Activation of PKC-
I in glomerular
mesangial cells is associated with specific NF-
B
subunit translocation
Anoop
Kumar,
Karen S.
Hawkins,
Meredith A.
Hannan, and
Michael B.
Ganz
Division of Nephrology, Veterans Affairs Medical Center, and
Section of Nephrology, Department of Medicine, Case Western Reserve
University, Cleveland, Ohio 44106
 |
ABSTRACT |
Changes
in expression and activity of protein kinase C (PKC) isoforms and early
transcription factors may account for alterations in cell behavior seen
in diabetes. We studied the expression of PKC-
I in rat
glomerular mesangial cells (MCs) cultured in normal or high glucose and
compared it with the temporal and spatial expression of dimeric
transcription factor (NF-
B) p50 and p65. The results show that in
unstimulated cells PKC-
I and NF-
B p50 are distributed
in the cytosol and, on stimulation, their distribution is perinuclear
and they are localized to the membrane. Serum-starved MCs cultured in
high-glucose medium exhibit a predominantly cytosolic localization of
PKC-
I and both p50 and p65 NF-
B. However, phorbol 12-myristate 13-acetate (PMA) stimulation of cells grown in the presence of high glucose resulted in membrane translocation of PKC-
I that was associated with nuclear translocation of
NF-
B p65, but not NF-
B p50. Moreover, the translocation to the
nucleus for NF-
B p65 was significantly higher in MCs exposed to high glucose compared with those exposed to normal glucose. These
observations indicate that the NF-
B p65, but not NF-
B p50,
expression and translocation pattern mirrors that of
PKC-
I, which may be one important pathway by which
signaling is enhanced in the high-glucose state.
protein kinase C; transcriptional factors; diabetes; nephropathy
 |
INTRODUCTION |
HYPERGLYCEMIA HAS BEEN
PROPOSED to be the main determinant in the initiation and
progression of diabetic microvascular complications including
nephropathy in both insulin-dependent and non-insulin-dependent diabetes mellitus (38, 40). The renal glomerular lesion in diabetes is characterized by glomerular hypertrophy that is followed by
the deposition of extracellular matrix (1). Activation of protein kinase C (PKC) is one of the major mechanisms involved in
high-glucose-induced glomerular injury, reactive oxygen production, and
subsequent lipid peroxidation (18, 19, 29). Activation of
specific PKC isoforms has also been shown to be associated with
translocation to different subcellular compartments (11, 42). After cellular stimulation, PKC isoforms are targeted to distinct subcellular loci, including plasma membrane, nucleus, and
cytoskeleton, suggesting specific biological roles (11, 17, 26,
39, 42). A preferential activation of PKC-
isoforms in
glomeruli isolated from diabetic rats has also been reported (27). Substantial evidence suggests that the accumulation
of extracellular matrix in diabetes is related to changes in glomerular mesangial cell (MC) phenotypic behavior (1, 32, 36).
Several studies support a role for PKC in high-glucose-induced early
signaling events causing increased extracellular matrix accumulation by MCs (1, 4, 37).
A large number of genes that play important roles in immune and stress
responses, inflammation, and apoptosis are regulated by the
dimeric transcription factor nuclear factor (NF)-
B (5, 6,
9). The oxidative stress seen in diabetes and hyperglycemia appears to cause increased expression of various genes that are NF-
B
dependent (8, 35). Hyperglycemia also activates NF-
B, in part by activation of PKC (28, 31). We sought to
determine whether expression of PKC-
I in rat MCs
correlates with a temporal and spatial change in the expression of
specific NF-
B isoforms. Unstimulated MCs exhibited similar
expression of PKC-
I and NF-
B p65 and p50 regardless
of the glucose medium in which the cells were grown. On stimulation
[phorbol 12-myristate 13-acetate (PMA)] PKC-
I
translocated to the membrane that was temporally associated with
nuclear translocation of NF-
B p65 but not NF-
B p50. Moreover, the
nuclear translocation of NF-
B p65 was more profound in high glucose.
These results indicate that PKC activation leads to a preferential
activation of NF-
B p65 that may be responsible for the activation of
other genes in hyperglycemic conditions.
 |
METHODS |
Isolation and culture of MCs.
MCs were isolated from the glomeruli of young adult male Sprague-Dawley
rat kidneys according to a previously described protocol (15). The protocol for harvesting the MCs was approved by
the Institutional Animal Care and Use Committee. Briefly, glomeruli were washed twice with Hanks' balanced salt solution (HBSS) and were
plated onto 75-cm2 tissue culture flasks. The culture media
contained DMEM with 5.6 mM glucose, 110 mg/ml sodium pyruvate, 100 U/ml
penicillin G, 100 µg/ml streptomycin, and 20% fetal bovine serum
(FBS). Routine identification of MCs was performed by indirect
immunofluorescence microscopy using rabbit IgG directed against
vascular smooth muscle myosin and mouse anti-rabbit FITC-conjugated
IgG. Cells showed uniformly strong positive staining of longitudinal
filaments, a pattern that is characteristic of MCs (34).
Confluent passaged MCs were subcultured onto 60-mm petri dishes for
further experiments. The passaged MCs (between passages 6 and 12) were grown in DMEM with either 5.6 (normal) or 30 mM (high) glucose. When the cells reached ~70% confluence, the medium was replaced with serum-free medium (0.5% FBS) with all other constituents, and the cells were grown for another 48 h. At the end of serum starvation, the cells in the PMA-treatment group were
incubated with 0.1 µM PMA for 10 min, and both groups of cells were
fractionated as described below.
Cell fractionation.
The cells were fractionated as previously described with few
modifications (16). Briefly, cells were washed twice with
ice-cold PBS, harvested by scraping in 1 ml of ice-cold PBS containing protease inhibitors (100 µM PMSF, 1 µM aprotinin, and 1 µM
pepstatin) into prechilled Eppendorf tubes and pelleted at 4°C. Cell
pellets were resuspended in 100 µl of ice-cold homogenization buffer
(60 mM Tris, pH 7.0, 0.25 M sucrose, 2 mM EDTA, and 1 mM
-mercaptoethanol) containing protease inhibitors (100 µM PMSF, 1 µM aprotinin, and 1 µM pepstatin). The cell suspension was then
subjected to three 30-s bursts of sonication, and cell lysis was
checked under a microscope. The homogenates were centrifuged at 12,000 g for 20 min at 4°C. The supernatants were collected as
cytosol fractions. An additional washing step with PBS (containing
protease inhibitors) was included to eliminate the possibility of
carryover of proteins from one fraction to another. The pellets were
washed once with PBS (containing protease inhibitors described above)
and resuspended in 500 µl of PBS (with protease inhibitors) and
centrifuged as above. The supernatants were discarded, and the pellets
were resuspended in 100 µl of homogenization buffer containing 1%
Triton X-100 and incubated on ice for 30 min with intermittent
vortexing. The samples were then centrifuged in a Beckman L8-60M
ultracentrifuge at 100,000 g for 30 min at 4°C. The
supernatants were collected as the membrane fractions. The pellets,
which contain mainly the cytoskeleton and nucleus, were washed and
resuspended in PBS (with protease inhibitors) and subjected to
centrifugation as described above. The supernatants were discarded, and
the pellets were resuspended in 100 µl of homogenization buffer
containing 1% SDS, boiled for 10 min, and briefly microfuged to
separate debris, and the supernatant was then collected as the
particulate fraction (21). The protein concentrations were
measured using the Micro BCA assay (Pierce, Rockford, IL).
Western blot analysis of PKC-
I and
NF-
B.
Equal amounts of protein samples along with appropriate molecular size
standards (Bio-Rad) were run on 10% SDS-PAGE under reducing conditions
in a Mini Protean II Dual Slab Cell (Bio-Rad). Proteins separated on
SDS gels were then transferred onto nitrocellulose membranes using a
Mini Transblot electrophoretic transfer cell (Bio-Rad). The membrane
was incubated in a blocking buffer (PBS, 0.05% Tween 20, 5% nonfat
dry milk; 1% BSA) for 1 h at room temperature and then reacted
with primary antibodies to PKC-
I, NF-
B p50, or
NF-
B p65 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1,000 dilution) in the blocking buffer for 3 h at room temperature. This
was followed by three washes with PBS-0.05% Tween 20 (PBS-Tween), incubation with horseradish peroxidase-conjugated secondary antibodies (1:4,000 dilution) in blocking buffer for 1 h at room temperature, and three additional washes with PBS-Tween. The reaction was detected using enhanced chemiluminescence (NEN Life Sciences Products, Boston,
MA). Rat brain lysate was used as a positive control to identify
PKC-
I. The specificity of bands for NF-
B p50 or
NF-
B p65 was tested using blocking peptides (Santa Cruz
Biotechnology) specific for the respective antibodies by preadsorption
with the blocking peptides. Translocation was determined by a
comparison of the respective bands in the cytosol, membrane and
particulate fractions, and densitometric analysis using Scion Image, a
Windows-compatible version of National Institutes of Health Image
software. Statistical analysis was done using the Statview program (SAS
Institute, Cary, NC).
Immunofluorescence labeling for
PKC-
I and NF-
B.
MCs cultured on chamber slides were serum starved as described above.
The cells in the PMA-treatment group were stimulated for 10 min at
37°C with 0.1 µM of PMA in 400 µl of serum-free medium. The
control (unstimulated) group received the same amount of medium. The
wells were washed three times with PBS and fixed in 3.7% formaldehyde
in PBS for 20 min at room temperature. The slides were then rinsed
three times with PBS, and the nonspecific binding sites were blocked
with 5% BSA in PBS for 60 min at room temperature. The cells were
incubated for 1 h at room temperature with rabbit
anti-PKC-
I-specific or goat anti-NF-
B p50- or NF-
B p65-specific antibodies (1:100) in 0.1% BSA in PBS. This was followed by three washes with PBS. Labeling was achieved using affinity-purified FITC or rhodamine-conjugated secondary antibodies (1:200) by incubation in the dark for 1 h at room temperature as previously described (34). Applying the different primary antibodies
simultaneously, we were able to perform double immunofluorescence.
After being washed, cells were incubated with a mixture of FITC or
rhodamine-conjugated secondary antibodies. Immunostaining was studied
using a fluorescence microscope (Eclipse E600, Nikon). Images were
collected separately and then merged using Scion Image.
 |
RESULTS |
Distribution of PKC-
I.
The expression pattern of PKC-
I in different fractions
as determined by Western blotting is represented in Fig.
1. In both the cytosol and membrane
fractions, a single band at 80 kDa was observed, which comigrated with
the rat brain positive control. In unstimulated cells,
PKC-
I was found predominantly in the cytosol fraction of
cells in either the normal- or high-glucose concentration condition
(96.7% in normal vs. 98.7% in high glucose; Fig.
2). In MCs cultured under normal glucose,
stimulation with PMA for 10 min resulted in the rapid translocation of
PKC-
I to the membrane fraction with a substantial
reduction in PKC-
I that was present in the cytosol. PMA
treatment for 60 min resulted in near complete activation of
PKC-
I; most PKC-
I translocated from the
cytosol to the membrane (Figs. 1 and 2). In MCs subcultured under
high-glucose conditions, a similar pattern was evident; there was an
increase in association with the membrane fraction by 10 min and an
even greater increase by 60 min (Figs. 1 and 2). Under both glucose concentrations, PKC-
I did not translocate to the
particulate (nuclear) fraction.

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Fig. 1.
Representative Western blot analysis of subcellular
fractions of mesangial cells (MCs) stained for protein kinase C
(PKC)- I. Subcellular fractions were prepared from MCs
without any treatment (unstimulated) or after stimulation with phorbol
12-myristate 13-acetate (PMA) for 10 or 60 min. Each lane was loaded
with 50 µg of total protein.
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Fig. 2.
Expression of PKC- I in MC fractions.
Western blots were prepared from subcellular fractions from MCs without
any treatment (untreated) or after treatment with PMA for 60 min. The
results represent the percentages of total PKC- I in
unstimulated MCs grown in 5.6 mM glucose.
|
|
Representative immunofluorescence images of MCs labeled with primary
antibody to PKC-
I are shown in Fig.
3. In unstimulated MCs grown in both
normal and high glucose, PKC-
I was distributed throughout the cytosol. When the cells were exposed to PMA,
PKC-
I translocated to perinuclear and plasma membrane
locations. PKC-
I localization was similar in MCs grown
in high glucose;
I in unstimulated cells was distributed
throughout the cytosol, with an intense staining and a punctate
appearance. On exposure to PMA, PKC-
I assumed a
perinuclear and plasma membrane distribution. In both glucose
concentrations, nuclei were not stained, as shown in Western blot
analysis.

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Fig. 3.
Representative immunofluorescence photomicrographs of MCs
demonstrating PKC- I. Immunofluorescence was performed on
MCs grown in normal or high glucose that were either unstimulated
(left) or PMA stimulated (right) for 10 min and
stained for PKC- I.
|
|
Distribution of NF-
B.
The distribution pattern of NF-kB p50 and p65 as determined by Western
blotting is shown in Fig. 4. NF-
B p50
was present predominantly in the cytosol of MCs in both normal and
high-glucose concentrations (85.9% in 5.6 mM vs. 86.2% in 30 mM).
NF-
B p50 in both unstimulated and stimulated and low and high
glucose remained predominantly in the cytosol fraction. NF-
B p50 was
not visibly evident in the particulate fraction (1.5% in normal
glucose vs. negligible amount in high glucose) (Figs. 4 and
5A). However, NF-
B p65 exhibited significantly more
particulate localization in unstimulated cells in both glucose
concentrations (Figs. 4 and 5B), in addition to cytosolic
localization. On PMA stimulation, the amount of p65 increased
significantly in the particulate fraction of cells in 30 mM glucose
compared with cells exposed to 5.6 mM glucose (Fig.
5B; 35.4 vs. 20.4%).

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Fig. 4.
Western blot analysis of MC subcellular fractions stained
for the dimeric transcription factor nuclear factor (NF)- B p50 and
NF- B p65 (top and bottom, respectively).
Subcellular fractions were the same fractions that were used for the
determination of PKC- I. Each lane was loaded with 25 µg of total protein.
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Fig. 5.
A: expression of NF- B p50 in particulate
fractions of MCs. Densitometric readings of Western blots of
particulate fractions of MCs grown in 5.6 and 30 mM glucose are shown.
The results represent the percentages of total NF- B p50 in
unstimulated MCs grown in 5.6 mM glucose. B: expression of
NF- B p65 in particulate fractions of MCs. Densitometric readings of
Western blots of particulate fractions of MCs grown in 5.6 and 30 mM
glucose are shown. The results represent the percentages of total
NF- B p65 in unstimulated MCs grown in 5.6 mM glucose.
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|
Immunostaining for PKC-
I and
NF-
B.
In data not shown, on PMA exposure p65 showed redistribution into
nuclear and perinuclear areas in a fashion similar to that shown in
Western blots. We performed double immunostaining using two antibodies
(immunolabeled with FITC and/or rhodamine) to determine colocalization
of PKC-
I and NF-
B p50 and NF-
B p65. The
double-staining pattern for PKC-
I and NF-
B p50, as
shown in Fig. 6, closely resembles the
pattern seen with single immunostaining. The results show that in
unstimulated cells PKC-
I and NF-
B p50 are distributed in the cytosol, and, on stimulation, their distribution is perinuclear and on the membrane. The results shown in Fig. 6 also show that PKC-
I and NF-
B p65 are distributed in the cytosolic
and membrane areas. However, MCs in both glucose concentrations show
nuclear staining for p65, which is similar to the results in Western
blots. On stimulation, the cells showed intense staining in the nucleus and perinuclear areas, indicating that there is increased nuclear translocation of p65 correlating with the spatial perinuclear localization PKC-
I.

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Fig. 6.
Representative photomicrographs of double staining of MCs
[unstimulated (left) or PMA stimulated (right)]
for PKC- I and NF- B. Red staining represents the
staining pattern for PKC- I, green staining represents
that for NF- B p50 or NF- B p65, and yellow staining reveals the
colocalization of proteins.
|
|
 |
DISCUSSION |
The present study demonstrates that PKC-
I
activation (translocation) was temporally associated with nuclear
translocation of NF-
B p65 but not NF-
B p50. In addition, the
temporal and spatial localization of NF-
B p65 was greatly
accentuated when cells were grown under high-glucose conditions. These
results may help to define the specific biological roles for isoforms of PKC and those of transcription factor NF-
B.
Several studies have shown that MCs express calcium-dependent classic
isoforms of PKC such as
,
I,
II, and
(16, 21, 33, 34). PKC-
I and
-
II are the isoforms of PKC most frequently associated
with alterations in MC phenotypic behavior such as proliferation and
matrix deposition (41). Previous studies from our
laboratory have shown that PKC-
II is temporally
associated with rapid MC proliferation during development (2,
34). With further renal maturation, this isoform becomes
downregulated in MCs (34). Therefore, in the present
investigation we concentrated on the PKC-
I isoform,
which is readily expressed in adult MCs.
High glucose has been found to induce numerous changes in the
phenotypic behavior of various cells, including glomerular MCs (13, 20, 22, 30). Hyperglycemia-induced glomerular injury has been shown to modulate MC biological behavior, which is reflected in early changes in membrane transport (1, 16, 33, 37). Cellular distribution studies have demonstrated that PKC isoforms exhibit tissue- and region-specific expression in different cells (3, 14). These findings have led to the notion that
different PKC isoforms may be activated by different agonists and/or at different intracellular sites, allowing for the selective activation of
isoforms in response to external stimuli.
Several studies have shown that the activity of PKC is elevated
in the retina, glomeruli, and heart of diabetic animals, in purified
microvessels from diabetic animals, and in endothelial and mesangial
cells cultured in high glucose or galactose (4, 14, 21, 23, 24,
29, 42). Our data in the present study showing that the
expression of PKC-
I is enhanced in the presence of high
glucose, and PMA stimulation causes membrane translocation of these PKC
isoforms, are consistent with previous observations (21, 25,
42). However, some studies have also demonstrated that exposure
of MCs to high glucose for 24-48 h in the presence of PMA results
in translocation of PKC isoforms to membrane and nuclear and/or
cytoskeletal compartments (12, 21).
Our observations in the present study also indicate that enhanced
expression of PKC isoforms is associated with nuclear translocation of
NF-
B p65. NF-
B is a major target of intracellularly induced oxidative stress (8, 35). The proteins of the NF-
B
family form the inactive heterodimeric complexes in the cytoplasm of cells. Activation of NF-
B results from the dissociation of these inactive complexes from their inhibitory proteins and translocation of
the active NF-
B complexes to the nucleus (5-7,
10). Such phenomena lead to changes in vascular homeostasis and
endothelial dysfunction manifested in diabetic vascular complications,
such as angiopathy, neuropathy, and nephropathy (8).
Unlike p65, NF-
B p50 does not translocate simultaneously to the
nucleus. It is therefore conceivable that signaling that leads to
changes in MC behavior is achieved through a PKC-
I and
NF-
B p65 pathway. These results indicate the possibility of
a dissociation of NF-
B p50 from the p50-p65 heterodimeric complex
during PKC activation after stimulation of MCs.
In summary, our results indicate that MCs cultured in high-glucose
medium exhibit enhanced membrane translocation of PKC-
I on stimulation with PMA. This was associated with nuclear translocation of NF-
B p65. These observations may enhance our understanding of
intracellular events associated with MC susceptibility to injury and
subsequent hyperactivity associated with diabetes.
 |
ACKNOWLEDGEMENTS |
This work was supported by American Heart Established Investigator
Grant 96001450 and a Veterans Affairs Merit Review (to M. B. Ganz).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. Kumar, Section of Nephrology, Research Service 151 (W), Cleveland VA
Medical Center, 10701 East Blvd., Cleveland, OH 44106 (E-mail: axk38{at}po.cwru.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 March 2001; accepted in final form 17 May 2001.
 |
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