Department of Pathology, Seoul National University College of Medicine, Seoul 110-799, Korea
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ABSTRACT |
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Abnormal lipid accumulation in glomeruli could be implicated in
the pathogenesis of glomerulosclerosis. Low-density lipoprotein (LDL)
stimulates collagen mRNA expression in cultured human mesangial cells
(HMC). To explore the possible molecular mechanisms by which LDL
promotes collagen gene expression, we examined the effects of LDL on
protein kinase C (PKC) activity and transforming growth factor-
(TGF-
) expression in relation to collagen gene regulation in HMC.
LDL (200 µg/ml) induced an acute increase in PKC activity, particularly PKC-
and -
, within 15 min, which decreased to
control value at 2 h. LDL stimulated TGF-
1, and
1(I) and
1(IV)
collagen mRNA expression within 30 min of incubation with HMC, and
levels remained elevated until hour 4.
LDL induced the secretion of TGF-
by HMC. This TGF-
was shown by
CCL-64 mink lung cell assay to be, in part, bioactive. The stimulatory
effects of LDL on collagen gene regulation in HMC were blocked by the
inhibition of PKC using GF-109203X (GFX) or the downregulation of PKC
using phorbol myristate acetate. Neutralizing antibody to TGF-
inhibited the increased collagen mRNA expression by HMC exposed to LDL.
The downregulation or inhibition of PKC did not affect the stimulatory
effect of LDL on TGF-
mRNA or protein expression. These results
suggest that in HMC, LDL stimulates collagen mRNA expression through
the rapid activation of PKC-
and -
and transcriptional
upregulation of TGF-
. Thus PKC and TGF-
may function as
independent key signaling intermediaries in the pathway by which LDL
upregulates collagen gene expression in HMC.
glomerulosclerosis; lipids; transmembrane signaling; collagen gene
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INTRODUCTION |
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THE ACCUMULATION of extracellular matrix proteins in the mesangial area is an important component of glomerulosclerosis. Glomerular mesangial cells, which are often compared with vascular smooth muscle cells (VSMC), are a major site of synthesis of matrix proteins in chronic glomerular disease.
Studies have suggested that lipid abnormalities may be involved in the progression of glomerular disease (reviewed in Ref. 15). Low-density lipoprotein (LDL) can upregulate collagen gene expression after incubation for 48 h with cultured human mesangial cells (HMC) (17). The stimulation by LDL of quiescent HMC, which in turn oversecretes matrix proteins, might require a number of coordinated cellular events. Information regarding a variety of extracellular signals is relayed across the cell membrane to regulate many intracellular signals via a signal-bifurcating pathway, protein kinase C (PKC) activation and Ca2+ mobilization (20).
PKC comprises of a family of 11 different gene products that are
classified into Ca2+-dependent or
conventional PKC (,
, and
) or
Ca2+-independent or novel PKC
(
,
,
,
, and µ) and atypical PKC groups
according to their structure and function (21). Some experiments have
shown that in cultured VSMC, LDL activates phosphoinositide turnover and PKC and that these responses are mediated by
LDL receptor (4, 23, 26). Other investigators, however, have argued
that LDL elevates intracellular calcium and pH but not phosphoinositide
turnover in VSMC and that these responses are not mediated
by LDL receptor (25). In HMC, specific receptor-mediated endocytosis of
LDL has been clearly demonstrated (10, 18). In cultured rat mesangial
cells (RMC), PKC activity was acutely and transiently activated by LDL,
and more prolonged exposure of cells to LDL increased both transforming
growth factor-
(TGF-
) bioactivity and fibronectin synthesis (28).
TGF- exists in mammals in three isoforms (
1,
2, and
3), and
its overproduction has been implicated in the pathogenesis of
glomerulosclerosis (16). The neutralization of TGF-
activity by
antiserum against TGF-
or the inhibition of TGF-
expression by
antisense oligonucleotides suppressed progression to glomerulosclerosis (1, 5). It has been suggested that TGF-
increases mRNA levels in
most matrix proteins in cultured cells (22), and in mouse mesangial
cells, TGF-
stimulates collagen and fibronectin synthesis (19). It
has been shown that RMC express TGF-
mRNA and secrete TGF-
proteins, mostly in latent form (13). The induction of TGF-
protein
and its mRNA expression in RMC seems to be mediated through a tyrosine
kinase-dependent mechanism but not through PKC (11). Studer et al.
(28), however, suggested that the activation of PKC signals increased
TGF-
bioactivity.
The present study was designed to investigate the effects of LDL on PKC
activity and TGF- in relation to collagen gene regulation in HMC. We
found that LDL stimulated PKC, particularly PKC-
and -
, and
TGF-
1 expression at earlier time points in HMC, and these increases
were intimately linked to the onset of enhanced collagen mRNA expression.
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MATERIALS AND METHODS |
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Reagents. A PKC assay kit was obtained
from Upstate Biotechnology, Lake Placid, NY. Polyclonal rabbit
anti-PKC- and -
1 were purchased from Santa Cruz Biotech (Santa
Cruz, CA). Monoclonal mouse anti-PKC-
was from Transduction
Laboratories (Lexington, KY). Phorbol myristate acetate (PMA),
protease inhibitors, and 10-nm gold-labeled goat anti-rabbit IgG was
from Sigma Chemical (St. Louis, MO). GF-109203X (GFX) was
obtained from Calbiochem (San Diego, CA), and
[
-32P]ATP was
from Amersham (Arlington Heights, IL). Monoclonal mouse anti-TGF-
1-
2-
3 was from Genzyme Diagnostics (Cambridge, MA), and polyclonal rabbit anti-TGF-
was from R & D (Minneapolis, MN).
TGF-
1 enzyme-linked immunosorbent assay (ELISA) kit was from Promega
(Madison, WI). Other reagents were from the sources previously reported
(17).
Culture of HMC. HMC were obtained from adult nephrectomy specimens, as previously described (18). Culture medium was made of DMEM supplemented with 20% fetal calf serum, 200 mM L-glutamine, and antibiotics (penicillin 100 U/ml, streptomycin 100 µg/ml, and amphotericin 0.25 µg/ml). For the present experiments, cells between the 5th and 7th passage were used.
LDL isolation. Human LDL (density, 1.019-1.063) and lipoprotein-deficient serum (density >1.215) were isolated from the plasma of normal volunteers by the method of sequential ultracentrifugation, as previously described (18). Isolated LDL was dialyzed for 24 h at 4°C against buffer a containing 0.15 M NaCl and 0.24 mM EDTA, pH 7.4. After dialysis, LDL was stored at 4°C under nitrogen and was used within 14 days.
Experimental conditions. HMC were grown to near confluence. The cells were synchronized to quiescence in serum-free DMEM containing 5 µg/ml insulin-transferrin-selenite for 48 h. After synchronization, experiments were performed by the addition of 200 µg LDL protein per ml serum-free DMEM to HMC for 0.25, 0.5, 1, 2, and 4 h at 37°C. In some experiments, 2 µM of GFX, a potent PKC inhibitor (29), or 0.5 µM of PMA was added to cells 30 min or 24 h prior to LDL administration. In a given experiment, simultaneous control monolayers were treated with serum-free DMEM alone.
Assay of PKC activity. Quiescent HMC
in 6-well plates were exposed to 200 µg/ml LDL for the required time
intervals, and PKC activity was assayed using QKRPSQRSKYL as a specific
substrate peptide as described by manufacturer (Upstate Biotech). Cells were washed rapidly three times with 25 mM Tris-buffered saline (TBS)
at 4°C. After 1 ml of extraction buffer [PKB: containing 25 mM Tris · HCl, pH 7.4, 2 mM EGTA, 10 mM
-mercaptoethanol, 20 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] was added to each well,
cells were harvested and homogenized. Aliquots of the homogenate were
taken to measure total PKC activity. For PKC assay, 10 µl of sample
was added to a microcentrifuge tube, where the same amounts of
substrate cocktail, inhibitor cocktail, and lipid activator were
present. After the addition of 10 µl diluted
[
-32P]ATP
mixture, the microcentrifuge tube was incubated at
30°C for 10 min, and a 25-µl aliquot was transferred onto the
center of numbered P81 phosphocellulose paper. Assay squares were
washed in 40 ml 0.75% phosphoric acid and then in 20 ml acetone, then transferred to a scintillation vial. After scintillation cocktail was
added, radioactivity was determined by a scintillation counter. Controls for endogenous phosphorylation of proteins in the sample extract were performed by substituting assay dilution buffer for substrate cocktail.
PKC specific activity (pmol phosphate incorporated into substrate peptide/min) was calculated as follows
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Cell fractionation and PKC
immunoblots. Quiescent HMC in 100-mm plates were
stimulated with 200 µg/ml LDL for 15 min or 30 min. Cells were washed
twice with ice-cold PBS. After 100 µl of buffer
A containing 20 mM Tris · HCl, pH
7.5, 5 mM EDTA, 1 mM PMSF, and 10 µg/ml leupeptin was added, cells
were harvested and sonicated. The homogenates were centrifuged at
32,000 rpm for 1 h at 4°C and the supernatants were retained as the
cytosolic fraction. The pellets were resuspended in 100 µl of
buffer B (buffer A with 1% Triton X-100), sonicated, left on ice for 1 h, and centrifuged again at 32,000 rpm for 1 h at 4°C. The
supernatants were collected and used as the membrane fraction. Protein
concentration was determined using a BCA protein assay reagent (Pierce,
Rockford, IL). Aliquots were then electrophoretically resolved in 10%
polyacrylamide gel in SDS buffer and transferred onto nitrocellulose
membranes as we have previously described (17). The blots were
incubated with rabbit anti-PKC- (1:1,000) or -
1 (1:500) or mouse
anti-PKC-
(1:250) at 4°C overnight. Bound primary antibody was
visualized by the subsequent incubation with horseradish
peroxidase-conjugated secondary antibody and enhanced chemiluminescence
system kit (Amersham). Densitometry was performed using an imaging
densitometer (Bio-Rad model GS-700). The specificity of primary
antibody reaction with sample was eliminated in the presence of peptide
that was used to generate the antibody.
Preparation of conditioned media and activation of
latent TGF-.
At the end of the experiment, the culture supernatants from HMC were
harvested in siliconized tubes in ice, centrifuged to remove cell
debris, and subjected to bioassay. To activate latent TGF-
, an
aliquot of medium was diluted to adjust protein concentration to
50-100 µg/ml and heated to 80°C for 8 min. Samples were
analyzed both before heating (to measure the active form) and after
heat activation of the latent form (to determine total TGF-
).
Quantitation of secreted
TGF-1.
The concentration of TGF-
1 in conditioned media was determined using
a TGF-
1 ELISA kit, according to the manufacturer's instructions
(Promega). Briefly, samples were activated with 1 N HCl for 15 min at
room temperature followed by neutralization with 1 N NaOH. Samples were
added to microtiter plates, which were coated with monoclonal
anti-TGF-
1. After incubation for 90 min at room temperature, the
plates were incubated with rabbit anti-human TGF-
1 antibody for 2 h.
Then goat anti-rabbit IgG conjugated to horseradish peroxidase was
added and incubated for 2 h. The assay was developed with the
peroxidase substrate for 3-4 min at room temperature followed by 1 M phosphoric acid. The absorbance was measured at 450 nm with a
Molecular Devices (Spectra Max 250). Standard curves for TGF-
1 were
prepared using serial dilutions of exogenous TGF-
1 (Promega).
Bioassay of TGF-
activity.
The biological activity of TGF-
was assayed using a modification of
method of Danielpour et al. (8). In brief, mink lung epithelial cells
(American Type Culture Collection CCL-64) were plated onto 24-well
plates in DMEM containing 0.2% lipoprotein-deficient serum. After 24 h, test samples were added, and 22 h later, cells were exposed to 0.5 µCi of [3H]thymidine
per well in a total of 0.5 ml/well for 2 h at 37°C. Cells were then
washed, treated with 0.5% trypsin/EDTA, harvested, and radioactivity
was measured using a Canberra scintillation counter. Data were
expressed as the percentage of thymidine incorporation in untreated
control mink lung cells.
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RESULTS |
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LDL increases 1(IV) and
1(I) collagen mRNA levels.
Starting 30 min after incubation with HMC, LDL at concentrations of 200 µg/ml stimulated mRNA expression of
1(IV) and
1(I) collagen;
expression remained high until hour 4 (Fig. 1). At 1 h, LDL had increased the
1(IV) and
1(I) collagen transcripts to 2.2- and 6.2 times the
levels in the control cells. At 2 h, the level of
1(IV) collagen
mRNA in HMC exposed to LDL had increased to 2.8-fold higher than in the
control cells. LDL also led to an increase in type IV collagen
expression as estimated by immunoblotting compared with controls (data
not shown).
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LDL induces TGF- mRNA expression
and the secretion of bioactive TGF-
.
Thirty minutes after the addition of LDL, mRNA levels of TGF-
1 had
increased to 1.3 times their levels in the control cells. After 2 h of
culture with LDL, the mRNA levels had increased to 1.6-fold compared
with control cells. The magnitude of this effect persisted until
hour 4 (Fig.
4).
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PKC inhibition or downregulation attenuates
LDL-induced increase in collagen mRNA levels, but not
TGF-1 expression.
The role played by PKC in LDL-induced increase in collagen gene
expression was assessed by inhibiting enzymatic activity of the kinase
in HMC. When the GFX-pretreated HMC were incubated with LDL for 2 h,
the LDL-induced increases in
1(IV) mRNA expression were
significantly inhibited (Fig.
8A).
Prior exposure of HMC to PMA for 24 h before LDL also blocked the
stimulatory effect of LDL on
1(IV) collagen mRNA expression (Fig.
8A). However, downregulation or
inhibition of PKC by PMA or GFX did not affect the action of LDL on
TGF-
1 mRNA expression (Fig. 8B);
nor did it affect the LDL-induced TGF-
1 secretion in media as
measured by ELISA.
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Anti-TGF- attenuates LDL-induced
increase in collagen mRNA levels.
The effects of pan-specific anti-TGF-
neutralizing antibody on
increases in collagen mRNA expression in HMC cultured with LDL are
shown in Fig. 9. When cells were incubated
with anti-TGF-
antibody (25 µg/ml) along with LDL for 2 h, the
expected increase in
1(I) collagen mRNA expression was abrogated.
However, the same concentration of control rabbit IgG had no effect on
LDL-induced increase in collagen mRNA levels. This suggests an
intermediate role for TGF-
in the effects of LDL.
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DISCUSSION |
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The experiments detailed in this report demonstrate that LDL rapidly
stimulates PKC- and -
and TGF-
expression in HMC. LDL also
increases mRNA levels of
1(I) and
1(IV) collagen at earlier time
points. The LDL-induced upregulation of collagen gene expression was
inhibited by downregulation or inhibition of PKC or by the application
of anti-TGF-
neutralizing antibody, suggesting that PKC and TGF-
are intimately linked to the onset of enhanced collagen mRNA expression.
The activation of PKC was seen within 15 min of exposure of HMC to LDL
and had disappeared at hour 2. LDL
increased membrane-associated PKC- and -
and cytosolic PKC-
but did not stimulate a change in PKC-
cytosol content. It has been
frequently observed that, upon activation by agonists, PKC is
translocated from the cytosolic to the membrane fraction of the cell
(21, 26). The lack of LDL-induced translocation of PKC-
and -
in
the present study suggests that cellular compartmental analysis by
immunoblot alone may not be sufficient to detect translocation of PKC.
The physiological significance of translocation, however, is not clear.
Nishizuka (20, 21) suggested that the translocation of PKC could be a
biochemical artifact caused by the presence of large quantities of
Ca2+ chelators to prevent the
proteolysis during the homogenization. It is tempting to speculate that
LDL activates PKC via a specific LDL receptor on the plasma membrane of
HMC. Lipid peroxidation products increased measurably after LDL was
incubated with HMC for 1 h (17). Thus the return of PKC activity to
control levels at 2 h might partly be related to gradual lipid
peroxidation of LDL by HMC, with reduced binding affinity to the LDL receptor.
The prolonged exposure of HMC to PMA prior to LDL administration
resulted in the downregulation of PKC and the subsequent abolition of
the stimulatory effects of LDL on collagen gene expression. Inhibition
of PKC by the addition of GFX to HMC 30 min prior to LDL administration
also caused a marked reduction in 1(I) and
1(IV) mRNA stimulation
by LDL. Thus the rapid activation of PKC by LDL seems to signal the
upregulation of collagen gene expression in HMC. In cultured human
umbilical vein endothelial cells, increased PKC activity also mediated
the onset of enhanced collagen IV mRNA expression (6).
We also found that exposure of HMC to LDL activated TGF- mRNA
synthesis and induced the secretion of bioactive TGF-
within the
first few hours of stimulation. A number of studies, including our own,
have indicated that changes in TGF-
mRNA levels in the glomeruli or
in mesangial cells are associated with a change in protein expression
(12, 24, 27). The degree of TGF-
protein expression was much greater
than that of mRNA expression as shown by us and others (24), suggesting
that the synthesis rate of TGF-
protein is fast or its degradation
rate is slow or both. In addition, some researchers observed, as we
did, that TGF-
mRNA levels could reflect TGF-
bioactivity (12,
24, 27), whereas others negated that possibility (2, 7).
Two-hour incubation of HMC with neutralizing antibody to TGF- along
with LDL inhibited both the expected increase in
1(I) collagen mRNA
expression and the secretion of biologically active TGF-
. These
observations indicate that anti-TGF-
antibody effectively neutralized the biologically active TGF-
secreted from activated HMC
by LDL, abrogating the expected collagen gene upregulation. It has been
demonstrated that in murine mesangial cells exposed to LDL or high
glucose levels, anti-TGF-
antibody prevents the expected increase in
collagen or fibronectin synthesis (28, 32). In experimental
glomerulonephritis, the administration of anti-TGF-
antibody
neutralized TGF-
activity, thus resulting in decreased proteoglycan
synthesis (5).
In the present study, the stimulatory effects of LDL on TGF- mRNA or
protein expression were not blocked by inhibition or downregulation of
PKC, suggesting that LDL stimulates TGF-
mRNA expression not
mediated through PKC activation. In support of our observation,
Hirakata et al. (11) demonstrated that PKC is not involved in the
induction of TGF-
mRNA expression in RMC. Furthermore, the
antimitotic effect of TGF-
in RMC and VSMC was not affected by
downregulation of PKC (3, 30). Others, however, have suggested that the
activation of PKC signals increases in TGF-
mRNA expression (9) or
TGF-
bioactivity (28). As suggested by Kaname et al. (13), the
induction of TGF-
mRNA expression in RMC may be mediated through
either a PKC-independent or -dependent pathway, and this requires
further confirmation.
Despite an early and transient rise in PKC activity, an LDL-induced
increase in TGF- and collagen mRNA levels was sustained over 4 h in
the present study. Thus TGF-
could be involved not only in the onset
of collagen gene upregulation by LDL but also in its maintenance in
HMC. LDL-induced enhanced collagen mRNA expression leads to increased
collagen synthesis (17). In view of the rapid and/or transient
induction of PKC, TGF-
, and collagen expression, the in vitro model
may not be representative of the in vivo situation of chronic
progressive glomerulosclerosis. Nonetheless, the in vitro data could
indirectly explain the potential mechanisms by which LDL stimulates
mesangial matrix in vivo eventually associated with glomerulosclerosis.
In summary, LDL rapidly stimulates PKC- and -
and TGF-
expression in HMC. The LDL-induced upregulation of collagen gene expression was inhibited by the downregulation or inhibition of PKC or
by the application of anti-TGF-
neutralizing antibody. These results
suggest that LDL stimulates collagen mRNA expression through the
activation of PKC-
and -
and transcriptional upregulation of
TGF-
in HMC. This effect of LDL might have a pathophysiological function in the pathogenesis of progressive glomerulosclerosis.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Ministry of Health and Welfare (Korea) and Seoul National University Hospital.
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. S. Lee, Dept. of Pathology, Seoul National Univ. College of Medicine, Chongno-gu, Yongon-dong 28, Seoul 110-799, Korea (E-mail: hyunsoon{at}plaza.snu.ac.kr).
Received 20 October 1998; accepted in final form 18 May 1999.
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