Protein kinase C-
mediates TNF-
-induced ICAM-1 gene
transcription in endothelial cells
Arshad
Rahman,
Khandaker N.
Anwar, and
Asrar B.
Malik
Department of Pharmacology, University of Illinois College of
Medicine, Chicago, Illinois 60612
 |
ABSTRACT |
We addressed the role of
protein kinase C (PKC) isozymes in mediating tumor necrosis factor-
(TNF-
)-induced oxidant generation in endothelial cells, a
requirement for nuclear factor-
B (NF-
B) activation and
intercellular adhesion molecule-1 (ICAM-1) gene transcription.
Depletion of the conventional (c) and novel (n) PKC isozymes following
24 h exposure of human pulmonary artery endothelial (HPAE) cells
with the phorbol ester, phorbol 12-myristate 13-acetate (500 nM),
failed to prevent TNF-
-induced oxidant generation. In contrast,
inhibition of PKC-
synthesis by the antisense oligonucleotide prevented the oxidant generation following the TNF-
stimulation. We
observed that PKC-
also induced the TNF-
-induced NF-
B binding to the ICAM-1 promoter and the resultant ICAM-1 gene transcription. We
showed that expression of the dominant negative mutant of PKC-
prevented the TNF-
-induced ICAM-1 promoter activity, whereas overexpression of the wild-type PKC-
augmented the response. These
data imply a critical role for the PKC-
isozyme in regulating TNF-
-induced oxidant generation and in signaling the activation of
NF-
B and ICAM-1 transcription in endothelial cells.
protein kinase C isoforms; oxidants; nuclear factor-
B; intercellular adhesion molecule-1; endothelium
 |
INTRODUCTION |
THE BASIS OF STABLE
POLYMORPHONUCLEAR leukocyte (PMN) adhesion to the vascular
endothelial cells involves the expression of intercellular adhesion
molecule-1 (ICAM-1; CD54) on the endothelial cell surface (36,
37). ICAM-1 mediates firm adhesion of PMN to the vascular
endothelium by serving as a counter-receptor for leukocyte
2-integrins, CD11a/CD18 and CD11b/CD18. Although ICAM-1 is expressed basally in endothelial cells, its expression is markedly induced by the inflammatory cytokines such as tumor necrosis factor-
(TNF-
) through activation of the transcription factor nuclear factor-
B (NF-
B) (14, 20).
The NF-
B/Rel family of transcription factors is composed of
the transcriptionally active p65/Rel A (25, 30), c-Rel
(42), and Rel B (31) and
transcriptionally silent p50/NF-
B1 (12, 17), and p52/
NF-
B2 (5, 32). All NF-
B proteins exist as an
inactive dimer in the cytoplasm bound to the inhibitory proteins of the
I
B family, I
B
, IkB
, I
B
, p100, p105, and IkB
(3, 41). TNF-
stimulation of cells results in the
phosphorylation, and subsequent ubiquitination-dependent degradation of
I
B
by the proteasome 26S (6, 7, 40). This
allows the NF-
B dimer to migrate to the nucleus, where it activates
transcription of the ICAM-1 gene. We have shown the critical
involvement of protein kinase C (PKC), a family of serine/threonine
kinases, in the activation of NF-
B and the transcription of ICAM-1
(29); however, the specific PKC isozyme(s) responsible for
this effect in endothelial cells has not been identified. PKC isozymes
with differential cellular distributions, substrate specificities, and
activator responsiveness are classified into three groups: conventional (cPKCs;
,
I,
II, and
), novel [nPKCs;
,
, µ,
,
and
/L (mouse/human)], and atypical [aPKCs;
, and
/
(mouse/human)] (13, 16). cPKCs are
Ca2+ dependent and are activated by diacylglycerol and
phorbol esters, whereas neither nPKCs nor aPKCs require
Ca2+ for activation (15, 16). The nPKC
isozymes are activated by diacylglycerol and phorbol esters, whereas
aPKCs are irresponsive to both diacylglycerol and phorbol esters
(16).
We showed that stimulation of human pulmonary artery endothelial (HPAE)
cells with TNF-
resulted in activation of PKC and oxidant generation
(29). We also showed that PKC activation and oxidant
generation were both necessary for NF-
B activation and ICAM-1
expression, since the inhibition of PKC by calphostin C and scavenging
of oxidants by antioxidants prevented the TNF-
-induced NF-
B
activation and ICAM-1 gene transcription (29). These
results demonstrated that TNF-
-activated oxidant generation in
endothelial cells occurred downstream of PKC activation
(29). In the present study, we have extended these
observations by showing that PKC-
, the aPKC isozyme in endothelial
cells, is critically involved in the mechanism of TNF-
-induced
oxidant generation and thereby in the signaling of activation of
NF-
B and ICAM-1 gene transcription.
 |
METHODS |
Cell culture.
HPAE cells, obtained from Clonetics (La Jolla, CA), were grown on
gelatin-coated flasks or plates in endothelial cell growth medium (EGM)
containing 10% FCS and 3.0 mg/ml of endothelial-derived growth factor
from bovine brain extract protein. Human recombinant TNF-
with a
specific activity of 2.3 × 107 was purchased from
Promega (Madison, WI). All experiments were made in cells under the
10th passage except where indicated otherwise.
Northern analysis.
Total RNA was isolated from HPAE cells with an RNeasy kit (Qiagen,
Chatsworth, CA) according to manufacturer's recommendations. Quantification and purity of RNA were assessed by
A260/A280 absorption, and an aliquot of RNA (20 µg) from samples with a ratio above 1.6 was fractionated using a 1%
agarose formaldehyde gel. The RNA was transferred to Duralose-UV
nitrocellulose membrane (Stratagene, La Jolla, CA) and covalently
linked by ultraviolet (UV) irradiation using a Stratalinker UV cross
linker (Stratagene). Human ICAM-1 (0.96-kb Sal
I-to-Pst I fragment) (39) and rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1.1-kb Pst
I fragment) were labeled with [
-32P]dCTP using
the random primer kit (Stratagene), and hybridization was carried out
as described (28). Briefly, the blots were prehybridized for 30 min at 68°C in QuikHyb solution (Stratagene) and hybridized for 2 h at 68°C with random-primed 32P-labeled
probes. After hybridization, the blots were washed 2× for 30 min at
room temperature in 2× SSC with 0.1% SDS followed by two washes for
15 min each at 60°C in 0.1× SSC with 0.1% SDS. Autoradiography was
performed with an intensifying screen at
70°C for 12-24 h. The
signal intensities were quantified by scanning the autoradiograms with
a laser densitometer (Howtek, Hudson, NH) linked to a computer analysis
system (PDI, Huntington Station, NY). The nitrocellulose membrane was
soaked for stripping the probe with boiled water or 0.1× SSC with
0.1% SDS.
Detection of oxidant generation.
Oxidant generation in HPAE cells was measured as described
(27). Briefly, confluent HPAE monolayers were stimulated
for 1 h with TNF-
(100 U/ml) in EGM containing 2% serum as
described above. Cells were washed 2× with EGM (2% serum) and stained
for 20 min with 1 µM 5(and
6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate
bis(acetoxymethyl) ester (C-DCDHF-DA; Molecular Probes, Eugene, OR) in
EGM (with 2% serum). Cultures were viewed with fluorescence microscopy
and photographed. Fluorescence was imaged using a Nikon Diaphot 200 microscope (Nikon, Garden City, NJ), and the results were quantified
using the Image Pro Plus software (Media Cybernetics, Silver Spring, MD).
Nuclear extract preparation.
Nuclear protein extracts were prepared, and an electrophoretic mobility
shift assay (EMSA) was performed as described (29). After
treatments, cells were washed twice with ice-cold Tris-buffered saline
(TBS) and resuspended in 400 µl of buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT),
and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)]. After 15 min,
Nonidet P-40 (NP-40) was added to a final concentration of 0.6%.
Nuclei were pelleted and resuspended in 50 µl of buffer C
(20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). After 30 min at 4°C, lysates were centrifuged, and
supernatants containing the nuclear proteins were transferred to new
vials. Protein concentration of the extract was measured using a
Bio-Rad protein determination kit (Bio-Rad, Hercules, CA).
Electrophoretic mobility shift assay.
EMSAs were performed as described (29). Briefly, 10 µg
of nuclear extract was incubated with 1 µg of poly(dI-dC) in a
binding buffer [10 mM Tris · HCl, pH 7.5, 50 mM NaCl, 0.5 mM
DTT, 10% glycerol (20 µl final volume)] for 15 min at room
temperature. Then, end-labeled double-stranded oligonucleotides
containing the NF-
B site of ICAM-1 promoter (30,000 cpm each) were
added, and the reaction mixtures were incubated for 15 min at room
temperature. The DNA-protein complexes were resolved in 5% native
polyacrylamide gel electrophoresis in low ionic strength buffer (0.25×
Tris-borate-EDTA). The oligonucleotide used for the gel shift analysis
was ICAM-1 NF-
B 5'-AGCTTGGAAATTCCGGAGCTG-3'.
This represents a 21-bp sequence of ICAM-1 promoter encompassing the
NF-
B binding site located 183 bp upstream of the transcription
initiation site (14); this sequence motif within the
oligonucleotide is underlined.
Reporter gene constructs, endothelial cell transfections, and
luciferase assay.
ICAM-1 promoter-firefly luciferase (LUC) plasmids containing wild-type
(ICAM-LUC) and mutated NF-
B site
(ICAM-1NF-
BmutLUC) were provided by
Dr. Zhaodan Cao (Tularik, San Francisco, CA) (14). The
pcDNA3 vector harboring tagged wild-type and dominant negative forms of
Xenopus laevis PKC-
were gifts of Dr. J. Moscat (Universidad Autonoma, Madrid, Spain). The PKC-
mutant
(4) is a kinase-deficient PKC-
generated by a
substitution of lysine-275 for tryptophan and thus lacks a functional
catalytic domain. The plasmid pNF-
B-LUC containing five copies of
consensus NF-
B sequences linked to a minimal E1B promoter-luciferase
gene was purchased from Stratagene. The expression vector pcDNA3
containing tagged dominant negative form of PKC-
, -
, and -
isozymes (38) was provided by Dr. I. B. Weinstein
(Columbia University, New York, NY). Transfections were performed with
Superfect (Qiagen) as described (28), with slight
modifications. Briefly, reporter DNA (1 µg) was mixed with 5 µl of
Superfect in 100 µl serum-free EGM (Clonetics). We used 0.2 µg
pTKRLUC plasmid (Promega) containing Renilla luciferase gene
driven by the constitutively active thymidine kinase promoter to
normalize the transfection efficiencies. Because we did not observe any
significant difference in transfection efficiencies in initial
experiments, we did not cotransfect the pTKRLUC construct in the later
experiments. After a 5- to 10-min incubation at room temperature, 0.6 ml EGM containing 10% FCS was added, and the mixture was applied onto
the cells that had been washed once with PBS. Three hours later, the
medium was changed to EGM containing 10% FCS, and the cells were grown
to confluence.
Using this protocol, we achieved a transient transfection efficiency of
20 ± 2% (mean ± SD; n = 3) for HPAE cells.
To determine transfection efficiency, HPAE cells were transfected with
an expression plasmid pGreen Lantern-1 containing green fluorescence
protein (GFP) gene (GIBCO-BRL; Life Technologies). Transfected
cells were subjected to fluorescence-activated cell sorting
(FACS) analysis for GFP expression to determine transfection
efficiency. In some experiments, we used the DEAE-dextran method
(22) with slight modifications. Briefly, 5 µg DNA were
mixed with 50 µg/ml DEAE-dextran in serum-free EGM, and the mixture
was added onto cells that were 70-80% confluent. After 1 h,
cells were incubated for 4 min with 10% dimethyl sulfoxide (DMSO) in
serum-free EGM. The cells were then washed 2× with EGM containing 10%
FCS and grown to confluence. Cell extracts were prepared and assayed
for luciferase activity using the Promega Biotech assay system
(Promega). Luciferase activity was normalized per microgram of protein
extract and expressed as relative light units (RLU). Protein content
was determined using a Bio-Rad protein determination kit.
We used Trypan blue (Sigma Chemical, St. Louis, MO) exclusion assay to
evaluate cell viability following transfection. Cells were washed
gently with 2× PBS and trypsinized, and the cells were resuspended and
washed with EGM containing 10% FCS. The cell suspension (10 µl) was
mixed with 10 µl of 1× Trypan blue solution, and 10 µl of the
resulting mixture were loaded onto a hemocytometer. Results showed that
>95% of the cells were viable.
Transfection of HPAE cells with oligonucleotides.
Phosphorothioate oligonucleotides to PKC-
sense (ATG CCC AGC AGG
ACC) and antisense (GGT CCT GCT GGG CAT) have been described elsewhere
(10); both are targeted to translation initiation codon of
PKC-
mRNA. Phosphorothioate antisense oligonucleotides to PKC-
(GTT CTC GCT GGT GAG TTT CA) are directed to the 3'-untranslated region
of PKC-
mRNA (8). HPAE cells were grown in 100-mm
dishes to 50% confluence. Transfections of oligonucleotides were
performed with Lipofectin (GIBCO-BRL) as described
(29).
Western blot analysis.
Confluent HPAE cells grown in six-well plates were stimulated for the
indicated time periods and harvested in 200 µl of lysis buffer (50 mM
Tris · HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 75 mM NaCl, 1%
Triton X-100, 0.5% SDS, 0.75% deoxycholate, supplemented with 50 µg/ml PMSF, 2 mM sodium orthovanadate, 5 µg/ml leupeptin, 5 µg/ml
aprotinin, 1.25 mM NaF, and 1 mM sodium pyrophosphate). Each cell
lysate (8-10 µg) was denatured by boiling for 5 min before being
loaded onto 12.5% SDS-polyacrylamide gel. Gels were run at 110 mV for
1.5 h at room temperature. Proteins were transferred to Immobilon
membranes (Millipore, Bedford, MA) in blotting buffer (25 mM Tris base,
192 mM glycine, and 10% methanol) at 400 mA for 1.5 h at 4°C.
Membranes were blocked for 1 h with 5% (wt/vol) nonfat dry milk
solution in TBST (10 mM Tris base, 150 mM NaCl, and 0.05% Tween 20)
before incubating the membrane for 1 h with rabbit polyclonal
anti-human I
B
or I
B
(Santa Cruz Biotechnology, Santa Cruz,
CA) diluted 1:1,000. Membranes were washed three times with TBST and
incubated for another hour with goat anti-rabbit horseradish
peroxidase-linked IgG (Amersham, Arlington Heights, IL) diluted
1:5,000. After another three washes, antibody-labeled protein was
detected by enhanced chemiluminescence (ECL kit, Amersham) according to
manufacturer's recommendations.
 |
RESULTS |
PKC isozymes present in HPAE cells.
We first identified the PKC isozymes present in HPAE cells and
determined their sensitivities to phorbol ester. Western blot analysis
showed that PKC-
, -
, -
, and -
isozymes were abundantly expressed (Fig. 1), whereas PKC-
I and
PKC-
were not detectable (data not shown). PKC-
appeared as a
doublet corresponding to 78- and 76-kDa bands, consistent with previous
observations in other cell types (26). Exposure of HPAE
cells to phorbol ester (500 nM for 24 h) resulted in depletion of
PKC-
, -
, and -
isoforms. PKC-
was the most sensitive to
phorbol ester, whereas residual levels of PKC-
and PKC-
(76 kDa)
remained detectable (Fig. 1). In contrast, phorbol ester treatment
failed to deplete the aPKC isozyme, PKC-
(Fig. 1).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of phorbol 12-myristate 13-acetate (PMA) in
depleting protein kinase C (PKC) isozymes in human pulmonary artery
endothelial (HPAE) cells. Confluent HPAE monolayers were treated
without ( ) or with (+) PMA (500 nM in 10% FBS/EGM) for 24 h.
Total cell lysate (10 µg/lane) was separated by 12.5% SDS-PAGE and
immunoblotted for indicated PKC isozymes as described in
METHODS. Results are representative of 2 separate
experiments. EGM, endothelial cell growth medium.
|
|
We also determined the effects of an antisense oligonucleotide directed
against the translation initiation codon of PKC-
mRNA (10,
11). PKC-
expression was analyzed by Western blotting following the transfection of the sense and antisense phosphorothioate oligonucleotides in HPAE cells. Antisense oligonucleotide concentration of 0.25 µM inhibited PKC-
expression (Fig.
2); thus this concentration was used in
subsequent studies for analysis of the role of PKC-
in ICAM-1
transcription. In contrast, the antisense oligonucleotide concentration
of 1 µM failed to prevent PKC-
expression (Fig. 2). The failure of
higher concentrations (1 µM) of oligonucleotides to inhibit
expression of PKC-
is consistent with the reported effect of PKC-
antisense oligonucleotide (18). One explanation may be
that the increasing oligonucleotide/Lipofectin ratio
interferes with the complex formation and hence oligonucleotide
transfection efficiency; however, increasing the incubation time or
Lipofectin concentration did not succeed because of a loss of cell
viability. Sense oligonucleotide had no significant effect on PKC-
expression (Fig. 2). In another control experiment, antisense
oligonucleotide to PKC-
failed to prevent synthesis of PKC-
(Fig.
2), indicating specificity of the antisense oligonucleotide.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 2.
Antisense oligonucleotide to PKC- inhibits PKC-
expression. HPAE cells were transfected with antisense oligonucleotide
to PKC- as described in METHODS. After 36-48 h,
cells were lysed and analyzed by Western blot analysis for the
expression of PKC- and PKC- isozymes; the same amount of lysate
(10 µg/lane) was subjected to 10% SDS-PAGE. Results are
representative of 3 experiments.
|
|
Inhibition of PKC-
expression prevents TNF-
-induced oxidant
generation.
We determined the effects of the phorbol ester-induced depletion of
cPKC and nPKC isozymes and the inhibition of PKC-
expression by
antisense oligonucleotide on the TNF-
-induced oxidant generation. Studies were made in cells stimulated with TNF-
for 1 h to
allow maximum oxidant production. Control cells exhibited low
fluorescence intensity, whereas stimulation of HPAE cells with TNF-
resulted in markedly increased fluorescence (Fig.
3). We observed oxidant production as
early as 5 min after TNF-
challenge of HPAE cells (data not shown).
Depletion of cPKC and nPKC isozymes by the phorbol ester treatment
failed to prevent the TNF-
-induced oxidant generation (Fig. 3).
However, antisense oligonucleotide to PKC-
markedly reduced the
oxidant generation, whereas sense oligonucleotides had little effect
(Fig. 4, A and B).
In another control experiment, the antisense oligonucleotide to PKC-
failed to prevent TNF-
-induced oxidant generation in endothelial
cells (Fig. 4, C and D).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of phorbol ester-induced depletion of cPKC and
nPKC isozymes on oxidant generation induced by tumor necrosis
factor- (TNF- ) in HPAE cells. Confluent HPAE monolayers were
treated without ( ) or with (+) PMA (500 nM in 10% FBS/EGM) for
24 h. Cells were stimulated with TNF- (100 U/ml) for 1 h
to yield maximum reactive oxygen species (ROS) production. Cells were
washed and then stained with 5(and
6)-carboxy-2',7'-dichlorodihydrofluorescein (C-DCDHF-DA, 1 µM) for 20 min and analyzed by fluorescence microscopy as described in
METHODS. A: fluorescent images of representative
control or TNF- -stimulated cells without ( ) or after (+) PMA
pretreatment (results are representative of 3 separate experiments).
B: relative fluorescent intensities for each condition in
A were determined, compiled, and partitioned into 4 brightness classes (1-4), with class 1 representing the lowest fluorescence intensity and class 4 representing the highest fluorescence intensity. The relative
fluorescence intensity for cells stimulated with TNF- was shifted to
the higher fluorescence intensity classes compared with control cells.
Pretreatment with PMA failed to prevent the TNF- -induced shift to
the higher fluorescence intensity classes. Results are representative
of 3 experiments.
|
|

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 4.
Antisense oligonucleotide to PKC- prevents
TNF- -induced oxidant generation in HPAE cells. HPAE cells were
transfected with sense or antisense oligonucleotide to PKC-
(A) or antisense oligonucleotide to PKC- (C)
as described in METHODS. After 36-48 h, cells were
stimulated for 1 h with TNF- (100 U/ml). Cells were washed and
then stained with C-DCDHF-DA (1 µM) for 20 min and analyzed by
fluorescence microscopy as described in METHODS.
A: representative fluorescence images of cells transfected
with sense or antisense oligonucleotide to PKC- after TNF-
stimulation (representative of 3 separate experiments). B:
relative fluorescent intensities for each condition in A. C: representative fluorescence images of cells transfected
with antisense oligonucleotide to PKC- after TNF- stimulation
(representative of 2 separate experiments). D: relative
fluorescent intensities for each condition in
C.
|
|
Effects of depletion of cPKC and nPKC isozymes and PKC inhibitors
on TNF-
-induced I
B
degradation.
We next evaluated the role of PKC isozymes in mediating I
B
degradation, a requirement for NF-
B activation (6, 7, 40). TNF-
stimulation of endothelial cells induced I
B
degradation, whereas it had no effect on I
B
(Figs.
5 and 6). Depletion of cPKC and nPKC
isozymes failed to prevent the TNF-
-induced I
B
degradation,
and expectedly it inhibited I
B
degradation in response to the
phorbol ester stimulation (Fig. 5). Moreover, pretreatment of HPAE
cells with calphostin C, the broad spectrum inhibitor of PKC isozymes
(19), prevented the TNF-
-induced I
B
degradation, whereas staurosporine, which inhibits cPKC and nPKC but not aPKC isozymes (23, 35), failed to prevent the TNF-
response
(Fig. 6). Taken together, these results
suggest that the TNF-
-induced I
B
degradation in HPAE cells is
independent of cPKC and nPKC isozymes and support the involvement of
the aPKC isozyme.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5.
Phorbol ester-induced depletion of conventional PKC
(cPKC) and novel PKC (nPKC) isozymes fails to prevent TNF- -induced
I B degradation. Confluent HPAE monolayers were treated without
( ) or with (+) PMA (500 nM in 10% FBS/EGM) for 24 h followed by
stimulation for 20-30 min with TNF- (100 U/ml) or PMA (100 nM).
Total cell lysate (10 µg/lane) was separated by 12.5% SDS-PAGE and
immunoblotted for I B and I B . Results are representative of
2 experiments.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
Differential effects of calphostin C and staurosporine on
TNF- -induced I B degradation. HPAE cells were pretreated with
calphostin C or staurosporine for 15 min before challenge with TNF-
(100 U/ml). Total cell lysate (10 µg) was separated by 12.5%
SDS-PAGE and immunoblotted for I B and I B . Results are
representative of 2 experiments.
|
|
Inhibition of PKC-
synthesis prevents TNF-
-induced NF-
B
binding to ICAM-1 promoter.
We performed the EMSA to determine the role of the aPKC isozyme,
PKC-
, in the mechanism of TNF-
-induced NF-
B binding to the
ICAM-1 promoter. Depletion of cPKC and nPKC isozymes prevented NF-
B
binding to ICAM-1 promoter in response to stimulation of HPAE cells
with the phorbol ester (Fig. 7). However,
the depletion of these isozymes failed to prevent TNF-
-induced
NF-
B binding to the ICAM-1 promoter (Fig. 7). In contrast,
inhibition of PKC-
expression by the antisense oligonucleotide
prevented the TNF-
-induced NF-
B binding to the ICAM-1 promoter
(Fig. 8). In a control experiment, inhibition of PKC-
expression by the antisense oligonucleotide failed to prevent the TNF-
response (Fig. 8).

View larger version (87K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of phorbol ester-induced depletion of cPKC and
nPKC isozymes on TNF- -induced nuclear factor- B (NF- B) binding
to intercellular adhesion molecule-1 (ICAM-1) promoter. Confluent HPAE
monolayers were treated without ( ) or with (+) PMA (500 nM in 10%
FBS/EGM) for 24 h and subsequently incubated for 1 h with
TNF- (100 U/ml) or PMA (100 nM). Nuclear extracts were prepared and
assayed for NF- B binding activity by electrophoretic mobility shift
assay (EMSA) using radiolabeled oligonucleotide containing the
ICAM-1- B site. Results are representative of 2 experiments.
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 8.
Antisense oligonucleotide to PKC- prevents
TNF- -induced NF- B binding to ICAM-1 promoter. HPAE cells were
transfected with antisense oligonucleotide to PKC- -AS or
PKC- -AS as described in METHODS. After
36-48 h, cells were stimulated for 1 h with TNF- (100 U/ml). Nuclear extracts were prepared and assayed for NF- B binding
activity by EMSA using radiolabeled oligonucleotide containing the
ICAM-1- B site. Results are representative of 2 experiments.
|
|
Inhibition of PKC-
prevents TNF-
-induced NF-
B activity,
ICAM-1 promoter activation and mRNA expression.
We evaluated the role of PKC-
in mediating NF-
B activity by
cotransfecting a plasmid, pNF-
BLUC, containing five copies of
consensus NF-
B sequence from Ig gene linked to a minimal adenovirus E1B promoter-luciferase reporter gene with constructs encoding dominant
negative forms of PKC-
(PKC-
mut), -
(PKC-
mut), -
(PKC-
mut), or -
(PKC-
mut), isozyme in endothelial cells. As
shown in Fig.
9A,
expression of only PKC-
mut prevented the
TNF-
-induced NF-
B activity, whereas
PKC-
mut, -
mut,
-
mut, or -
mut
failed to prevent the response.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
A: inhibition of NF- B activity by
expression of dominant negative mutant of PKC- . Human umbilical vein
endothelial cells (HUVEC) were cotransfected with plasmid pNF- BLUC
containing 5 copies of consensus NF- B sequence from Ig gene linked
to a minimal adenovirus E1B promoter-luciferase reporter gene with
constructs encoding dominant negative form of PKC-
(PKC- mut), -
(PKC- mut), -
(PKC- mut), or -
(PKC- mut) isozymes using the DEAE-dextran
method (23). Cells were stimulated for 6 h with
TNF- (100 U/ml) before harvesting the cells. Cytoplasmic extracts
were prepared and luciferase activity was determined. Luciferase
activity is expressed as relative light units (RLU) per microgram
protein. Data are means ± SE (n = 3 for each
condition). B: effects of expression of PKC- dominant
negative mutant and wild-type PKC- on TNF- -induced ICAM-1
promoter activity. The construct ICAM-1LUC or ICAM-1
NF- BmutLUC (inset) containing the
wild-type or NF- B mutant version of ICAM-1 promoter was
cotransfected with wild-type (PKC- wt) or
dominant negative (PKC- mut) into HPAE cells
using Superfect as described in METHODS. Cells were
stimulated for 6-8 h with TNF- (100 U/ml) before harvesting the
cells. Cytoplasmic extracts were prepared, and luciferase activity was
determined. Luciferase activity is expressed as RLU per microgram
protein or as fold increase relative to untreated control of each
condition (inset). Data are means ± SE
(n = 4 to 6 for each condition).
|
|
We next determined the effects of PKC-
on NF-
B-dependent ICAM-1
promoter activity. We cotransfected the wild-type (ICAM-1LUC) or
NF-
B mutant (ICAM-1NF-
BmutLUC) versions of
the ICAM-1 promoter-luciferase reporter gene construct with the
PKC-
wt or PKC-
mut
in HPAE cells and then determined luciferase activity. As shown in Fig.
9B, expression of PKC-
mut
prevented the TNF-
-induced ICAM-1 promoter activity, whereas overexpression of PKC-
wt significantly
augmented the TNF-
response. These data indicate the critical role
of PKC-
in activating the ICAM-1 promoter. Mutation of the
downstream NF-
B site prevented the TNF-
-induced ICAM-1 promoter
activation (Fig. 9B, inset). Moreover, the
expression of PKC-
mut or overexpression of
PKC-
wt failed to modify the TNF-
response
(Fig. 9B, inset). These data indicate that
PKC-
activates ICAM-1 promoter through an NF-
B-dependent pathway.
To further address the role of PKC-
in the mechanism of the
response, we showed that the antisense oligonucleotide to PKC-
prevented TNF-
-induced ICAM-1 mRNA expression (by ~50%), whereas
the sense oligonucleotide had no effect (Fig.
10).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 10.
Antisense oligonucleotide to PKC- prevents ICAM-1
mRNA expression. HPAE cells were transfected with indicated
concentrations of antisense oligonucleotide to PKC- or PKC- as
described in METHODS. After 36-48 h, cells were
stimulated for 2 h with TNF- (100 U/ml). Total RNA was isolated
and analyzed by Northern hybridization with a human ICAM-1 cDNA, which
hybridizes to a 3.3-kb transcript. Blots were stripped and reprobed to
determine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA
expression as a measure of RNA loading. A: autoradiogram.
B: relative intensities of ICAM-1 mRNA signals. Results are
representative of 3 experiments.
|
|
 |
DISCUSSION |
The PKC isozymes are structurally related proteins having
different cofactor and substrate specificities (15, 24).
On the basis of their ability to phosphorylate effector proteins at
serine and threonine residues, they can signal diverse responses in a
cell- and stimulus-specific manner (18, 21). In a previous study, we showed that PKC activation induces the generation of oxidants
in endothelial cells, which in turn mediates the activation of NF-
B
and transcription of ICAM-1 gene (29). In the present study, we have identified the atypical endothelial PKC isozyme, PKC-
, in transducing the TNF-
-induced oxidant generation and the
downstream activation of NF-
B and ICAM-1 transcription.
We used the approaches described below to address the contributions of
PKC-
, -
, -
, and -
isozymes, which are abundantly expressed
in HPAE cells, in mediating the TNF-
-activated responses. To address
the role of PKC-
, -
, and -
isozymes in mediating oxidant
production following TNF-
challenge, we depleted these isozymes by
exposing HPAE cells to phorbol ester for 24 h in the standard
manner. To study the role of the phorbol ester-insensitive aPKC
isozyme, PKC-
, we used the antisense oligonucleotide, which inhibits
synthesis by binding to the translation initiation codon of PKC-
mRNA (10, 11). We showed that depletion of cPKCs and
nPKCs failed to prevent TNF-
-induced oxidant production in endothelial cells, whereas the inhibition of PKC-
synthesis by the
antisense oligonucleotide prevented the oxidant production. Thus the
results indicate an important role of the aPKC isozyme PKC-
in
activating oxidant generation induced by TNF-
stimulation of
endothelial cells. Generation of oxidants following TNF-
stimulation may be mediated by the activation of endothelial NADPH oxidase (1). Components of the NADPH oxidase complex were present
in endothelial cells, and stimulation with TNF-
resulted in their translocation and activation (A. Rahman, unpublished results); thus a
possible mechanism of oxidant generation may involve PKC-
-induced phosphorylation of the p47 subunit of NADPH oxidase, resulting in its
translocation to the plasma membrane where it interacts with the
cytochromeb558 to form the active
NADPH oxidase complex (9).
Because the present results implicate PKC-
in the mechanism of
TNF-
-induced oxidant production, we next addressed the possibility that the oxidant production mediated by PKC-
was responsible for the
NF-
B activation and ICAM-1 gene transcription in endothelial cells.
We showed that inhibition of PKC-
expression using the antisense
oligonucleotide prevented both NF-
B activation and ICAM-1
transcription, whereas in control experiments, the sense oligonucleotides had no inhibitory effect. Moreover, expression of the
dominant negative form of PKC-
prevented TNF-
-induced NF-
B
activity, whereas the expression of dominant negative forms of PKC-
,
-
, and -
isozymes failed to prevent the response. The expression
of the PKC-
dominant negative mutant also prevented ICAM-1 promoter
activity in response to TNF-
challenge, and overexpression of
wild-type PKC-
augmented this response. Mutation of the downstream NF-
B binding site on the ICAM-1 promoter prevented the
TNF-
-induced ICAM-1 promoter activation. Taken together, these
results indicate the essential role of the PKC-
isozyme in mediating
TNF-
-induced NF-
B activation and ICAM-1 transcription.
In the present study, we also addressed the mechanism of the
PKC-
-induced activation of NF-
B. The results showed that
inhibition of PKC-
expression prevented I
B
degradation and
NF-
B binding to the ICAM-1 promoter induced by TNF-
. These
results can be explained on the basis of PKC-
-induced oxidant
generation promoting the degradation of I
B
and activation of
NF-
B (33, 34). Another possibility is that PKC-
can
directly phosphorylate NF-
B p65, since the dominant negative mutant
of PKC-
was shown to inhibit NF-
B p65 phosphorylation, resulting
in the loss of NF-
B p65 transcriptional activity (2).
Hence, PKC-
may induce NF-
B activity by 1) activation
of the oxidant signaling pathway and downstream induction of NF-
B
binding to ICAM-1 promoter and 2) direct phosphorylation of
NF-
B p65 resulting in increased transcriptional activity of the
bound NF-
B.
In summary, we have shown that 1) phorbol ester-induced
depletion of cPKC and nPKC isozymes failed to prevent TNF-
-induced oxidant generation and NF-
B activation; 2) inhibition of
PKC-
synthesis by the antisense oligonucleotide prevented
TNF-
-induced oxidant generation, NF-
B activation, and ICAM-1 gene
transcription; 3) expression of the dominant negative mutant
form of PKC-
inhibited the TNF-
-induced NF-
B activity; and
4) expression of the dominant negative mutant form of
PKC-
also inhibited the TNF-
-induced ICAM-1 promoter activity,
whereas overexpression of wild-type PKC-
augmented the response.
Thus the aPKC isozyme PKC-
plays a critical role in mediating
TNF-
-induced oxidant generation, NF-
B activation, and resultant
ICAM-1 gene transcription in endothelial cells. The results point to
the endothelial cell PKC-
as an important target in preventing the
proinflammatory effects of TNF-
such as ICAM-1 expression and
neutrophil adhesion.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grants HL-27016, HL-46350, and HL-45638.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. Rahman, Dept. Pharmacology (M/C 868), Univ. of Illinois College of
Medicine, 835 South Wolcott Ave., Chicago, IL 60612-7343 (E-mail: ARahman{at}uic.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 20 December 1999; accepted in final form 27 April 2000.
 |
REFERENCES |
1.
Al-Mehdi, AB,
Zhao G,
Dodia C,
Tozawa K,
Costa K,
Muzykantov V,
Ross C,
Blecha F,
Dinauer M,
and
Fisher AB.
Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+.
Circ Res
83:
730-737,
1998[Abstract/Free Full Text].
2.
Anrather, J,
Csizmadia V,
Sores MP,
and
Winkler H.
Regulation of NF-
B RelA phosphorylation and transcriptional activity by p21(ras) and protein kinase C zeta in primary endothelial cells.
J Biol Chem
274:
13594-13603,
1999[Abstract/Free Full Text].
3.
Baldwin, AS.
The NF-
B and I
B proteins: new discoveries and insights.
Annu Rev Immunol
14:
649-683,
1996[ISI][Medline].
4.
Berra, E,
Diaz-Meco MT,
Lozano J,
Frutos S,
Municio MM,
Sanches P,
Sanz L,
and
Moscat J.
Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta.
EMBO J
14:
6157-6163,
1995[Abstract].
5.
Bours, V,
Burd PR,
Brown K,
Villlobos J,
Park S,
Ryseck RP,
Bravo R,
Kelly K,
and
Siebenlist U.
A novel mitogen-inducible gene product related to p50/p105-NF-
B participates in transactivation through a
B site.
Mol Cell Biol
12:
685-695,
1992[Abstract].
6.
Brown, K,
Gerstberger S,
Carlson L,
Franzoso G,
and
Siebenlist U.
Control of I
B
proteolysis by site-specific, signal-induced phosphorylation.
Science
267:
1485-1488,
1995[ISI][Medline].
7.
Chen, ZJ,
Parent L,
and
Maniatis T.
Site-specific phosphorylation of I
B
by a novel ubiquitination-dependent protein kinase activity.
Cell
84:
853-862,
1996[ISI][Medline].
8.
Dean, NM,
McKay R,
Condon TP,
and
Bennett CF.
Inhibition of protein kinase C-
in human A549 cells by antisense oligonucleotide inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters.
J Biol Chem
269:
16416-16424,
1994[Abstract/Free Full Text].
9.
DeLeo, FR,
and
Quinn MT.
Assembly of phagocyte NADPH oxidase: molecular interaction of oxidase proteins.
J Leukoc Biol
60:
677-691,
1996[Abstract].
10.
Dominguez, I,
Diaz-Meco T,
Municio MM,
Berra E,
Garcia de Harreros A,
Cornet ME,
Sanz L,
and
Moscat J.
Evidence for a role of protein kinase C
subspecies in maturation of Xenopus laevis oocytes.
Mol Cell Biol
12:
3776-3783,
1992[Abstract].
11.
Folgueira, L,
McElhinny JA,
Bren GD,
MacMorran WS,
Diaz-Meco MT,
Moscat J,
and
Paya V.
Protein kinase C-
mediates NF-
B activation in human immunodeficiency virus-infected monocytes.
J Virol
70:
223-231,
1996[Abstract].
12.
Ghosh, S,
Gifford AM,
Riviere LR,
Tempst P,
Nolan GP,
and
Baltimore D.
Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal.
Cell
62:
1019-1029,
1990[ISI][Medline].
13.
Hofmann, J.
The potential for isoenzyme-selective modulation of protein kinase C.
FASEB J
11:
649-669,
1997[Abstract/Free Full Text].
14.
Hou, J,
Baichwal V,
and
Cao Z.
Regulatory elements and transcription factors controlling basal and cytokine-induced expression of gene encoding ICAM-1.
Proc Natl Acad Sci USA
91:
11641-11645,
1994[Abstract/Free Full Text].
15.
Hug, H,
and
Sarre TF.
Protein kinase C isoenzymes: divergence in signal transduction?
Biochem J
291:
329-343,
1993[ISI][Medline].
16.
Jaken, S.
Protein kinase C isoezymes and substrates.
Curr Opin Cell Biol
8:
168-173,
1996[ISI][Medline].
17.
Kieran, M,
Blank V,
Logeat F,
Vandekerckhove J,
Lottspeich F,
Le Bail O,
Urban MB,
Kourilsky P,
Baeuerle PA,
and
Israel A.
The DNA binding subunit of NF-kappa B is identical to factor KBF1 and homologous to the rel oncogene product.
Cell
62:
1007-1018,
1990[ISI][Medline].
18.
Korchak, HM,
Rossi MW,
and
Kilpatrick LE.
Selective role for
-protein kinase C in signaling for O2
· generation but not degranulation or adherence in differentiated HL60 cells.
J Biol Chem
273:
27292-27299,
1998[Abstract/Free Full Text].
19.
Larivee, P,
Levine S,
Martinez A,
Wu T,
Logan C,
and
Shelhamur JH.
Platelet activating factor induces airway mucin release via activation of protein kinase C: evidence for translocation of protein kinase C to membranes.
Am J Respir Cell Mol Biol
11:
199-205,
1994[Abstract].
20.
Ledebur, HC,
and
Parks TP.
Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells: essential roles of a variant NF-
B site and p65 homodimers.
J Biol Chem
270:
933-943,
1995[Abstract/Free Full Text].
21.
Li, Q,
Subbulakshmi V,
Fields AP,
Murray NR,
and
Cathcart MK.
Protein kinase C-
regulates human monocyte O2
· production and low density lipoprotein lipid oxidation.
J Biol Chem
274:
3764-3771,
1999[Abstract/Free Full Text].
22.
Lopata, MA,
Cleveland DW,
and
Sollner-Webb B.
High level of transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.
Nucleic Acids Res
12:
5707-5717,
1984[Abstract].
23.
Marte, B,
Meyer T,
Stabel S,
Standke GJ,
Jaken S,
Fabbro D,
and
Hynes NE.
Protein kinase C and mammary cell differentiation: involvement of protein kinase alpha in the induction of beta-casein expression.
Cell Growth Differ
5:
239-247,
1994[Abstract].
24.
Mellor, H,
and
Parker PJ.
The extended protein kinase C superfamily.
Biochem J
332:
281-292,
1998[ISI][Medline].
25.
Nolan, GP,
Ghosh S,
Liou HC,
Tempest P,
and
Baltimore D.
DNA binding and I
B inhibition of the cloned p65 subunit of NF-
B, a rel-related polypeptide.
Cell
64:
961-969,
1991[ISI][Medline].
26.
Ogita, K,
Miyamoto S,
Yamaguchi K,
Koide H,
Fujisawa N,
Kikkawa U,
Sahara S,
Fukumi Y,
and
Nishizuka Y.
Isolation and characterization of delta subspecies of protein kinase C from rat brain.
Proc Natl Acad Sci USA
89:
1592-1596,
1992[Abstract].
27.
Rahman, A,
Kefer J,
Bando M,
Niles WD,
and
Malik AB.
E-selectin expression in human endothelial cells by TNF-
-induced oxidant generation and NF-
B activation.
Am J Physiol Lung Cell Mol Physiol
275:
L533-L544,
1998[Abstract/Free Full Text].
28.
Rahman, A,
Anwar KN,
True AL,
and
Malik AB.
Thrombin-induced p65 homodimer binding to downstream NF-
B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion.
J Immunol
162:
5466-5476,
1999[Abstract/Free Full Text].
29.
Rahman, A,
Bando M,
Kefer J,
Anwar KN,
and
Malik AB.
Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene transcription.
Mol Pharmacol
55:
575-583,
1999[Abstract/Free Full Text].
30.
Ruben, SM,
Dillon PJ,
Schreck R,
Henkel T,
Chen CH,
Maher M,
Baeuerle PA,
and
Rosen CA.
Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B (Abstract).
Science
254:
11,
1991[ISI][Medline].
31.
Ryseck, RP,
Bull P,
Takamiya M,
Bours V,
Siebenlist U,
Dobrzanksi P,
and
Bravo R.
RelB, a new Rel family transcription activator that can interact with p50-NF-kappa B.
Mol Cell Biol
12:
674-684,
1992[Abstract].
32.
Schmid, RM,
Perkins ND,
Duckett CS,
Andrews PC,
and
Nabel GJ.
Cloning of an NF-kappa B subunit which stimulates HIV transcription in synergy with p65.
Nature
352:
733-736,
1991[ISI][Medline].
33.
Schreck, R,
Albermann K,
and
Baeuerle PA.
Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells.
Free Radic Res Commun
17:
221-237,
1992[ISI][Medline].
34.
Schreck, R,
Rieber P,
and
Baeuerle PA.
Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-
B transcription factor and HIV-1.
EMBO J
10:
2247-2258,
1991[Abstract].
35.
Seynaeve, CM,
Kazanietz M,
Blumberg PM,
Sausville EA,
and
Worland PJ.
Differential inhibition of protein kinase C isoenzymes by UCN-01, a staurosporine analogue.
Mol Pharmacol
45:
1207-1214,
1994[Abstract].
36.
Smith, CW,
Marlin SD,
Rothlein R,
Toman C,
and
Anderson DC.
Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecules-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro.
J Clin Invest
83:
2008-2017,
1989[ISI][Medline].
37.
Smith, CW,
Rothlein R,
Hughes BJ,
Mariscalo MM,
Rudloff HE,
Schmalstieg FC,
and
Anderson FC.
Recognition of an endothelial determinant for CD18-dependent human neutrophil adherence and transendothelial migration.
J Clin Invest
82:
1746-1756,
1988[ISI][Medline].
38.
Soh, JW,
Lee EH,
Prywes R,
and
Weinstein IB.
Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response elememt.
Mol Cell Biol
19:
1313-1324,
1999[Abstract/Free Full Text].
39.
Staunton, DE,
Marlin SD,
Stratowa C,
Dustin ML,
and
Springer TA.
Primary structure of ICAM-1 demonstrates interaction between members of immunoglobulin and integrin supergene families.
Cell
54:
925-933,
1988.
40.
Traenckner, EBM,
Pahl HL,
Henkel T,
Schmidt KN,
Wilk S,
and
Baeuerle PA.
Phosphorylation of human I
B
on serines 32 and 36 controls I
B
proteolysis and NF-
B activation in response to diverse stimuli.
EMBO J
14:
2876-2883,
1995[Abstract].
41.
Whiteside, ST,
and
Israel A.
I kappa B proteins: structure, function and regulation.
Semin Cancer Biol
8:
75-82,
1997[ISI][Medline].
42.
Wilhelmsen, KC,
Eggleton K,
and
Temin H.
Nucleic acid sequences of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homologue, the proto-oncogene c-rel.
J Virol
52:
172-182,
1984[ISI][Medline].
Am J Physiol Cell Physiol 279(4):C906-C914
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society