Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha

Thomas Ferro1, Paul Neumann2, Nancy Gertzberg2, Richard Clements2, and Arnold Johnson1,2

1 Research Service, Stratton Veterans Affairs Medical Center, and 2 Vascular Biology Research Group, Albany Medical College, Albany, New York 12208


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that protein kinase C-alpha (PKC-alpha ) mediates tumor necrosis factor-alpha (TNF-alpha )-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-alpha mRNA (Northern cDNA blot), protein (Western immunoblot), and activity (translocation and phosphorylation of myristoylated arginine-rich C kinase substrate). Incubation of PEM with TNF-alpha (1,000 U/ml) for 4 h resulted in increases in 1) PKC-alpha protein, 2) cytoskeletal-associated PKC-alpha , 3) PKC-alpha activity, and 4) permeability to albumin. The TNF-alpha -induced increase in PKC-alpha protein, PKC-alpha activity, and permeability was prevented by a 4-h pretreatment with PKC-alpha antisense oligonucleotide but not by the scrambled nonsense oligonucleotide. The TNF-alpha -induced increase in permeability to albumin was prevented by myristoylated protein kinase C inhibitor (an inhibitor of PKC-alpha /beta , 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-alpha still prevented barrier dysfunction induced by 4 h of TNF-alpha treatment. The data indicate that prolonged activation of PKC-alpha , maintained by a translation-dependent pool of PKC-alpha protein, mediates TNF-alpha -induced increases in endothelial permeability in PEM.

antisense; edema; messenger ribonucleic acid; transcription


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha , beta 1,2, and gamma  isotypes. The activation of the classical isotypes is calcium and diacylglycerol dependent. The novel group includes the epsilon , delta , eta , and theta  isotypes, the activation of which is calcium independent. The atypical group includes the iota , lambda  and zeta  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-alpha (TNF-alpha ) is a mediator of sepsis syndrome and the adult respiratory distress syndrome (2, 22, 40). TNF-alpha 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-alpha are mediated by the activation of PKC. However, the role of a specific isotype of PKC in the above responses to TNF-alpha were not studied. The isotypes PKC-alpha and PKC-epsilon are the most prevalent in endothelium (34, 35), and the isotypes beta , gamma , delta , zeta  and eta  are not consistently detected (34, 35). We showed that TNF-alpha induces the translocation of PKC-alpha and/or PKC-beta in pulmonary artery endothelium (11). Therefore, in the present study, the role of PKC-alpha in the TNF-alpha -induced increase in pulmonary microvascular endothelial permeability was investigated.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha mRNA

Isolation of RNA. RNA was extracted using TRI Reagent (Molecular Research Center; Cincinnati, OH). The phenolic layer of the extraction preparation containing cellular proteins was stored at 4°C for later purification. The total RNA was resuspended in stabilized formamide (FORMAzol, Molecular Research Center) and stored at -20°C before electrophoresis.

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-alpha (pbPKC-alpha 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 [alpha -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 · min-1 · 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/ml alpha 1-antitrypsin, 30 µg/ml antipain, 1.5 KIU/ml aprotinin, 100 µM benzamidine, 150 nM calpeptin, 15 µg/ml leupeptin, 5 µg/ml pepstatin A, and 3 mM 4-(2-aminoethyl)benzenesulfonylfluoride-HCl (AEBSF, Calbiochem; San Diego, CA)] was added to the cells. The dishes were scraped with a rubber policeman, and cells were removed to an ultracentrifuge tube. The wells were rinsed with an additional 1 ml of buffer A. The pooled samples were lysed on ice with an ultrasonicator, setting 6.5, 3 × 20 s each.

The 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 alpha 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-alpha antibody (1:20,000 dilution in Blotto), which recognizes amino acid residues 659---672 of PKC-alpha , was used. In addition, anti-PKC-zeta antibody was used to verify specificity of PKC-alpha 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 [gamma -32P]ATP-mediated phosphorylation of the peptide substrate myristoylated arginine-rich C kinase substrate (MARCKS) (14) using an original assay that we developed for this study.

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 [gamma -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-alpha . MPKCI contains the pseudosubstrate sequence from PKC-alpha /beta (42), and we cannot detect PKC-beta 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-alpha , 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-alpha (1,000 U/ml) with and without the PKC inhibitors before preparation for the assays. The lysis or assay buffer was added to the PEM after aspiration of the treated culture medium.

TNF-alpha and inactivated TNF-alpha . Highly purified recombinant human TNF-alpha from E. coli (Genzyme; Cambridge, MA) with a specific activity of 900 × 105 U/ml was used (10). The TNF-alpha had less than 14 pg of endotoxin contamination per 106 units of TNF-alpha activity by standard Limulus assay. To control for endotoxin contamination and the possible effects of the TNF-alpha vehicle, TNF-alpha 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-alpha or posttreated 0.5, 1.0, 2.0, or 3.0 h after TNF-alpha . As previously mentioned, MPKCI contains the pseudosubstrate sequence from PKC-alpha /beta and in addition is myristolated (NH2 terminus) to allow intracellular availability of the peptide (42).

Antisense oligonucleotides. Translation of PKC-alpha RNA was inhibited using a phosphorothioated PKC-alpha antisense oligonucleotide (Promega; Madison, WI) complementary to a region two bases upstream from the initiation codon of PKC-alpha [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-alpha 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-alpha mRNA was reduced 75-85% below baseline levels as indicated by Northern blot analysis. The literature indicates that PKC-alpha protein has a long half-life (from 6 to >24 h), which requires that PKC-alpha 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-alpha antisense induced an ~80% reduction in constitutive PKC-alpha (data not shown); therefore, we chose a short-term 4-h antisense incubation period because our goal was to prevent the TNF-alpha -induced increase in PKC-alpha protein and to not affect the constitutive PKC-alpha 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Viability of PEM: Trypan Blue Exclusion and Cell Counts

We previously showed that TNF-alpha and calphostin do not affect cell viability (20, 32). Treatment with oligonucleotides with or without TNF-alpha did not affect PEM viability because the level of cell counts and trypan blue exclusion was similar among the groups (data not shown).

TNF-alpha Induces Increases in PKC-alpha Protein

Figure 1A shows a representative Western blot for PKC-alpha protein in total lysate after TNF-alpha treatment for 4.0 h. There are two major bands in each lane corresponding to a molecular mass of 80 and 50 kDa. The 80- and 50-kDa densities represent the PKC-alpha native enzyme and the catalytically active degradation product PKM-alpha , respectively (8, 37).


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Fig. 1.   Tumor necrosis factor-alpha (TNF) causes increase in protein kinase C-alpha (PKC-alpha ) protein. Pulmonary microvessel endothelial monolayers (PEM) were coincubated with control (Con) vehicle or TNF for 0.5, 2.0, or 4 h. A: typical Western blot of total PEM lysate in control and 4.0-h TNF groups. B: mean data of relative quantities of 80-kDa PKC-alpha and 50-kDa PKM-alpha protein, which were based on densities of 80- and 50-kDa bands. Exp, experimental. * Different from control group.

Figure 1B shows the time course for the change in relative density units for the 80-kDa PKC-alpha and 50-kDa PKM-alpha proteins in total lysate at 0.5, 2.0, and 4.0 h after TNF-alpha . TNF-alpha for 0.5 h induced little change in 80-kDa PKC-alpha but significantly decreased 50-kDa PKM-alpha . There were increases in both the 80-kDa PKC-alpha and 50-kDa PKM-alpha proteins after 2.0 and 4.0 h of TNF-alpha treatment. The data indicate that TNF-alpha treatment for 4 h induces the protracted increase of total 80-kDa PKC-alpha and PKM-alpha in PEM lysate.

TNF-alpha Induces Translocation of 80-kDa PKC-alpha

Figure 2 shows the mean data for the proportions of PKC-alpha protein in membrane (Mem), cytoskeleton (Cytoskel), and cytosol (Cyto) after treatment with control vehicle or 4.0-h TNF-alpha . In the 4.0-h TNF-alpha group, there was a decrease of 80-kDa PKC-alpha in the membrane fraction that was associated with the increase in the cytosol fraction (Mem/Cyto 0.06 > Mem/Cyto 0.03, P < 0.05), i.e., a lesser ratio of membrane to cytosol compared with that in the control group. In the 4.0-h TNF-alpha group, there was an increase of 80-kDa PKC-alpha in the cytoskeletal fraction that was associated with a lesser increase in the cytosol fraction (Cytoskel/Cyto 0.01 < Cytoskel/Cyto 0.11, P < 0.05), i.e., a greater ratio of cytoskeletal to cytosol compared with that in the control group. The data indicate, similar to previous studies, that acute TNF-alpha -induced PKC activation is associated with translocation of 80-kDa PKC-alpha (8). The translocation is characterized by association of PKC-alpha with the cytoskeleton in parallel with a decrease in membrane PKC-alpha .


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Fig. 2.   PKC-alpha (80 kDa) translocates to cytoskeleton in response to TNF. PEM were coincubated with control vehicle or 4.0-h TNF-alpha . PKC-alpha (80 kDa) was assessed in membrane, cytoskeletal and cytosol fractions using immunoblot. Products of cytoskeletal/cytosol 80-kDa PKC-alpha  × total 80-kDa PKC-alpha and membrane/cytosol 80-kDa PKC-alpha  × total 80-kDa PKC-alpha are shown. * Different from control group.

TNF-alpha Induces Membrane and Cytoskeletal Association of 50-kDa PKM-alpha

Figure 3 shows the mean data for the proportions of 50-kDa PKM-alpha protein in membrane, cytoskeleton, and cytosol after treatment with control vehicle or 4.0-h TNF-alpha . In the 4.0-h TNF-alpha group, there was an increase of 50-kDa PKM-alpha in the membrane fraction that was associated with no change in the cytosol fraction (Mem/Cyto 0.3 < Mem/Cyto 1.0, P < 0.05), i.e., a greater ratio of membrane to cytosol compared with that in the control group. In the 4.0-h TNF-alpha group, there was an increase of 50-kDa PKM-alpha in the cytoskeletal fraction that was associated with no change in the cytosol fraction (Cytoskel/Cyto 0.20 < Cytoskel/Cyto 0.35, P < 0.05), i.e., a greater ratio of cytoskeleton to cytosol compared with that in the control group. The data indicate, similar to previous studies, that prolonged activation of PKC-alpha promotes an increase in 50-kDa PKM-alpha [i.e., the PKC-alpha degradation product, (8)] characterized by an increase of 50-kDa PKM-alpha in both the cytoskeletal and membrane compartments.


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Fig. 3.   PKM-alpha (50 kDa) increases in membrane and cytoskeleton in response to TNF. PEM were coincubated with control vehicle or 4.0-h TNF. PKM-alpha (50 kDa) was assessed in membrane, cytoskeletal, and cytosol fractions using immunoblot. Products of cytoskeletal/cytosol 50-kDa PKM-alpha  × total 50-kDa PKM-alpha and membrane/cytosol 50-kDa PKM-alpha  × total 50-kDa PKM-alpha are shown. * Different from control group.

Antisense to PKC-alpha Prevents TNF-alpha -Induced Increase in PKC-alpha Protein

Figure 4A shows a representative (n = 8) Northern blot for PKC-alpha mRNA after TNF-alpha treatment for 2 h. There are three demonstrable bands for each lane, corresponding to 8.1, 3.5, and 3.1 kb. The 8.1- and 3.5-kb densities represent the unprocessed RNA and the mRNA for PKC-alpha , respectively, whereas the 3.1-kb density represents probable binding of the probe to other PKC isoforms (5). The data indicate that there is a pool of 3.5-kb PKC-alpha mRNA that increases in response to 2.0 h of TNF-alpha treatment in PEM. There was no increase in 3.5-kb PKC-alpha mRNA after 0.5 and 4.0 h of TNF-alpha treatment (data not shown).


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Fig. 4.   Antisense to PKC-alpha prevents increase in PKC-alpha protein in response to TNF. PEM were incubated with control (CON) vehicle, antisense (Anti), or nonsense (Non) to PKC-alpha for 4 h, followed by incubation with control vehicle or TNF for 2 h. A: representative blot of 8 experiments depicting PKC-alpha mRNA quantified using Northern blot. B: graphic quantification of PKC-alpha protein levels (n = 6) after 4 h of TNF. * Different from control group.

Figure 4B shows the graphic quantification of the densities of the bands for the 80-kDa PKC-alpha protein. There was a significant increase in 80-kDa PKC-alpha at 4.0 h in the TNF-alpha and nonsense (Non) + TNF-alpha groups compared with that in the control group. There was no change in PKC-alpha in the antisense-treated groups and in the Non group at 4 h. These data indicate that TNF-alpha induces an increase in the pool of PKC-alpha protein at 4 h that is specifically inhibited by PKC-alpha antisense.

TNF-alpha Induces Activation of PKC-alpha

Figure 5A shows lysate activity isolated from PEM that were treated with vehicle, 0.5-h TNF-alpha or 4.0-h TNF-alpha . In separate paired studies, MPKCI was added to the lysate mixture. There was no change in PKC activity in response to 0.5-h TNF-alpha ; however, there was an increase in PKC activity in response to 4.0-h TNF-alpha . There were significant deceases in PKC activity in the MPKCI + TNF-alpha groups compared with that in the time-matched TNF-alpha groups. The data indicate that the isotype PKC-alpha mediates constitutive and TNF-alpha -induced increase in PKC activity because MARCKS contains the pseudosubstrate sequence from PKC-alpha /beta (42) and we cannot detect PKC-beta protein.


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Fig. 5.   TNF-induced increase in PKC activity is mediated by PKC-alpha . 32P activity of myristoylated arginine-rich C kinase substrate (MARCKS) peptide was assessed in lysate fractions of PEM. A: PEM that were coincubated with control vehicle, 0.5-h TNF or 4.0-h TNF. B: in separate positive control studies, PEM were incubated with control vehicle, 0.5-h phorbol 12-myristate 13-acetate (PMA) or 4.0-h PMA. In paired studies, effect of addition of myristoylated PKC inhibitor (MPKCI) to reaction mixture during assay of PKC was measured. Hatched oval, separate treatment time. * Different from control group. # Different from TNF group or PMA group.

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-alpha , that PKC-alpha is the primary isotype that mediates prolonged PKC activation in PEM.

Figure 6 shows lysate activity isolated from PEM that were treated with PKC-alpha antisense or scrambled-nonsense, followed by treatment with vehicle or TNF-alpha for 4 h. There were significant increases in 32P activity in MARCKS peptide in the TNF-alpha and Non + TNF-alpha groups compared with the activity in the control group. There was no increase in activity in the antisense (Anti) + TNF-alpha group compared with the control group. The activity values in the Anti and Anti + TNF-alpha groups were less than the values in the TNF-alpha group. Moreover, initial studies (n = 2) indicate that neither TNF-alpha nor PKC-alpha antisense had an effect on the phosphorylation of the PKC-epsilon -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-alpha ) 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-alpha mediates the increases in PKC activity in response to TNF-alpha .


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Fig. 6.   Antisense to PKC-alpha prevents TNF-induced increase in PKC-alpha activity. PEM were coincubated with control vehicle, antisense, or nonsense to PKC-alpha for 4 h, followed by incubation with control vehicle or TNF for 4.0 h. 32P activity of MARCKS peptide was assessed in lysate fractions of PEM. * Different from control group. # Different from TNF group.

Antisense to PKC-alpha Does Not Affect PKC-zeta

Figure 7 shows the graphic quantification of the densities of the band for the atypical isotype PKC-zeta . PEM were treated with PKC-alpha antisense or scrambled nonsense, followed by treatment with TNF-alpha or vehicle for 4.0 h. There was no significant change in PKC-zeta in any of the groups. These data indicate that PKC-alpha antisense does not cause nonspecific changes in PKC-zeta protein levels.


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Fig. 7.   Antisense to PKC-alpha does not affect PKC-zeta protein. PEM were incubated with control vehicle, antisense, or nonsense to PKC-alpha for 4 h, followed by incubation with control vehicle or TNF for 4 h. Graphic quantification of PKC-zeta protein levels after 4 h of TNF is shown.

Inhibition of PKC-alpha Prevents TNF-alpha -Induced Increases in Albumin Clearance Rate in PEM

Figure 8 shows the permeability response of PEM that were treated with MPKCI. In addition, Fig. 8 shows the permeability response of PEM that were transfected with antisense and scrambled nonsense to PKC-alpha for 4 h, followed by treatment with TNF-alpha or vehicle for 4 h. In the TNF-alpha and Non + TNF-alpha groups, there were significant increases in albumin clearance rate compared with the value in the control group. There was no change in albumin clearance rate in the Lipofectin, Anti, Non, MPKCI, MPKCI + TNF-alpha , and Anti + TNF-alpha groups. The data indicate that PKC-alpha mediates the increase in permeability in response to TNF-alpha .


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Fig. 8.   Inhibition of PKC-alpha prevents barrier dysfunction induced by TNF. PEM were coincubated with control vehicle, MPKCI and/or TNF for 4 h. In separate studies, PEM were preincubated for 4 h with control vehicle, antisense or nonsense to PKC-alpha followed by incubation with control vehicle or TNF for 4 h. PEM were assayed for endothelial albumin clearance rate. * Different from control group.

Prolonged PKC Activation Mediates TNF-alpha -Induced Increases in Albumin Clearance Rate in PEM

Figure 9 shows the effect of calphostin when cotreated (TNF-alpha  + Cal-0.0 h) or posttreated 0.5, 1.0, 2.0, or 3.0 h after TNF-alpha . The assay for permeability was measured 4 h after the TNF-alpha treatment. The cotreatment of calphostin with TNF-alpha or 0.5, 1.0, 2.0, or 3.0 h after TNF-alpha is sufficient to prevent the increase in permeability. Moreover, our data indicate that only a 0.5-, 1.0-, 2.0-, 3.0-, and 4.0-h incubation with TNF-alpha is sufficient to increase endothelial permeability (data not shown). Thus the data clearly indicate that the prolonged TNF-alpha -induced increase in permeability is reversible and PKC-alpha dependent.


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Fig. 9.   Effect of TNF on barrier function is reversed by PKC inhibition. PEM were coincubated with vehicle, calphostin, and/or TNF for 4 h. In separate studies, calphostin (Cal) was added 0.5, 1.0, 2.0, and 3.0 h after TNF. Permeability was measured at 4.0 h after TNF. * Different from control group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study indicates TNF-alpha -induced activation of PKC-alpha mediates the increase in protein permeability of PEM. PKC-alpha mediated the increase in permeability because 1) TNF-alpha induced the activity of PKC-alpha (i.e., assay of translocation and phosphorylation), 2) two structurally different PKC inhibitors, calphostin and MPKCI, prevented the effects of TNF-alpha , and 3) antisense to PKC-alpha prevented the effects of TNF-alpha . The data indicate that the cellular effects of the PKC inhibitors used in the present study (i.e., calphostin, MPKCI, and PKC-alpha 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-alpha . Finally, there was similar cell viability among all groups. We previously showed in vivo that TNF-alpha -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-alpha ; 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-alpha , a specific PKC isotype, during TNF-alpha -induced barrier dysfunction.

In the present study, TNF-alpha induced the association of PKC-alpha and PKM-alpha with the cytoskeleton. It is not clear what the mechanisms are for activation of PKC-alpha or the identity of the targets for activated PKC-alpha and PKM-alpha during the increase in endothelial permeability in response to TNF-alpha . TNF-alpha has been shown to bind to the TNF-alpha receptor p55 and activate phosphatidylcholine (PC)-phospholipase C (PLC) via the activity of the TNF-alpha 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-alpha by binding of DAG and calcium to the regulatory domains of PKC-alpha . The TNF-alpha -induced activation scenario (TNF-alpha right-arrow PLCright-arrowDAG + Ca2+) in concert with our finding of sustained levels of PKC-alpha protein promotes the prolonged increase in PKC-alpha activity in response to TNF-alpha . 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-alpha with the cytoskeletal fraction, which supports the hypothesis that prolonged endothelial barrier dysfunction is dependent on the maintained PKC-alpha -induced alterations in cytoskeletal targets. Moreover, the increase in the association of PKM-alpha with the cytoskeleton may also contribute to the long-term effects of phosphorylation of critical cytoskeletal targets.

In the present study, TNF-alpha induced the increase in the association of PKM-alpha 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-alpha 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-alpha -induced PKC-alpha activation, unphosphorylated membrane-bound MARCKS sequesters calmodulin, which results in suppression of activity of the calmodulin. In response to TNF-alpha , a possible pathway is PKM-alpha -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-alpha -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-alpha -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-alpha /epsilon 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-alpha 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-alpha -induced barrier dysfunction is also mediated by nitric oxide (11, 19). The notion that PKC-alpha 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-alpha protein pool would be required for the prolonged activation of PKC-alpha (8, 29, 37). PKC-alpha antisense treatment prevented the increase in PKC-alpha protein, activity, and permeability in response to TNF-alpha , indicating that increased translation of PKC-alpha mRNA into PKC-alpha protein is the probable mechanism for maintenance of the PKC-alpha protein pool. The increase in PKC-alpha protein was associated with a significant but small increase in the pool of 3.5-kb PKC-alpha mRNA, which supports the concept of both transcriptional and posttranscriptional regulation of the PKC-alpha protein pool. Interestingly, the 8.1-kb PKC-alpha RNA did not increase in response to TNF-alpha , 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-alpha protein without a substantial change in PKC-alpha mRNA. Our demonstration for dependence on an ongoing translation-dependent pool of PKC-alpha 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-alpha and oligonucleotides on the protein levels of PKC-zeta or on the phosphorylation of the PKC-epsilon -specific substrate. However, considering the complexities of signal transduction pathways, the data do not rule out a possible downstream effect of PKC-alpha activity on the activity of other PKC isotypes (25, 41).

Our data are consistent with increased degradation of PKC-alpha because TNF-alpha induced an increase in the membrane and cytoskeletal association of PKM-alpha . The literature indicates that the calcium lipid-dependent protease calpain-µ degrades PKC-alpha to the catalytically active PKM-alpha by cleaving off the regulatory domain. Thus the increase in membrane- and cytoskeletal-associated PKM-alpha protein may be the primary site for the prolonged increase in PKC-alpha activity. In addition, PKM-alpha is downregulated by further degradation by calpain-m (8, 37), which is consistent with the acute decrease in membrane-associated PKM-alpha in the present study. The early decrease in PKM-alpha probably occurs because the rate of PKM-alpha degradation is greater than the rate of synthesis of new PKC-alpha , consistent with the notion that the pool of PKC-alpha must be maintained for the prolonged activity of PKC-alpha .

The present study indicates the continued activation of PKC-alpha is required for the prolonged increase in permeability in response to TNF-alpha because posttreatment with calphostin 3.0 h after TNF-alpha still prevented the increase in permeability. Our data indicate the continued activation of PKC-alpha in response to TNF-alpha because of the 1) persistence of the in vitro phosphorylation of MARCKS that was specifically inhibited by PKC-alpha antisense and MPKCI, 2) translocation of PKC-alpha to the cytoskeleton, and 3) association of PKM-alpha with the cytoskeleton and membrane. Ross and Joyner (35) demonstrated the translocation of PKC-alpha to the membrane in response to TNF-alpha 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-alpha prevented the increase in permeability to TNF-alpha , indicating that the early activation of PKC (e.g., possibly PKC-alpha ) is required for endothelial barrier dysfunction in response to TNF-alpha . Finally, PKC inhibition with MPKCI did not affect basal levels of endothelial permeability, indicating that PKC-alpha 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-alpha induces the prolonged dysfunction of the endothelial barrier, which is dependent on the concomitant continued increase in PKC-alpha activity. The protracted response to TNF-alpha is inhibited by PKC-alpha antisense, indicating that the dynamic regulation of the PKC-alpha protein pool is a primary factor in the response to TNF-alpha . The development of strategies that target ongoing PKC-alpha 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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