and fragmentation, as well as caspases activation
(data not shown), suggesting that exogenous TGF-
1 per se
does not induce tubular epithelial cells undergoing apoptotic cell
death. However, pre-incubation of HKC cells with TGF-
1 dramatically potentiated tubular epithelial cell apoptosis induced by a well known
death stimulus, staurosporine. As shown in Fig.
1, staurosporine induced tubular
epithelial cell apoptosis in a time- and dose-dependent manner. After treatment with 1 µM staurosporine for
24 h, about 20% of HKC cells underwent nuclear condensation and
fragmentation as visualized by H-33258 staining (Fig. 1E).
Pretreatment of HKC cells with 2 ng/ml of TGF-
1 for 16 h
drastically increased the percentage of cells undergoing apoptosis
to ~40%, as assessed by H-33258 staining (Fig. 1, D and
F). TGF-
1 potentiation of HKC cell apoptosis induced by
staurosporine was apparently dependent on the duration of
pre-incubation; simultaneous incubation of HKC cells with TGF-
1 and
staurosporine did not significantly increase cell death compared with
staurosporine treatment alone (Fig. 1G). The ability of
TGF-
1 to enhance staurosporine-induced cell death was also
dose-dependent and was effective at a concentration as low
as 0.5 ng/ml (Fig. 1H).
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Fig. 1.
TGF- 1 potentiates
renal tubular epithelial cell apoptosis induced by staurosporine.
A-D, nuclear condensation of apoptotic
cells shown by fluorescent dye H-33258 staining. HKC cells were
pretreated with or without 2 ng/ml of TGF-
1 for 16 h and
followed by incubating with or without 1 µM staurosporine
for 24 h. A, control HKC cells; B, TGF-
1
alone; C, staurosporine alone; D, TGF-
1 plus
staurosporine. Arrowheads indicate apoptotic cells.
E, time course of HKC cell apoptosis following staurosporine
treatment. HKC cells were treated with 1 µM staurosporine
for various periods of time as indicated. *, p < 0.05 versus control. F, graphic presentation of
apoptotic cell index (%) in different treatment groups. Pretreatment
with TGF-
1 for 16 h markedly augmented tubular epithelial cell
apoptosis induced by staurosporine. *, p < 0.05 versus control; **, p < 0.05 versus staurosporine alone. G, TGF-
1
potentiation of tubular epithelial cell death was dependent on the
duration of pre-incubation. HKC cells were pretreated with TGF-
1 for
various periods of time as indicated, followed by incubating with 1 µM staurosporine for 24 h. *, p < 0.05 versus staurosporine alone. H, dose
dependence of TGF-
1 potentiation of tubular epithelial cell death.
HKC cells were pretreated for 16 h with various concentrations of
TGF-
1 as indicated and followed by incubating with 1 µM staurosporine for 24 h. *, p < 0.05 versus staurosporine alone.
1 potentiation of tubular epithelial cell
death, we employed two additional approaches to detect cell apoptosis
after various treatments of HKC cells, namely TUNEL staining and DNA
laddering analysis. As shown in Fig. 2,
TGF-
1 pre-incubation for 16 h markedly enhanced HKC cell
apoptosis induced by staurosporine. TUNEL staining revealed a more than
2-fold induction of apoptosis in the TGF-
1-pretreated group as
compared with that with staurosporine treatment alone (Fig. 2,
A-D). Similarly, DNA laddering analysis also
exhibited a greater DNA fragmentation observed in TGF-
1-pretreated
cells as compared with that in the cells treated with staurosporine
alone (Fig. 2E, lanes 3 and 4).
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Fig. 2.
TGF- 1 potentiates
renal tubular epithelial cell death induced by staurosporine. HKC
cells were pretreated with or without 2 ng/ml of TGF-
1 for 16 h
and followed by incubating with 1 µM staurosporine for
24 h. A-D, TUNEL staining.
A-C, representative micrographs show
TUNEL-positive cells. A, control HKC cells; B,
staurosporine alone; C, TGF-
1 plus staurosporine.
Arrowheads indicate apoptotic cells. D,
graphic presentation of HKC cell apoptosis after various treatments.
Data are expressed as -fold induction over control HKC cells. *,
p < 0.05 versus control; **,
p < 0.05 versus staurosporine alone.
E, DNA laddering analysis shows DNA fragmentation in HKC
cells after various treatments with TGF-
1 or/and staurosporine.
Lane 1, control HKC cells; lane 2, TGF-
1
alone; lane 3, staurosporine alone; lane 4,
TGF-
1 plus staurosporine.
1 Accelerates Cleavage and Activation of Pro-caspase-9 but
Not Caspase-8--
To elucidate how TGF-
1 potentiates tubular
epithelial cell death, we examined the activation of pro-caspases in
HKC cells after incubation with staurosporine alone or following
pretreatment with TGF-
1. Fig. 3 shows
the kinetics of pro-caspase-9 activation in HKC cells upon treatment
with death stimulus. Staurosporine induced rapid, marked pro-caspase-9
cleavage to produce an active form in tubular epithelial cells in a
time-dependent fashion. Of interest, pre-incubation with
TGF-
1 dramatically accelerated this activation process in HKC cells.
At 1 h after staurosporine treatment, whereas pro-caspase-9
activation was barely detectable in HKC cells, the level of active
caspase-9 in TGF-
1-pretreated cells was markedly increased and
reached about 20-fold of that in control HKC cells (Fig. 3,
A and B). The active caspase-9 peaked at 3 h
(about 60-fold) following addition of death stimulus in TGF-
1-preteated cells, whereas it reached the highest level at 6 h in non-pretreated HKC cells. These results suggest that
TGF-
1 potentiates tubular epithelial cell apoptosis by accelerating pro-caspase-9 activation after death challenge.
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Fig. 3.
TGF- 1 accelerates
the activation of pro-caspase-9, but not caspase-8, following
staurosporine treatment. HKC cells were pretreated with 2 ng/ml of
TGF-
1 for 16 h and followed by incubating with 1 µM staurosporine. At different time points as indicated,
cells were collected, and cell lysates were subjected to Western blot
analysis with specific antibodies. A, Western blot
demonstrates the kinetics of pro-caspase-9 activation. Samples were
immunoblotted with antibodies against cleaved caspase-9 and actin,
respectively. B, graphical presentation of cleaved caspase-9
levels after normalization to actin in tubular epithelial cells
following various treatments. Solid circle, staurosporine
alone; open circle, TGF-
1 plus staurosporine. Western
blot (C) and graphical presentation (D) of the
kinetics of pro-caspase-8 activation in HKC cells are shown. An
identical pattern of pro-caspase-8 activation is observed in HKC cells
pretreated either with or without TGF-
1. Solid circle,
staurosporine alone; open circle, TGF-
1 plus
staurosporine.
1 did not change the kinetics of
pro-caspase-8 cleavage and its subsequent activation. The pattern of
pro-caspase-8 activation after treatment with death stimulus was
essentially identical in the tubular epithelial cells pretreated with
or without TGF-
1 (Fig. 3, C and D).
1 Accelerates the Activation of Downstream Effector
Pro-caspase-3--
We further investigated the effect of TGF-
1 on
the activation of downstream effector pro-caspase in tubular epithelial
cells after death challenge. As shown in Fig.
4, pro-caspase-3 activation following
staurosporine treatment in HKC cells was also significantly accelerated
by TGF-
1 pretreatment. At 3 h after addition of death stimulus,
active caspase-3 level in TGF-
1-pretreated HKC cells was more than
11-fold of that in untreated cells (Fig. 4, A and B). Significant acceleration on pro-caspase-3 activation by
TGF-
1 pretreatment was also found at other time points in HKC cells (Fig. 4). Dose dependence studies revealed that TGF-
1, at a
concentration of 0.5 ng/ml, was sufficient for enhancing pro-caspase-3
activation (Fig. 4, C and D). A higher dose of
TGF-
1 did not result in a further significant increase in the levels
of active caspase-3 in HKC cells (Fig. 4).
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Fig. 4.
TGF- 1 accelerates
the activation of downstream effector caspase-3 after staurosporine
treatment. HKC cells were pretreated with 2 ng/ml of TGF-
1 for
16 h and followed by incubating with 1 µM
staurosporine for various periods of time as indicated. A,
Western blot demonstrates the kinetics of pro-caspase-3 activation.
Cell lysates were blotted with specific antibodies against either
full-length or cleaved caspase-3, respectively. B, graphical
presentation of the relative abundance of cleaved caspase-3 (-fold
induction relative to control cells) after normalization to
pro-caspase-3 in HKC cells following various treatments. C
and D, effects of different concentrations of TGF-
1 on
accelerating pro-caspase-3 activation. HKC cells were pretreated for
16 h with various concentrations of TGF-
1 as indicated,
followed by incubation with staurosporine for 6 h. C,
Representative Western blot. D, graphical presentation of
the relative abundance of cleaved caspase-3 in HKC cells.
1 Activates Both Smad-2 Signaling and p38 MAP Kinase in
Renal Tubular Cells--
To decipher the signal pathway(s) responsible
for TGF-
1 potentiation of renal tubular cell apoptosis, we first
investigated the potential signaling events initiated by TGF-
1 in
HKC cells. As shown in Fig. 5, incubation
of HKC cells with TGF-
1 induced rapid activation of Smad signaling.
The phosphorylated state of Smad-2 increased as early as 10 min, peaked
at 30 min, and gradually returned toward basal level at 3 h after
TGF-
1 stimulation (Fig. 5, A and C). As Smad-2
is an effector intermediate signaling molecule for TGF-
1, its
phosphorylation and activation manifest that the Smad signaling is
probably one of the major signal transduction pathways that is readily
activated and potentially mediates the cellular actions of TGF-
1 in
renal tubular epithelial cells.
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Fig. 5.
TGF- 1
activates Smad-2 and p38 MAP kinase in renal tubular epithelial
cells. Kinetics of Smad-2 (A) and p38 MAP kinase
(B) activation induced by TGF-
1 are shown. HKC cells were
treated with 2 ng/ml of TGF-
1 for various periods of time as
indicated. Cell lysates were immunoblotted with antibodies against
either phospho-specific Smad-2 and phospho-specific p38 MAP kinase or
total Smad-2/3 and total p38 MAP kinase, respectively. C,
graphical presentation of the kinetics of Smad-2 and p38 MAP kinase
activation induced by TGF-
1 in HKC cells. Solid circle,
p-Smad-2; open circle, p-p38 MAP kinase.
1 on
other signal pathways in renal tubular epithelial cells. Fig.
5B shows the activation of p38 MAP kinase upon TGF-
1
stimulation in HKC cells. Treatment of HKC cells with TGF-
1 induced
p38 MAP kinase phosphorylation at 1 h, and this induction was
largely sustained throughout the entire experimental scheme of 48 h (Fig. 5, B and C). Under the same conditions,
TGF-
1 did not significantly activate the ERK1/2 and JNK, the
other two subfamilies of the MAP kinases (see Fig. 8). These results
suggest that both Smad signaling and p38 MAP kinase are activated in
renal tubular epithelial cells upon stimulation by TGF-
1.
1 Potentiation of Cell Death--
To investigate
the potential role of Smad signaling in mediating TGF-
1 potentiation
of renal tubular cell death, we studied the effects of blocking Smad
signaling on cell apoptosis by overexpressing inhibitory Smad-7. To
this end, stable cell lines were established after
transfecting with either pSmad-7
expression plasmid or pcDNA3 empty vector. As shown in Fig. 6,
HK-pSmad-7 cell line robustly expressed Smad-7, as demonstrated
by Western blot analysis (Fig. 6A) and immunofluorescence
staining (Fig. 6B). Interestingly, overexpression of Smad-7
completely abolished Smad-2 activation following TGF-
1 incubation in
the HK-pSmad-7 cells (Fig. 6C). There was no significant
increase in the levels of phosphorylated Smad-2 in the HK-pSmad-7 cells
after TGF-
1 treatment, in contrast to the HK-pcDNA3 cells (Fig.
6C), suggesting that inhibitory Smad-7 can effectively blunt
TGF-
1-initiated Smad signaling. Of note, overexpression of Smad-7
did not affect p38 MAP kinase activation induced by TGF-
1 in renal
epithelial cells (Fig. 6D).
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Fig. 6.
Overexpression of inhibitory Smad-7 abrogates
Smad-2 activation induced by TGF- 1.
A, Western blot confirms Smad-7 overexpression in the stably
transfected cell line HK-pSmad-7. Cell lysates were derived from the
cell lines established by stably transfecting pcDNA3 and pSmad-7
plasmids and immunoblotted with antibodies against Smad-7 and actin,
respectively. B, immunofluorescence staining for Smad-7 in
HK-pcDNA3 and HK-pSmad-7 cell lines. C, abrogation of
TGF-
1-induced Smad-2 activation in the Smad-7-overexpressed cell
line (HK-pSmad-7). Cell lysates were immunoblotted with
antibodies against phospho-specific Smad-2 and total Smad-2/3,
respectively, at various time points as indicated after incubation with
2 ng/ml of TGF-
1. Smad-2 activation by TGF-
1 in control
HK-pcDNA3 cells was presented as a positive control. D,
Smad-7 overexpression did not affect p38 MAK kinase activation by
TGF-
1. Western blot demonstrates an identical pattern of p38 MAP
kinase activation by TGF-
1 in control HK-pcDNA3 and the
Smad-7-overexpressed HK-pSmad-7 cell lines.
1 on
staurosporine-induced apoptosis in HK-pSmad-7 and control HK-pcDNA3
cell lines. As shown in Fig. 7, no
significant difference in apoptotic index was found between
HK-pcDNA3 and HK-pSmad-7 cells. Likewise, it was virtually
identical in the abundance of the cleaved, active caspase-3 in the
HK-pcDNA3 and HK-pSmad-7 cells after incubation with
staurosporine for various periods of time (Fig. 6, C
and D). Thus, overexpression of inhibitory Smad-7 in renal
tubular cells abrogates Smad-2 activation but does not abolish TGF-
1 potentiation of cell death, indicating that TGF-
1 promotes cell apoptosis by a mechanism independent of Smad signaling.
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Fig. 7.
Overexpression of Smad-7 does not affect
TGF- 1 potentiation of renal tubular cell
apoptosis. A, representative micrographs show cell
apoptosis in pcDNA3- and pSmad-7-transfected renal tubular HKC
cells. Cells were treated with 2 ng/ml of TGF-
1 for 16 h
followed by incubation with 1 µM staurosporine.
B, graphical presentation of apoptotic index (%) in
pcDNA3- and pSmad-7-transfected cells. Data are presented as
mean ± S.E. (n = 3). C, overexpression
of Smad-7 did not affect caspase-3 activation induced by TGF-
1 and
staurosporine. D, quantitative determination of caspase-3
activation following various treatments as indicated. The relative
levels of cleaved caspase-3 were presented after normalization with
procaspase-3.
1 Promotion of Renal Tubular Cell Apoptosis Is Dependent on
p38 MAP Kinase Activation--
We also investigated the possible role
of p38 MAP kinase in renal tubular cell apoptosis. Treatment of
HKC cells with SC68376, a specific inhibitor of p38 MAP kinase,
abolished TGF-
1-induced p38 MAP kinase phosphorylation and
activation in a dose-dependent manner (Fig.
8A). SC68376 at a
concentration of 20 µM completely abrogated p38 MAP
kinase activation but did not influence Smad-2 phosphorylation induced
by TGF-
1. Of note, both TGF-
1 and p38 MAP kinase inhibitor
SC68376 failed to significantly affect the phosphorylation status of
the ERK1/2 and JNK in renal epithelial HKC cells (Fig. 8, C
and D).
View larger version (35K):
[in a new window]
Fig. 8.
SC68376 specifically inhibits p38 MAP kinase
activation by TGF- 1 in renal tubular
epithelial cells. A, SC68376 inhibits p38 MAP kinase
activation by TGF-
1 in a dose-dependent manner. HKC
cells were pretreated with different doses of SC68376 as indicated for
30 min prior to incubation with 2 ng/ml of TGF-
1. Cell lysates were
immunoblotted with either phospho-specific or total p38 MAP kinase,
respectively. B, SC68376 at a high concentration (20 µM) does not influence Smad-2 activation by TGF-
1 in
renal epithelial cells. C and D, neither TGF-
1
nor p38 MAP kinase inhibitor SC68376 affects ERK-1/2 (C) and
JNK (D) phosphorylation in renal epithelial cells.
1-induced caspase-3 activation in renal
tubular epithelial cells but did not affect basal caspase-3 activation
induced by staurosporine alone. Consistently, inhibition of p38 MAP
kinase activation by SC68376 also obliterated TGF-
1 potentiation of
cell apoptosis in a time- and dose-dependent fashion (Fig.
9, C and D). These results indicate that TGF-
1 promotes renal tubular epithelial cell apoptosis by a p38 MAP kinase-dependent mechanism.
View larger version (23K):
[in a new window]
Fig. 9.
Blockade of p38 MAP kinase activation by
SC68376 abolishes TGF- 1 potentiation of
caspase-3 activation and cell apoptosis. SC68376 inhibits TGF-
1
potentiation of caspase-3 activation in renal epithelial HKC cells, as
demonstrated by Western blot analysis (A) and graphical
presentation (B). C, SC68376 abolishes TGF-
1
potentiation of renal tubular cell apoptosis in a
time-dependent manner. HKC cells were treated without or
with 2 ng/ml of TGF-
1 in the absence or presence of 20 µM SC68376, followed by treated with staurosporine for
various periods of time as indicated. Solid circle,
staurosporine alone; solid triangle, TGF-
1 and
staurosporine; open circle, SC68376 plus TGF-
1 and
staurosporine. D, SC68376 abolishes TGF-
1 promotion of
cell apoptosis in a dose-dependent way. HKC cells were
treated with 2 ng/ml of TGF-
1, 1 µM staurosporine, and
different doses of SC68376 as indicated. Data are presented as
mean ± S.E. (n = 3). *, p < 0.05 versus staurosporine alone; **, p < 0.05 versus TGF-
1 plus staurosporine.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 has long been alleged to play a critical role in
the tubular atrophy characterized by epithelial cell loss (3, 9), there
is little direct evidence demonstrating that TGF-
1 actually causes
tubular epithelial cells to die in vitro. Incubation of
cultured tubular epithelial cells with TGF-
1 at the concentration
comparable with physiologic or pathophysiologic circumstances resulted
in little or no appreciable cell death (see Figs. 1 and 2). This
observation suggests that TGF-
1 signaling is certainly not
sufficient to trigger tubular epithelial cells to commit suicide. In
this study, we have demonstrated that TGF-
1 plays an imperative role
in promoting tubular epithelial cell death, not by directly inducing
cell apoptosis but rather by sensitizing these cells to a hit from
secondary death cues. These findings are consistent with a
two-hit model of tubular epithelial cell death, in which the
primary role of hyperactive TGF-
1 signaling perhaps is to render
tubular cells readily susceptible to a second hit from other death stimuli.
1 up-regulation without secondary hit may
eventually leave tubular epithelial cells to survive. However,
sustained activation of TGF-
1 signaling followed by secondary death
challenges, a situation that presumably occurs under chronically
injured conditions (32), could have lethal consequence to the tubular
cells. Our two-hit working model is supported by the in vivo
observation that the expression of both TGF-
1 and its type I
receptor is specifically up-regulated in renal tubules of the
obstructed kidneys (12, 32). Furthermore, this tubule-specific
induction of TGF-
1 axis is an early event that takes place at 1 day
after ureteral obstruction (32), a timing that precedes significant
cell apoptosis in the kidneys. These spatial and temporal correlations
between the activation of TGF-
1 signaling and tubular epithelial
cell apoptosis imply that hyperactive TGF-
1 may play a critical role
in promoting tubular cell death in the diseased kidneys, probably by
priming these cells to commit suicide upon secondary death challenges.
1 signaling. For example, in kidneys with persistent and complete ureteral obstruction, chronic hypoxia induced by a compromised interstitial blood flow could
result in cellular ATP deprivation that might serve as an apoptosis
trigger (33, 34). Likewise, infiltration of inflammatory cells as seen
in the obstructed kidneys may contribute to cell death by producing
pro-death ligand or cytokines (35-37). Furthermore, disruption of
normal cell-to-cell and cell-to-matrix interactions in the diseased
kidneys provides a hostile environment for tubular cell survival that
ultimately leads cells to die. In this context, the suppression of
E-cadherin expression in the obstructed kidneys, as reported previously
(31), may act as a secondary hit that results in tubular cell death.
E-cadherin is an epithelial cell adhesion receptor found within
adherens-type junctions. Although it is widely recognized that
E-cadherin plays an essential role in the maintenance of structural
integrity of renal tubular epithelia (38), recent studies also
implicate it as a major survival factor for tubular epithelial cells
(39). In addition, the progressive deposition of extracellular matrix
as seen in the chronically diseased kidneys will eventually disrupt
normal cell-matrix interactions of renal tubules (40-42). Such
disturbance of the microenvironment that surrounds the tubular cells
could potentially function as a second hit for renal epithelial cell
apoptosis. Collectively, there are a wide variety of factors present in
the diseased kidneys that could conceivably work in concert with
TGF-
1 to lead tubular epithelial cells to commit to die in
vivo.
1 only accelerates the activation of
pro-caspase-9, but not pro-caspase-8, suggests that its action is
probably mediated via a pathway involving mitochondria (43, 44). Both
caspase-9 and caspase-8 are recognized as initial caspases whose
activation triggers a cascade of activation process of downstream
effector caspases such as caspase-3 (45, 46). However, the upstream
pathways leading to activation of caspase-9 and caspase-8 are believed
to be different. Although caspase-8 activation is involved in death
receptor pathway such as FasL and Fas, pro-caspase-9 cleavage and
subsequent activation is coupled with the events associated with the
release of cytochrome C from mitochondria and activation of Apaf-1
(47-49). The specific acceleration of caspase-9 activation by TGF-
1
suggests that its pro-apoptotic signaling is implicated in a pathway
involved in mitochondria, although the details remain to be unraveled.
Of note, TGF-
1 apparently only accelerates pro-caspase-9 activation
but not increases the magnitude of active caspase-9 peak level (Fig.
3). For instance, TGF-
1 pre-incubation followed by treatment with
death stimulus produced the peak of active caspase-9 3 h earlier
over the control cultures. This phenomenon could be attributable to the
assumption that endogenous pro-caspase-9 may be fully activated upon
stimulation with death challenges in tubular epithelial cells.
1 potentiation of renal tubular cell apoptosis. It has been
well documented that TGF-
1, through binding to its type II and type
I receptors, elicits a wide range of cellular responses that modulate
cell proliferation, differentiation, and apoptosis (10, 25, 50). Many
of the signaling responses initiated by TGF-
1 are primarily mediated
by intermediate signaling molecules Smad proteins (51-53). It appears
clear that TGF-
1 activates at least two separate intracellular
signal transduction pathways that result in both Smad and p38 MAP
kinase activation in renal tubular cells (Fig. 5). Of interest, despite
early and marked activation of Smad-2, Smad signaling appears not to
mediate TGF-
1 promotion of cell death (see Figs. 5 and 7). This is
in contrast to the TGF-
1-induced apoptosis in podocytes, in which
Smad-7 is demonstrated to play a critical role (23). Of note, Smad-7 is
an inhibitory Smad that can functionally antagonize
receptor-regulated-Smad (such as Smad-2 and -3) signaling by
competitively binding to TGF-
receptors (10). Although not
determined yet, it is reasonable to speculate that overexpression of
inhibitory Smad-7 may also block Smad-3 activation in renal epithelial
cells, in view of the fact that Smad-2 and Smad-3 are highly homologous
and often activated simultaneously by TGF-
1 (51). Of interest,
neither endogenous Smad-7 in renal tubular epithelial cells is induced by TGF-
1 (data not shown), nor does overexpression of exogenous Smad-7 affect cell death/survival under basal conditions. Furthermore, although overexpression of Smad-7 abolished Smad-2 phosphorylation and
activation in response to TGF-
1 stimulation (Fig. 6), it failed to
obliterate renal tubular cell apoptosis (Fig. 7). Therefore, unlike in
podocytes, it appears obvious that TGF-
1 promotes renal tubular cell
apoptosis via a mechanism independent of Smad signaling.
1 promotion of
cell apoptosis and p38 MAP kinase activation (see Figs. 8 and 9). The
p38 MAP kinase belongs to a subfamily of the MAP kinases and is
activated primarily in response to stress, as well as cytokine
stimulation (54, 55). Earlier studies suggest that different
subfamilies of MAP kinases play a distinct role in regulating cell
apoptosis, with ERK-MAP kinase as pro-survival and p38 MAP kinase and
JNK as pro-death signals (56-58). Thus, the ratio among different MAP
kinases may dictate cell fate in certain conditions. In this regard, we
found no significant alterations in ERK-MAP kinase and JNK,
suggesting that the p38 MAP kinase activation likely plays
a predominant role in mediating TGF-
1-induced cell death. The
requirement of p38 MAP kinase activation for mediating TGF-
1-induced
apoptosis is illustrated by abrogating cell death after blockade of its
activation (Fig. 9). Of note, a sustained, but not transient, p38 MAP
kinase activation may also be of importance in inducing cell apoptosis,
because a long period of pre-incubation with TGF-
1, which induces
sustained p38 MAK kinase activation (Fig. 5B), is required
for optimal potentiation of staurosporine-induced cell death (Fig.
1G). Thus it is concluded that a p38 MAP
kinase-dependent, Smad-independent signaling mediates
TGF-
1-induced renal tubular cell apoptosis.
1 dramatically
potentiates cell apoptosis triggered by other death cues by
accelerating the activation of caspase-9 and caspase-3 in tubular epithelial cells. Thus, hyperactive TGF-
1 signaling promotes renal
interstitial fibrogenesis not only by its well documented profibrotic
actions but also by its potentiation of tubular epithelial cell
apoptosis. Moreover, our results unravel a novel mode of TGF-
1
action in the regulation of cell death/survival, in which it primarily
renders the tubular epithelial cells readily susceptible to secondary
death cues rather than directly inducing them to die. This action of
TGF-
1 in promoting cell death appears to be mediated by a mechanism
dependent of p38 MAP kinase but independent of Smad signaling.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. P. ten Dijke for generously providing Smad-7 expression vector.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK-02611, DK-54922, and DK-61408 (to Y.L.).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 postdoctoral fellowships from the American Heart
Association Pennsylvania-Delaware Affiliate.
§ To whom correspondence should be addressed: Dept. of Pathology, University of Pittsburgh School of Medicine, S-405 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15261. Tel.: 412-648-8253; Fax: 412-648-1916; E-mail: liuy@msx.upmc.edu.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M300777200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-1, transforming growth factor-
1;
MAP, mitogen-activated protein;
ERK, extracellular signal-regulated kinase;
JNK, c-Jun N-terminal
kinase;
TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP nick-end labeling
staining;
Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
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