High Glucose Stimulates Synthesis of Fibronectin via a Novel
Protein Kinase C, Rap1b, and B-Raf Signaling Pathway*
Sun
Lin
,
Atul
Sahai§,
Sumant S.
Chugh§,
Xiaomin
Pan,
Elisabeth I.
Wallner§,
Farhad R.
Danesh§,
Jon W.
Lomasney, and
Yashpal S.
Kanwar§¶
From the Departments of Pathology and
§ Medicine, Northwestern University Medical School,
Chicago, Illinois 60611
Received for publication, April 23, 2002, and in revised form, July 12, 2002
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ABSTRACT |
The molecular mechanism(s) by which high glucose
induces fibronectin expression via G-protein activation in the kidney
are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and
activation, and the relevance of protein kinase C (PKC) and Raf
pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a
dose-dependent increase in Rap1b expression in glomeruli of
diabetic rat kidneys. Similarly, in vitro exposure of MCs
to HG led to an up-regulation of Rap1b with concomitant increase in
fibronectin (FN) mRNA and protein expression. The up-regulation of
Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and
bisindolymaleimide, while also reducing HG- induced FN expression in
non-transfected MCs. Overexpression of Rap1b by transfection with
pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis;
however, the PKC inhibitors had no significant effect in
reducing FN expression in Rap1b-transfected MCs.
Transfection of Rap1b mutants S17N (Ser 
Asn) or T61R (Thr Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf
and Raf-1 expression was investigated to assess whether Rap1b effects
are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was
increased in MCs transfected with Rap1b. HG also caused activation of
Rap1b, which was largely unaffected by anti-platelet-derived growth
factor (PDGF) antibodies. HG-induced activation of Rap1b was specific,
since Rap2b activation and expression of Rap2a and Rap2b were
unaffected by HG. These findings indicate that hyperglycemia and
HG cause an activation and up-regulation of Rap1b in renal glomeruli
and in cultured MCs, which then stimulates FN synthesis. This effect
appears to be PKC-dependent and PDGF-independent, but
involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix
synthesis, a hallmark of diabetic nephropathy.
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INTRODUCTION |
Diabetic nephropathy is a common complication of both type-I and
-II diabetes mellitus, and it is characterized by excessive accumulation of extracellular matrix
(ECM)1 proteins in the renal
glomerulus (1). The major ECM proteins include various types of
collagens, laminin, fibronectin, and proteoglycans, and they are an
integral part of the capillary basement membrane and mesangial matrix,
the latter being situated in the intercapillary region of the
glomerulus (2). Several animal and human studies have revealed an
altered synthesis or expression of various mesangial ECM proteins,
especially of fibronectin, type-I, -III, and -IV collagens, and
proteoglycans in diabetic nephropathy (1, 3). Similarly, in
vitro cell culture studies indicate that high glucose induces an
increased synthesis of various ECM proteins expressed in glomerular
mesangial and epithelial cells (4). The initial events involved in high
glucose induction include activation of specific diacylglycerol
(DAG)-sensitive protein kinase C (PKC) and mitogen-activated protein
kinase (MAPK) signaling pathways (5-7), which are apparently regulated
by transforming growth factor (TGF-
) and platelet-derived growth
factor (PDGF), advanced glycosylation end products (AGEs), and reactive
oxygen species (ROS) (8-11). In addition, the Smad family of proteins, regulated by TGF-, also play a critical role in modulating the effecter events (12). The downstream effects include increased expression of proto-oncogenes, like c-fos and
c-jun, and activation of transcription factors such as AP1,
a heterodimer of c-fos and c-jun, and NF
B
(13), which then conceivably can affect a number of target genes,
e.g. collagen and fibronectin. The mechanism(s) that are
known for the high glucose-induced increased expression of fibronectin
include the involvement of the PKC pathway modulated by ROS, TGF-,
and nitric oxide (NO) (14). In addition, it is known that interaction
of AGEs with its receptors (RAGE) leads to an activation of the PKC
followed by that of transcription factors, which then affect the target
genes and cause increased expression of fibronectin (9, 15).
Fibronectin, a large glycoprotein consisting of two similar polypeptide
chains, is a key component of the mesangial matrix (16). It may exist
in a soluble dimeric form or as oligomers of fibronectin or a highly
insoluble fibrillar form in the extracellular matrix. The latter form
has been shown to modulate various biological processes such as cell
adhesion, migration, and differentiation (17). Fibronectin matrix
assembly is a highly ordered stepwise process, and many domains along
its monomer have been shown to be important for its formation and
ultimate proper incorporation into the mesangial ECM (16, 17).
Conceivably, excessive production and a disordered incorporation or
assembly of fibronectin or of other matrix proteins, secondary to the
phenotypic change in the mesangial cell or due to glycoxidative- or
ROS-induced damage to the proteins, is expected to result in an
abnormal accumulation of ECM in diabetic nephropathy with the evolution
of Kimmelstiel-Wilson lesions that progress to glomerulosclerosis and
end stage renal disease with kidney failure (1-3). The information
regarding the mechanism(s) of excessive synthesis of ECM proteins is
available in the literature; further insights into the mechanism(s)
involved in the evolution of diabetic glomerulosclerosis still remains to be explored, which may be helpful in developing future therapies in
the amelioration of this disease process.
Previously, by suppressive subtractive hybridization procedures a small
GTP-binding protein, Rap1b, was found to be up-regulated in the kidneys
of diabetic newborn mouse (18). Similarly, up-regulation of Rap1b was
seen in diabetic rats as well (18). The up-regulation was observed in
several other organs, but Rap1b expression in the kidney was increased
in parallel to the alterations in the blood levels of glucose,
suggesting that glucose by itself, rather than any cytokine,
e.g. insulin-like growth factor, may be responsible for the
increased Rap1b expression. Indeed, an increased Rap1b expression was
seen in the embryonic metanephric explants exposed to high glucose
ambience (18). Based on these observations the present study was
undertaken to determine the effect of hyperglycemia on the expression
of Rap1b in the glomerular compartment of kidneys of diabetic rats as
well as to assess the effects of high glucose on the expression and
activation of Rap1b in cultured rat glomerular mesangial cells (MCs)
and the underlying mechanisms involved in the stimulation of
fibronectin synthesis.
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EXPERIMENTAL PROCEDURES |
Animals and Induction of Diabetes--
A diabetic state was
induced in 4-week-old Spargue-Dawley rats (Harlan Company) by a single
intraperitoneal injection of streptozotocin (100 mg/kg of body weight;
Sigma) in a citrate buffer, pH 4.6 (18). A week later the rats with
blood glucose level >200 mg/dl were regarded as having a diabetic
state, and their kidneys were processed for isolation of glomeruli,
Rap1b protein, and mRNA expression and immunohistochemical studies.
Control rats were injected with citrate buffer only.
Immunohistochemical Staining--
The kidneys from both diabetic
and control rats were snap-frozen in liquid nitrogen chilled isopentane
and embedded in OCT compound (Sakura, Torrance, CA).
Four-micrometer-thick cryostat sections were prepared. The sections
were overlaid with primary monoclonal antibody directed against Rap1b
at a recommended dilution of 1:50 (Santa Cruz Biotechnology, Inc.) The
sections were then stained with avidin-biotin peroxidase by following
the instructions provided by the vendor (Ultra Streptavidin Detection
System, Signet Laboratories Inc.).
Preparation of Isolated Glomeruli--
The glomeruli were
isolated from kidneys of diabetic rats with different blood glucose
level 5 days following the injection of streptozotocin by the
differential sieving method with minor modifications (19). Briefly,
cortices were dissected, and then they were then gently minced with a
razor blade and pressed through 106-µm pore size steel mesh screen
with a spatula. The paste was collected from the bottom of the screen
and was mixed with 5 volumes of sterile phosphate-buffered saline and
then filtered through a 180-µm pore size mesh, and finally the
glomeruli were collected on the surface of a third mesh with a 75-µm
pore size. The purity of the glomerular preparation was examined by
light microscopy.
Cell Culture Studies--
Glomerular MCs were isolated from
kidneys of Sprague-Dawley rats (Harlan Laboratories), and they were
used to establish primary cell cultures as described previously (20).
The cells were grown in Dulbecco's modified Eagle's medium/Ham's
F-12 medium (Sigma) supplemented with 10% heat-inactivated fetal calf
serum, 100 units/ml penicillin and streptomycin, and 0.3 unit/ml
insulin. They were maintained in 75-cm2 flasks in a
humidified atmosphere of 5% CO2 and 95% air at 37 °C,
and at ~80% confluence they were trypsinized, propagated, and
utilized between passages 5 and 7. At a given passage, the cell
cultures were replaced with fresh serum-free Opti-MEM medium (Invitrogen). After 48 h, the medium was supplemented with
varying concentrations of D-glucose (10-30
mM), and the quiescent mesangial cell cultures were
maintained for 24 h. The L-glucose- (30 mM) and 5 mM D-glucose-treated
cells served as controls.
Northern Blot Analysis--
Total RNAs were prepared from both
the isolated glomeruli and MCs by the acid guanidinium
isothiocyanate-phenol-chloroform extraction method (21). About 10 µg
of RNA from various experiments was subjected to 1.5% agarose gel
electrophoresis containing 2.2 M formaldehyde and
capillary-transferred to the Hybond N+ nylon membrane
(Amersham Biosciences). After cross-linking of RNA to the membrane,
prehybridization and hybridization of different blots was carried out
with various [32P]dCTP-labeled (1 × 106
cpm/ml) cDNA probes (18, 22). The cDNA probes for Rap1a, Rap2a,
and Rap2b were generated from plasmid constructs provided by Martina
Schmidt of Institut fur Pharmakologie, Universittsklinikum Essen,
Germany (23). The Rap1b cDNA probe was generated as described previously in our laboratory (18). The fibronectin (type-III) 590-bp
cDNA probe was generated by PCR using a sense (5'-CCC CGC CCT GGT
GTC ACG GAG GCC-3') and an antisense (5'-GGC ACT GAC GAA GAG CCC TTA
CAG-3') primers and single-stranded cDNA prepared from rat kidney
mRNA. Following the preparation of autoradiograms, the membrane
blots were stripped and re-hybridized with radiolabeled -actin
cDNA probe. The integrity of RNA was monitored by visualization of
the intact 18 and 28 S bands on the membrane blots stained with 0.05%
methylene blue.
Immunoprecipitation Studies--
For glomerular
immunoprecipitation, 12 diabetic rats with different blood glucose
levels and 4 control normoglycemic rats received an intraperitoneal
injection of [35S]methionine (1 mCi/kg of body weight).
After 24 h, the radiolabeled glomeruli were isolated and briefly
sonicated to release the cells. The glomerular cells were collected by
centrifugation at 1,000 × g and processed for
immunoprecipitation as described below. Similarly, control and
glucose-treated MCs in culture were labeled with
[35S]methionine (10 µCi/ml) in a methionine-free
Dulbecco's modified Eagle's medium at 37 °C. After 12 h, the
cells were washed with the culture medium and lysed in an
immunoprecipitation buffer (IP buffer: 20 mM Tris-HCl, pH
7.4, containing 100 mM NaCl, 1 mM
CaCl2, 1 mM MgCl2, 10 mM benzamidine HCl, 10 mM 
-amino-
-caproic acid, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 1%
Triton X-100) by vigorous shaking at 4 °C for 2 h. The lysates
were centrifuged at 12,000 × g for 30 min at 4 °C,
and the protein concentration in the supernatant was determined by
Bradford assay (Bio-Rad) and adjusted to 1 mg/ml. The supernatants (500 µl) from control and high glucose-treated cell cultures were mixed
with monoclonal anti-Rap1b antibody (Santa Cruz Biotechnology, Inc.)
with gentle agitation at 4 °C for 2 h, followed by addition of
50 µl of 50% protein A-Sepharose 4BTM (Amersham
Biosciences) and incubation extended for another 1 h at 4 °C.
The protein A-SepharoseTM beads were then washed with IP
buffer. 25 µl of 2× SDS-sample buffer was added to the washed beads,
boiled for 5 min, and subjected to 15% SDS-PAGE. The gels were then
dried and autoradiograms prepared (18, 22).
Western Blot Analysis--
Protein expression of fibronectin,
Rap1b, B-raf, and Raf-1 were assessed by Western blot analysis (18).
For fibronectin, the culture medium was collected, and the protein
concentration was adjusted to 1 mg/ml. Equal amounts (~25 µg)
protein (control versus high glucose) were loaded into the
gel wells and subjected to 5% SDS-PAGE under reducing conditions. The
gel proteins were then electroblotted onto a nitrocellulose membrane.
The membrane was then immersed in a blocking solution containing 5%
nonfat milk in TBS-T (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl,
and 0.1% Tween 20) for 60 min, followed by successive incubations with
the fibronectin antibody (Invitrogen) and anti-rabbit IgG conjugated
with horseradish peroxidase (Amersham Biosciences) for 1 h each at
22 °C with an intermediate wash with TBS-T. Following the final wash
the autoradiograms were developed using an enhanced chemiluminescence
(ECL) Western blot kit (Amersham Biosciences).
For Rap1b protein expression, MCs were lysed in a lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 2 mM NaF, 1 mM PMSF, 1 µg/ml leupeptin, and 2 µg/ml aprotinin). The
lysate was centrifuged at 12,000 × g for 30 min at
4 °C, and the protein concentration in the supernatant was adjusted
to 1 mg/ml. The lysates from various cell cultures were subjected to 15% SDS-PAGE and transferred to nitrocellulose membranes. After blocking, the membranes were successively incubated with anti-Rap1b and
anti-mouse IgG conjugated with horseradish peroxidase (Amersham Biosciences) and autoradiograms developed using the ECL system.
For the determination of B-Raf and Raf-1 protein expression, the MCs in
culture flasks were immersed in 1 ml of TME lysis buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 25 mM NaF, 100 µM
Na3VO4, and 1 mM dithiothreitol).
After freezing and thawing on ice for 30 min, the cells were scrapped
from the flasks lysed by using a Dounce homogenizer for 5 min at
4 °C. The homogenates were centrifuged at 1,000 × g
for 10 min. The supernatant was saved and centrifuged at 100,000 × g for 90 min at 4 °C. The pellet (membrane proteins)
was solubilized with 1% Triton X-100 and protein concentration
adjusted to 1 mg/ml. Equal amounts of protein (20 µg) from various
experiments were loaded onto the gel wells and subjected to 10%
SDS-PAGE, followed by Western blot analyses using anti-B-Raf and
anti-Raf-1 antibodies (Santa Cruz Biotechnology).
Generation of Rap1b Eukaryotic Expression Construct and Stable
Transfectants--
A full-length Rap1b cDNA in PCR II vector (18)
and sense
(5'-GGGGGGGGATCCACCATGGTCATGCGTGAGTACAAGCT-3')
and antisense (5'-GGGGGGCTCGAGTCACTTGTCATCGTCGTCCTT
GTAGTCAAGCAGCTGACA-3') primers were used to generate the
expression construct by PCR. The GC clamps (GGGGGG), BamHI
site (GGATCC) and Kozak's sequence (ACCATGG)
were introduced into the sense primer, while the antisense primer
included XhoI site (CTCGAG) and FLAG epitope
(N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C). The restriction sites are in
bold, the Kozak's sequence is italicized, and the FLAG epitope is
underscored. The generated PCR product was digested with
BamHI and XhoI, cloned into pCR II vector
(Invitrogen), gel-purified, and then subcloned into BamHI-
and XhoI-digested pcDNA3.1 and designated as
pcDNA3.1/Rap1b. They were then transfected into MCs using
LIPOFECTAMINETM2000 reagent, and stable transfectants were
selected by growing cells in 800 µg/ml G418. The surviving clone of
cells were then propagated in the presence of relatively low
concentration of G418 (100 µg/ml) and used for further studies. To
assess the Rap1b expression in the cells, RT-PCR, immunoprecipitation,
and Western blot analysis were performed (18, 22).
PKC Inhibitor Studies--
The MCs cells were transfected with
pcDNA3.1/Rap1b as described above. Both transfected and
non-transfected MCs were maintained in a fresh serum-free Opti-MEM
containing 5 or 30 mM D-glucose in the medium.
Various PKC inhibitors, such as calphostin C and bisindolymaleimide,
were individually added into the culture medium at a concentration
range of 10-100 nM and 5-20 µM,
respectively. The MCs were treated for 12 h. After confirming the
viability of MCs by trypan blue exclusion assay, both media and the
cells were collected as described above. The MCs were processed for Rap1b and fibronectin mRNA expression by Northern blot analysis, while the medium for the fibronectin protein expression was
processed by Western blot analysis. In addition, Rap1b protein
expression in MCs labeled with [35S]methionine was
assessed by immunoprecipitation followed by SDS-PAGE autoradiography.
Rap1b Mutangenesis Studies--
A full-length Rap1b cDNA was
cloned into pcDNA3.1 vector lacking FLAG epitope and was
generated as described above. Using this pcDNA3.1/Rap1b plasmid two
mutants were generated by employing a QuikChange Site-Directed
Mutagenesis kit (Stratagene) following vendor's instructions.
In the first Rap1b mutant, serine 17 was substituted to asparagine and
designated as Rap1b/S17N. The second mutation included substitution of
threonine 61 to arginine, and the mutant was designated as Rap1b/T61R.
For generation of Rap1b/N17 and Rap1b/R61, the following respective
primers were used: 5'-GGAGGTGTTGGGAAGAATGCTCTGACTGTACAG-3' and
5'-CCTGGATACTGCAGGAAGGGAGCAGTTTACAGCCATG-3'. After confirming the
nucleotide sequence, the mutants were transfected into MCs and
maintained overnight in an environment of 5% CO2 in a
Lab-Tek chamber slides system (Nunc). The D-glucose
concentration in the medium was adjusted to either 5 mM
(low glucose) or 30 mM (high glucose), and cultures were
maintained for an additional 24 h. Cells were then rinsed with PBS
and fixed with 3.7% paraformaldehyde in PBS, followed by incubation in
0.1 M glycine in PBS for 15 min. They were permeabilized
with 0.2% Triton X-100 (w/v) for 10 min. After another PBS wash, the
cells were treated with blocking solution containing 1% BSA, 0.1%
Tween 20, and 5 mM MgCl2 for 20 min. They were
then successively incubated with anti-fibronectin and fluorescein
isothiocyanate-conjugated anti-rabbit IgG and examined with UV light
microscope. The medium from MC cultures was also collected to determine
the fibronectin protein expression. In addition, MCs from the above
experiments were harvested and processed for B-Raf and Raf-1 protein
expression as described above.
Rap1b and Rap2b Activation Assays--
A pGEX-RalGDS (RBD)
plasmid encoding glutathione S-transferase (GST) fusion
protein containing the RBD of RalGDS proteins was used for the
activation assays, and this plasmid was a generous gift from Dr.
Johannes L. Bos of the Utrecht University, the Netherlands (24-27).
After transformation, the plasmid was propagated using Escherichia coli (DH5
) host, and recombinant protein
production was induced with
isopropyl-1-thio--D-galactopyranoside as
previously described (18, 22). Following induction for 3 h, the
bacteria were collected and lysed by sonication in a bacterial lysis
buffer (50 mM Tris, 200 mM NaCl, 2 mM MgCl2, 1 mM PMSF, 10% glycerol, 1% Nonidet P-40, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor). After sedimenting the disrupted
cells by centrifugation, the supernatant was passed through an affinity glutathione-SepharoseTM column and eluted with the buffer
containing 50 mM Tris-HCl, pH 8.0, 10 mM
glutathione, 0.25 mM dithiothreitol, and protease inhibitors. The eluted GST-Ral fusion protein was subjected to 12.5%
SDS-PAGE to confirm the purity of the protein, following which it was
dialyzed against 50 mM Tris-HCl, 100 mM NaCl,
and 5% glycerol and stored at 
70 °C in 10% glycerol. The
recombinant fusion protein was immobilized on glutathione-Sepharose
4BTM beads (Pharmacia Biosciences) to be used for the
activation assays (24-27). To assess Rap1b or Rap2b activation, the
D-glucose with varying concentrations (5-40
mM), was added to the medium and MC cultures maintained for
5-60 min at 37 °C, followed which the cells were harvested and
disrupted with the lysis RIPA buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1%
Nonidet P-40, 1 mM Na3V04, 1 mM PMSF, 10 µg/ml leupeptin, and 1 µg/ml aprotinin) and
the lysate centrifuged for 10 min at 10,000 × g. The
resulting supernatants were incubated with freshly prepared glutathione-Sepharose 4BTM beads coupled to recombinant
protein for 1 h to allow the association of activated Rap1b or
Rap2b with the effector-GST fusion protein. The beads were washed three
times with the RIPA buffer, suspended in sample buffer, and subjected
to 12.5% SDS-PAGE. The gel proteins were electroblotted onto nylon
membranes and processed for Western blot analysis using anti-Rap1b and
-Rap2b antibodies and the ECL system. The Rap1b activation was also
assessed following inclusion of anti-PDGF neutralizing antibody (1-10
µg/ml) in the MC culture medium. Similarly, Rap1b activation was
assessed following transfection of MCs with wild type versus
mutant Rap1b as described above.
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RESULTS |
Rap1b Expression in Renal Tissues and
Glomeruli--
Immunohistochemical studies revealed a mild Rap1b
expression in cortical and juxtamedullary glomeruli as well as in the
inner medullary collecting tubules of the kidneys of normoglycemic rats (Fig. 1B,
arrowheads). The Rap1b expression notably increased in
kidneys of hyperglycemic rats, particularly in the glomerular compartment (Fig. 1A, arrowheads). To confirm the
Rap1b expression in glomerular compartment of the kidney, glomeruli
from hyperglycemic rats with different blood glucose levels, ranging
between 200 and 400 mg/dl, were isolated by the differential sieving
method and processed for Northern blot analysis and immunoprecipitation studies. By the sieving method the preparation was found to be made up
of almost 100% glomeruli (Fig. 1C). Northern blot analysis of glomeruli of normoglycemic rat with blood glucose level of 120 mg/dl
revealed a faint ~2.3-kb Rap1b mRNA transcript (Fig. 1D). In hyperglycemic rats, an increase in the Rap1b
mRNA expression, proportional to the blood glucose levels (200-400
mg/dl), was observed (Fig. 1D). The -actin expression was
unaffected by the blood glucose levels (Fig. 1F). Similarly,
immunoprecipitation studies revealed a dose-dependent
increase in the intensity of the ~21-kDa band, corresponding to the
size of Rap1b protein, in SDS-PAGE autoradiograms (Fig. 1G),
suggesting that both protein and mRNA expression increase in
proportion to the rise in blood glucose levels in diabetic rats.
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Fig. 1.
Rap1b expression in the intact kidney and
isolated glomeruli of diabetic rats. Immunohistochemical staining
reveals a mild Rap1b expression in the glomeruli of normal rat kidney
(B, arrowheads). The Rap1b expression is notably
increased in the glomeruli of diabetic rat kidney (A,
arrowheads). C includes a preparation of isolated
glomeruli. The preparation is made up pure population of isolated
glomeruli. Northern blot analysis shows a faint band, corresponding to
an ~2.3-kb size of Rap1b mRNA transcript (D,
arrow) in the glomeruli of normoglycemic rat with blood
glucose level of 120 mg/dl (D, first lane). The
Rap1b mRNA expression notably increased in glomeruli of
hyperglycemic rats, and the increase seems to be proportional to the
blood glucose levels, ranging from 200 to 400 mg/dl (D,
second through fourth lanes). The
-actin expression is unchanged in glomeruli of hyperglycemic rats
(F). E indicates the intactness of the RNAs and
their loading of equal amounts of total RNA in various lanes. In
parallel to the increase in mRNA, the Rap1b protein expression also
increases in proportion to blood glucose levels in diabetic rats, as
assessed by immunoprecipitation followed by SDS-PAGE autoradiography
(G).
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Effect of HG on Rap1b and Fibronectin Expression in Mouse
MCs--
Under basal conditions with 5 mM glucose
concentration in the medium, a single faint ~2.3-kb Rap1b mRNA
transcript was detected by Northern blot analysis (Fig.
2A). Exposure of MCs to HG
(10-30 mM) induced a dose-dependent increase
in the expression of Rap1b, with a maximal response of ~20-fold
increase at 30 mM. The -actin expression was unaffected
(Fig. 2B). An increase in the protein expression of de
novo synthesized Rap1b was also seen with 30 mM
glucose in the culture medium by immunoprecipitation methods (Fig.
2D). To assess the specificity of HG response, MCs were exposed to 30 mM L-glucose. No increase in the
expression was noted, and the intensity of the ~21-kDa
Rap1b band was similar to that of the cells treated with 5 mM D-glucose (Fig. 2D).
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Fig. 2.
Northern blot analyses depicting the effect
of different concentrations of D-glucose (5-30
mM) on Rap1b and fibronectin expression in cultured
glomerular MCs. The mRNA expression of Rap1b and fibronectin
(A and E, arrows) in MCs increases
proportionally to D-glucose concentration in the culture
medium. The -actin expression remains unchanged by HG treatment
(B and F). C and G indicate
the intactness of the RNAs and their loading of equal amounts of total
RNA in various lanes. D represents the experiments in which
glucose-treated MCs were labeled with [35S]methionine
followed by immunoprecipitation of cell lysates with anti-Rap1b
antibody. The expression of de novo synthesized Rap1b
protein increases in high D-glucose ambience
(D). In parallel, the fibronectin protein expression also
increases in high D-glucose ambience, as assessed by
Western blot analysis (H). No increase in the Rap1b or
fibronectin protein expression is seen in MCs exposed to high
L-glucose ambience (D and H).
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In a protocol similar to the assessment of Rap1b, the effect of HG on
fibronectin mRNA was also determined. At 5 mM, a faint ~8-kb fibronectin mRNA transcript was seen (Fig. 2E).
Exposure of MCs to HG for 24 h caused a significant
dose-dependent increase in the fibronectin mRNA
expression, which paralleled the increase in Rap1b mRNA expression
observed under similar experimental conditions (Fig. 2, A
and E). MCs exposed to HG also resulted in a significant increase in fibronectin protein expression as determined by Western blot analysis (Fig. 2H). Incubation of MCs with 30 mM L-glucose had no stimulating effect on
fibronectin protein expression, and the intensity of the ~220-kDa
fibronectin band was similar to that seen in the control cells treated
with 5 mM D-glucose (Fig. 2H).
Effect of Calphostin C and Bisindolylmaleide on Glucose-induced
Rap1b Expression in MCs--
MCs were exposed to 5-30 mM
D-glucose in the absence or presence of various
concentrations of PKC inhibitors, calphostin C and bisindolylmaleide
(Fig. 3). The treated cells were then
processed for the isolation of total RNA and radiolabeled cellular
proteins for the Northern blot analysis and immunoprecipitation
procedures, respectively. An increase in the Rap1b mRNA and protein
expression was observed in HG ambience (Fig. 3, A and
D, lane 2 versus lane 1). Both the PKC
inhibitors, calphostin C and bisindolylmaleide, caused a
dose-dependent diminution in the Rap1b expression in cells
treated with HG (Fig. 3, A and D, lanes
2-5). The Rap1b expression was almost undetectable at high
concentrations of PKC inhibitors, i.e. 100 nM
calphostin C and 20 µM bisindolylmaleide (Fig. 3,
A and D, lane 5). The expression of
-actin remained unchanged in MCs treated either with calphostin C or
bisindolylmaleide in HG ambience (Fig. 3, C and
G, lanes 1-5).
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Fig. 3.
Effect of PKC inhibtors on Rap1b expression
in MCs exposed to HG ambience (30 mM). Rap1b mRNA
is significantly increased in the MCs exposed to HG ambience compared
with cells treated with 5 mM glucose (A and
E, lane 2 versus lane 1).
Both the PKC inhibitors, calphostin C and bisindolylmaleide, reduce the
Rap1b mRNA expression in a dose-dependent manner in MCs
exposed to HG ambience (A and E, lanes
3-5). The -actin expression in MCs treated with in HG or PKC
inhibitors (C and G) is unaffected. B
and F indicate the intactness of the RNA and their loading
of equal amounts of total RNA in various lanes. D and
H represent the expression of
[35S]methionine-labeled and immunprecipitated Rap1b
protein in MCs exposed to HG ambience and treated with PKC inhibitors.
In parallel to mRNA expression, the protein expression increases in
MCs exposed to HG ambience and decreases in a
dose-dependent manner with the treatment of PKC inhibitors
(D and H).
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Role of PKC in HG-induced Fibronectin Synthesis in MCs
Overexpressing Rap1b--
First a mammalian Rap1b cDNA expression
construct was synthesized by cloning the wild type Rap1b cDNA into
pcDNA 3.1 and designated as pcDNA3.1/Rap1b. The MCs were then
transfected with pcDNA3.1/Rap1b, and stable transfectants were
selected by using antibiotic G418. These stable transfectants
(Rap1b(VT+)) exhibited a 30-fold increase in Rap1b mRNA
expression compared with control cells transfected with empty vector
(Vector(VT)), as assessed by RT-PCR analysis (Fig.
4A). The authenticity of
stable transfectants was confirmed by immunoprecipitation and Western
blot analyses, and both revealed a high expression of de
novo synthesized (Fig. 4B) and native Rap1b proteins
(Fig. 4C).
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Fig. 4.
Characterization of Rap1b transfectant cell
lines. A pcDNA 3.1/Rap1b recombinant plasmid was transfected
into MCs, and a stable MC line was generated by using G418 selection.
The authenticity of stable transfectants overexpressing Rap1b was
confirmed by RT-PCR, immunoprecipitation, and Western blot methods. By
RT-PCR, a 520-bp size product is observed in non-transfected cells
(A, Vector (VT), lane 2,
arrow), and expression of Rap1b mRNA is significantly
increased in MCs transfected with Rap1b (A,
Vector(VT+), lane 3,
arrow). Similarly, by immunoprecipitation and
Western blot methods the respective expression of de novo
synthesized and native Rap1b is increased in overexpressing MCs
transfectants (B and C,
Vector(VT+), lane 2) compared with
non-transfected cells (B and C,
Vector(VT), lane 1).
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Since activation of PKC by HG has been shown to mediate the stimulation
of fibronectin synthesis both in vitro and in
vivo models of diabetes (28-30), the role of PKC in HG-induced
fibronectin expression in control MCs as well as MCs transfected with
Rap1b was investigated. Both control MCs and Rap1b-transfected cells were exposed to 5-30 mM glucose for 24 h in the
absence or presence of a PKC inhibitor calphostin C (10-100
nM) or bisindolylmaleide (5-20 µM), and
fibronectin mRNA expression in the cells and protein expression in
the culture medium were assessed by Northern and Western blot analyses,
respectively. Similar to the previous observations (Fig. 2,
E and H), HG induced a significant increase in
both fibronectin mRNA and protein expression in control
non-transfected MCs (Fig. 5, lane 2 versus lane 1). In control MCs, both the PKC inhibitors, calphostin C and bisindolylmalede, inhibited the HG-induced stimulation of fibronectin mRNA and protein expression in a
dose-dependent manner (Fig. 5, lanes 6-8). By
contrast, both the PKC inhibitors had no significant effect in reducing
HG-induced increase fibronectin mRNA and protein levels in cells
overexpressing Rap1b (Fig. 5, lanes 3-5). The -actin
expression in MCs was unaffected by treatments with HG and PKC
inhibitors (Fig. 5, C and G). These results
suggest that PKC-dependent mechanism(s), potentially
involving Rap1b, mediate HG-induced stimulation in fibronectin
synthesis in cultured MCs, and also Rap1b may be downstream of PKC in
the induction of fibronectin expression.
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Fig. 5.
Role of PKC in high glucose-induced FN
expression in non-tranfected MCs and cells overexpressing Rap1b.
The PKC inhibitors (calphostin C and bisindolylmaleide) in
non-transfected MCs reduce the FN mRNA and protein expression in
high glucose ambience in a dose-dependent manner
(A, D, E, and H,
lanes 6-8) compared with the control (A,
D, E, and H, lanes 1 and
2). While in MCs overexpressing Rap1b, the PKC inhibitors
did not significantly reduce the FN mRNA or protein expression
(A, D, E, and H,
lanes 3-5). The expression of -actin is unaffected by
high glucose treatment in both non-transfected or transfected MCs
(C and G). B and F indicate
the intactness of the RNAs and their loading of equal amounts of total
RNA in various lanes.
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Effect of Rap1b Mutation in HG-induced Fibronectin
Synthesis--
MCs were transfected either with pcDNA3.1 empty
vector alone or pcDNA3.1/Rap1b (WT) or mutant (Rap1b/S17N, Ser Asn) or (Rap1b/T61R, Thr Arg) in the presence of low and high
glucose in the culture medium. Then cellular expression of fibronectin
and that secreted in the medium were determined by
immunofluorescence and Western blotting procedures, respectively.
Minimal cellular expression of fibronectin was observed at low glucose
(5 mM) concentration in the medium (Fig.
6A). At high glucose (30 mM) concentration, its expression markedly increased, and
it was seen diffusely distributed in the cytoplasm (Fig. 6, B
versus A). A remarkable reduction in the HG-induced cellular
fibronectin was observed in cells transfected with mutant Rap1b/S17N or
Rap1b/T61R (Fig. 6, C and D versus B). No
distinguishable differences between the effects of these mutants on the
reduction of fibronectin expression were observed. Transfection with
pcDNA3.1/Rap1b (WT) resulted in a marked increase in expression of
cellular fibronectin compared with the MCs exposed to HG alone (Fig. 6,
F versus B). However, transfection with pcDNA3.1 empty vector induced minimal increase in the fibronectin expression compared
with the cells incubated with HG alone (Fig. 6, E versus B).
Similar results were observed for the fibronectin secreted in the
medium (Fig. 6G). An increased fibronectin expression was observed with the HG (Fig. 6G, lane 1 versus lane
2). A reduction in the expression was seen with transfection of
the mutants (Fig. 6G, lanes 3 and 4 versus lane 2). A mild increase in the expression was
observed with transfection of vector alone (E-VE) (Fig.
6G, lane 5), while a remarkable increase in the
fibronectin protein expression in culture media of the MCs was seen
with the transfection of wild type pcDNA3.1/Rap1b (WT) (Fig.
6G, lane 6 versus lane 2).
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Fig. 6.
Effect of Rap1b mutants on HG-induced FN
expression in mesangial cells. HG induces increased expression of
FN in non-transfected cells, as assessed by immunofluorescence
microscopy (B versus A) and Western
blot analysis (G, lane 2 versus
lane 1, arrow). Transfection with the empty
pcDNA3.1 vector results in a mild increase in the cellular
(E) as well as FN secreted in the culture medium
(G, E-VE, lane 5). While a marked
increase in the FN expression is seen with the transfection of
pcDNA3.1/Rap1b (F and G, WT,
lane 6). Whereas, transfection with Rap1b/S17N or Rap1b/T61R
mutants markedly diminishes the FN expression (C and
D, and G, S17N and T61R,
lanes 3 and 4).
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Effect of Rap1b Transfection on B-Raf and Raf-1 Expression--
To
delineate which one of the Raf pathways is involved in Rap1b-mediated
responses, expression of B-Raf and Raf-1 in MCs exposed to HG was
determined. The cells were transfected either with empty pcDNA3.1
vector (E-VE), or wild type pcDNA3.1/Rap1b (WT), or with Rap1b
mutants Rap1b/S17N (S17N) and Rap1b/T61R (T61R) and exposed to HG,
followed by the assessment of B-Raf and Raf-1 expression by Western
blot analysis (Fig. 7). In B-Raf
experiments, two bands of ~94 and ~68 kDa were observed in cells
transfected with empty vector (E-VE) (Fig. 7A, lane
1). The ~68-kDa band was faint, but was detectable. This band
disappeared in cells transfected with S17N or T61R Rap1b mutants, and
the upper ~94 kDa band was unaffected (Fig. 7A,
lanes 2 and 3). The transfection of MCs with WT
resulted in an increase in the intensity of both the bands (Fig.
7A, lane 4). However, there was a marked increase
in the intensity of the ~68-kDa band, suggesting that the B-Raf
pathway is affected by Rap1b-mediated responses in the presence of HG
(Fig. 7A, lane 4). In contrast to the results of
B-Raf, the expression of Raf-1 was unaffected. In cells
transfected with empty vector (E-VE), a ~74-kDa band was
observed (Fig. 7B, lane 1), and its intensity was
unaffected by transfection of the mutants S17N or T61R (Fig. 7B, lanes 2 and 3). Similarly, no
increase in the intensity of this band was observed with the
transfection of WT Rap1b. Instead, a mild decrease in its intensity was
observed (Fig. 7B, lane 4), suggesting that, most
likely, the Raf-1 pathway is not involved and rather the Rap1b may have
a negative effect on the expression of Raf-1 in the HG milieu.
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Fig. 7.
Effect of Rap1b transfection on B-Raf
(A) and Raf-1 (B) expression in high
glucose milieu. After transfection of MCs with WT Rap1b, an
increased intensity of both ~94-kDa and ~68-kDa bands of B-Raf is
observed compared with the control cells transfected with empty vector
(E-VE) (A, lane 4 versus
lane 1, arrows). The band intensity of ~68 kDa,
however, is markedly increased. This ~68-kDa band disappears in cells
transfected with S17N or T61R Rap1b mutants, while the intensity of the
upper ~94-kDa band is unaffected (A, lanes 2 and 3). In contrast to the results of B-Raf, the expression
of the Raf-1 ~74-kDa band is not increased with the transfection of
WT Rap1b compared with cells transfected with the empty vector
(E-VE), instead, a small decrease in its intensity is
observed (B, lane 4 versus lane
1, arrow). The intensity of the Raf-1 band is
unaffected by transfection of the mutant S17N or T61R (B,
lanes 2 and 3).
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Effect of High Glucose on Rap1b Activation--
Rap1b activation
was performed by following the method of Bos and his colleagues
(23-27). The assay is based on the differential affinity of the RBD of
RalGDS for Rap1GTP versus Rap1GDP, and thus only the
activated form of Rap1bGTP will bind to RBD containing fusion protein,
which then can be analyzed by SDS-PAGE and Western blotting procedures.
The MCs exposed to 5-40 mM glucose resulted in a marked
increase in Rap1b-GTP in a dose-dependent manner, with a
~7-fold stimulation at 30 mM glucose (Fig.
8A, panel a). Next,
the time course of activation of Rap1b by HG was determined, and the
MCs were exposed to 30 mM glucose for 0-60 min. The time course analyses revealed a significant activation of Rap1b within 5 min
of incubation with HG, which increased over a period of 60 min (Fig.
8A, panel b). Maximal ~25-fold stimulation in
Rap1b was observed at 30 min of incubation.
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Fig. 8.
Effect of HG on activation of Rap1b and
Rap2b. A dose-dependent increase in Rap1b-GTP is seen
in MCs exposed to 5-40 mM D-glucose with
maximal stimulation at 30 mM D-glucose
(A, panel a). The time course studies indicate
the activation of Rap1b within 5 min of exposure to HG and increases
over a period of 60 min (A, panel b). The
activation of Rap1b was largely unaffected by anti-PDGF antibody even
at the highest concentration of 10 µg/ml in the presence of 30 mM D-glucose (A, panel
C). The Rap1b activation is markedly enhanced in MCs transfected
with WT Rap1b compared with the non-transfected (NT) cells
(A, panel d, lane 4 versus
lane 1), whereas a significant reduction in the Rap1b
activation is observed in MCs transfected with mutants (S17N and T61R)
(A, panel d, lanes 2 and
3). A very mild increase in the Rap1b activation is also
observed in cells transfected with empty vector (E-VE)
(A, panel d, lane 5). No significant
activation of Rap2b is observed with HG treatment (B).
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Since PDGF is produced by cells in response to HG (31) and has been
shown to cause an activation of Rap1b in other cell types (32), the
role of PDGF in HG-induced stimulation of Rap1b was investigated. The
MCs were incubated with HG (30 mM) for 30 min in the
absence or presence of PDGF neutralizing antibody (1-10 µg/ml), and
Rap1b activation was assessed. The addition of PDGF antibody in the
incubating medium had no effect in reducing Rap1b activation induced by
HG irrespective of the antibody concentration used (Fig. 8A,
panel c). No cytotoxicity of anti-PDGF antibody was noted as
assessed by trypan blue exclusion test. These results suggest that the
HG-induced activation of Rap1b is, most likely, independent of the
effects of PDGF.
Specificity of Rap1b Activation by HG--
To assess the
specificity of Rap1b activation by HG, the cells were transfected with
wild type Rap1b (WT) or mutants (S17N and T61R) or by the empty vector
(E-VE). Compared with non-transfected (NT) MCs, the cells
transfected with wild type Rap1b (WT) had a significant increase in the
Rap1b activation after 30 min treatment with HG (Fig. 8A,
panel d, lane 4 versus lane 1). By contrast, the
cells transfected with Rap1b mutants (S17N and T61R) had no significant
effect on the activation of Rap1b under HG conditions, rather activity
was markedly diminished (Fig. 8A, panel d,
lanes 2 and 3). The cells transfected with empty
vector (E-VE) showed a small increase in the activity of Rap1b (Fig.
8A, panel d, lane 5), which was
reminiscent of the increase in its protein expression (Fig.
6G, lane 5).
To further define the specificity of HG response to Rap1b, the
activation of Rap2b and the mRNA expression of Rap1a, Rap2a, and
Rap2b were assessed in MCs incubated with 5-30 mM glucose. No significant activation of Rap2b with a transcript size of ~4.8 kb
(33) was observed in MCs exposed to HG up to a concentration of 40 mM glucose (Fig. 8B). Exposure to HG (30 mM) also had no significant effect on the mRNA
expression of Rap2a and Rap2b when compared with their expression in
the MCs exposed to 5 mM glucose (Fig.
9, B and C). However, Rap1a,
which exhibits ~80% homology to Rap1b, showed a significant increase
in the mRNA levels in MCs treated with HG in a
dose-dependent manner similar to Rap1b (Fig.
9A). Although there is a high degree of homology between the
Rap1a and Rap1b, their transcript sizes substantially differ, i.e. ~2.3 and ~1.6 kb, respectively (34). The -actin
levels were unaffected by HG treatment (Fig. 9, D-F).
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Fig. 9.
Northern blot analyses depicting effect of HG
on Rap1a, Rap2a, and Rap2b mRNA expression in MCs exposed to 5-30
mM D-glucose. A dose-dependent
increase in Rap1a expression is seen (A), while no
significant increase in the expression of Rap2a or Rap2b is observed in
cells treated with HG (B and C). The -actin
expression remains unchanged (D-F). G-I
indicate the intactness of the RNAs and their loading of equal amounts
of total RNA in various lanes.
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DISCUSSION |
The clinical course of diabetic nephropathy is characterized by
hyperfiltration, microalbuminuria progressing to overt proteinuria, and
azotemia culminating into end stage renal disease. The pathologic changes that may correlate with the functional abnormalities include thickening of the glomerular basement membrane and increased deposition of mesangial matrix. During the late stages, changes in the mesangial matrix become markedly accentuated with the formation of intercapillary mesangial nodules in the renal glomerulus. These nodular lesions are
believed to be the result of hyperplasia followed by hypertrophy of MCs
accompanied with certain phenotypic alterations and accumulation of
excessive ECM proteins (1-4). Since among the various cell types of
the glomerulus the MCs seems to be predominantly affected by the high
glucose ambience; thus in the present investigation, like in previous
studies, it was utilized as the model culture system to study the
mechanism(s) involved in the HG-induced activation of Rap1b and
synthesis of fibronectin, an ECM protein. The utilization of in
vitro MCs culture system was considered suitable for the present
investigation, since our initial studies suggested that Rap1b is
expressed in vivo in the glomerular compartment of the kidney, and its up-regulation is modulated by the hyperglycemic state
in diabetic rats (Fig. 1). Further support for the use of in
vitro model system was derived from the fact that Rap1b was found
to be expressed in the cultured MCs (Fig. 2).
The Rap1b is a Ras-related (Ras-proximate) GTP-binding protein, and it
belongs to a superfamily consisting many members that regulate cell
proliferation, differentiation, intracellular vesicular trafficking,
cytoskeletal rearrangement, cell cycle events, and glucose transport
(35-37). In addition, they can modulate the expression of ECM
proteins, e.g. oncogenic transformation of fibroblasts by
v-Src and v-Ras is shown to be associated with
down-regulation of fibronectin (38). Among the various family members,
the amino acid sequence of Rap1b and Ras are homologous in their
putative effector domain that includes a stretch of 32-40 amino acid
residues (39). But Rap1 has been shown to antagonize the Ras functions, such as in the Ras-induced transformation of NIH 3T3 cells (40) and
activation of c-Raf-1 protein kinase-dependent MAP kinase cascade in Rat-1 cells (41). The findings that there is a concomitant increased expression of Rap1b and fibronectin under high glucose ambience (Fig. 2) would support the concept of antagonistic actions of
Rap1b and Ras GTPases, since overexpression of the Ras results in
down-regulation of fibronectin (see above). This effect of D-glucose seems to be specific, since the cells exposed to
L-glucose did not alter the expression of either of Rap1b
or fibronectin (Fig. 2). The increased expression or activation of
Rap1b can occur by a wide variety of stimuli, including various growth
factors, phospholipase C, and second messengers, which include
Ca2+, cAMP, and DAG, some of which conceivably can affect
the ECM synthesis via PKC pathway (35-37). Nevertheless, to assess
whether Rap1b can directly affect the expression of ECM protein like
fibronectin, a Rap1b expression construct was prepared, and a stable
mesangial cell line overexpressing Rap1b was generated (Fig. 4). The
fact that MC transfectant overexpressing Rap1b exhibited an
up-regulation of fibronectin (Fig. 5 versus Fig. 2), in
contrast to previously reported effect of Ras on fibronectin
expression, further strengthens the notion that their effects are
antagonistic. Using the Rap1b transfectants the studies were initiated
to assess whether or not the increased fibronectin expression by high
glucose was mediated via PKC pathway. The role of PKC in HG-induced
increased expression of ECM proteins is known, and it is believed that
PKC pathway in MCs can be activated by a wide array of glucose
byproducts, including hexosamine-6-phosphate, diacylglycerol (DAG) and
AGEs, as well other endogenous biological compounds, such as
angiotensin II and ROS (6-11, 42-44). Since these endogenous products
are derived from various cellular sources, it would indicate that the
involvement of PKC pathway may not be restricted to MCs, and indeed
HG-induced PKC-mediated increased expression of fibronectin has been
reported in endothelial cells as well as peritoneal mesothelial cells
(45, 46). The results obtained in this investigation also point toward the fact that HG mediates an increased expression of fibronectin via
the PKC-dependent pathway, but also involving the small
GTPase protein, i.e. Rap1b. The fact that PKC inhibitors
reduce Rap1b mRNA and protein expression in MCs exposed to HG
support such a contention (Fig. 3). Similar results have been reported
in other systems as well, where PKC inhibitors were shown to inhibit
the thrombin induced second phase Rap1b activation in blood platelets (47). The blood platelet experiments also indicated that Rap1b activation is downstream of PKC signaling pathway. To attest that Rap1b
is involved downstream of the PKC pathway, transfection experiments
were carried out. The treatment of MC Rap1b transfectants with PKC
inhibitors (calphostin C and bisindolylmaleide) did not result in any
significant change in the fibronectin expression, suggesting that the
effects of Rap1b are downstream of the PKC effect (Fig. 5). To
further confirm the contribution of Rap1b in HG-induced
PKC-dependent effects, mutagenesis experiments were carried out.
The mutagenesis experiments have been performed in the past to assess
the function of both Ras and Rap1 with conflicting results. The
disparity in the findings may be related to the fact that their effects
may be cell-specific, and these GTPases do not bind to the same guanine
nucleotide exchange factors (GEFs). For instance, the RasN17 mutant
tightly binds to Ras-GEFs, while RapN17 has very little affinity to
associate with Rap1-GEF (48). Moreover, the GEF for Ras is SOS, while
that for Rap1 is C3G, which may account for the differential effects of
Ras versus Rap1. Although C3G is the GEF for Rap1, the
Rap1A(S17N) does not act as a dominant negative mutant on the
C3G-mediated activation of Rap1 in vivo (48). Similarly,
cell specificity is exhibited in the activation experiments where
RapN17 has been shown to inhibit the activation of B-Raf by PKA in
COS-7 cells, while in others, e.g. RK13 or PC12, activation
could not be elucidated (49, 50). Nevertheless, several studies were
able to demonstrate the effects of dominant negative of Rap1N17 mutant.
The mutant could block the ability of nerve growth factor to stimulate
the sustained phase of ERK activation in PC12 cells (51). The Rap1N17
mutant has also been shown to interfere in the endogenous Rap1 and thus
alters the Dictylostelium discoideum morphology by affecting
the cytoskeleton; the effect on the latter could conceivably affect the
phagocytosis as well (52). In another study, this mutant was shown to
affect the Rap1 adaptor protein CrkL, as a result it inhibited the
1-integrin-fibronectin-mediated adhesion of
hematopoietic cells (53). In view of the above convincing dominant
negative effects in several studies, a mouse Rap1b/S17N mutant was
constructed, and its effect on the glucose-mediated increased
expression of fibronectin was assessed (Fig. 6). In line with the above
observations, a markedly decreased expression of fibronectin was
observed in mesangial cells. Another mutant, Rap1b/T61R, was also
constructed, since residue Thr61 plays a key role in
transformation suppressor activity in NIH 3T3 cells transfected with
K-ras. Interestingly, this mutant also had a negative effect
on the fibronectin, which supports the notion that Ras and Rap1b have
antagonistic actions as overexpression of v-Ras or
v-Raf leads to down-regulation of fibronectin (38). Taken
together, these results suggest that both Ser17 and
Thr61 are sites that significantly modulate the functions
of Rap1b. Since there is a certain degree of overlap in Rap1b and
Ras-mediated signaling events following their activation, the status of
B-Raf and Raf-1 following glucose-mediated induction of Rap1b
expression was determined utilizing Rap1b mutants.
There are three isoforms of Raf kinase in mammals, and they include
Raf-1, A-Raf, and B-Raf, and they are non-redundant proteins serving
distinct functions, since each yields a different phenotype in
knock-out mice (54). At present, all three isoforms share Ras as an
upstream activator and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) as a commonly accepted downstream substrate. However, since Raf-1 is ubiquitously expressed and A-Raf and
B-Raf have restricted distribution, it is likely that the latter
two serve specialized functions in specific locations in the mammalian
system (55, 56). Most of the extracellular signals, e.g.
PDGF and EGF, which activate Rap1 also induce Raf-1 activation. The
activation of the latter is related to the binding of Rap1 with the
cysteine-rich and Ras binding domains of Raf-1, and such
domain-specific interactions are believed to competitively inhibit Ras
functions (57). On the other hand Rap1 exhibits an additive effect on
the Ki-Ras-stimulated B-Raf activity and downstream MAPK cascade
(41), suggesting that these two small G proteins, i.e. Ras
and Rap1, have differing actions with the involvement of different Raf
isoforms. Intriguingly, in other external stimuli, such as nerve growth
factor, which affects both Ras and Rap1, they divergently modulate
Raf-1 and Raf-B activities so that the early activation of ERKs is
Ras-Raf-1-dependent, while sustained activation is
dependent upon Rap1-B-Raf cascade (58, 59). The activation of ERKs may
selectively involve Rap1-B-Raf pathway as exemplified by experiments
with depolarization-mediated calcium influx in PC12 cells (60). Along
these lines, it seems that in the present studies with MCs the HG
selectively induced the expression of B-Raf, while Raf-1 was unaffected
(Fig. 7). In fact a mild negative effect on the Raf-1 expression was
observed upon transfection with wild type Rap1b. The notion that HG
mediates B-Raf expression was strengthened by the mutational analysis
where transfection with S17N and T61R Ra1b mutants resulted in the
disappearance of the ~68-kDa band in Rap1b autoradiograms. The
dominant negative effect of the S17N mutant on the B-Raf activation has
also been reported in PC12 cells subjected EGF stimulation (50). The
above discussion suggests that small G-proteins are activated in
non-renal cells in response to a number of growth factors, thus the
next critical question that needs to be addressed is whether glucose can directly or indirectly, e.g. via growth factors, induce
the activation of Rap1b (Fig. 8).
The activation of small GTPases has been the subject of many recent
reviews (36, 37, 51, 58, 61) and following discussion briefly
summarizes certain features relevant to the activation of Rap1. A wide
range of stimuli in different cell types has been reported to activate
Rap1. The stimulus may be thrombin as in platelets, in lymphocyte by
B-cell antigen receptor activation, and platelet-activating factor in
neutrophils. In mesenchymal fibroblasts, Rap1 activation has been shown
to occur by growth factors, such as EGF and PDGF. The Rap1 activation
is usually rapid, and it is largely abolished by inhibitors of
phospholipase C, while sparing the activation by cAMP. The second
messenger generated by phospholipase C activation includes
Ca2+ and DAG. Both exhibit a certain degree of cell
specificity, i.e. Ca2+ is involved in
thrombin-induced Rap1 activation in platelets, while DAG mediates
B-cell antigen receptor-induced activation. Thus, these three distinct
second messengers, Ca2+, DAG, and cAMP, which activate
Rap1, have been clearly identified, and they play a pivotal role in
various signal transduction processes. Beside the stimuli described
above, glucose could conceivably induce activation of Rap1b. Among the
various second messengers, DAG may be responsible for its activation in
MCs exposed to high glucose, since DAG is de novo
synthesized in hyperglycemic state and, in part, also by the hydrolysis
of phosphatidylcholine (62). DAG is known to activate PKC, which is
followed by increased TGF--mediated ECM production (1, 3, 4, 6, 8,
15). This idea seems to be plausible, since fibronectin, an ECM
protein, expression was reduced by PKC inhibitors in non-transfected
MCs (Fig. 5). However, in addition to PKC the possibility that protein
kinase A (PKA) may be involved should be considered as well, since PKA has been described to increase the transcription of type-IV collagen, another ECM protein, in MCs induced with HG (63). This transcriptional increase may be related to the phosphorylation of cAMP-responsive element (CRE)-binding protein (CREB) (64). Thus, in this scenario of
complex biological signaling system (65), it is conceivable that in
addition to PKC, PKA may also be involved, which could explain the
increase in fibronectin expression, since this ECM protein contains CRE
in its promoter region, and cAMP is known to activate Rap1. Both Rap1a
and Rap1b have been shown to be phosphorylated by
cAMP-dependent PKA in intact cells and cell-free systems
(66). The PKA-dependent activation of B-Raf in PC12 cells
has been described (58); however, the present findings suggest that
transfection of MCs with Rap1b, a G-protein activated downstream of
PKC, also leads to the activation of B-Raf (Fig. 7). Here, it would be
of interest to determine whether PKC and cAMP-dependent PKA
can exert a synergistic effect activating Rap1b, and this will
certainly be the subject of future investigations. Nevertheless, this
cAMP-dependent pathway may be potentially involved in the
HG-mediated intracellular events, since, in contrast to Ras in the
plasma membrane, the Rap1 is localized to the perinuclear Golgi region
and a protein activator known as Epac, exchange protein activated by
cAMP, is also co-distributed in the perinuclear region with Rap1b as
well (67, 68).
Like Rap1b expression its activation was dose-dependent
(Fig. 8A, panel a) and rapid, i.e.
occurring within minutes (<5 min) of exposure to HG (Fig.
8A, panel b). Since Rap1 is localized in the
perinuclear Golgi region, that is at a distance from the plasma
membrane, and the de novo synthesis of cytokines,
e.g. TGF-, EGF or PDGF, would take some time to reach the
perinuclear region, it is most likely that these cytokines may not be
involved for Rap1b induction. Nevertheless, the role of PDGF in
cellular proliferation linked to up-regulation of small GTPases has
been elucidated in other studies, where an ~2-fold increased
expression of Rap1b was observed in response to the exposure of smooth
muscle cells to PDGF (69, 70). With respect to PDGF, its up-regulation has been reported in glomeruli of diabetic rats as well as mesangial cells exposed to HG (71, 72). In human MCs, HG has been shown to induce
an overexpression of TGF- through activation of PDGF loop, which in
turn leads to cellular proliferation and mesangial ECM production (31,
73). Such effects were reversed in MCs treated with neutralizing
anti-PDGF antibodies (31). However, in the present study the anti-PDGF
antibody failed to reverse the HG-induced activation of Rap1b (Fig.
8A, panel c), suggesting that the PDGF is not
involved in the Rap1b-B-Raf pathway that downstream modulates the
ECM-fibronectin synthesis or expression. It is also interesting to note
that like the results of Rap1b expression studies (Fig. 6) certain
amino acid residues, i.e. Ser17 or
Thr61 are also critical for HG activation of Rap1b, since
overexpression of dominant negative Rap1b/S17N and/T61R notably reduced
the Rap1bGTP (Fig. 8A, panel d). Also, similar to
the expression studies, the Rap1b activation was enhanced in cells
overexpressing Rap1b. The HG-induced activation was GTPase-specific,
since Rap2b was unaffected (Fig. 8B), suggesting that the
latter may not involve the cAMP- dependent PKA pathway. This
is also the case in studies with platelets where Rap2B, unlike Rap1a
and Rap1b, was found not to be phosphorlyated by PKA (74).
Analogous to the above findings, the expression studies revealed no
change in Rap2a or Rab2b mRNA levels (Fig. 9) in MCs subjected to
HG ambience. Interestingly, Rap1a expression exhibited a
dose-dependent response to HG treatment, suggesting that
the homologous Rap1a and Ra1b proteins respond to the HG milieu in a
similar manner and add to the list of other Ras-related GTPases,
e.g. Rad and Gem (75, 76), that are relevant to pathogenesis
of diabetic complications. Moreover, Rad has also been shown to
contribute to insulin resistance as well (77). Above all, the findings of this investigation support the notion that the small GTP-binding proteins, like Rap1b, are relevant to the pathogenesis and progression of diabetic nephropathy, which is characterized by excessive synthesis of ECM proteins, glomerulosclerosis, and ultimately renal failure (Fig.
10) (78-80).
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Fig. 10.
Schematic sketch depicting the potential
mechanism(s) of HG-induced extracellular matrix synthesis in kidney
MCs. The bold arrows represent data relevant to the
present investigation. The thin arrows indicate the known
pathways that are involved in the HG-induced increased expression of
fibronectin in MCs. The dotted arrows represent conceivable
connections between the effectors and the substrates that need to be
further investigated.
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ACKNOWLEDGEMENTS |
We are thankful to Dr. Johannes L. Bos,
Martina Schmidt, and Michael R. Gold for providing us with Rap1a,
Rap2a, and Rap2b cDNAs and RBD plasmid construct.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK28492, DK60635, and HL063407.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.
¶
To whom correspondence should be addressed: Dept. of
Pathology, Northwestern University Medical School, 303 East Chicago
Ave., Chicago, IL 60611. Tel.: 312-503-0084; Fax:
312-503-0627; E-mail: y-kanwar@northwestern.edu.
Published, JBC Papers in Press, August 23, 2002, DOI 10.1074/jbc.M203957200
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ABBREVIATIONS |
The abbreviations used are:
ECM, extracellular matrix;
AGE, advanced glycation end products;
ROS, reactive oxygen species;
DAG, diacylglycerol;
GEF, guanine nucleotide
exchange factor;
PKC, protein kinase C;
Rap1b, Ras-proximate
GTP-binding protein 1b;
PKA, protein kinase A;
TGF-, transforming
growth factor ;
PDGF, platelet-derived growth factor;
EGF, epidermal
growth factor;
CRE, cAMP-responsive element;
CREB, cAMP-responsive
element-binding protein;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal regulated kinase;
MC, mesangial cell;
HG, high
glucose;
PMSF, phenylmethylsulfonyl fluoride;
RT, reverse
transcriptase;
PBS, phosphate-buffered saline;
GST, glutathione
S-transferase;
RBD, Rap binding domain;
WT, wild type;
FN, fibronectin.
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REFERENCES |
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