Department of Pediatrics, Northwestern University, Chicago, Illinois 60611
Submitted 25 February 2003 ; accepted in final form 14 May 2003
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ABSTRACT |
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transforming growth factor- signal transduction; cross talk; gene regulation; extracellular matrix accumulation; glomerulosclerosis
Members of the TGF- superfamily transmit their signal via heteromeric
complexes of transmembrane serine/threonine kinases, the type I and type II
receptors (T
RI and T
RII). The Smads are a series of proteins that
function downstream from the TGF-
family receptors to transduce signal
to the nucleus (2,
34,
38). The receptor-regulated or
pathway-restricted Smads (R-Smads), Smad2 and Smad3, contain a SSXS
phosphorylation site in their COOH-terminal end that is a direct target of
T
RI. Upon ligand binding, the R-Smads are phosphorylated and associate
with the common partner Smad, Smad4. The resulting heteromultimer translocates
to the nucleus where it regulates expression of TGF-
target genes by
direct binding to DNA and/or interaction with other transcription factors
(2,
34,
38). The inhibitory Smads,
Smad6 and Smad7, may participate in a negative feedback loop to control
TGF-
responses by competitive interaction with T
RI
(18,
21,
34,
36). R-Smad and Smad4 are
composed of Mad-homology (MH)1 and MH2 domains separated by a variable linker
region. Smad3 and Smad4 can bind directly to DNA through their MH-1 domain
(2,
34,
38).
Although most studies of TGF- signal transduction have focused on
Smad activity, the data also suggest a role for more classical growth factor
signaling, such as protein kinase C (PKC). The PKC family is composed of at
least 11 serine/threonine kinases. These are grouped according to the
biochemical requirement for their activation. The conventional PKCs (cPKCs),
including PKC
, PKC
I, PKC
II, and PKC
, depend on
calcium and phospholipids. The novel PKCs, PKC
, PKC
, PKC
,
and PKC
, do not require Ca2+ but are phospholipid
dependent. The atypical enzymes, PKC
and PKC
, require neither
Ca2+ nor phospholipid. PKC isoenzymes are expressed in a
tissue-specific fashion and their subcellular localization varies depending on
the cell type (22).
Several studies have implicated TGF-1 and PKC as mediators
of ECM accumulation in diabetic animal models and in mesangial cells cultured
in high glucose. In streptozotocin-induced diabetic rats, a model for type 1
diabetes, administration of LY333531, a PKC
inhibitor, prevented
increased expression of mRNA for TGF-
1, fibronectin, and type
IV collagen (28). In
db/db mice, a model for type 2 diabetes, the same inhibitor
prevented ECM expansion (27).
PKC is involved in hyperglycemia-stimulated TGF-
1 promoter
activity in mesangial cells
(51). This could be the
mechanism leading to increased TGF-
1 mRNA expression and
protein synthesis that have been observed in murine mesangial cells cultured
in high glucose. Thus high glucose could mediate its effect through
PKC-induced TGF-
1 activation leading to increased ECM
production (56).
Conversely, even without high concentration of glucose,
TGF-1 could exert its effect on expression of some of the ECM
components through PKC activation. Halstead et al.
(13) showed that treatment of
a human carcinoma cell line with the PKC inhibitor calphostin C blocked
TGF-
1-induced increases in plasminogen activator inhibitor-1
(PAI-1) and fibronectin mRNA expression. More recently, it has been suggested
that stabilization of elastin mRNA in lung fibroblasts by TGF-
requires
Smads, PKC
, and the MAP kinase p38. These data suggest potential
synergy between classical TGF-
and PKC signaling cascades. In support of
this notion, Yakymovych et al.
(53) recently showed that
Smad2 and Smad3, the targets of TGF-
receptors, can be phosphorylated in
their MH1 domain by PKC.
We previously showed that TGF-1-induced collagen I gene
expression is Smad3 dependent in human mesangial cells. Here, we investigated
whether PKC might be involved in collagen I accumulation in response to
TGF-
1. With the use of specific PKC isozyme inhibitors, we
showed that PKC
, but not PKC
or PKC
I, mediates collagen I
production by TGF-
1 in human mesangial cells. We demonstrated
that TGF-
1 activates PKC
in these cells and that this
activation plays a role in TGF-
1-stimulated Smad
transcriptional activity and collagen I gene transcription.
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MATERIALS AND METHODS |
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Cell culture. Human mesangial cells were isolated from glomeruli by differential sieving of minced normal human renal cortex obtained from anonymous surgery or autopsy specimens. The cells were grown in DMEM/Ham's F-12 medium, supplemented with 20% heat-inactivated FBS, glutamine, penicillin/streptomycin, sodium pyruvate, HEPES buffer, and 8 µg/ml insulin (Invitrogen Life Technologies, Carlsbad, CA) as previously described (44), and were used between passages 5 and 8.
Protein kinase assay. Cells were switched to medium containing 1%
FBS and then treated with 1 ng/ml TGF-1 for various time
periods leading up to simultaneous harvest in RIPA buffer (50 mM
Tris·HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.5% deoxycholate; 0.1%
SDS) containing protease inhibitors (1 mM PMSF, 1 mM EDTA, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin). After clarification by
centrifugation, the protein content was determined by Bradford protein assay
(BioRad, Hercules, CA). Immunoprecipitation was performed with 2 µg
anti-PKC
or anti-PKC
antibody and 30 µl protein G-sepharose
for 1 h at 4°C. Immunocomplexes were incubated for 10 min at room
temperature with a PKC
substrate peptide (which can be phosphorylated by
both PKC
and PKC
) (Upstate, Waltham, MA) and
[
32P] ATP in 10 mM HEPES, pH 7.0; 10 mM DTT; and 10 mM
MgCl2. The reactions were then spotted onto P81 phosphocellulose
paper and washed four times with 1% phosphoric acid and once with acetone. The
amount of incorporated radioactivity into the substrate was determined by
scintillation counting.
Immunocytochemistry. Mesangial cells were grown to 60% confluence
on eight-well culture slides coated with 1 mg/ml gelatin. The cells were
switched to medium containing 1% FBS and then treated with 1 ng/ml
TGF-1 for different durations before simultaneous formalin
fixation and permeabilization with Triton X-100. The cells were then stained
with 1 µg/ml anti-PKC antibody according to the manufacturer's
instructions. The staining was detected with Oregon Green 514-conjugated
secondary antibodies from Molecular Probes (Eugene, OR) and evaluated under a
fluorescent microscope.
Preparation of cell lysates and Western blot analysis. Cells were
switched to medium containing 1% FBS and pretreated for 1 h with calphostin C
(100 nM), rottlerin (5 µM), Gö 6976 (10 nM), or DMSO as vehicle
control. The cells were then incubated for 24 h with 1 ng/ml
TGF-1, followed by lysis at 4°C in RIPA or lysis buffer
(10 mM Tris·HCl, pH 8.0; 150 mM NaCl; 1% Nonidet P-40) containing
protease and phosphatase inhibitors (1 mM sodium orthovanadate, 50 mM sodium
fluoride, 40 mM
-glycerophosphate). Lysates were clarified by
centrifugation at 18,000 g for 10 min. Proteins were separated by
SDS-PAGE (6 or 10% acrylamide gels), transferred onto a PVDF membrane
(Millipore, Bedford, MA), and immunoblotted with anti-type I collagen,
anti-Smad1/2/3, or anti-Smad4 antibody (0.2 µg/ml). The blots were
developed with chemiluminescence reagents according to the manufacturer's
protocol (Santa Cruz Biotechnology). Autoradiograms were scanned with an Arcus
II Scanner (AGFA) in transparency mode and densitometric analysis was
performed using the National Institutes of Health Image 1.61 program for
Macintosh.
Cell fractionation. Cells were scraped into a detergent-free
buffer (20 mM Tris·HCl, pH 7.5; 0.5 mM EDTA; 0.5 mM EGTA; 10 mM
-mercaptoethanol) containing protease and phosphatase inhibitors. The
cells were then disrupted by 15 strokes of a Dounce homogenizer. After
centrifugation at 100,000 g, the supernatant, representing the
cytosolic fraction, was saved; the pellet, representing the particulate
fraction, was resuspended in buffer; and Triton X-100 was added to be 0.5%
final concentration. After 30-min incubation on ice, the pellet was
centrifuged at 18,000 g for 10 min to remove insoluble material. The
supernatant was saved as the soluble membrane fraction. After determination of
the protein content, each fraction was analyzed by immunoblotting with
anti-PKC
antibody (0.2 µg/ml) as described above.
RNA isolation and Northern blot. Cells were plated in 100-mm
culture dishes. Three days later, the cells were switched to medium containing
1% FBS. They were preincubated with PKC inhibitors for 1 h before addition of
1 ng/ml TGF-1 or control vehicle for 24 h. Total RNA was
harvested using Trizol (Invitrogen Life Technologies) and analyzed by Northern
blot as described previously
(40). The same blots were
successively rehybridized with additional probes after confirmation of
complete stripping. cDNAs for human
1(I) [clone Hf677
(8)] and
2(I)
collagen [clone Hf1131 (4)]
chains were obtained from Dr. Y. Yamada. Quantification of the bands on
autoradiograms was performed using densitometric analysis. The signals
obtained by hybridization with these probes were corrected for loading using
the signal obtained with a bovine cDNA for 28S ribosomal RNA provided by Dr.
H. Sage.
Transient transfection and luciferase assay. The day before the
transfection, 6.5 to 8 x 104 cells were seeded in six-well
plates. Eighteen hours later, cells were switched to 1% FBS medium and
transfected with the indicated constructs along with 0.5 µg of
CMV-SPORT--galactosidase (Gibco BRL) as a control of transfection
efficiency. Transfection was performed with the Fugene6 transfection reagent
(Roche Applied Science, Indianapolis, IN) as previously described
(39). After 3 h, 1 ng/ml
TGF-
1 or control vehicle was added to the cells. In some
experiments, the transfected cells were pretreated for 1 h with PKC inhibitors
before addition of TGF-
1. Twenty-four hours later, the cells
were harvested in 300 µl reporter lysis buffer (Promega). Luciferase and
-galactosidase activities were measured as previously described
(39). Luciferase assay results
were normalized for
-galactosidase activity. Experimental points were
performed in triplicates in several independent experiments. One arbitrary
unit was set up as the ratio between luciferase and
-galactosidase for
cells cotransfected with the promoter-reporter construct and the empty
expression vector and incubated with control vehicles (for
TGF-
1 and PKC inhibitors).
Plasmid constructs. The 376COL1A2-LUC construct containing the
sequence 376 bp of the 2(I) collagen (COL1A2) promoter and
58 bp of the transcribed sequence fused to the luciferase (LUC) reporter gene
was previously described (39).
The SBE-LUC (54) reporter
construct was kindly provided by Dr. B. Vogelstein. The vectors expressing the
indicated Smad3 variants (32)
were kindly provided by Drs. H. F. Lodish and X. Liu. The Gal4-Smad constructs
(10) were kindly provided by
Dr. M. P. de Caestecker.
Statistical analysis. Statistical differences between experimental groups were determined by analysis of variance using StatView 4.02 software program for Macintosh. Values of P < 0.05 by Fisher's protected least significant difference (PLSD) were considered significant. Difference between two comparative groups was further analyzed by unpaired Student's t-test.
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RESULTS |
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Activation of PKC by
TGF-
1. Inhibition of
TGF-
1-stimulated collagen I expression by blockade of
PKC
suggested that TGF-
1 could activate PKC
.
Thus we examined the timing of PKC
activation by TGF-
1
in human mesangial cells. Cells were treated with 1 ng/ml
TGF-
1 for different time periods leading up to simultaneous
harvest. Lysates were immunoprecipitated with an anti-PKC
or -PKC
antibody. Immunocomplexes were used for an in vitro kinase assay.
TGF-
1 stimulates PKC
in a time-dependent manner
(Fig. 3). PKC
activity
began to increase 5 min after adding TGF-
1, although not
significantly. Maximal activity was detected at 60 min and increased activity
was sustained for up to 24 h. In contrast, PKC
activity was not affected
by TGF-
1 treatment. Incubation for 15 min with 100 nM PMA was
used as a positive control for PKC activation (not shown).
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Next, we examined whether increased in vitro PKC kinase activity
correlates with changes in cellular localization. PKC
translocates to
the plasma membrane with TGF-
1 treatment in a time-dependent
manner (Fig. 4). At 5 min,
staining at the cell periphery slightly increased, whereas staining decreased
in the nucleus. Staining at the plasma membrane was more apparent at 60 min,
when PKC
activity was more robust (see
Fig. 3). Membrane localization
remained elevated for up to 24 h of treatment. Nuclear staining began
increasing at 15 min and remained elevated for up to 24 h. Translocation to
the cell periphery in response to TGF-
1 was also demonstrated
by immunocytochemistry studies on mesangial cells transfected with a green
fluorescent protein-PKC
construct (data not shown). In contrast to
PKC
, PKC
did not translocate to the plasma membrane in response
to TGF-
1, whereas translocation of both isozymes was detected
following 15 min of treatment with PMA
(Fig. 4). Because increased
PKC
activity and membrane association between 5 and 30 min were subtle,
we sought to further analyze early PKC
activation. We performed Western
blot analysis of cells separated into membrane and cytosolic fractions. An
increase in membrane-associated PKC
was detectable as early as 5 min
after adding TGF-
1 (Fig.
5), corresponding to the low levels of increased activity shown in
Fig. 3. Together, these data
suggest that PKC
, but not PKC
, is activated in human mesangial
cells in response to TGF-
1.
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TGF-1-induced PKC
activity modulates collagen I gene expression. Because PKC
is
activated by TGF-
1, and inhibition of PKC
blocked
TGF-
1-stimulated collagen I production, we investigated
whether PKC
modulates collagen I gene expression. Cells were pretreated
for 1 h with PKC inhibitors before addition of 1 ng/ml TGF-
1
for 24 h. Steady-state mRNA levels for
1(I) collagen
(COL1A1) and
2(I) collagen (COL1A2) were measured by
Northern blot. As shown in Fig.
6, both calphostin C and rottlerin inhibited
TGF-
1-induced COL1A1 and COL1A2 mRNA expression. Rottlerin
also decreased basal collagen I mRNA levels in agreement with its effect on
basal protein synthesis (see Fig.
1). In contrast, the cPKC inhibitor Gö 6976 slightly
increased basal mRNA expression with minimal effect on TGF-
1
fold induction. To further define a role for PKC
in
TGF-
1-induced collagen I gene expression, we performed
transient transfection experiments with 376COL1A2-LUC, a construct containing
the sequences from -376 to +58 of the human COL1A2 promoter in front of the
luciferase reporter gene (39).
The transfected cells were pretreated with PKC inhibitors for 1 h.
TGF-
1 was then added for 24 h and luciferase activity was
determined. Similar to the results with protein and mRNA, calphostin C and
rottlerin blocked TGF-
1-induced COL1A2 promoter activity,
whereas Gö 6976 did not affect the response
(Fig. 7). Together, these
results suggest that PKC
plays a role in basal collagen I expression
and is necessary for the transcriptional response to
TGF-
1.
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Regulation of Smad by PKC. We previously showed that Smad3
is required for TGF-
1-stimulated COL1A2 gene
transcription. Therefore, we investigated whether the inhibitory effect of
PKC
blockade on collagen I expression is due to modulation of Smad3
expression and/or activity. Rottlerin decreased basal expression of Smad3,
although not consistently, suggesting the inhibitory effect of rottlerin on
collagen I production could, at least in part, be due to decreased Smad3
expression (data not shown). To determine whether PKC
inhibition
impaired TGF-
1-induced collagen I expression by decreasing
Smad3 levels, cells were pretreated with 1 µM PMA for 24 h to deplete
cellular PKCs. The cells were then transfected with 376COL1A-LUC and a
construct expressing wild-type Smad3 (Flag-N-Smad3)
(32) or an empty vector (pEXL)
before treatment with TGF-
1. Similar to the effect of
incubation with rottlerin, prolonged exposure to PMA not only decreased basal
COL1A2 promoter activity but also completely blocked the promoter induction by
TGF-
1 (Fig.
8A). Overexpression of Smad3 restored the response to
TGF-
1. Figure
8B shows a Western blot analysis indicating that chronic
PMA treatment led to PKC
downregulation and decreased Smad3 protein
levels. Together, these data suggest that the inhibitory effect of PKC
blockade or depletion on COL1A2 transcription could be partially due
to the downregulation of Smad3 expression.
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Because it has been shown that PMA-activated PKC modulates Smad3 DNA
binding activity (53), we
investigated whether rottlerin decreases Smad protein transcriptional activity
as well as decreasing Smad3 levels. Cells were cotransfected with a reporter
construct containing five Gal4 binding sites in front of the luciferase gene
and a construct expressing the Gal4 DNA binding domain fused to either
full-length Smad3 (Gal4-Smad3) or Smad4 (266-552) [Gal4-Smad4(N)].
These constructs enable us to study the transcriptional activity of Smads
independently of their DNA binding activity. We previously showed that both
constructs are responsive to TGF-
1 in mesangial cells
(41). The cells were
pretreated for 1 h with the PKC
inhibitor and then incubated with
TGF-
1 for 24 h. Rottlerin blocked
TGF-
1-stimulated Smad3 transcriptional activity
(Fig. 9). Smad4 transcriptional
activity was also decreased by rottlerin pretreatment. These data suggest that
Smad activity is modulated by PKC
signaling in response to
TGF-
1. To further support these data, we evaluated the effect
of PKC
inhibition on the activity of the SBE-LUC reporter construct
containing four copies of the CTCTAGAC sequence that has been shown to bind
recombinant Smad3 and Smad4
(54). Cells were cotransfected
with SBE-LUC and an empty vector (pEXL) or a construct expressing wild-type
Smad3. The cells were pretreated with rottlerin for 1 h and then incubated
with TGF-
1 for 24 h. As expected, TGF-
1
stimulated the activity of the SBE-LUC reporter indicating activation of Smad
proteins (Fig. 10, pEXL
histograms). TGF-
1-induced activity of endogenous Smads was
decreased by rottlerin pretreatment. Moreover, blockade of PKC
almost
completely inhibited transcriptional activity of overexpressed Smad3. These
results further confirm that blockade of PKC
inhibits Smad3 activity.
To investigate whether PKC
modulation of Smad3 activity is dependent on
phosphorylation at the T
RI-specific target site of Smad3, mesangial
cells were cotransfected with a mutated Smad3 construct (Smad3D) in which the
three COOH-terminal serine residues are replaced by three aspartic acid
residues (32). Inhibition of
PKC
drastically decreased luciferase activity induced by Smad3D. These
data suggest that the inhibitory effect of rottlerin on Smad3 transcriptional
activity is not due to blocking of phosphorylation at the COOH-terminal
T
RI target site. Of note, Smad3D can function as a transcriptional
activator in the absence of TGF-
1 as previously demonstrated
(32).
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Finally, we investigated whether the inhibitory effect of rottlerin on Smad
activity modulates COL1A2 promoter activity. Mesangial cells were
cotransfected with the 376COL1A2-LUC and an expression vector for Smad3,
Smad3D, or the corresponding empty vector. One hour after adding rottlerin or
control vehicle, the cells were incubated with TGF-1 for 24
h. Similar to the results obtained with the SBE-LUC reporter construct,
inhibition of PKC
partially blocked ligand-independent and
ligand-dependent, Smad3-mediated COL1A2 promoter activity
(Fig. 11). This inhibition did
not require phosphorylation at the COOH-terminal SSXS phosphorylation site of
Smad3. Taken together, these results suggest that
TGF-
1-induced PKC
activity contributes to increased
COL1A2 gene transcription by modulating Smad3 expression and
transcriptional activity.
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DISCUSSION |
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With the use of an in vitro kinase assay, immunocytochemistry, and
immunoblotting on cytosol/membrane fractions, we demonstrated that, in human
mesangial cells, PKC is activated by TGF-
1 in a
time-dependent manner, beginning at 5 min with maximal activation at 60 min.
Similarly, Perillan and colleagues
(37) showed that
TGF-
1 causes PKC
translocation to the membrane in
rat-reactive astrocytes. In contrast, Studer et al.
(46) suggested that TGF-
does not stimulate PKC(s) in rat mesangial cells, whereas Uchiyama-Tanaka et
al. (48) showed a rapid and
transient stimulation in murine mesangial cells. However, it has been reported
that TGF-
variably affects vascular smooth muscle cell PKC translocation
depending on the embryonic lineage
(52). Thus discrepancies among
these studies could be due to cell- or species-specific responses as well as
to culture conditions.
PKC translocation to specific intracellular compartments is variable
depending on the isoform, stimulus, and/or cell type
(22,
35). In our
immunocytochemistry experiments, PKC staining increases at the membrane
as well as at the nuclear area after 60 min of treatment with
TGF-
1.
Inhibition of PKC by rottlerin blocked
TGF-
1-stimulated collagen I production. This is not due to a
nonspecific effect of rottlerin on cell activity because levels of Sp1 and
Smad4 [2 proteins whose expression is not modulated by TGF-
1
but that are involved in TGF-
1-induced collagen I expression
(41)] are not affected by the
PKC
inhibitor. The inhibitory effect of rottlerin on collagen I
production is due, at least in part, to inhibition of COL1A1 and
COL1A2 gene transcription because rottlerin also inhibited mRNA
expression and promoter activity. Taken together with the data showing changes
of PKC
, but not PKC
, translocation and kinase activity, our
findings suggest that PKC
stimulates collagen I expression in response
to TGF-
1 in human mesangial cells. Recently, Rosenbloom and
colleagues (29,
30) suggested a role for
PKC
in mediating increased fibronectin transcription and elastin mRNA
stabilization by TGF-
in human lung fibroblasts. Thus TGF-
could
stimulate ECM accumulation in several cell types, in part, by modulating
PKC
activity.
In contrast to rottlerin and low concentration of the general PKC inhibitor
calphostin C, Gö 6976, an inhibitor of cPKCs, did not affect collagen I
production or COL1A2 promoter activity in response to TGF-1.
However, in some of our experiments, Gö 6976 increased basal collagen I
protein and
2(I) mRNA expression. This result suggests that
cPKC isoenzymes might inhibit collagen I expression in unstimulated cells.
In our experiments, rottlerin not only blocked TGF-1
stimulation of COL1A2 transcription but also decreased promoter
activity in unstimulated cells. Similarly, a recent report showed blockade of
COL1A1 gene transcription by inhibition of PKC
in scleroderma
fibroblasts (23). These
findings suggest that PKC
is involved in maintaining basal
COL1A1 and COL1A2 gene expression as well as playing a role
in gene activation by TGF-
1. Because mesangial cells have
been shown to produce TGF-
1
(25), it is also possible that
the inhibitory effect of rottlerin on basal collagen I expression might be due
to blockade of the autocrine/paracrine stimulation by
TGF-
1.
To determine the mechanism by which a TGF-1-induced PKC
pathway modulates collagen I expression, we investigated whether PKC
modulates Smad expression and/or activity. Both depletion of PKC by chronic
PMA treatment and inhibition of PKC
by rottlerin slightly decreased
Smad3 protein levels. These data suggest that PKC
could play a role in
maintaining basal Smad3 expression. Downregulation of endogenous Smad3 by
rottlerin or chronic PMA correlates with decreased COL1A2 promoter activity
and inhibition of the TGF-
1 response. Because the inhibition
following PKC depletion by PMA is overcome by ectopic expression of Smad3,
this observation suggests that blockade of PKC
might inhibit
TGF-
1-induced collagen I gene transcription, at least in part
by decreasing Smad3 expression.
Although the effect of rottlerin on COL1A2 could be partly due to the
downregulation of Smad3 expression, our data with the Gal4 assay system and
the transfection experiments with overexpressed Smad3 demonstrate that
blockade of PKC also inhibits Smad3 transcriptional activity. Thus
TGF-
1-induced PKC
activity could stimulate Smad
activity, leading to increased COL1A2 gene expression. Because
blockade of PKC
similarly decreased transcriptional activity of Smad3
and Smad3D, PKC
likely modulates Smad3 activity independently of
phosphorylation of the specific T
RI COOH-terminal target site.
With the use of different cell lines (Mv1Lu and NIH-3T3 cells) stably
expressing tagged Smad proteins, Yakymovych et al.
(53) showed that cell
treatment with the PKC activator PMA resulted in phosphorylation of Smad2 and
Smad3. The phosphorylation did not affect nuclear translocation but it
abrogated direct DNA binding by Smad3. This phosphorylation-dependent
mechanism involving PKC could selectively downregulate certain
TGF-1 signals. In contrast, our data indicate that blocking
the PKC
pathway decreases TGF-
1-induced collagen I
production and promoter activity as well as Smad activity, suggesting a
positive effect of PKC
on the TGF-
1 signal leading to
collagen I accumulation. Thus the same signaling pathway could inhibit or
activate the Smad pathway depending on the cell type and/or stimuli.
In this paper, we showed modulation of the Smad pathway by PKC in response
to TGF-1. Previously, we demonstrated that inhibition of
TGF-
1-activated ERK1/2 decreased Smad transcriptional
activity (19). In several cell
lines, PKC has been linked to ERK activation in response to some stimuli
(3,
9,
15,
17,
49,
55). For example, Axmann et
al. (3) demonstrated that
calcium-dependent PKCs are required for ERK1/2 phosphorylation by
TGF-
1 in rat lung fibroblasts. Our laboratory showed that
TGF-
1 stimulation of Smad3 phosphorylation outside the
COOH-terminus serines is dependent on ERK activation
(20). However, this
observation may not explain the role of PKC in our system because the
requirement of PKC for ERK activation is dependent on the cell types and
stimuli. For example, in neuronal cells, PKC
mediates activation of ERK
by fibroblast-derived growth factor and nerve growth factor but not by
epidermal growth factor (9).
Thus TGF-
1-induced PKC
in human mesangial cells could
modulate Smad activity directly and/or be dependent on ERK activation. On the
other hand, activation of PKC
by TGF-
1 might itself
require activation of other signaling pathways. It has been shown that the
phosphatidylinositol 3-kinase (PI3K) associates with PKC
in the
erythroleukemia cell line TF-1 stimulated with cytokines
(11) and that phosphorylation
of PKC
in response to serum in the human embryonic kidney 293 cells was
PI3K dependent (31).
In summary, we showed that, in human mesangial cells,
TGF-1 stimulates PKC
translocation and activity.
PKC
positively regulates Smad3 and Smad4 transcriptional activity and
is required for increased collagen I production, suggesting that activation
and interaction of multiple signaling pathways contribute to the pathogenesis
of glomerular matrix accumulation.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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