1 Research Service, Stratton Veterans Affairs Medical Center, and 2 Vascular Biology Research Group, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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We tested the hypothesis that protein
kinase C- (PKC-
) mediates tumor necrosis factor-
(TNF-
)-induced alterations in permeability of pulmonary microvessel
endothelial monolayers (PEM). The permeability of PEM was assessed by
the clearance rate of Evans blue-labeled albumin. PEM lysates were
analyzed for PKC-
mRNA (Northern cDNA blot), protein (Western
immunoblot), and activity (translocation and phosphorylation of
myristoylated arginine-rich C kinase substrate). Incubation of PEM with
TNF-
(1,000 U/ml) for 4 h resulted in increases in 1)
PKC-
protein, 2) cytoskeletal-associated PKC-
, 3)
PKC-
activity, and 4) permeability to albumin. The
TNF-
-induced increase in PKC-
protein, PKC-
activity, and
permeability was prevented by a 4-h pretreatment with PKC-
antisense
oligonucleotide but not by the scrambled nonsense oligonucleotide. The
TNF-
-induced increase in permeability to albumin was prevented by
myristoylated protein kinase C inhibitor (an inhibitor of PKC-
/
,
100 µM) and calphostin (an inhibitor of the classic and novel PKC
isotypes, 200 nM). The treatment with calphostin from 0.5 to 3.0 h
after TNF-
still prevented barrier dysfunction induced by 4 h of
TNF-
treatment. The data indicate that prolonged activation of
PKC-
, maintained by a translation-dependent pool of PKC-
protein,
mediates TNF-
-induced increases in endothelial permeability in PEM.
antisense; edema; messenger ribonucleic acid; transcription
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INTRODUCTION |
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PROTEIN KINASE C (PKC) is a serine/threonine kinase
characterized by 11 different PKC isotypes, which differ in substrate utilization and mechanisms of activation (5, 29, 34, 35). The classical
group includes the ,
1,2, and
isotypes. The activation of the classical isotypes is calcium and diacylglycerol dependent. The novel group includes the
,
,
, and
isotypes, the activation of which is calcium independent. The atypical
group includes the
,
and
isotypes. The mechanism of
activation of the atypical group is independent of both calcium and
diacylglycerol and involves other lipid-dependent pathways (35). The
literature indicates that the prolonged activation (~2 h) of PKC is
associated with the proteolytic degradation of PKC (8, 29, 37). Thus maintenance of the pool of PKC protein is necessary to support the
continued activated state of PKC (2, 6, 18).
Tumor necrosis factor- (TNF-
) is a mediator of sepsis syndrome
and the adult respiratory distress syndrome (2, 22, 40). TNF-
induces an increase in pulmonary vascular permeability in vivo (22) in
the isolated lung (15) and in pulmonary microvessel endothelial
monolayers (17). We previously demonstrated in lungs of guinea pig (36)
and bovine pulmonary artery endothelium (20, 32) that superoxide
generation, alteration in nitric oxide, glutathione depletion,
glutathione oxidation, increased vascular pressures and resistance, and
pulmonary edema in response to TNF-
are mediated by the activation
of PKC. However, the role of a specific isotype of PKC in the above
responses to TNF-
were not studied. The isotypes PKC-
and PKC-
are the most prevalent in endothelium (34, 35), and the isotypes
,
,
,
and
are not consistently detected (34, 35). We showed
that TNF-
induces the translocation of PKC-
and/or PKC-
in
pulmonary artery endothelium (11). Therefore, in the present study, the
role of PKC-
in the TNF-
-induced increase in pulmonary
microvascular endothelial permeability was investigated.
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MATERIALS AND METHODS |
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Reagents
All reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted.Pulmonary Microvessel Endothelial Cell Culture
Bovine pulmonary microvessel endothelial cells derived from fresh calf lungs were obtained at 4th passage (Vec Technologies; Rensselaer, NY) and serially cultured from 4 to 12 passages (10, 11, 20, 32). The culture medium contained DMEM (GIBCO BRL; Grand Island, NY) supplemented with 20% fetal bovine serum (HyClone Laboratories; Logan, UT), 15 µg/ml endothelial cell growth supplement (Upstate Biotechnology; Lake Placid, NY), and 1% nonessential amino acids (GIBCO BRL).Pulmonary microvessel endothelial monolayers (PEM) were maintained in 5% CO2 plus humidified air at 37°C and reached confluence within two to three population doublings, which took 4-5 days. The preparations were identified as endothelial monolayers by 1) the characteristic "cobblestone" appearance using contrast microscopy, 2) the presence of factor VIII-related antigen (indirect immunofluorescence), 3) the uptake of acylated low-density lipoproteins, and 4) the absence of smooth muscle actin (indirect immunofluorescence).
Assay of Endothelial Permeability
Polycarbonate micropore membranes (13-mm diameter, 0.8-µm pore size; Corning Costar; Cambridge, MA) were gelatinized (type II calf skin gelatin) as previously described (10, 26), mounted on plastic cylinders (9-mm ID; Adaps; Dedham, MA), and sterilized by ultraviolet light for 12-24 h. Endothelial cells (2 × 105 in 0.50 ml of DMEM) were then seeded to the gelatinized membranes and cultured for 3-5 days (37°C, 5% CO2) to allow the cells to be confluent.The experimental apparatus for the study of transendothelial transport in the absence of hydrostatic and oncotic pressure gradients has been described (10, 26). In brief, the system consisted of two compartments separated by a microporous polycarbonate membrane lined with the endothelial cell monolayer as previously described. The luminal (upper) compartment (0.7 ml) was suspended in the abluminal (lower) compartment (25 ml). The lower compartment was stirred continuously for complete mixing. The entire system was kept in a water bath at a constant temperature of 37°C. The fluid height in both compartments was the same to eliminate convective flux.
Endothelial permeability was characterized by the clearance rate of albumin labeled with Evans blue dye using our adaptation (10) of the original technique described by Patterson et al. (31). Hanks' balanced salt solution (HBSS) containing 0.5% BSA and 20 mM HEPES buffer was used on both sides of the monolayer. The albumin was labeled with Evans blue dye by addition (20 µl) of stock Evans blue (20 mg/ml in HBSS) to the upper compartment (680 µl). The absorbance of free Evans blue in the luminal and abluminal compartments was always <1% of the total absorbance of Evans blue. At the beginning of each study, an upper compartment sample (700 µl) was diluted 1:100 to determine the initial absorbance of the luminal compartment. Samples (300 µl) were taken from the lower compartment (abluminal) every 5 min for 60 min. The absorbance (620 nm) of the samples was measured in a microplate spectrophotometer (Molecular Devices; Sunnyvale, CA). The clearance rate of Evans blue albumin was determined by least-squares linear regression between 10 and 60 min for the control and experimental groups (39).
Assay of Cell Viability
Trypan blue exclusion. PEM were seeded (2 × 106 cells/well) and grown until confluent in 100-mm well dishes. After respective drug procedures, PEM were washed with PBS (2 ml) followed by treatment with 0.05% trypsin (1 ml) for 1.0 min at 37°C. Then the cells were washed and suspended in PBS (5 ml). An aliquot of cell suspension (50 µl) was combined with 0.4% trypan blue (50 µl) for 3 min. Then 20 µl of the mixture were counted for cells using a hemocytometer. Cell viability was defined by the following formula: cell viability = (cells excluding trypan blue/total cells) × 100.Cell number. Total cell number of PEM was determined as above in conjunction with the trypan exclusion studies.
Detection of PKC- mRNA
Northern blot. RNA was electrophoresed (8-12 µg/lane) in agarose (1.2%)-formaldehyde (0.41 M) gels (0.625 cm thick) for ~1.5 h at 75 V and 2 A. RNA was transferred to nylon membranes [Gene Screen Plus NEN (Boston, MA) or Hybond N+, Amersham (Arlington Heights, IL)] via downward capillary blotting for ~1.5 h with Northern/Southern Transfer Solution (Molecular Research Center). The membranes were then either baked for 20 min at 80°C or air-dried overnight at room temperature to fix the RNA to the membranes. Before hybridization, the membranes were stained with methylene blue (Molecular Research Center), scanned, and digitized to confirm equal loading and/or transfer.
Hybridization probes. A plasmid constructed for bovine PKC-
(pbPKC-
2, ATCC 37705) with a 0.531-kb cDNA insert was used (15, 34).
The plasmids were grown in Escherichia coli, cut using the
restriction enzyme EcoR I (per ATCC bulletin for plasmid 37705) with the resultant nucleotide inserts purified using agarose gel electrophoresis before radiolabeling. The probes were labeled with
either [
-32P]dCTP (Amersham) or digoxigenin
(Dig)-11-dUTP (Boehringer Mannheim; Indianapolis, IN) using
random-primed labeling reactions.
Hybridization. Membranes were prehybridized for 60 min at
65°C in hybridization solution [0.25 M
Na2HPO4, pH 7.2, 1 mM EDTA, 20% SDS, and 0.5%
of either Hammerstein Casein (VWR Scientific; Boston, MA) or blocking
reagent (Boehringer Mannheim)]. The membranes were
then hybridized for at least 16 h at 65°C with hybridization solution (20 ml/100 cm2 membrane) containing
106
counts · min1 · ml
1
of [32P]cDNA or 2.5 ng/ml Dig-cDNA. Membranes
were washed 3 × 20 min at 60°C with 20 mM
Na2HPO4 (pH 7.2) containing 1 mM EDTA and 1% SDS and either sealed in hybridization bags for autoradiography at
70°C ([32P]cDNA) or further
processed for chemiluminescent detection (Dig-cDNA, Engler-Blum
protocol) using CDP-Star (Boehringer Mannheim) as substrate.
Assay of PKC Protein
Preparation of PEM lysate fractions. PEM were seeded (1 × 106 cells) into 100-mm plastic culture dishes and incubated for 3-4 days until confluent (4 × 106 cells). After interventions, the PEM were washed on ice two times with ice-cold PBS containing 15 mM EDTA and 6 mM EGTA. Then 1 ml of cold extraction buffer A [150 mM Tris, 6 mM EGTA, 15 mM EDTA, 150 mM 2-mercaptoethanol (2-ME), 5 µg/mlThe whole cell lysate was centrifuged at 110,000 g for 1 h at
4°C. Supernatants (cytosolic fraction) were collected and
concentrated with Centricon filters (10 kDa cutoff) at 5,000 g
for 1 h at 4°C. The pellets were resuspended in 2 ml of cold
extraction buffer B (50 mM Tris, 2 mM EGTA, 5 mM EDTA, 0.1%
Triton X-100, 50 mM 2-ME, 1.67 µg/ml 1-antitrypsin, 10 µg/ml antipain, 0.5 KIU/ml aprotinin, 33.3 µM benzamidine, 50 nM
calpeptin, 5 µg/ml leupeptin, 1.67 µg/ml pepstatin A, and 1 mM
AEBSF) and sonicated 3 × 20 s each on ice. The suspensions were
centrifuged at 110,000 g for 1 h at 4°C, and the
supernatants (soluble membrane fraction) were collected and
concentrated with Centricon filters. The pellets were resuspended in
buffer C (0.2% SDS in buffer B) and sonicated 3 × 20 s each on ice. The suspensions were centrifuged at 16,000 g for 5 min at 4°C and the supernatants (insoluble
cytoskeletal) were collected and all samples were stored at
70°C.
Total protein. Lysate total protein concentrations were determined in diluted samples with Coomassie blue plus protein assay reagent (Pierce; Rockford, IL).
SDS-PAGE. The samples were diluted 1:1 in 2× Laemmli buffer containing 2-ME (10%) and boiled for 5 min. The lysate proteins were separated by SDS-PAGE on either 7.5 or 10% gels using standard procedures (Mini-Protean II Electrophoresis Cell Manual, Bio-Rad; Hercules, CA). All lanes were loaded so that each lane contained 8-12 µg of total protein.
Western blot. The polyacrylamide gels were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore; Bedford, MA) using standard procedures (Mini-Trans Blot Electrophoretic Transfer Cell Manual, Bio-Rad). The PVDF membranes were blocked with Blotto [5% nonfat dry milk in Tween 20-Tris buffered saline solution (TTBS; 0.1% Tween 20, 10 mM Tris, and 100 mM NaCl, pH 7.5)].
Primary and secondary antibodies. The rabbit polyclonal primary
IgG anti-PKC- antibody (1:20,000 dilution in Blotto), which recognizes amino acid residues 659
672 of PKC-
, was used. In addition, anti-PKC-
antibody was used to verify specificity of PKC-
antisense oligonucleotide. Goat anti-rabbit IgG conjugated to
alkaline phosphatase was used as the secondary antibody. All incubations of the membranes were done for 30 min at 37°C. The unbound antibodies were removed by washing five times with TTBS for 5 min at room temperature. The PVDF membranes were developed with
CDP-Star and exposed to X-omat AR, Ortho-G or BioMax ML (Kodak) film
for 0.5-15 min.
Quantitation of Autoradiographs
The autoradiographs were digitized by scanning (Scan Jet 4C with transparency adapter; Hewlett Packard Canberra; Downers Grove, IL), and the bands were quantified with Sigma Scan Pro (SPSS Scientific Software; San Rafael, CA) (10). Each band was analyzed by fill area (manual threshold 100) multiplied by intensity to give total intensity units. All Western blots were corrected for interblot density variability by dividing the density of each lane by the mean of the total densities for each blot. For the Northern blots, the total intensity units of each sample was divided by that of the 18S ribosomal band to correct for the total RNA load. There were 5-10 separate experiments for each group.Quantitation of PKC Activity
PKC activity in vitro using cell lysate was measured by the [Preparation of sample. PEM were seeded (400,000 cells/well) and
grown until confluent in 35-mm well dishes. After respective treatments, PEM were washed three times with ice-cold PBS (2 ml/well) and scraped with 400 µl of ice-cold homogenization buffer (10 mM
potassium phosphate, pH 6.8, 10 mM EDTA, 10 mM 2-ME, 15 µg/ml leupeptin, and 3 mM AEBSF). The cell suspensions were immediately snap-frozen in liquid nitrogen and stored at 70°C. To assay, cell suspensions were lysed on ice with an ultrasonicator (setting 6.5, 3 × 10 s), centrifuged at 16,000 g (5 min, 4°C), and
the supernatant was analyzed.
Activity of sample. The samples (8 µl) were added to wells in
every fourth row or column of a round-bottomed 96-well microplate. Then
20 µl of reaction mixture [300 µM MARCKS PSD peptide (Alexis; San
Diego, CA), 40 µM Tris, pH 7.4, 20 mM magnesium acetate, and 200 µM
[-32P]ATP (1,500 counts · min
1 · pmol
1,
Amersham)] were added to each sample with either an 8- or 12-channel pipette with adequate mixing and incubated for 12 min at room temperature. Each sample was also incubated with reaction mixture without MARCKS peptide for background phosphorylation measurement. The
reaction was terminated by spotting aliquots (15 µl) with a
multichannel pipette onto a grid of 1-in. squares drawn on a sheet of
phosphocellulose filter paper (Whatman P81, Fisher Scientific; Pittsburgh, PA). After drying, the filter paper was washed 3 × 20 min in phosphoric acid (75 mM), neutralized 15 min in sodium phosphate
buffer (100 mM, pH 7.4), and allowed to dry. The separated squares
containing spotted samples, as well as blank squares from the same
washed sheet, were placed in scintillation vials containing Ecoscint A
(10 ml, National Diagnostic; Atlanta, GA), and radioactivity was
measured using a beta-counter (Packard 2500 TR). Lysate total protein
concentrations were determined from the remaining supernatant with
Coomassie blue plus protein assay reagent.
In some studies, to verify (i.e., positive control) PKC-mediated
phosphorylation of the MARCKS, the PEM were treated with the pan-PKC
activator phorbol 12-myristate 13-acetate (PMA, 1 µM). In some
samples, myristoylated PKC inhibitor-(19-27) (MPKCI; 100 µM;
Calbiochem) was added to the cell suspension to verify that
phosphorylation activity was specific for PKC-. MPKCI contains the
pseudosubstrate sequence from PKC-
/
(42), and we cannot detect
PKC-
protein in our PEM lysate. The assay is specific for PKC
because 1) MARCKS contains a consensus sequence specific for
PKC-mediated phosphorylation (14), 2) MPKCI totally prevented the increase in activity in response to TNF-
, and 3) MPKCI
attenuated PMA (1 µM)-induced phosphorylation (20, 32) of MARCKS.
Treatments
PEM cultured in 60-mm dishes were used for the isolation of mRNA and proteins. PEM cultured in 35-mm dishes were used for isolation of PKC activity. PEM cultured in 13-mm wells were used for the permeability assay. The PEM were treated with TNF-TNF- and inactivated TNF-
. Highly purified recombinant
human TNF-
from E. coli (Genzyme; Cambridge, MA) with a
specific activity of 900 × 105 U/ml was used (10).
The TNF-
had less than 14 pg of endotoxin contamination per
106 units of TNF-
activity by standard Limulus
assay. To control for endotoxin contamination and the possible effects
of the TNF-
vehicle, TNF-
was heat inactivated by boiling for 45 min (10).
PKC inhibitors. The inhibitors used were calphostin C (200 nM)
and MPKCI (100 µM, Calbiochem). Calphostin C inhibits the
phosphorylation activity of the classic and novel isotypes of PKC by
binding to the regulatory diacylglycerol-binding domain (23).
Calphostin C was cotreated with TNF- or posttreated 0.5, 1.0, 2.0, or 3.0 h after TNF-
. As previously mentioned, MPKCI contains the
pseudosubstrate sequence from PKC-
/
and in addition is
myristolated (NH2 terminus) to allow intracellular
availability of the peptide (42).
Antisense oligonucleotides. Translation of PKC- RNA was
inhibited using a phosphorothioated PKC-
antisense oligonucleotide (Promega; Madison, WI) complementary to a region two bases upstream from the initiation codon of PKC-
[nucleotide 49 5'-GTC
CCT CGC CGC CTC CTG-3' nucleotide 32 (44)]. The control
oligonucleotide was the scrambled nonsense nucleotide (5'-TGC CTC
CGC GCC TCC CGT-3'). Specificity of the sequence to bovine
PKC-
and scrambled nonsense was verified by using the Entrez-GenBank
data base of the National Institutes of Health.
Transfection of oligonucleotides in PEM. Transfection of
oligonucleotides was accomplished by using the cationic liposome carrier Lipofectin (GIBCO BRL) using a protocol previously described by
Busuttil et al. (4), which indicated 100% uptake of the liposomes
within 2 h. A stock oligonucleotide solution (0.3 nmol/µl) was
diluted 1:10 with serum-free DMEM to a concentration of 0.03 nmol/µl.
The Lipofectin was prepared in serum-free DMEM at 10 µl/ml. The
Lipofectin (1.5 ml) was added to the PEM followed by 10 µl of the
oligonucleotides, for a final oligonucleotide concentration of 0.2 nmol/ml. The dish was swirled five times and incubated at 37°C for
4 h, a period over which the PKC- mRNA was reduced 75-85%
below baseline levels as indicated by Northern blot analysis. The
literature indicates that PKC-
protein has a long half-life (from 6 to >24 h), which requires that PKC-
mRNA is inhibited for extended
periods of time with antisense to achieve a reduction in constitutive
levels of protein (7). Indeed, an 18-h pretreatment with PKC-
antisense induced an ~80% reduction in constitutive PKC-
(data
not shown); therefore, we chose a short-term 4-h antisense incubation
period because our goal was to prevent the TNF-
-induced increase in
PKC-
protein and to not affect the constitutive PKC-
protein levels.
Statistics
A one-way ANOVA was used to compare values among the treatments. If significance among treatments was noted, a Bonferroni multiple comparison test was used to determine significant differences among the groups (39). A t-test was used when appropriate. Each PEM well and flask represents a single experiment. All data are reported as means ± SE. Significance was at P < 0.05. There are 5-10 samples per group in all studies. ![]() |
RESULTS |
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Cell Viability of PEM: Trypan Blue Exclusion and Cell Counts
We previously showed that TNF-TNF- Induces Increases in PKC-
Protein
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Figure 1B shows the time course for the change in relative
density units for the 80-kDa PKC- and 50-kDa PKM-
proteins in total lysate at 0.5, 2.0, and 4.0 h after TNF-
. TNF-
for 0.5 h
induced little change in 80-kDa PKC-
but significantly decreased 50-kDa PKM-
. There were increases in both the 80-kDa PKC-
and 50-kDa PKM-
proteins after 2.0 and 4.0 h of TNF-
treatment. The
data indicate that TNF-
treatment for 4 h induces the protracted increase of total 80-kDa PKC-
and PKM-
in PEM lysate.
TNF- Induces Translocation of 80-kDa
PKC-
|
TNF- Induces Membrane and Cytoskeletal Association
of 50-kDa PKM-
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Antisense to PKC- Prevents
TNF-
-Induced Increase in PKC-
Protein
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Figure 4B shows the graphic quantification of the densities of
the bands for the 80-kDa PKC- protein. There was a significant increase in 80-kDa PKC-
at 4.0 h in the TNF-
and nonsense (Non) + TNF-
groups compared with that in the control group. There was no
change in PKC-
in the antisense-treated groups and in the Non group
at 4 h. These data indicate that TNF-
induces an increase in the
pool of PKC-
protein at 4 h that is specifically inhibited by
PKC-
antisense.
TNF- Induces Activation of PKC-
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Figure 5B shows lysate activity isolated from PEM that were
treated with vehicle, 0.5-h PMA or 4.0-h PMA. In paired studies, MPKCI
was added to the lysate mixture. Note that the scale for the PKC
activity in Fig. 5B is five times the scale for the PKC activity in Fig. 5A. There was an increase in PKC activity that progressively declined in response to PMA between 0.5-h PMA and 4.0-h
PMA. Thus the data confirm adequate positive control assay conditions
and that long-term treatment with PMA will eventually result in
depletion of PKC activity (11, 25, 26). The increase in PKC activity in
response to 0.5-h PMA was only partially inhibited by MPKCI; however,
the increase in PKC activity in response to 4.0-h PMA was completely
inhibited by MPKCI. The data derived from the 0.5-h MPKCI + PMA group
confirm that the 0.5-h culturing condition did not negatively affect
the ability of MPKCI to inhibit PKC activity. Interestingly, the
complete inhibition of PKC activity in the 4.0-h MPKCI + PMA group
suggests, similar to TNF-, that PKC-
is the primary isotype that
mediates prolonged PKC activation in PEM.
Figure 6 shows lysate activity isolated
from PEM that were treated with PKC- antisense or
scrambled-nonsense, followed by treatment with vehicle or TNF-
for 4 h. There were significant increases in 32P activity in
MARCKS peptide in the TNF-
and Non + TNF-
groups compared with
the activity in the control group. There was no increase in activity in
the antisense (Anti) + TNF-
group compared with the
control group. The activity values in the Anti and Anti + TNF-
groups were less than the values in the TNF-
group. Moreover, initial studies (n = 2) indicate that neither TNF-
nor
PKC-
antisense had an effect on the phosphorylation of the
PKC-
-specific substrate (Calbiochem)
H-Glu-Arg-Met-Arg-Pro-Arg-Lys-Arg-Gln-Gly-Ser-Val-Arg-Arg-Arg-Val-OH [(control) 162.5 pmol
[32P]ATP · min
1 · mg
1
vs. (TNF-
) 152.0 pmol
[32P]ATP · min
1 · mg
1
vs. (Anti) 142.0 pmol
[32P]ATP · min
1 · mg
1].
Thus the data indicate that PKC-
mediates the increases in PKC
activity in response to TNF-
.
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Antisense to PKC- Does Not Affect
PKC-
|
Inhibition of PKC- Prevents
TNF-
-Induced Increases in Albumin Clearance Rate in PEM
|
Prolonged PKC Activation Mediates TNF--Induced
Increases in Albumin Clearance Rate in PEM
|
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DISCUSSION |
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The present study indicates TNF--induced activation of PKC-
mediates the increase in protein permeability of PEM. PKC-
mediated
the increase in permeability because 1) TNF-
induced the
activity of PKC-
(i.e., assay of translocation and phosphorylation), 2) two structurally different PKC inhibitors, calphostin and
MPKCI, prevented the effects of TNF-
, and 3) antisense to
PKC-
prevented the effects of TNF-
. The data indicate that the
cellular effects of the PKC inhibitors used in the present study (i.e.,
calphostin, MPKCI, and PKC-
antisense) were specific for PKC
activity because they are structurally dissimilar, with three different
distinct mechanisms of action. In addition, the scrambled nonsense
oligonucleotides and the appropriate vehicles had no significant effect
on the response to TNF-
. Finally, there was similar cell viability
among all groups. We previously showed in vivo that TNF-
-induced
pulmonary edema was associated with activation of PKC and was prevented by the PKC inhibitor calphostin (36). Moreover, we (21) and others (12,
25, 26, 38, 43) have demonstrated in vitro that PKC activation
increases pulmonary arterial endothelial permeability and mediates
endothelial injury in response to inflammatory mediators such as
vascular endothelial growth factor (43), H2O2
(21), and thrombin (12, 25, 26, 38). The present data agree with the
results of Ferro et al. (10), Ishii et al. (17), and Goldblum et al.
(13), indicating an increase in permeability of pulmonary arterial
endothelial monolayers in response to TNF-
; however, the role of a
PKC isotype on endothelial permeability was not investigated. Thus this
study is the first to demonstrate a role for PKC-
, a specific PKC
isotype, during TNF-
-induced barrier dysfunction.
In the present study, TNF- induced the association of PKC-
and
PKM-
with the cytoskeleton. It is not clear what the mechanisms are
for activation of PKC-
or the identity of the targets for activated
PKC-
and PKM-
during the increase in endothelial permeability in
response to TNF-
. TNF-
has been shown to bind to the TNF-
receptor p55 and activate phosphatidylcholine (PC)-phospholipase C
(PLC) via the activity of the TNF-
receptor-associated death domain
in the mouse pre-B cell line 70Z/3 and the mouse fibrosarcoma cell
lines L929 and Wehi164 (27). The activation of PC-PLC will generate
diacylglycerol lipase (DAG) and inositol 1,4,5-trisphosphate, resulting
in release of calcium from the sarcoplasmic reticulum and activation of
PKC-
by binding of DAG and calcium to the regulatory domains of
PKC-
. The TNF-
-induced activation scenario
(TNF-
PLC
DAG + Ca2+) in concert with our
finding of sustained levels of PKC-
protein promotes the prolonged
increase in PKC-
activity in response to TNF-
. PKC is known to
phosphorylate substrates that are critical for maintenance of the
endothelial cytoskeleton such as caldesmon77 (38), myosin
light chain (6), vimentin (38), zonula occuden-associated proteins (9),
and MARCKS (14). MARCKS has been shown to directly cross-bridge
F-actin. The phosphorylation of MARCKS can alter its interaction with
actin, possibly resulting in a change in paracellular permeability (14,
33). The present study shows the sustained increased localization of
PKC-
with the cytoskeletal fraction, which supports the hypothesis
that prolonged endothelial barrier dysfunction is dependent on the
maintained PKC-
-induced alterations in cytoskeletal targets.
Moreover, the increase in the association of PKM-
with the
cytoskeleton may also contribute to the long-term effects of
phosphorylation of critical cytoskeletal targets.
In the present study, TNF- induced the increase in the association
of PKM-
with the membrane. PKC phosphorylates membrane-bound substrates such as NADPH oxidase (6), endothelial nitric oxide synthase
(eNOS) (16, 24, 43), and MARCKS (14). PKM-
can mediate the
phosphorylation of membrane-bound MARCKS, which precipitates barrier
dysfunction via activation of myosin light chain kinase (MLCK) (12) and
eNOS (16, 24, 43). Before TNF-
-induced PKC-
activation,
unphosphorylated membrane-bound MARCKS sequesters calmodulin, which results in suppression of activity of the calmodulin. In response to TNF-
, a possible pathway is PKM-
-mediated
phosphorylation of MARCKS, which induces the release of calmodulin,
resulting in increased activity of eNOS and MLCK. Goldblum et al. (13) demonstrated that TNF-
-induced barrier dysfunction was prevented by
stabilization of actin polymers using phallicidin, supporting a role
for MLCK and actinomyosin in barrier dysfunction (13). We showed that
PKC activation mediates TNF-
-induced alterations in nitric oxide
activity, peroxynitrite generation, and glutathione oxidation (20, 32,
36). Huang and Yuan (16) have shown that increases in microvascular
permeability in response to PMA were mediated by nitric oxide. Wu et
al. (43) have demonstrated vascular endothelial growth factor-induced
activation of PKC mediates nitric oxide-dependent venular
hyperpermeability. PKC-
/
has been shown to induce
transcription of eNOS in human vein endothelial cells (24). Finally,
activation of NADPH oxidase and superoxide generation can be mediated
by PKC (6, 28). Thus a downstream target for PKM-
activation are
pathways leading to generation of reactive nitrogen species via
membrane-bound eNOS and NADPH oxidase, which is consistent with our
previous work indicating that TNF-
-induced barrier dysfunction is
also mediated by nitric oxide (11, 19). The notion that PKC-
mediates the activation of eNOS and generation of nitric oxide during
endothelial dysfunction is presently under intense investigation in our laboratory.
The literature indicates that activation of PKC is associated with an
increased degradation rate of PKC; thus maintenance of the PKC-
protein pool would be required for the prolonged activation of PKC-
(8, 29, 37). PKC-
antisense treatment prevented the increase in
PKC-
protein, activity, and permeability in response to TNF-
,
indicating that increased translation of PKC-
mRNA into PKC-
protein is the probable mechanism for maintenance of the PKC-
protein pool. The increase in PKC-
protein was associated with a
significant but small increase in the pool of 3.5-kb PKC-
mRNA,
which supports the concept of both transcriptional and
posttranscriptional regulation of the PKC-
protein pool.
Interestingly, the 8.1-kb PKC-
RNA did not increase in response to
TNF-
, which suggests alternative initiation, termination, or
splicing events during transcription and posttranscription processing
as initially noted by Parker et al. (30). It is possible that the
8.1-kb RNA did not accumulate because it was quickly processed (e.g.,
spliced) into the 3.5-kb RNA. The associated level of degradation of
the 3.5-kb RNA allowed the 3.5-kb RNA to accumulate to detectable levels. Iwamoto et al. (18) demonstrated in T cells that anti-CD3 antibody induced an increase in PKC-
protein without a substantial change in PKC-
mRNA. Our demonstration for dependence on an ongoing translation-dependent pool of PKC-
protein to mediate a response is
in accord with results of other studies that indicate
translation-dependent PKC activity (1, 3). We could not detect a
consistent effect of TNF-
and oligonucleotides on the protein levels
of PKC-
or on the phosphorylation of the PKC-
-specific substrate.
However, considering the complexities of signal transduction pathways, the data do not rule out a possible downstream effect of PKC-
activity on the activity of other PKC isotypes (25, 41).
Our data are consistent with increased degradation of PKC- because
TNF-
induced an increase in the membrane and cytoskeletal association of PKM-
. The literature indicates that the calcium lipid-dependent protease calpain-µ degrades PKC-
to the
catalytically active PKM-
by cleaving off the regulatory domain.
Thus the increase in membrane- and cytoskeletal-associated PKM-
protein may be the primary site for the prolonged increase in PKC-
activity. In addition, PKM-
is downregulated by further degradation
by calpain-m (8, 37), which is consistent with the acute decrease in
membrane-associated PKM-
in the present study. The early
decrease in PKM-
probably occurs because the rate of PKM-
degradation is greater than the rate of synthesis of new PKC-
,
consistent with the notion that the pool of PKC-
must be maintained
for the prolonged activity of PKC-
.
The present study indicates the continued activation of PKC- is
required for the prolonged increase in permeability in response to
TNF-
because posttreatment with calphostin 3.0 h after TNF-
still
prevented the increase in permeability. Our data indicate the continued
activation of PKC-
in response to TNF-
because of the 1)
persistence of the in vitro phosphorylation of MARCKS that was
specifically inhibited by PKC-
antisense and MPKCI, 2)
translocation of PKC-
to the cytoskeleton, and 3)
association of PKM-
with the cytoskeleton and membrane. Ross and
Joyner (35) demonstrated the translocation of PKC-
to the membrane
in response to TNF-
in human umbilical vein endothelial cells as
opposed to the cytoskeleton in the present study; however, differences among cell types can explain the different translocation patterns between these studies. In addition, cotreatment and posttreatment with
calphostin from 0.5 to 2 h after TNF-
prevented the increase in
permeability to TNF-
, indicating that the early activation of PKC
(e.g., possibly PKC-
) is required for endothelial barrier dysfunction in response to TNF-
. Finally, PKC inhibition with MPKCI
did not affect basal levels of endothelial permeability, indicating
that PKC-
is not a primary determinant of constitutive barrier
function; however, other isoforms of PKC may indeed have a role in
constitutive barrier function.
In summary, our study indicates for the first time that TNF- induces
the prolonged dysfunction of the endothelial barrier, which is
dependent on the concomitant continued increase in PKC-
activity.
The protracted response to TNF-
is inhibited by PKC-
antisense,
indicating that the dynamic regulation of the PKC-
protein pool is a
primary factor in the response to TNF-
. The development of
strategies that target ongoing PKC-
activity may provide novel
directions for therapy of the systemic inflammation syndrome and
associated acute lung injury.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Julie White and Dr. Min-Fu Tsan for technical assistance and cooperation.
![]() |
FOOTNOTES |
---|
This work was supported by the Department of Veterans Affairs Medical Research Service Merit Review (A. Johnson and T. J. Ferro) and National Heart, Lung, and Blood Institute Grants RO1-HL-48406-07 (A. Johnson) and RO1-HL-59901-01 (A. Johnson). R. Clements was supported by a predoctoral training grant (National Institutes of Health T32-HL-07194) in the Department of Physiology and Cell Biology of Albany Medical College.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Johnson, 151, 113 Holland Ave., Stratton VA Medical Center, Albany, NY 12208 (E-mail: jmurd{at}msn.com).
Received 29 September 1999; accepted in final form 23 December 1999.
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