1 Vascular Biology Institute and Division of Nephrology, Department of Medicine, University of Miami School of Medicine, Miami, Florida 33136; and 2 Division of Nephrology and Hypertension, Vanderbilt University Medical Center, Vanderbilt, Tennessee 37232
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ABSTRACT |
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We found that peroxisome
proliferator-activated receptor- (PPAR
) mRNA was reduced by 77%
in glomeruli of diabetic mice. Because mesangial cells play an
important role in diabetic nephropathy, we examined regulation of type
I collagen expression by PPAR
and transforming growth
factor-
1 (TGF-
1) in mouse mesangial cells
in the presence of 6 and 25 mM glucose. Mesangial cells contained
functionally active PPAR
. Exposure to 25 mM glucose resulted in
reduced PPAR
expression and transcriptional activity, accompanied by
increased type I collagen expression. Restoration of PPAR
activity
to normal levels in cells cultured in 25 mM glucose, by transfection
with a PPAR
expression construct and treatment with the PPAR
agonist troglitazone, returned type I collagen levels toward normal
values. Activation of PPAR
by troglitazone also decreased type I
collagen mRNA and blocked TGF-
1-mediated upregulation of
type I collagen mRNA and protein. Moreover, PPAR
activation
suppressed basal and activated TGF-
1 responses in mesangial cells. This action was blocked by transfection of cells with
a dominant-negative PPAR
construct. In summary, PPAR
suppresses the increased type I collagen mRNA and protein expression mediated by
TGF-
1 in mesangial cells.
peroxisome proliferator-activated receptor-; transforming growth
factor-
1; diabetic nephropathy
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INTRODUCTION |
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THE PEROXISOME
proliferator-activated receptor (PPAR) is a family of ligand-activated
transcription factors (19). Three PPAR subtypes (,
,
and
) have been identified and cloned. PPARs regulate gene
transcription by directly binding to repeat-1 sites [peroxisome
proliferator response elements (PPRE)] in targeted gene promoters or
upstream enhancer regions as heterodimeric complexes with the retinoid
X receptor (RXR). PPAR
is expressed at high levels in the liver,
kidney, and heart and at lower levels in the retina and pancreatic
islets (5, 19). PPAR
activation results in
proliferation of peroxisomes and induction of hepatic genes involved in
fatty acid
-oxidation (5). PPAR
is ubiquitously expressed in many tissues, but its function and the genes it regulates have not been completely elucidated (5). PPAR
is
expressed at high levels in adipose tissue and is a key regulator of
adipocyte differentiation. In addition, PPAR
has also been found to
play an important role in regulation of systemic insulin sensitivity. Severe insulin resistance and diabetes mellitus have been shown in
patients with PPAR
mutations (2). Thiazolidinediones
(TZD), insulin-sensitizing agents, have been recognized as ligands for PPAR
(20). Recently, PPAR
was also found in a
variety of nonadipose tissues and cells, including skeletal muscle,
heart, kidney, bone marrow stromal cells, neutrophils, lymphocytes, and
macrophages (8, 9, 17, 37, 39).
PPAR expression in macrophages and lymphocytes regulates
inflammatory responses in a negative fashion (17, 39),
suggesting a function of this receptor distinct from its metabolic
activity. Further evidence for these nonmetabolic functions was
obtained in studies of diabetic rats, where it was found that
troglitazone, one of the TZD family of PPAR
ligands, prevented
albuminuria and increased glomerular extracellular matrix (ECM) gene
expression (14). These effects were obtained in the
absence of changes in systemic blood glucose levels. A similar
improvement in glomerular lesions was also found in Zucker rats, a
model of non-insulin-dependent diabetic glomerulosclerosis, and in 5/6
nephrectomized rats (3, 23, 24). Taken together, these
data indicate that TZD may have direct effects on glomeruli by
mechanisms other than their insulin-sensitizing activity. The finding
that PPAR
and RXR were expressed in rat glomerular mesangial cells
further supports this possibility (1, 28).
The pathological hallmark of diabetic glomerular lesions is ECM
accumulation, including type I collagen (7, 35). Although endothelial, epithelial, and mesangial cells are involved in glomerular ECM turnover, mesangial cells appear to be the major source of pathological glomerular ECM (21). An imbalance between ECM
synthesis and degradation by mesangial cells contributes directly to
the development and progression of diabetic glomerular lesions
(22). Nuclear receptors, including estrogen receptors, the
retinoic acid receptor, and RXR, are known to regulate
expression of ECM and metalloproteinase genes in various tissues and
cells, including mesangial cells (27, 29, 32). Although a
PPAR response element (PPRE) has not been found upstream of ECM
genes, there is evidence suggesting that PPAR
may participate in
regulation of ECM genes, especially type I collagen. First, peroxisome
proliferator reduced type I collagen and elastin gene expression in
lung fibroblasts (25). Second, ligand-induced activation
of PPAR
inhibited type I collagen promoter activity and decreased
1 type I collagen mRNA levels in hepatic stellate cells
(26). Finally, TZD treatment decreased glomerular ECM gene
expression and protein accumulation in diabetic rats and in 5/6
nephrectomized rats (14, 23). Mesangial cells,
fibroblasts, and activated stellate cells share several aspects of ECM
turnover (18). Therefore, we hypothesized that one of the
functions of PPAR
in mesangial cells was to regulate type I collagen expression.
Transforming growth factor- (TGF-
) is one of the key regulators
of ECM genes in mesangial cells. Elevated glomerular
TGF-
1 levels have been shown to make a significant
contribution to the pathogenesis of diabetic glomerular lesions,
including accumulation of type I collagen (15, 34).
Recently, glomerular TGF-
1 expression was found to be
decreased in diabetic rats and 5/6 nephrectomized rats treated with
troglitazone (14, 23). We postulated that the effect of
PPAR
on type I collagen expression might be mediated by its
inhibition of TGF-
1.
We found that PPAR expression was reduced in mesangial cells in
conditions associated with an increase in type I collagen expression,
namely, cells cultured in 25 mM glucose and glomeruli isolated from
animals with diabetic nephropathy. In addition, we found that
activation of PPAR
resulted in suppression of type I collagen
expression. Thus one possible mechanism by which PPAR
could suppress
type I collagen expression in mesangial cells is via inhibition of
TGF-
responses.
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MATERIALS AND METHODS |
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Mesangial cell culture.
Mesangial cells from C57 mice were isolated and characterized as
previously described (6). Cells were grown in DMEM-Ham's F-12 mixture supplemented with 20% fetal bovine serum (FBS), 100 U/ml
streptomycin, and 100 U/ml penicillin. Cells were maintained in culture
medium containing 6 or 25 mM glucose. To test the effect of a PPAR
ligand on collagen I expression, cells were switched to DMEM-Ham's
F-12 mixture containing 0.1% BSA and 2-8 µM troglitazone. Because 20 µM troglitazone was associated with mesangial cell toxicity (20% loss of adherence), we chose 8 µM troglitazone for most experiments. At this concentration, there was no increase in
cellular trypan blue staining compared with cells treated with vehicle
(0.1% DMSO).
Transfection and reporter gene assay.
Transient transfection was performed in cultured mesangial cells using
Transfast according to the manufacturer's instructions (Promega,
Madison, WI). To examine whether PPAR was functionally active in
mesangial cells, cells were transfected with a PPAR
reporter
construct containing three copies of the PPRE from the acyl-CoA oxidase
gene linked to the thymidine kinase promoter and luciferase vector or
an RL-TK empty vector in the presence or absence of the PPAR
ligand
troglitazone. Cotransfection with a
-galactosidase vector served as
an internal control. Cells were harvested 24-48 h after
transfection. Luciferase and
-galactosidase activities were measured
as previously described (32). Overexpression of PPAR
was achieved by transfecting the full-length rabbit PPAR
obtained
from 5'- and 3'-rapid amplification of cDNA ends and cloned into the
pcDNA3 expression vector. Mouse dominant-negative PPAR
was
constructed by mutation of highly conserved hydrophobic and charged
residues (L466/L467) in helix 12 of the ligand-binding domain to
alanine (L466A/L467A), as previously reported in human PPAR
dominant-negative mutation (2, 10). To test the effect of
our dominant-negative PPAR
construct on basal and ligand-activated PPAR
transcriptional activity in mesangial cells, cells were transfected with a mouse dominant-negative PPAR
construct together with a PPAR reporter construct in the presence or absence of the PPAR
ligand troglitazone. We further examined the dominant-negative function of this construct by cotransfecting it with a full-length PPAR
cDNA expression vector. Cells transfected with empty pcDNA3 vector served as a control. To determine the role of PPAR
activation/inhibition on TGF-
responses in mesangial cells, the
TGF-
reporter construct p3TP-Lux [a gift from Dr. J. Massague
(38)] was introduced into mesangial cells together with a
PPAR
expression vector, a mouse dominant-negative PPAR
vector, or
a control vector in the presence or absence of PPAR
ligands and
recombinant human TGF-
1 (4 ng/ml; R&D Systems,
Minneapolis, MN). The same amount of DNA was used for each transfection
throughout the experiments. The transfection efficiency remained at
28-32%, as determined by transfection of C57 mesangial cells with
-galactosidase.
Mouse model of diabetes. We reported that diabetic C57 mice had mild glomerular lesions, while diabetic C57 Os/+ mice, in which the number of nephrons was reduced 50% by the Os mutation, developed moderate glomerulosclerosis (40). Therefore, C57 Os/+ mice were selected for this study. Diabetes was induced within 2 wk by 7-14 intraperitoneal injections of streptozotocin (25-50 µg/g body wt) or vehicle (citrate buffer) (40). Mice were weighed, and blood glucose levels were determined at baseline and weekly thereafter. Mice with stable hyperglycemia between 300 and 400 mg/dl were included in the study. Mice were kept without insulin treatment and allowed free access to food and water. After 12 wk, mice were killed according to University of Miami School of Medicine-approved procedures. The left kidney was perfused with a solution containing collagenase and RNase inhibitors for glomerular microdissection (30, 40). The right kidney was perfused with fixative containing 4% freshly prepared paraformaldehyde and was used for microscopic studies.
RT-PCR.
Total RNA was extracted from cells or microdissected glomeruli using
TriReagent (MRC, Cincinnati, OH). Reverse transcription was performed
on 0.3-2 µg of total RNA with oligo(dT)15 primer and
avian myeloblastosis virus reverse transcriptase (Roche, Indianapolis, IN). Expression of PPAR in mesangial cells and diabetic glomeruli was determined by PCR using a specific set of primers:
5'-GACATCCAAGACAACCTGCTG (sense) and 5'-GCAATCAATAGAAGGAACACG
(antisense). The amplified samples were separated on a 3% agarose gel
and analyzed by densitometry (NIH Image). To determine the linear range
of the PCR, the intensity of amplified products was plotted against the
cycle number as previously described (data not shown). The optimum
number of PCR cycles for PPAR
was 32 for the mesangial cells and 35 for the glomeruli. Because we found that the glomerular expression of
-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) remained stable in diabetic mice compared with nondiabetic mice,
-actin or
GAPDH was used as a housekeeping gene control. Expression levels of
1 type I collagen in mesangial cells were quantitated by
competitive PCR using the primers 5'-GTGAACCTGGCAAACAAGGT (sense) and
5'-CTGGAGACCAGAGAAGCCAC (antisense). The
1 type I
collagen mutant was created by deletion of a 140-bp fragment from the
original PCR product using the primers 5'-GAATCTGGACGTGAAAGAATGGCGATCG
(sense) and 5'-CGATCGCCATTCTTTCACGTCCAGATTC (antisense). Competitive
PCR was performed by addition of decreasing amounts of mutant to sample
tubes, as previously described (30). Data are expressed as
the ratio of type I collagen to GAPDH mRNA.
Western blots.
Mesangial cells were grown to 80% confluence in 10-cm petri dishes.
Cells were scraped off the plates and lysed in ice-cold buffer (20 mM
Tris, 140 mM NaCl, 3 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate, 2 mM NaVO4, 10% glycerol, pH 7.4, 1% Triton X-100) with
protease inhibitors (1.5 µM aprotinin, 20 µM leupeptin, 50 µM
phenylmethylsulfonyl fluoride, 1.5 µM benzamidine). The insoluble material was removed by centrifugation at 20,000 g for 30 min at 40°C. Samples containing equal amounts of protein were
resolved by SDS-PAGE and transferred to nitrocellulose membranes. After incubation with rabbit anti-PPAR antibody (Santa Cruz Biotechnology, Santa Cruz, CA), the blots were washed and incubated with
peroxidase-conjugated secondary antibody, and protein bands were
analyzed using a chemiluminescence kit (Santa Cruz Biotechnology).
ELISA.
Mesangial cells (3 × 104) were plated in 24-well
plates in medium supplemented with 20% FBS. Cells were transfected
with a PPAR expression vector, a dominant-negative PPAR
vector,
or a control vector. After 2 h, medium containing 10% FCS and 8 µM troglitazone or 0.1% DMSO was added. TGF-
1 (4 ng/ml) or vehicle (4 mM HCl and 1 mg/ml BSA) was added 12 h later.
After 24 h, the medium was replaced with medium containing 0.1%
FBS, and
-aminopropionitrile (80 µg/ml) was added. Supernatants
were collected, and cells were lysed in 0.5 ml of 5 M guanidine-0.1 M
Tris · HCl (pH 8.6) at 48 h in the presence of 20 µM
phenylmethylsulfonyl fluoride, 2.5 µM EDTA, and 10 µM
N-ethylmaleimide. The amount of type I collagen was
determined in the supernatants and cell layers by ELISA
(16). Briefly, 96-well ELISA plates were coated with 100 µl of sample or mouse type I collagen standards and incubated at
37°C for 2 h. After they were blocked with PBS + 0.25%
BSA + 0.05% Tween 20, the plates were incubated with rabbit
anti-type I collagen diluted 1:2,000 (Biodesign International,
Kennebunk, ME) at 4°C overnight. A 1:2,000 dilution of a biotinylated
goat anti-rabbit IgG was used as secondary antibody (Biosource
International, Camarillo, CA). Final values were normalized for cell number.
Statistics. Values are means ± SE. ANOVA and Student's t-test were used. P < 0.05 was considered to be statistically significant.
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RESULTS |
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Diminished PPAR expression in glomeruli of diabetic mice.
Diabetic C57 Os/+ mice developed moderate glomerulosclerosis
after 3 mo of untreated diabetes (40). PPAR
expression
was examined by RT-PCR in glomeruli that had been individually
microdissected (30) from control and diabetic C57 Os/+
mice. The diabetic mice had persistently elevated glucose levels, which
averaged 333 ± 32.66 mg/dl. PPAR
mRNA levels were
conspicuously reduced in diabetic glomeruli (77% reduction compared
with nondiabetic controls; Fig. 1).
Functionally active PPAR
is expressed in mesangial cells. To
determine whether mesangial cells express PPAR
, total RNA from C57
mouse mesangial cells was examined by RT-PCR. A 258-bp PPAR
fragment
was obtained by PCR (Fig. 2A).
The specificity of the PCR amplification was confirmed by sequence
analysis (data not shown). Western blot analysis confirmed the presence
of PPAR
in mesangial cells (Fig. 2B).
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Diminished PPAR expression in mesangial cells exposed to 25 mM
glucose.
PPAR
mRNA levels were significantly reduced in mesangial cells
cultured in 25 mM glucose (Fig. 3,
A and B). Similarly, Western blot analysis
revealed that the PPAR
protein levels were reduced in mesangial
cells cultured in 25 mM glucose (Fig. 3C). To test whether
the decrease of PPAR
expression was associated with diminished PPAR
responses in cells cultured in 25 mM glucose, cells were transfected with the PPAR reporter construct or the empty RL-TK vector
and studied in the presence or absence of troglitazone. In agreement
with PPAR
mRNA and protein levels, the PPAR
response was
decreased (65%) in mesangial cells cultured in 25 mM glucose (Fig.
3D). Although troglitazone still induced a PPAR
response in cells cultured in 25 mM glucose, the levels did not reach that in
unstimulated cells cultured in 6 mM glucose (Fig. 3D).
Transfection of cells cultured in 25 mM glucose with a PPAR
expression vector restored PPAR
transcription to levels comparable
to that in cells cultured in 6 mM glucose, and this was further
activated by troglitazone (Fig. 3D). PPAR
transcriptional
activity remained low in cells cultured in 25 mM glucose that had been
transfected with the empty pcDNA3 vector (data not shown).
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Decreased type I collagen expression by PPAR activation.
To determine the effects of PPAR
activation on type I collagen
expression, 1 × 105 mesangial cells cultured in
six-well plates were collected 6-24 h after addition of
troglitazone or DMSO. Type I collagen mRNA levels were determined by
competitive PCR. Addition of a PPAR
agonist for 24 h inhibited
type I collagen mRNA expression in a dose-dependent fashion (Fig.
4, A and B). To
determine whether the suppressive effect of the PPAR
agonist on type
I collagen expression in mesangial cells was mediated via the PPAR
pathway, mesangial cells were transfected with a dominant-negative
PPAR
expression vector in the presence or absence of troglitazone. The suppressive effect of troglitazone on mesangial cell type I
collagen expression was blocked by the transfected dominant-negative PPAR
expression vector (Fig. 4C). Furthermore, we found
that type I collagen expression was significantly increased in
mesangial cells transfected with dominant-negative PPAR
expression
vector (Fig. 4, C and D). Transfection of cells
with an empty pcDNA3 vector had no effect on type I collagen expression
(data not shown). These data indicated that inhibition of mesangial
cell type I collagen expression with troglitazone was mediated via the
PPAR
transcriptional pathway.
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Decreased type I collagen expression by restoration of PPAR
expression in mesangial cells cultured in 25 mM glucose.
To examine the effect of a decrease in PPAR
transcription on type I
collagen expression, total RNA extracted from cells cultured in 25 mM
glucose was analyzed by competitive PCR. Levels of
1 type I collagen mRNA were significantly increased in cells cultured in
25 mM glucose compared with cells cultured in 6 mM glucose (Fig.
5, A and B). Type I
collagen accumulation was also significantly increased in cells
cultured in 25 mM glucose: 8.8 ± 0.76 vs. 4.9 ± 0.3 ng/104 cells (P < 0.01; Fig.
5C). Interestingly, the addition of troglitazone did not
decrease type I collagen expression in the presence of 25 mM glucose
(Fig. 5, B and C). To determine whether the lack of suppression of type I collagen expression by troglitazone was due to
low levels of PPAR
expression, cells were transfected with a PPAR
expression vector. This expression vector induces increased PPAR
transcriptional activity, especially in the presence of troglitazone.
As shown in Fig. 5, B and C, restoration of
PPAR
transcriptional responses in these cells significantly reduced the increased type I collagen expression.
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Inhibition of TGF-1-mediated type I collagen
expression by PPAR
activation.
Because TGF-
has been shown to stimulate type I collagen expression
in mesangial cells, TGF-
1 (4 ng/ml) was added to the mesangial cells in the presence or absence of troglitazone. As shown in
Fig. 6, A and B,
TGF-
1 induced a twofold increase in type I collagen
expression, while troglitazone abrogated this effect. Type I collagen
production was also stimulated by TGF-
1, and this was
also inhibited by troglitazone (Fig. 6C).
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Inhibition of a TGF- response element by PPAR
activation.
To further examine the effect of PPAR
activation on
TGF-
1 responses, mesangial cells were transfected with
the TGF-
reporter construct p3TP-Lux. TGF-
1 (4 ng/ml)
or vehicle was added to transfected cells in the presence or absence of
a PPAR
agonist. Troglitazone significantly inhibited the basal
TGF-
responses (Fig. 7A).
TGF-
1 induced a 9.7-fold increase in reporter activity,
and troglitazone caused a 50% reduction in this activity. To test
whether the effect of troglitazone on the TGF-
responses was
mediated via the PPAR pathway, a dominant-negative PPAR
expression
vector or a pcDNA3 empty vector was cotransfected with the TGF-
reporter construct, and the responses were examined in the presence or
absence of troglitazone. Transfection with a dominant-negative PPAR
abrogated the ability of troglitazone to suppress TGF-
responses
(Fig. 7B). Furthermore, we found that mesangial cells
transfected with a dominant-negative PPAR
expression vector
exhibited significantly increased TGF-
responses at baseline.
Transfecting cells with a pcDNA3 empty vector did not change the
TGF-
responses (8.9-fold increase of luciferase activity in the
presence of TGF-
1) or the inhibition of TGF-
responses (47% inhibition) by troglitazone. These data suggest that
inhibition of mesangial cell TGF-
responses by troglitazone was
mediated via the PPAR
transcriptional pathway.
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DISCUSSION |
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We found that mouse glomerular mesangial cells express
functionally active PPAR. PPAR
mRNA and protein were expressed by mesangial cells. Treatment with the PPAR
agonist troglitazone increased the PPAR transcriptional responses. In addition, we found
that overexpression of PPAR
increased PPAR responses in mouse
mesangial cells. A dominant-negative PPAR
expression vector, developed by mutating the highly conserved hydrophobic and charged residues L466/L467 in helix 12 of the ligand-binding domain to alanine
(L466A/L467A), significantly reduced basal and ligand-activated PPAR
responses. Furthermore, transfection with the dominant-negative PPAR
expression vector inhibited the increase of PPAR
transcriptional activity induced by PPAR
overexpression. Gurnell et al.
(10) also found decreased basal and ligand-activated
PPAR
responses in 293 cells transiently transfected with human
L468A/E471A dominant-negative PPAR
. The effects of dominant-negative
PPAR
may be related to its recruitment of corepressors, delay of
ligand-dependent corepressor release, or impairment of the recruitment
of coactivators (10). Nevertheless, expression of PPAR
in mesangial cells, together with expression of 9-cis
retinoic acid receptor, may play an important role in mesangial cell
gene regulation (1, 28). This speculation is supported by
the finding by Nicholas et al. (28) that the angiotensin
II-mediated increase in plasminogen activator inhibitor-1 (PAI-1)
expression in mesangial cells was inhibited by the PPAR
ligand troglitazone.
Mesangial cells are key players in the accumulation of ECM in diabetic
nephropathy (21, 22, 36). An imbalance between matrix
synthesis and degradation by mesangial cells has been thought to
directly contribute to the pathogenesis of diabetic glomerulosclerosis. Recently, Isshiki et al. (14) found that troglitazone, one
of the PPAR ligands, prevented increased glomerular ECM expression in
streptozotocin-induced diabetic rats without lowering blood glucose
levels. In addition, troglitazone treatment reduced glomerulosclerosis in Zucker fatty rats and 5/6 nephrectomized rats (3, 23, 24). These data suggest that PPAR agonists may have direct effects on glomerular extracellular turnover, other than through their
insulin-sensitizing and metabolic activities.
We found that activation of PPAR by troglitazone decreased type I
collagen expression in mouse mesangial cells. Inasmuch as troglitazone
has also been shown to have antioxidant properties (12),
it was necessary to determine whether this response was directly
related to its activity as a PPAR
ligand. This was assessed by
transfecting mesangial cells with a mouse dominant-negative PPAR
vector. We found that this dominant-negative PPAR
vector completely
abrogated the suppressive effect of troglitazone on mesangial cell type
I collagen expression, suggesting that the inhibition was mediated
through the PPAR
pathway. Our observations also showed that the
reduction of basal PPAR response levels by the dominant-negative
PPAR
was associated with an increase in type I collagen expression.
This indicates that one function of PPAR
may be to downregulate type
I collagen expression in mesangial cells.
The mechanism by which PPAR controls type I collagen gene
transcription is unknown. Because a typical direct repeat-1 PPAR binding site is not present in type I collagen gene promoter, it is
likely that this effect of PPAR
is mediated indirectly through other
pathway(s). It is well established that TGF-
is one of the key
mediators of mesangial cell ECM accumulation (31). Because
we found that activation of PPAR
by troglitazone suppressed TGF-
1-mediated type I collagen expression in mesangial
cells, we speculated that one of the mechanisms by which PPAR
might regulate mesangial cell type I collagen gene expression could be via
inhibition of TGF-
1. The findings that increased PPAR
activity in mesangial cells inhibited TGF-
responses, while
decreased PPAR
activity led to increased TGF-
responses, provided
further support for this possibility. Therefore, the expression levels of type I collagen in mesangial cells may be determined, at least in
part, by the balance between TGF-
and PPAR
levels. Our
observation that reducing basal PPAR
activity by transfecting
mesangial cells with a dominant-negative PPAR
vector was associated
with increased TGF-
responses and type I collagen expression
provides further evidence for this balance in determining net cellular
responses. The mechanism by which PPAR
inhibited TGF-
responses
is not clear. Because the TGF-
reporter construct p3TP-Lux, used in our study, contains activator protein-1 (AP-1) and PAI-1 promoter sites, we speculate that the action of PPAR
may be via its
interaction with AP-1 and other transcription factors. This speculation
is supported by the findings that the activation of PPAR
decreased AP-1 transcriptional activity and attenuated angiotensin II-mediated upregulation of PAI-1 (4, 28).
It has been found that TGF-1 and type I collagen
expression is increased in diabetic glomeruli and mesangial cells
cultured in 25 mM glucose (11, 15, 34). We found that this
increase was associated with decreased PPAR
expression. These data
contradict the previous report that PPAR
expression was increased in
glomeruli of diabetic rats (28). This difference may be
caused by the use of different species and different types and degrees
of glomerular lesions. Furthermore, our in vitro data on mouse
mesangial cells agree with our in vivo data. Namely, mesangial cells
exposed to 25 mM glucose had decreased PPAR
expression and PPAR
responses, which were associated with increased type I collagen
expression. Similarly, others have found that elevated glucose levels
lead to decreased PPAR
expression in cultured macrophages
(33). Interestingly, the macrophages isolated from type II
diabetic patients also showed decreased PPAR
expression
(33). When a PPAR
expression vector was transfected
into mesangial cells cultured in 25 mM glucose, PPAR
activity was
restored to levels comparable to those in cells cultured in 6 mM
glucose. Furthermore, the addition of troglitazone resulted in
significantly reduced levels of type I collagen.
Mesangial cells cultured in 25 mM glucose were previously found
to have increased TGF-1 expression and TGF-
responses
(13). These data, together with our findings, support the
postulate that PPAR
and TGF-
may play an important role in
regulation of type I collagen expression. The fact that long-term
treatment of diabetic rats and 5/6 nephrectomized rats with
troglitazone decreased TGF-
1 and ECM gene expression in
glomeruli, thereby preventing development and progression of
glomerulosclerosis, further supports the importance of counterbalancing
interaction(s) between PPAR
and TGF-
in vivo (14,
23).
Thus activation of PPAR in mesangial cells may be a useful way to
inhibit the prosclerotic actions of TGF-
in these cells. However,
because PPAR
levels were significantly decreased in mouse mesangial
cells cultured in high glucose and in diabetic mouse glomeruli with
moderate sclerotic lesions, a higher level of TZDs or a stronger
PPAR
ligand may be required to achieve this therapeutic effect.
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. Zheng, 1600 NW 10th Ave., Rm. 1009B (R-104), Miami, FL 33136 (E-mail: fzheng{at}med.miami.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.
10.1152/ajprenal.00189.2001
Received 22 June 2001; accepted in final form 28 September 2001.
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