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Am J Physiol Cell Physiol 279: C906-C914, 2000;
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Vol. 279, Issue 4, C906-C914, October 2000

Protein kinase C-zeta mediates TNF-alpha -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We addressed the role of protein kinase C (PKC) isozymes in mediating tumor necrosis factor-alpha (TNF-alpha )-induced oxidant generation in endothelial cells, a requirement for nuclear factor-kappa B (NF-kappa 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-alpha -induced oxidant generation. In contrast, inhibition of PKC-zeta synthesis by the antisense oligonucleotide prevented the oxidant generation following the TNF-alpha stimulation. We observed that PKC-zeta also induced the TNF-alpha -induced NF-kappa 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-zeta prevented the TNF-alpha -induced ICAM-1 promoter activity, whereas overexpression of the wild-type PKC-zeta augmented the response. These data imply a critical role for the PKC-zeta isozyme in regulating TNF-alpha -induced oxidant generation and in signaling the activation of NF-kappa B and ICAM-1 transcription in endothelial cells.

protein kinase C isoforms; oxidants; nuclear factor-kappa B; intercellular adhesion molecule-1; endothelium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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-alpha (TNF-alpha ) through activation of the transcription factor nuclear factor-kappa B (NF-kappa B) (14, 20).

The NF-kappa 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-kappa B1 (12, 17), and p52/ NF-kappa B2 (5, 32). All NF-kappa B proteins exist as an inactive dimer in the cytoplasm bound to the inhibitory proteins of the Ikappa B family, Ikappa Balpha , IkBbeta , Ikappa Bgamma , p100, p105, and IkBepsilon (3, 41). TNF-alpha stimulation of cells results in the phosphorylation, and subsequent ubiquitination-dependent degradation of Ikappa Balpha by the proteasome 26S (6, 7, 40). This allows the NF-kappa 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-kappa 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; alpha , beta I, beta II, and gamma ), novel [nPKCs; delta , epsilon , µ, theta , and eta /L (mouse/human)], and atypical [aPKCs; zeta , and lambda /iota (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-alpha resulted in activation of PKC and oxidant generation (29). We also showed that PKC activation and oxidant generation were both necessary for NF-kappa B activation and ICAM-1 expression, since the inhibition of PKC by calphostin C and scavenging of oxidants by antioxidants prevented the TNF-alpha -induced NF-kappa B activation and ICAM-1 gene transcription (29). These results demonstrated that TNF-alpha -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-zeta , the aPKC isozyme in endothelial cells, is critically involved in the mechanism of TNF-alpha -induced oxidant generation and thereby in the signaling of activation of NF-kappa B and ICAM-1 gene transcription.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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 [alpha -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-alpha (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-kappa 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-kappa B 5'-AGCTTGGAAATTCCGGAGCTG-3'. This represents a 21-bp sequence of ICAM-1 promoter encompassing the NF-kappa 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-kappa B site (ICAM-1NF-kappa 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-zeta were gifts of Dr. J. Moscat (Universidad Autonoma, Madrid, Spain). The PKC-zeta mutant (4) is a kinase-deficient PKC-zeta generated by a substitution of lysine-275 for tryptophan and thus lacks a functional catalytic domain. The plasmid pNF-kappa B-LUC containing five copies of consensus NF-kappa 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-alpha , -delta , and -epsilon 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-zeta 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-zeta mRNA. Phosphorothioate antisense oligonucleotides to PKC-alpha (GTT CTC GCT GGT GAG TTT CA) are directed to the 3'-untranslated region of PKC-alpha 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 Ikappa Balpha or Ikappa Bbeta (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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , -delta , -epsilon , and -zeta isozymes were abundantly expressed (Fig. 1), whereas PKC-beta I and PKC-iota were not detectable (data not shown). PKC-delta 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-alpha , -epsilon , and -delta isoforms. PKC-alpha was the most sensitive to phorbol ester, whereas residual levels of PKC-epsilon and PKC-delta (76 kDa) remained detectable (Fig. 1). In contrast, phorbol ester treatment failed to deplete the aPKC isozyme, PKC-zeta (Fig. 1).


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  		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-zeta mRNA (10, 11). PKC-zeta 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-zeta expression (Fig. 2); thus this concentration was used in subsequent studies for analysis of the role of PKC-zeta in ICAM-1 transcription. In contrast, the antisense oligonucleotide concentration of 1 µM failed to prevent PKC-zeta expression (Fig. 2). The failure of higher concentrations (1 µM) of oligonucleotides to inhibit expression of PKC-zeta is consistent with the reported effect of PKC-beta 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-zeta expression (Fig. 2). In another control experiment, antisense oligonucleotide to PKC-zeta failed to prevent synthesis of PKC-delta (Fig. 2), indicating specificity of the antisense oligonucleotide.


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  		Fig. 2.   Antisense oligonucleotide to PKC-zeta inhibits PKC-zeta expression. HPAE cells were transfected with antisense oligonucleotide to PKC-zeta as described in METHODS. After 36-48 h, cells were lysed and analyzed by Western blot analysis for the expression of PKC-zeta and PKC-delta isozymes; the same amount of lysate (10 µg/lane) was subjected to 10% SDS-PAGE. Results are representative of 3 experiments.

Inhibition of PKC-zeta expression prevents TNF-alpha -induced oxidant generation. We determined the effects of the phorbol ester-induced depletion of cPKC and nPKC isozymes and the inhibition of PKC-zeta expression by antisense oligonucleotide on the TNF-alpha -induced oxidant generation. Studies were made in cells stimulated with TNF-alpha for 1 h to allow maximum oxidant production. Control cells exhibited low fluorescence intensity, whereas stimulation of HPAE cells with TNF-alpha resulted in markedly increased fluorescence (Fig. 3). We observed oxidant production as early as 5 min after TNF-alpha challenge of HPAE cells (data not shown). Depletion of cPKC and nPKC isozymes by the phorbol ester treatment failed to prevent the TNF-alpha -induced oxidant generation (Fig. 3). However, antisense oligonucleotide to PKC-zeta 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-alpha failed to prevent TNF-alpha -induced oxidant generation in endothelial cells (Fig. 4, C and D).


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  		Fig. 3.   Effects of phorbol ester-induced depletion of cPKC and nPKC isozymes on oxidant generation induced by tumor necrosis factor-alpha (TNF-alpha ) 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-alpha (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-alpha -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-alpha was shifted to the higher fluorescence intensity classes compared with control cells. Pretreatment with PMA failed to prevent the TNF-alpha -induced shift to the higher fluorescence intensity classes. Results are representative of 3 experiments.



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  		Fig. 4.   Antisense oligonucleotide to PKC-zeta prevents TNF-alpha -induced oxidant generation in HPAE cells. HPAE cells were transfected with sense or antisense oligonucleotide to PKC-zeta (A) or antisense oligonucleotide to PKC-alpha (C) as described in METHODS. After 36-48 h, cells were stimulated for 1 h with TNF-alpha (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-zeta after TNF-alpha 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-alpha after TNF-alpha 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-alpha -induced Ikappa Balpha degradation. We next evaluated the role of PKC isozymes in mediating Ikappa Balpha degradation, a requirement for NF-kappa B activation (6, 7, 40). TNF-alpha stimulation of endothelial cells induced Ikappa Balpha degradation, whereas it had no effect on Ikappa Bbeta (Figs. 5 and 6). Depletion of cPKC and nPKC isozymes failed to prevent the TNF-alpha -induced Ikappa Balpha degradation, and expectedly it inhibited Ikappa Balpha 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-alpha -induced Ikappa Balpha degradation, whereas staurosporine, which inhibits cPKC and nPKC but not aPKC isozymes (23, 35), failed to prevent the TNF-alpha response (Fig. 6). Taken together, these results suggest that the TNF-alpha -induced Ikappa Balpha degradation in HPAE cells is independent of cPKC and nPKC isozymes and support the involvement of the aPKC isozyme.


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  		Fig. 5.   Phorbol ester-induced depletion of conventional PKC (cPKC) and novel PKC (nPKC) isozymes fails to prevent TNF-alpha -induced Ikappa Balpha 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-alpha (100 U/ml) or PMA (100 nM). Total cell lysate (10 µg/lane) was separated by 12.5% SDS-PAGE and immunoblotted for Ikappa Balpha and Ikappa Bbeta . Results are representative of 2 experiments.



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  		Fig. 6.   Differential effects of calphostin C and staurosporine on TNF-alpha -induced Ikappa Balpha degradation. HPAE cells were pretreated with calphostin C or staurosporine for 15 min before challenge with TNF-alpha (100 U/ml). Total cell lysate (10 µg) was separated by 12.5% SDS-PAGE and immunoblotted for Ikappa Balpha and Ikappa Bbeta . Results are representative of 2 experiments.

Inhibition of PKC-zeta synthesis prevents TNF-alpha -induced NF-kappa B binding to ICAM-1 promoter. We performed the EMSA to determine the role of the aPKC isozyme, PKC-zeta , in the mechanism of TNF-alpha -induced NF-kappa B binding to the ICAM-1 promoter. Depletion of cPKC and nPKC isozymes prevented NF-kappa 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-alpha -induced NF-kappa B binding to the ICAM-1 promoter (Fig. 7). In contrast, inhibition of PKC-zeta expression by the antisense oligonucleotide prevented the TNF-alpha -induced NF-kappa B binding to the ICAM-1 promoter (Fig. 8). In a control experiment, inhibition of PKC-alpha expression by the antisense oligonucleotide failed to prevent the TNF-alpha response (Fig. 8).


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  		Fig. 7.   Effects of phorbol ester-induced depletion of cPKC and nPKC isozymes on TNF-alpha -induced nuclear factor-kappa B (NF-kappa 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-alpha (100 U/ml) or PMA (100 nM). Nuclear extracts were prepared and assayed for NF-kappa B binding activity by electrophoretic mobility shift assay (EMSA) using radiolabeled oligonucleotide containing the ICAM-1-kappa B site. Results are representative of 2 experiments.



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  		Fig. 8.   Antisense oligonucleotide to PKC-zeta prevents TNF-alpha -induced NF-kappa B binding to ICAM-1 promoter. HPAE cells were transfected with antisense oligonucleotide to PKC-zeta -AS or PKC-alpha -AS as described in METHODS. After 36-48 h, cells were stimulated for 1 h with TNF-alpha (100 U/ml). Nuclear extracts were prepared and assayed for NF-kappa B binding activity by EMSA using radiolabeled oligonucleotide containing the ICAM-1-kappa B site. Results are representative of 2 experiments.

Inhibition of PKC-zeta prevents TNF-alpha -induced NF-kappa B activity, ICAM-1 promoter activation and mRNA expression. We evaluated the role of PKC-zeta in mediating NF-kappa B activity by cotransfecting a plasmid, pNF-kappa BLUC, containing five copies of consensus NF-kappa B sequence from Ig gene linked to a minimal adenovirus E1B promoter-luciferase reporter gene with constructs encoding dominant negative forms of PKC-alpha (PKC-alpha mut), -epsilon (PKC-epsilon mut), -delta (PKC-delta mut), or -zeta (PKC-zeta mut), isozyme in endothelial cells. As shown in Fig. 9A, expression of only PKC-zeta mut prevented the TNF-alpha -induced NF-kappa B activity, whereas PKC-alpha mut, -epsilon mut, -delta mut, or -zeta mut failed to prevent the response.


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  		Fig. 9.   A: inhibition of NF-kappa B activity by expression of dominant negative mutant of PKC-zeta . Human umbilical vein endothelial cells (HUVEC) were cotransfected with plasmid pNF-kappa BLUC containing 5 copies of consensus NF-kappa B sequence from Ig gene linked to a minimal adenovirus E1B promoter-luciferase reporter gene with constructs encoding dominant negative form of PKC-alpha (PKC-alpha mut), -epsilon (PKC-epsilon mut), -delta (PKC-delta mut), or -zeta (PKC-zeta mut) isozymes using the DEAE-dextran method (23). Cells were stimulated for 6 h with TNF-alpha (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-zeta dominant negative mutant and wild-type PKC-zeta on TNF-alpha -induced ICAM-1 promoter activity. The construct ICAM-1LUC or ICAM-1 NF-kappa BmutLUC (inset) containing the wild-type or NF-kappa B mutant version of ICAM-1 promoter was cotransfected with wild-type (PKC-zeta wt) or dominant negative (PKC-zeta mut) into HPAE cells using Superfect as described in METHODS. Cells were stimulated for 6-8 h with TNF-alpha (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-zeta on NF-kappa B-dependent ICAM-1 promoter activity. We cotransfected the wild-type (ICAM-1LUC) or NF-kappa B mutant (ICAM-1NF-kappa BmutLUC) versions of the ICAM-1 promoter-luciferase reporter gene construct with the PKC-zeta wt or PKC-zeta mut in HPAE cells and then determined luciferase activity. As shown in Fig. 9B, expression of PKC-zeta mut prevented the TNF-alpha -induced ICAM-1 promoter activity, whereas overexpression of PKC-zeta wt significantly augmented the TNF-alpha response. These data indicate the critical role of PKC-zeta in activating the ICAM-1 promoter. Mutation of the downstream NF-kappa B site prevented the TNF-alpha -induced ICAM-1 promoter activation (Fig. 9B, inset). Moreover, the expression of PKC-zeta mut or overexpression of PKC-zeta wt failed to modify the TNF-alpha response (Fig. 9B, inset). These data indicate that PKC-zeta activates ICAM-1 promoter through an NF-kappa B-dependent pathway. To further address the role of PKC-zeta in the mechanism of the response, we showed that the antisense oligonucleotide to PKC-zeta prevented TNF-alpha -induced ICAM-1 mRNA expression (by ~50%), whereas the sense oligonucleotide had no effect (Fig. 10).


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  		Fig. 10.   Antisense oligonucleotide to PKC-zeta prevents ICAM-1 mRNA expression. HPAE cells were transfected with indicated concentrations of antisense oligonucleotide to PKC-zeta or PKC-alpha as described in METHODS. After 36-48 h, cells were stimulated for 2 h with TNF-alpha (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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B and transcription of ICAM-1 gene (29). In the present study, we have identified the atypical endothelial PKC isozyme, PKC-zeta , in transducing the TNF-alpha -induced oxidant generation and the downstream activation of NF-kappa B and ICAM-1 transcription.

We used the approaches described below to address the contributions of PKC-alpha , -delta , -epsilon , and -zeta isozymes, which are abundantly expressed in HPAE cells, in mediating the TNF-alpha -activated responses. To address the role of PKC-alpha , -delta , and -epsilon isozymes in mediating oxidant production following TNF-alpha 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-zeta , we used the antisense oligonucleotide, which inhibits synthesis by binding to the translation initiation codon of PKC-zeta mRNA (10, 11). We showed that depletion of cPKCs and nPKCs failed to prevent TNF-alpha -induced oxidant production in endothelial cells, whereas the inhibition of PKC-zeta synthesis by the antisense oligonucleotide prevented the oxidant production. Thus the results indicate an important role of the aPKC isozyme PKC-zeta in activating oxidant generation induced by TNF-alpha stimulation of endothelial cells. Generation of oxidants following TNF-alpha 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-alpha resulted in their translocation and activation (A. Rahman, unpublished results); thus a possible mechanism of oxidant generation may involve PKC-zeta -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-zeta in the mechanism of TNF-alpha -induced oxidant production, we next addressed the possibility that the oxidant production mediated by PKC-zeta was responsible for the NF-kappa B activation and ICAM-1 gene transcription in endothelial cells. We showed that inhibition of PKC-zeta expression using the antisense oligonucleotide prevented both NF-kappa 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-zeta prevented TNF-alpha -induced NF-kappa B activity, whereas the expression of dominant negative forms of PKC-alpha , -delta , and -zeta isozymes failed to prevent the response. The expression of the PKC-zeta dominant negative mutant also prevented ICAM-1 promoter activity in response to TNF-alpha challenge, and overexpression of wild-type PKC-zeta augmented this response. Mutation of the downstream NF-kappa B binding site on the ICAM-1 promoter prevented the TNF-alpha -induced ICAM-1 promoter activation. Taken together, these results indicate the essential role of the PKC-zeta isozyme in mediating TNF-alpha -induced NF-kappa B activation and ICAM-1 transcription.

In the present study, we also addressed the mechanism of the PKC-zeta -induced activation of NF-kappa B. The results showed that inhibition of PKC-zeta expression prevented Ikappa Balpha degradation and NF-kappa B binding to the ICAM-1 promoter induced by TNF-alpha . These results can be explained on the basis of PKC-zeta -induced oxidant generation promoting the degradation of Ikappa Balpha and activation of NF-kappa B (33, 34). Another possibility is that PKC-zeta can directly phosphorylate NF-kappa B p65, since the dominant negative mutant of PKC-zeta was shown to inhibit NF-kappa B p65 phosphorylation, resulting in the loss of NF-kappa B p65 transcriptional activity (2). Hence, PKC-zeta may induce NF-kappa B activity by 1) activation of the oxidant signaling pathway and downstream induction of NF-kappa B binding to ICAM-1 promoter and 2) direct phosphorylation of NF-kappa B p65 resulting in increased transcriptional activity of the bound NF-kappa B.

In summary, we have shown that 1) phorbol ester-induced depletion of cPKC and nPKC isozymes failed to prevent TNF-alpha -induced oxidant generation and NF-kappa B activation; 2) inhibition of PKC-zeta synthesis by the antisense oligonucleotide prevented TNF-alpha -induced oxidant generation, NF-kappa B activation, and ICAM-1 gene transcription; 3) expression of the dominant negative mutant form of PKC-zeta inhibited the TNF-alpha -induced NF-kappa B activity; and 4) expression of the dominant negative mutant form of PKC-zeta also inhibited the TNF-alpha -induced ICAM-1 promoter activity, whereas overexpression of wild-type PKC-zeta augmented the response. Thus the aPKC isozyme PKC-zeta plays a critical role in mediating TNF-alpha -induced oxidant generation, NF-kappa B activation, and resultant ICAM-1 gene transcription in endothelial cells. The results point to the endothelial cell PKC-zeta as an important target in preventing the proinflammatory effects of TNF-alpha 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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-kappa 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-kappa B and Ikappa B proteins: new discoveries and insights. Annu Rev Immunol 14: 649-683, 1996[Web of Science][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[Web of Science][Medline].

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-kappa B participates in transactivation through a kappa B site. Mol Cell Biol 12: 685-695, 1992[Abstract/Free Full Text].

6.   Brown, K, Gerstberger S, Carlson L, Franzoso G, and Siebenlist U. Control of Ikappa Balpha proteolysis by site-specific, signal-induced phosphorylation. Science 267: 1485-1488, 1995[Abstract/Free Full Text].

7.   Chen, ZJ, Parent L, and Maniatis T. Site-specific phosphorylation of Ikappa Balpha by a novel ubiquitination-dependent protein kinase activity. Cell 84: 853-862, 1996[Web of Science][Medline].

8.   Dean, NM, McKay R, Condon TP, and Bennett CF. Inhibition of protein kinase C-alpha 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 Czeta subspecies in maturation of Xenopus laevis oocytes. Mol Cell Biol 12: 3776-3783, 1992[Abstract/Free Full Text].

11.   Folgueira, L, McElhinny JA, Bren GD, MacMorran WS, Diaz-Meco MT, Moscat J, and Paya V. Protein kinase C-zeta mediates NF-kappa 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[Web of Science][Medline].

13.   Hofmann, J. The potential for isoenzyme-selective modulation of protein kinase C. FASEB J 11: 649-669, 1997[Abstract].

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.

16.   Jaken, S. Protein kinase C isoezymes and substrates. Curr Opin Cell Biol 8: 168-173, 1996[Web of Science][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[Web of Science][Medline].

18.   Korchak, HM, Rossi MW, and Kilpatrick LE. Selective role for beta -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-kappa 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-alpha 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/Free Full Text].

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.

25.   Nolan, GP, Ghosh S, Liou HC, Tempest P, and Baltimore D. DNA binding and Ikappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide. Cell 64: 961-969, 1991[Web of Science][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/Free Full Text].

27.   Rahman, A, Kefer J, Bando M, Niles WD, and Malik AB. E-selectin expression in human endothelial cells by TNF-alpha -induced oxidant generation and NF-kappa 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-kappa 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[Free Full Text].

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/Free Full Text].

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[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[Web of Science][Medline].

34.   Schreck, R, Rieber P, and Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J 10: 2247-2258, 1991[Web of Science][Medline].

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.

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.

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 Ikappa Balpha on serines 32 and 36 controls Ikappa Balpha proteolysis and NF-kappa B activation in response to diverse stimuli. EMBO J 14: 2876-2883, 1995[Web of Science][Medline].

41.   Whiteside, ST, and Israel A. I kappa B proteins: structure, function and regulation. Semin Cancer Biol 8: 75-82, 1997[Web of Science][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[Abstract/Free Full Text].

Am J Physiol Cell Physiol 279(4):C906-C914
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