1 Toxicological Sciences, Environmental Health Sciences, and 3 Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205; and Sections of 4 Pulmonary and Critical Care Medicine and 2 Nephrology, Department of Internal Medicine, Yale University School of Medicine and the Veterans Affairs Connecticut Healthcare Systems, New Haven, Connecticut 06520
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ABSTRACT |
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Transforming growth factor-1
(TGF-
1) is a potent inducer of extracellular matrix
protein synthesis and a key mediator of renal fibrosis. However, the
intracellular signaling mechanisms by which TGF-
1
stimulates this process remain incompletely understood. In this report,
we examined the role of a major stress-activated intracellular
signaling cascade, belonging to the mitogen-activated protein kinase
(MAPK) superfamily, in mediating TGF-
1 responses in rat
glomerular mesangial cells, using dominant-negative inhibition of
TGF-
1 signaling receptors. We first stably transfected
rat glomerular mesangial cells with a kinase-deleted mutant TGF-
type II receptor (T
R-IIM) designed to inhibit
TGF-
1 signaling in a dominant-negative fashion. Next,
expression of T
R-IIM mRNA was confirmed by Northern
analysis. Cell surface expression and ligand binding of
T
R-IIM protein were demonstrated by affinity cross-linking with 125I-labeled-TGF-
1.
TGF-
1 rapidly induced p38 MAPK phosphorylation in
wild-type and empty vector (pcDNA3)-transfected control mesangial cells. Interestingly, transfection with dominant-negative
T
R-IIM failed to block TGF-
1-induced p38
MAPK phosphorylation. Moreover, dominant-negative T
R-IIM
failed to block TGF-
1-stimulated pro-
1(I) collagen mRNA expression and cellular protein synthesis, whereas TGF-
1-induced extracellular signal-regulated kinase
(ERK) 1/ERK2 activation and antiproliferative responses were blocked by
T
R-IIM. In the presence of a specific inhibitor of p38
MAPK, SB-203580, TGF-
1 was unable to stimulate
pro-
1(I) collagen mRNA expression in the control and
T
R-IIM-transfected mesangial cells. Finally, we
confirmed that both p38 MAPK activation and pro-
1(I)
collagen stimulation were TGF-
1 effects that were
abrogated by dominant-negative inhibition of TGF-
type I receptor.
Thus we show first demonstration of p38 MAPK activation by
TGF-
1 in mesangial cells, and, given the rapid kinetics,
this TGF-
1 effect is likely a direct one. Furthermore,
our findings suggest that the p38 MAPK pathway functions as a component
in the signaling of pro-
1(I) collagen induction by
TGF-
1 in mesangial cells.
transforming growth factor- receptor; signal transduction; mitogen-activated protein kinase; matrix
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INTRODUCTION |
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TRANSFORMING GROWTH
FACTOR-1 (TGF-
1) is a member of a
superfamily of multifunctional cytokines that participates in a wide array of biological activities such as development and wound repair, as
well as pathological processes (2). TGF-
1
is a potent inducer of extracellular matrix (ECM) protein synthesis and
accumulation and has been implicated as the key mediator of
fibrogenesis in a variety of tissues (3). In the kidney,
the critical role of TGF-
1 has been well recognized in
several renal diseases characterized by progressive glomerular ECM
accumulation leading to the development of glomerulosclerosis. In
kidneys of both patients and experimental animals with
glomerulonephritis, increased TGF-
1 expression has been
observed (4, 32). Moreover, suppression of excess ECM deposition and glomerulosclerosis by neutralizing TGF-
1
antibody has been shown in experimental glomerulonephritis
(4). Enhanced glomerular expression of
TGF-
1 has also been shown in both human and experimental
diabetic nephropathy (45). Furthermore, in vivo
transfection of the TGF-
1 gene into the rat kidney has
been shown to induce glomerulosclerosis (24). Similarly,
the development of glomerulosclerosis has been described in mice
expressing the transgene for TGF-
1 under the control of
a murine albumin promoter (Alb/TGF-
1) and that have
elevated circulating levels of TGF-
1 (25).
TGF-1 induces the synthesis of a number of ECM proteins,
including types I and III collagen, fibronectin, laminin, and
proteoglycans (23, 36). Type I collagen is a major
structural component of the ECM, which is synthesized by fibroblasts
and vascular smooth muscle cells and in the kidney by glomerular
mesangial cells, considered to be centrally involved in progressive
glomerular ECM accumulation associated with chronic sclerotic processes
(12, 18). We and others have previously reported increased
collagen synthesis by cultured glomerular mesangial cells in response
to TGF-
1 (9, 18). Although mesangial cells
in culture express types I, III, IV, and V collagen, ~95% of the
total collagen synthesized is type I, which has been identified in both
the secreted and cell-associated fractions, and it has been suggested
that the process of cell culture induces an inflammatory or sclerosing phenotype in the glomerular mesangial cell (19, 39).
Induction of type I collagen in the glomerular mesangium has been well
demonstrated in experimental models of glomerulosclerosis. For
instance, in the Alb/TGF-
1 transgenic mice that develop
glomerulosclerosis, markedly increased immunostaining of type I
collagen, and type III collagen to a lesser degree, is seen in the
glomerulus within capillary loops and the mesangium, whereas near
normal expression of type IV collagen is observed (30).
Despite the ever-growing body of data demonstrating
TGF-1 effects on ECM induction and development of
glomerulosclerosis, the intracellular signaling mechanisms by which
TGF-
1 stimulates this process remain poorly understood.
Molecular cloning has revealed that biological actions of
TGF-
1 are mediated by two transmembrane Ser/Thr kinases,
types I and II, which are coexpressed by most cells, including
glomerular mesangial cells (9, 28). The initiation of
signaling involves the binding of TGF-
1 to TGF-
type
II receptor (T
R-II), a constitutively active Ser/Thr kinase, resulting in the recruitment and phosphorylation of TGF-
type I
receptor (T
R-I) to produce a heteromeric signaling complex, which in
turn activates downstream signaling pathways (40, 44). Recently, an emerging body of evidence implicates the intracellular mitogen-activated protein kinase (MAPK) as an important
TGF-
1 signaling pathway (1, 6, 20).
The MAPK is a major signaling system used by eukaryotic cells to transduce extracellular signals to intracellular responses (38, 41). The activation of the MAPKs requires a well-coordinated cascade of three protein kinase reactions that transduce signals by sequential phosphorylation and activation of the next kinase in their respective pathways (38). The MAPK cascades display evolutionary conservation and are implicated to play essential roles in the signal transduction of many biological events such as the regulation of cell growth, differentiation, and apoptosis and cellular responses to environmental stresses. The best characterized of the MAPKs is the ERK cascade, which consists of the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) and which is protypically activated by mitogenic stimuli (31). The other two MAPK family members, the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) and the p38 MAPK, are activated predominantly by cellular stresses or inflammatory signals (13, 26).
TGF-1 has been demonstrated in various cell
types to be capable of activating each of the three major MAPK
superfamily members. We have recently demonstrated the requirement of
the ERK pathway in signaling anti-apoptotic effects of
TGF-
1 to promote cell survival of macrophages
(6). This investigation was thus undertaken to determine
whether TGF-
1 activates specific MAPK signaling pathway(s) in cultured rat mesangial cells and whether this MAPK activation mediates TGF-
1-induced stimulation of type I
collagen, using the strategy of dominant-negative inhibition of
TGF-
1 signaling receptors. We have successfully used
this strategy previously to inhibit T
R-II-mediated signaling in
glomerular endothelial cells and in macrophages (6, 8). In
the present study, we show that TGF-
1 rapidly induced
the phosphorylation of p38 MAPK in cultured rat glomerular mesangial
cells. However, transfection with dominant-negative mutant of T
R-II
(T
R-IIM) failed to block the
TGF-
1-induced p38 MAPK phosphorylation and the
TGF-
1-stimulated pro-
1(I) chain of type I
procollagen mRNA and cellular protein synthesis. Moreover, a specific
chemical inhibitor of p38 MAPK, SB-203580, blocked
TGF-
1-induced pro-
1(I) collagen mRNA. Our data, taken together, indicate that TGF-
1 directly and
rapidly activates the p38 MAPK and that the p38 MAPK functions as a
component in TGF-
1 signaling of pro-
1(I)
collagen induction in mesangial cells.
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EXPERIMENTAL PROCEDURES |
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Reagents.
Recombinant human TGF-1 was obtained from R & D Systems
(Minneapolis, MN). The phospho-p38 MAPK
(Thr180/Tyr182), p38 MAPK, and
phospho-activating transcription factor-2 (ATF-2) (Thr71)
rabbit polyclonal antibodies, purified ATF-2 fusion protein, phospho-p44/42 MAPK (Thr202/Tyr204),
phospho-SAPK/JNK (Thr183/Tyr185), and SAPK/JNK
rabbit polyclonal antibodies were purchased from New England Biolabs
(Beverly, MA). The specific inhibitor of p38 MAPK, SB-203580, was
obtained from Calbiochem (San Diego, CA), and the MAPK kinase (MEK) 1 inhibitor PD-098059 was from New England Biolabs.
Constructs.
A truncated TGF- type II receptor construct (T
R-IIM),
lacking the cytoplasmic Ser/Thr kinase domain but containing the full transmembrane-spanning and extracellular domains, was generated by PCR
using a rat T
R-II cDNA as the template, as previously described
(8), with the following modifications. Primer sequences were sense primer 5'-GTTTGAATTCGACGGGGGCTGCCATG-3' and
antisense primer
5'-TTCTACTGTTACCGTGTCCATCACCACCATCATCACTAGCGGCCGCGGGCC-3' and contained the sequences for the restriction enzymes
EcoR I and Not I, respectively (underlined), for
directional cloning and a stop codon in the antisense primer. The PCR
amplified product was cloned into pcDNA3 (Invitrogen). Confirmation
that the clone contained correct directionality and in-frame sequences
of the PCR product was obtained by complete sequencing using the
dideoxy chain termination technique with Sequenase 2.0 (Amersham).
Cell culture.
Glomerular mesangial cells were isolated from the renal cortex of
~150-g Sprague-Dawley rats as previously described (9), using the standard sieving technique with the following modifications. After collagenase digestion, the cells were plated in RPMI 1640 medium
(GIBCO-BRL), supplemented with 20% FBS (HyClone), 5 U/ml penicillin,
and 5 µg/ml streptomycin, and incubated in a humidified atmosphere of
5% CO2 and 95% air at 37°C. After 96 h, the serum in the medium was changed to 15% FBS and cells propagated. All newly
isolated cells were characterized by immunostaining for anti-vimentin
(Dako) and anti-myosin antibodies (Zymed) and negative staining for
cytokeratin (Boehringer-Mannheim) and von Willebrand's factor
(Dianova) as well as negative fluorescent acetylated low-density lipoprotein uptake (Biomedical Technologies). Cell labeling was performed according to the manufacturer's protocol. Once the cells were established in culture, they were maintained in RPMI 1640 with
15% FBS, 5 U/ml penicillin, and 5 µg/ml streptomycin. Cells between
passages 5 and 15 were used for the experiments. In experiments involving exogenous TGF-1 treatment, cells grown to 90%
confluence were placed in RPMI medium containing 0.5% FBS in the
presence or absence of human TGF-
1 (R & D). In
experiments using the p38 and MEK1 inhibitors, cells were preincubated
for 1 h in the absence or presence of 10 µM SB-203580 or
PD-098059, respectively, before treatment with or without exogenous
TGF-
1.
Stable transfection of rat mesangial cells.
To generate clones that stably expressed TR-IIM or
T
R-IM, cells were transfected using Lipofectin
(GIBCO-BRL) as follows. Cells grown to ~50% confluency on six-well
plates were incubated with 1-5 µg DNA (T
R-IIM or
T
R-IM constructs ligated in pcDNA3) in RPMI and
5-10 µl Lipofectin suspension at 37°C in a 5% CO2 atmosphere. Control cells were incubated with pcDNA3 (not containing T
R-IIM) and Lipofectin. After a 24-h incubation, RPMI
medium containing 20% FBS was added to each well to make a final
concentration of 10% FBS and was incubated for another 24 h.
Next, the DNA- and Lipofectin-containing medium was changed to 10% FBS
in RPMI (no antibiotics) and incubated for 24 h. To select for
stable transfectants, cells were treated with 400 µg/ml geneticin
(GIBCO-BRL) in RPMI medium containing 15% FBS, and the medium was
changed every 2-3 days. G418-resistant clones emerging ~14 days
after lipofection were subcloned using ring cylinders, expanded, and maintained in RPMI medium containing 15% FBS, 200 µg/ml geneticin, 5 U/ml penicillin, and 5 µg/ml streptomycin. Four independent stably
transfected clones containing T
R-IIM, two clones
containing T
R-IM, and three clones containing empty
vector were expanded and used in all subsequent experiments, and data
from representative clones are presented. Confirmation of respective
mRNA expression of T
R-IIM and T
R-IM was
obtained by RT-PCR and Northern analyses.
Northern blot analysis.
Total RNA was isolated by cell lysis with TRIzol (GIBCO-BRL) according
to the manufacturer's instructions and was size-fractionated (10 µg/lane) on a 1% agarose-2% formaldehyde gel in 20 mM MOPS, 5 mM
sodium acetate, and 1 mM EDTA (pH 7.2). mRNA was transferred and
ultravioletly linked to a nylon membrane (Gene Screen Plus; Dupont).
The blots were prehybridized for 2 h at 65°C in 1% BSA (Sigma),
7% SDS, 0.5 M phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) and
hybridized overnight in the same solution containing the appropriate
32P-labeled probe at 65°C. The blots were then washed two
times in solution containing 0.5% BSA, 5% SDS, 40 mM phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) for 30 min each at 65°C followed by
four 15-min washes with 1% SDS, 40 mM phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) at 65°C. The blots were exposed to Kodak X-AR 5 film. The TR-II and T
R-I cDNA probes were previously described
2.8-kb rat T
R-II full-length cDNA and Xba I fragment of
rat T
R-I cDNA (9, 10). The human
pro-
1(I) collagen, fibronectin 1, and plasminogen
activator inhibitor (PAI)-1 cDNA probes were obtained from
ATCC. To control for relative equivalence of RNA loading, the
blots were hybridized with 32P-labeled
-actin cDNA
(Clontech) probe or an oligonucleotide probe corresponding to the 18S
rRNA as previously described (10).
Covalent labeling of TGF- receptors.
Cells grown on 100-mm plates were affinity labeled with 400 pM
125I-labeled TGF-
1 (Amersham) in the
presence or absence of 100 nM unlabeled TGF-
1 (R & D)
and covalently cross-linked using disuccinimidyl suberate (Pierce), as
previously described (8, 9). The cells were subsequently
lysed with 100 µl of 1% Triton X-100, 10 mM Tris (pH 7.4), 1 mM
EDTA, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ ml aprotinin, and 10 µg/ml pepstatin, and sample loading buffer
(sucrose, 0.01% bromphenol blue, 2%
-mercaptoethanol, and 5 mM
EDTA) was added (1:1 vol/vol) and boiled for 5 min followed by 12%
SDS-PAGE. A sample of rainbow-colored protein molecular weight markers
(Amersham) was loaded in an adjacent lane. The gel was stained with
Coomassie brilliant blue (Bio-Rad) to visualize equivalence in protein
loading and destained before autoradiography.
[3H]thymidine and [3H]leucine
incorporation.
Cells (104) were plated in 24-well dishes, incubated in
medium containing 15% FBS, and grown to subconfluence. Medium was then changed to serum-free RPMI for 48 h, followed by incubation in 0.5% FBS in the presence or absence of TGF-1 (2 ng/ml).
After 45 h, the medium was removed, and cells were exposed for
3 h to 1 µCi/ml [3H]thymidine in RPMI 1640 medium
containing 2% bovine platelet-poor plasma-derived serum at 37°C. The
cells were washed three times with RPMI and then extracted three times
with ice-cold 6% TCA followed by solubilization in 1 N NaOH and were
counted in a Packard liquid scintillation counter. For
[3H]leucine incorporation, [3H]leucine (1 µCi/ml) was added to the cells 40 h after initiation of
TGF-
1 treatment. At the end of an additional 8-h
incubation, the supernatant was removed, and the cell proteins were
precipitated on the plate with ice-cold 6% TCA and washed three times
with fresh TCA. The cells were then washed one time with
water-saturated ether, dried, solubilized in 1 N NaOH, and subjected to
scintillation counting. For all experiments, cell numbers were
determined in replicate plates. Data shown are mean values of
triplicate determinations ± SE, corrected for cell number, and
are expressed as percentage of baseline incorporation in cells not
treated with TGF-
1. Each of the experiments was repeated
three times (n = 3) and gave essentially the same results.
Western blot analysis. Total cellular extracts were obtained 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 a 12% SDS-PAGE and then electroblotted onto nitrocellulose membranes (Bio-Rad). The membranes were incubated with phospho-p38 MAPK, phospho-p44/42 MAPK, or phospho-SAPK/JNK rabbit polyclonal antibodies (1:1,000) for 1.5 h followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody for 1.5 h. Signal development was carried out using LumiGLO (New England Biolabs) and was exposed to X-ray film. All of the assays were repeated three times. As a control, all of the blots were subjected to immunoblotting for corresponding nonphospho-p38 MAPK, p44/42 MAPK, or SAPK/JNK rabbit polyclonal antibodies.
p38 MAPK activity assays.
Kinase assays were performed as described by Marais et al.
(27) 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 PMSF and sonicated. Protein concentrations were
determined as described for Western analysis. Total protein (200 µg)
samples were incubated with phospho-p38 MAPK rabbit polyclonal antibody
(1:50) overnight on a rocker at 4°C. Protein A-Sepharose beads
(Pharmacia Biotech) were then added to immunoprecipitate (IP) the
activated p38 MAPK complex. The IP pellets were incubated with 1 µg
ATF-2 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 (DTT), 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% wt/vol SDS, 10% glycerol, 50 mM DTT,
and 0.1% wt/vol bromphenol blue]. The samples were analyzed on a 12%
SDS-PAGE and electroblotted as described for Western blot. p38 MAPK
activity was assayed by detection of phosphorylated ATF-2 using a
phospho-ATF-2 rabbit polyclonal antibody (1:1,000). After overnight
incubation with the primary antibody at 4°C, the membrane was
incubated for 1 h with an HRP-conjugated anti-rabbit secondary
antibody (1:2,000) 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 [3H]thymidine and [3H]leucine incorporation assays was determined by the Student's t-test for paired data. P values <0.05 were considered significant. Data are presented as means ± SE of triplicate determinations.
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RESULTS |
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Expression of dominant-negative TR-IIM in rat
mesangial cells.
First, to demonstrate that the stably transfected rat mesangial cells
expressed T
R-IIM mRNA, Northern blot analysis of total RNA isolated from wild-type and transfected mesangial cells was performed using the T
R-II cDNA probe. As shown in Fig.
1A, a 5.5-kb band was detected
in all cells, corresponding to endogenously expressed T
R-II mRNA.
However, an additional band of ~1.8 kb in size was detected only in
the cells transfected with T
R-IIM, and not in wild-type
cells or in mock-transfected cells with empty vector pcDNA3. Next, cell
surface expression of T
R-IIM protein and ligand binding
were determined by affinity cross-linking with 125I-labeled
TGF-
1. As shown in Fig. 1B, an intensely
labeled band of ~46 kDa in molecular mass was detected in the cells
transfected with T
R-IIM, in addition to the ~89- and
69-kDa bands corresponding to endogenously expressed T
R-II and
T
R-I, respectively.
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Regulated expression of TGF- receptor mRNA by
TGF-
1.
We next determined whether the expression of TGF-
receptors in rat
mesangial cells was regulated by TGF-
1. Northern blot analysis of total RNA isolated from control mesangial cells transfected with empty vector pcDNA3 demonstrated a dose-dependent decrease in
T
R-II mRNA abundance (Fig.
2A), but not T
R-I mRNA
(Fig. 2B), in response to treatment with exogenous
TGF-
1. In mesangial cells transfected with
dominant-negative T
R-IIM, this dose-dependent decrease
in T
R-II mRNA expression was blocked. The lower molecular mass mRNA
corresponding to T
R-IIM was again detected only in the
cells transfected with T
R-IIM. Thus our data demonstrate differential regulation of the expression of the two signaling receptors by TGF-
1.
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Stimulation of pro-1(I) collagen mRNA expression by
TGF-
1.
TGF-
1 induces ECM protein synthesis in mesangial cells.
As shown in Fig. 3A, treatment
with exogenous TGF-
1 increased pro-
1(I) collagen mRNA expression in a dose-dependent fashion in control mesangial cells transfected with empty vector pcDNA3. Similarly, exogenous TGF-
1 also increased fibronectin (Fig.
3B) and PAI-1 (Fig. 3C) mRNA expression in a
dose-dependent fashion in the empty vector-transfected cells.
Remarkably, in mesangial cells transfected with dominant-negative
T
R-IIM, a dose-dependent increase in
pro-
1(I) collagen mRNA expression was also observed upon
treatment with exogenous TGF-
1. However,
TGF-
1 induced expression of fibronectin and PAI-1
mRNA was inhibited in cells transfected with T
R-IIM.
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Effect of TGF-1 on mesangial cell
[3H]thymidine and [3H]leucine
incorporation.
Treatment of wild-type rat mesangial cells in culture with exogenous
TGF-
1 (2 ng/ml for 48 h) significantly inhibited
[3H]thymidine incorporation to 18 ± 10% of
baseline (Fig. 4A). Inhibition of [3H]thymidine incorporation was similarly observed in
empty vector transfected cells treated with exogenous
TGF-
1. Conversely, treatment with exogenous
TGF-
1 increased [3H]leucine incorporation
to 140 ± 9% of baseline in wild-type mesangial cells (Fig.
4B) and in empty vector transfected cells. In transfected cells carrying T
R-IIM, the inhibitory effect on
[3H]thymidine incorporation by exogenous
TGF-
1 was blocked (Fig. 4A) but the
stimulatory effect on [3H]leucine incorporation was not
blocked (Fig. 4B).
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Activation of p38 MAPK in rat mesangial cells by
TGF-1.
Previous reports, including studies from our laboratory, have suggested
that TGF-
1 exerts its biological effects via the MAPK
signaling pathway in certain cell types (1, 6, 20). We
first determined the levels of p38 MAPK protein expression in rat
mesangial cells treated with exogenous TGF-
1 (2 ng/ml) by Western analyses, using phospho-p38 MAPK and p38 MAPK antibodies. The phospho-p38 MAPK antibodies detect specifically the phosphorylated forms of p38 MAPK, whereas the p38 MAPK antibodies detect total (phosphorylation state-independent) p38 MAPK proteins. As shown in Fig. 5, increases in phosphorylation
of p38 MAPK were observed within 30 min of stimulation with exogenous
TGF-
1 in the wild-type rat mesangial cells. Moreover,
the time-dependent increases in phosphorylated p38 MAPK were also
observed in mesangial cells transfected with dominant-negative
T
R-IIM but not in cells transfected with
dominant-negative T
R-IM.
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Induction of ERK1/ERK2 phosphorylation in rat mesangial cells by
TGF-1.
To determine whether TGF-
1 activated the ERK1/ERK2
pathway in cultured rat mesangial cells, we performed Western analyses using phospho-p44/42 MAPK polyclonal antibodies. The phospho-p44/42 MAPK antibodies detect specifically the phosphorylated forms of ERK1/ERK2 proteins. As shown in Fig. 7,
increases in phosphorylation of ERK1 and ERK2 proteins were observed in
wild-type mesangial cells as early as 30 min after stimulation with
exogenous TGF-
1 (2 ng/ml). However, this rapid induction
of ERK1/ERK2 phosphorylation was blocked in the
T
R-IIM-transfected mesangial cells.
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Effect of TGF-1 on SAPK/JNK phosphorylation in rat
mesangial cells.
We also determined whether TGF-
1 activated the SAPK/JNK
pathway by Western analyses using phospho-SAPK/JNK rabbit polyclonal antibodies in cultured rat mesangial cells. In contrast to the p38 MAPK
and the ERK1/ERK2, there were no appreciable increases in the
phosphorylation of SAPK/JNK within the same time periods of
TGF-
1 treatment (Fig. 8).
As a control, the same blots were immunoblotted with SAPK/JNK rabbit
polyclonal antibodies to detect levels of total (phosphorylation
state-independent) SAPK/JNK proteins.
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Inhibition of TGF-1-induced
pro-
1(I) collagen expression by SB-203580, a specific
inhibitor of p38 MAPK.
Given that TGF-
1 induced increases in both
pro-
1(I) collagen mRNA expression and p38 MAPK
phosphorylation and transfection with a dominant-negative
T
R-IIM failed to inhibit both of these TGF-
1-effects, we posed the question whether the p38
MAPK pathway was involved in mediating TGF-
1-induced
pro-
1(I) collagen mRNA expression. In the presence of
SB-203580, a specific inhibitor of p38 MAPK, exogenous
TGF-
1 was unable to induce pro-
1(I)
collagen mRNA expression in either wild-type (WT) or
T
R-IIM-transfected mesangial cells (Fig.
9), whereas, PD-098059, a specific
inhibitor of MEK1 that prevents the activation of ERK1/ERK2 pathway,
failed to inhibit TGF-
1-induced pro-
1(I)
collagen mRNA expression. At 10 µM concentrations of PD-098059 in
mesangial cells, we have observed inhibition of ERK activation by
TGF-
1. Moreover, in cells transfected with a
dominant-negative T
R-IM, TGF-
1-induced pro-
1(I) collagen mRNA expression was inhibited as was
TGF-
1-induced p38 MAPK phosphorylation.
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DISCUSSION |
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The critical role of TGF-1 in the pathogenesis of
glomerulosclerosis has been well recognized, and although it is well
known that TGF-
1 stimulates the transcription and
synthesis of ECM proteins, including type I collagen, the intracellular
signaling mechanisms by which TGF-
1 stimulates this
process remain poorly understood. This study examined the role of the
MAPK signaling cascades in mediating TGF-
1 responses in
rat glomerular mesangial cells using dominant-negative inhibition of
TGF-
signaling receptors. Given that heteromeric complex formation
and phosphorylation of T
R-I by T
R-II are thought to be essential
for propagation of TGF-
1 signals, a mutant
kinase-deleted T
R-IIM is predicted to inhibit
T
R-II-dependent signals by virtue of competition for T
R-I binding
(5, 8, 43). We have previously used this strategy of
dominant-negative inhibition of T
R-II-mediated signaling in
glomerular endothelial cells and in macrophages (6, 8). Thus we stably transfected cultured rat glomerular mesangial cells with
dominant-negative mutant T
R-IIM. The mRNA expression of T
R-IIM in the transfected mesangial cells was
demonstrated by Northern blot analysis (Fig. 1A). We next
confirmed that the T
R-IIM protein was expressed at the
cell surface and that it strongly bound the TGF-
1 ligand
by affinity labeling and cross-linking with
125I-labeled-TGF-
1 (Fig. 1B). We
then examined the effects on TGF-
1 responses by
dominant-negative inhibition of the T
R-II-dependent signaling
pathway in mesangial cells.
We first observed differential regulation of the expression of the two
signaling receptors by TGF-1 in mesangial cells. We had
previously shown that the T
R-II mRNA abundance in mesangial cells
was reduced within 24 h by exogenous TGF-
1,
suggesting that TGF-
1 negatively regulates expression of
its own receptor (9). The present study demonstrated that
exogenous TGF-
1 downregulated T
R-II mRNA in a
dose-dependent fashion, an effect blocked by transfection of
T
R-IIM but not T
R-I mRNA (Fig. 2). Thus modulation of
relative levels of expression of the two receptors may play an
important role in determining how cells interpret extracellular signals.
We next observed dose-dependent induction of PAI-1 mRNA and fibronectin
mRNA by exogenous TGF-1 in the wild-type and empty vector pcDNA3-transfected control mesangial cells. As predicted, both
of these TGF-
1 effects were inhibited in transfected
cells carrying dominant-negative T
R-IIM. However,
pro-
1(I) collagen mRNA increased in response to
exogenous TGF-
1 in both control and
T
R-IIM-transfected cells (Fig. 3). Moreover,
transfection of the dominant-negative T
R-IIM in
mesangial cells blocked the growth inhibitory effects of
TGF-
1, as determined by [3H]thymidine
incorporation, but failed to block the induction of protein synthesis
in mesangial cells, as determined by [3H]leucine
incorporation (Fig. 4). Remarkably, this apparent differential inhibition of TGF-
1 responses by T
R-IIM
was also noted when we examined the effects of TGF-
1 on
activation of the major MAPK pathways.
As shown in Fig. 5, treatment of mesangial cells with exogenous
TGF-1 resulted in a rapid induction of p38 MAPK
phosphorylation that was not inhibited by transfection of the
dominant-negative T
R-IIM. In contrast, exogenous
TGF-
1 also rapidly induced phosphorylation of ERK1/ERK2
in mesangial cells, but this induction was blocked by the
dominant-negative T
R-IIM (Fig. 7). Thus our findings
indicate that overexpression of the dominant-negative
T
R-IIM selectively inhibits some TGF-
1
effects, such as growth inhibition and ERK1/ERK2 activation, but not
its induction of pro-
1(I) collagen mRNA or p38 MAPK
activation. Two earlier studies had suggested seemingly separate
signaling pathways for TGF-
1-mediated growth inhibition and ECM induction. Chen et al. (5) overexpressed a
dominant-negative T
R-II in Mv1.Lu cells and reported abolition of
TGF-
1-mediated inhibition of both cell proliferation and
pRB phosphorylation but not TGF-
1-mediated induction of
ECM. Saitoh et al. (37) observed that phosphorylation of
Ser172 and Thr176 residues of T
R-I was
essential for the TGF-
1-mediated growth inhibitory
effect but not for TGF-
1 induction of ECM in Mv1.Lu cells. Indeed, the apparent disparity observed in all of these studies
may be due to different threshold levels of receptor activity (or
receptor protein density) and the signal transduction requirement for
the various TGF-
1 actions. Alternatively, it is also
possible that the differential inhibition by T
R-IIM is
because certain TGF-
1 responses involve T
R-I-mediated
signals that do not require T
R-II kinase-dependent phosphorylation.
For instance, the Ser172 and Thr176 residues of
T
R-I, which were shown to be essential for growth inhibition by
TGF-
1, are not phosphorylated (37). A
molecular mechanism remains to be elucidated in this case.
In contrast to the ERK pathway, p38 MAPK was not activated primarily by
mitogens but by cellular stresses and inflammatory cytokines in various
cell types. The p38 MAPK was first isolated as a 38-kDa protein that
was rapidly tyrosine phosphorylated by bacterial lipopolysaccharide
stimulation (15). p38 MAPK activation has also been
observed in response to other environmental stress such as heat shock
and hyperosmolarity and in inflammatory responses such as tumor
necrosis factor--stimulated neutrophils and human umbilical vein
endothelial cells (11, 34, 47). In addition, a number of
growth factors and cytokines have now also been shown to be capable of
inducing the activation of p38 MAPK, including granulocyte macrophage
colony-stimulating factor, fibroblast growth factor, and insulin-like
growth factor, as well as TGF-
1 (33). Despite growing evidence that TGF-
1 induces activation
of p38 MAPK in a number of cell types such as fibroblasts, neutrophils, and smooth muscle cells, paucity of data exists for glomerular mesangial cells (17, 21, 35). In the present studies,
given the rapid kinetics observed for TGF-
1-induced
activation of p38 MAPK (within 15 min of TGF-
1
treatment) in mesangial cells, the effect of TGF-
1 on
the p38 MAPK pathway is likely a direct one, perhaps through
TGF-
-activated kinase 1 (TAK1; see Refs. 16, 29, 46). Because, in our studies, the
transfection of dominant-negative T
R-IIM failed to
inhibit TGF-
1-induced p38 MAPK phosphorylation, we
sought to confirm that the p38 MAPK activation in mesangial cells was
indeed TGF-
1 induced. Here, we used dominant-negative inhibition of T
R-I by transfection of mesangial cells with a truncated T
R-IM construct lacking the GS and the kinase
domains. The activation of p38 MAPK by TGF-
1 was
completely abrogated by dominant-negative T
R-IM,
indicating that signaling by T
R-I is required for this
TGF-
1 response.
Given that TAK1 can also lead to the activation of JNK and in a number
of cell types, including Hep G2, Chinese hamster ovary, and Madin-Darby
canine kidney cell lines, TGF-1 has been shown to
activate SAPK/JNK. We also determined if TGF-
1 induced
SAPK/JNK activation (16, 42). However, no apparent
activation of JNK was observed within the same time periods of
TGF-
1 treatment in mesangial cells (Fig. 8). The failure
of rapid activation of JNK by TGF-
1 was also observed in
the studies by Hanafusa et al. (16) in
C2C12 cells.
Based on our data that dominant-negative TR-IIM
selectively inhibits some but not all TGF-
1 effects in
mesangial cells and the associated findings that the
T
R-IIM failed to block TGF-
1-mediated induction of pro-
1(I) collagen mRNA and protein
synthesis, as well as activation of p38 MAPK, we posed the question
whether the p38 MAPK pathway was involved in mediating
TGF-
1-induced pro-
1(I) collagen. We
hypothesized that blockade of the p38 MAPK cascade would result in
failure of TGF-
1 to induce pro-
1(I) collagen mRNA. Thus we used a specific chemical inhibitor of the p38
MAPK cascade known as SB-203580. As shown in Fig. 9, in the presence of
SB-203580, exogenous TGF-
1 was unable to induce
pro-
1(I) collagen mRNA expression in mesangial cells. In
contrast, blockade of the ERK cascade by PD-098059, a specific
inhibitor of MEK1 that prevents downstream activation of ERK1/ERK2, did
not prevent TGF-
1 induction of pro-
1(I)
collagen mRNA. Thus our data demonstrate the critical involvement of
the p38 MAPK in the TGF-
1 signaling pathway in
glomerular mesangial cells. Moreover, our findings suggest that the p38
MAPK functions as a component in the signaling of
pro-
1(I) collagen induction by TGF-
1.
Furthermore, as with our findings of inhibition of
TGF-
1-induced p38 MAPK activation by dominant-negative
T
R-IM, we also observed inhibition of
TGF-
1-induced pro-
1(I) collagen mRNA
expression in cells expressing the mutant T
R-IM and
indicate that signaling by T
R-I is required also for this
TGF-
1 response.
Reports in the literature suggesting a possible role of the p38 MAPK
cascade in the pathogenesis of glomerulosclerosis such as in diabetic
complications are only beginning to come forth. For instance, Igarashi
et al. (22) recently reported activation of p38 MAPK in
rat aortic smooth muscle cells by hyperglycemia and in aorta from
streptozotocin-induced diabetic rats, implicating involvement of the
p38 MAPK pathway in the development of diabetic vascular complications.
Interestingly, their studies showed that even a moderately elevated
glucose concentration (16.5 mM) significantly increased p38 MAPK but
not ERK1/ERK2 activities. Increased levels of phosphorylated p38 MAPK
were also detected in glomeruli isolated from rats with
streptozotocin-induced diabetes compared with nondiabetic control
rats and insulin-treated diabetic rats (14). In addition, activation of p38 MAPK was detected in isolated glomeruli exposed to
high glucose concentrations of 25 mmol/l compared with 5.6 mmol/l
(14). To our knowledge, this is the first demonstration of
rapid activation of the p38 MAPK by TGF-1 in glomerular
mesangial cells, and the data suggest the MAPKs as important
TGF-
1 signaling pathways involved in the tissue injury
response. Our findings suggest that TGF-
1 induces
pro-
1(I) collagen expression via the p38 MAPK-dependent
pathway. Furthermore, this TGF-
1-induced collagen
expression requires T
R-I-mediated signaling but is not dependent on
T
R-II kinase.
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ACKNOWLEDGEMENTS |
---|
This work was supported in part by National Institutes of Health (NIH) Grant R01 DK-57661-01, Grant-in-Aid no. 0051319T from the American Heart Association (AHA), and a Veterans Affairs Career Development Award to M. E. Choi. A. M. K. Choi was supported by NIH Grants R01 HL-55330, R01 HL-60234, and R01 AI-42365, and an AHA Established Investigator Award. B. Y. Chin was supported by a Laboratory Scholarship from Toxicological Sciences at the Johns Hopkins University School of Hygiene and Public Health.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: M. E. Choi, Univ. of Pittsburgh, Renal-Electrolyte Division, 3550 Terrace St., A919 Scaife Hall, Pittsburgh, PA 15261 (E-mail: choim{at}pitt.edu).
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.
Received 21 August 2000; accepted in final form 2 November 2000.
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