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Am J Physiol Heart Circ Physiol 279: H1679-H1689, 2000;
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Vol. 279, Issue 4, H1679-H1689, October 2000

PKCepsilon modulates NF-kappa B and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes

Richard C. X. Li1, Peipei Ping1,2, Jun Zhang2, William B. Wead2, Xinan Cao2, Jiuming Gao2, Yuting Zheng2, Shuang Huang3, Jiahuai Han3, and Roberto Bolli1,2

1 Experimental Research Laboratory, Division of Cardiology, 2 Department of Physiology and Biophysics, University of Louisville and the Jewish Hospital Heart and Lung Research Institute, Louisville, Kentucky 40202; and 3 Division of Immunology, Scripps Institute of Molecular Medicine, La Jolla, California 92037


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously shown that protein kinase C (PKC)-epsilon , nuclear factor (NF)-kappa B, and mitogen-activated protein kinases (MAPKs) are essential signaling elements in ischemic preconditioning. In the present study, we examined whether activation of PKCepsilon affects the activation of NF-kappa B in cardiac myocytes and whether MAPKs are mediators of this signaling event. Activation of PKCepsilon (+108% above control) in adult rabbit cardiomyocytes to a degree that has been previously shown to protect myocytes against hypoxic injury increased the DNA-binding activity of NF-kappa B (+164%) and activator protein (AP)-1 (+127%) but not that of Elk-1. Activation of PKCeta did not have an effect on these transcription factors. Activation of PKCepsilon also enhanced the phosphorylation activities of the p44/p42 MAPKs and the p54/p46 c-Jun NH2-terminal kinases (JNKs). PKCepsilon -induced activation of NF-kappa B and AP-1 was completely abolished by inhibition of the p44/p42 MAPK pathway with PD98059 and by inhibition of the p54/p46 JNK pathway with a dominant negative mutant of MAPK kinase-4, indicating that both signaling pathways are necessary. Taken together, these data identify NF-kappa B and AP-1 as downstream targets of PKCepsilon , thereby establishing a molecular link between activation of PKCepsilon and activation of NF-kappa B and AP-1 in cardiomyocytes. The results further demonstrate that both the p44/p42 MAPK and the p54/p46 JNK signaling pathways are essential mediators of this event.

protein kinase Cepsilon ; activator protein-1; nuclear factor-kappa B


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROTEIN KINASE C (PKC) is a family of serine-threonine kinases that may be classified into three subfamilies (29), which include the classical isoforms (alpha , beta , gamma ), the novel isoforms (epsilon , delta , eta , theta ), and the atypical isoforms (zeta , lambda , iota ). Activation of PKC has been shown to be an important signaling step in various biological processes (13, 29, 35), including the development of ischemic preconditioning (PC) (24, 27, 52). Recent studies from our laboratory (32, 33) and others (15, 23) have demonstrated that PKC-mediated cardioprotection is isoform specific and that the epsilon -isoform of PKC plays an essential role in the development of PC in rabbit myocardium. Among the 10 PKC isoforms expressed in the rabbit heart (32), the epsilon -isoform is translocated and activated during ischemic PC (32, 33). Inhibition of this isoform completely blocks the delayed cardioprotection (33), supporting the concept that activation of this signaling molecule is necessary for late PC to become manifest.

Although PKCepsilon appears to play an essential role in ischemic PC (15, 23, 32, 33), the downstream signaling events triggered by the activation of this specific isozyme during the development of the late phase of PC remain largely unknown. Considerable evidence indicates that the development of late PC involves the synthesis of new proteins (34), the upregulation of stress-responsive genes (16, 20), and the activation of transcription factors (25, 48). In noncardiac cells, PKC isoforms are known to promote the activation of various transcription factors, such as nuclear factor (NF)-kappa B (3, 4, 10, 12, 19, 45), activator protein (AP)-1 (8, 39, 43), and Elk-1 (38). In cardiac tissue, activation of NF-kappa B has been shown to be a necessary event during ischemic PC (25, 48), and activation of AP-1 has been observed after brief ischemia (5). However, the molecular link between the epsilon -isozyme of PKC and ischemia-activated transcription factors has never been established in cardiac cells. We hypothesized that transcription factors are downstream signaling targets of PKCepsilon in cardiomyocytes. Accordingly, in this study, we examined whether selective activation of the epsilon -isozyme of PKC via transfection with recombinant adenovirus encoding this enzyme (30, 31) leads to targeted activation of transcription factors implicated in late PC, i.e., NF-kappa B and AP-1.

Mounting evidence indicates that the three subfamilies of mitogen-activated protein (MAP) kinases (MAPKs), the p44/p42 MAPKs, the p38 MAPKs, and the p54/p46 c-Jun NH2-terminal kinases (JNKs), are important upstream regulators for the induction of various transcription factors (12, 37, 44). The role of each MAPK subfamily in the activation of transcription factors appears to be cell-type specific. In noncardiac cells, it has been shown that p44/p42 MAPKs activate AP-1, Elk-1, and NF-kappa B (7, 8, 18, 44, 53) and that p54/p46 JNKs activate AP-1 (18, 44, 47). Recent studies have shown that various subfamilies of MAPKs are activated in the ischemic myocardium (28, 41). Interestingly, MAPKs have also been implicated as downstream signaling targets of PKC in late PC (30, 31); specifically, activation and subsequent nuclear translocation of the p44 and p42 MAPKs were observed in the same rabbit model of late PC, where activation of both PKCepsilon (32) and NF-kappa B (48) has been demonstrated. Moreover, activation of the epsilon -isozyme of PKC has been found to result in increased phosphorylation activity of both the p44/p42 MAPKs and the p54/p46 JNKs in adult rabbit cardiac cells (30, 31). Nevertheless, whether MAPKs function as intermediate molecules transducing signals from PKCepsilon to transcription factors has not been established. Therefore, we further investigated whether activation of MAPKs is a necessary signaling step for the PKCepsilon -mediated activation of transcription factors in cardiac myocytes.

The rabbit was selected to study the role of MAPKs in PKCepsilon -triggered activation of transcription factors because this is the species in which activation of PKCepsilon and NF-kappa B during ischemic PC has been demonstrated (30-32, 48). To determine whether this signaling event is specific to the activation of the epsilon -isoform of PKC or is shared by other PKC isoforms, we also examined PKCeta , another isoform in the novel PKC subfamily. The results demonstrate that PKCepsilon (but not PKCeta ) activates the transcription factors NF-kappa B and AP-1 in adult rabbit cardiac myocytes and that both the p44/p42 MAPK and the p54/p46 JNK signaling pathways mediate the activation of these two factors.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials and Reagents

M199 medium, fetal calf serum (FCS), penicillin, and streptomycin were from GIBCO/BRL (Gaithersburg, MD). Mouse monoclonal antibodies for the p42/p44 MAPKs, the p38 MAPKs, NF-kappa B p65, NF-kappa B p50, c-Jun, and Jun-D were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal phosphor-antibodies for Tyr 204 p44/42 MAPK, Tyr 180/182 p38 MAPK, and Thr 183/184 JNK were from New England Biolab (Beverly, MA). Horseradish peroxidase-labeled sheep anti-mouse secondary antibody and enhanced chemiluminescence (ECL) detecting reagent were from Amersham (Princeton, NJ). Reagents for SDS-polyacrylamide gel were from Bio-Rad. Poly (dl/dc) and T4 polynucleotide kinase was from Pharmacia Biotech (Piscataway, NJ). Type II collagenase was from Worthington Biochemical (Lakewood, NJ). Double-stranded oligonucleotides containing AP-1 consensus sequences, oligonucleotides containing NF-kappa B consensus sequences, and oligonucleotides containing Elk-1 consensus sequences were from Promega (Madison, WI). [gamma -32P]ATP was purchased from DuPont New England Nuclear (Boston, MA). PD98059 and GF109203X were from Calbiochem (San Diego, CA). All other reagents were from Sigma Chemical (St. Louis, MO).

Isolation of Adult Rabbit Cardiac Myocytes

Adult rabbit cardiac myocytes were isolated by use of a modification of the method of Hadded et al. (17). This method yielded 80-85% rod-shaped cardiac cells, which generated an average total of 4-6 × 107 cells per rabbit heart. This is the same method that we have previously used to study PKCepsilon -induced activation of MAPKs in rabbit cardiomyocytes (30, 31). Briefly, the myocytes were plated onto laminin-coated 100-mm dishes at 37°C at subconfluency (2 × 106 cells/100-mm dish); incubated in M199 medium with 2% FCS, penicillin, and streptomycin; and cultured overnight. The medium was replaced with serum-free M199 medium supplemented with taurine (5 mM), creatine (5 mM), and carnitine (5 mM) for 24 h before adenovirus transfection.

Determination of PKCepsilon Isoform-Selective Phosphorylation Activity

PKCepsilon phosphorylation activity in total cardiac cell lysates was determined as previously described (31). Briefly, cardiac proteins were extracted by use of glass-glass homogenization in buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1% Nonidet NP-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 16 µg/ml benzamidine hydrochloride, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A. Total cardiac cell protein samples were immunoprecipitated with PKCepsilon antibodies and then subjected to a phosphorylation assay with the use of a PKCepsilon -selective substrate (ERMRPRKRQGSVRRRV). We found that the basal PKCepsilon activity of rabbit cardiac myocytes is 27.24 ± 1.52 pmol · min-1 · mg protein-1 (n = 6, where n is the no. of rabbits).

Construction of Recombinant Adenoviruses

Recombinant adenoviruses encoding rabbit active PKCepsilon , dominant negative PKCepsilon , and active PKCeta were constructed in our laboratory as previously described (30, 31). The hemagglutinin (HA) epitope enabled us to determine the expression of transgenic proteins. Preliminary data showed that the HA epitope, consisting of a nine-amino acid sequence, had no effect on the protein expression or on the enzymatic activity of rabbit PKCepsilon and PKCeta . Recombinant adenovirus encoding a dominant negative mutant of MAPK kinase (MKK)-4 (DN-MKK4) was generated in the laboratory of Huang and Han. This HA-tagged DN-MKK4 has been proven to be effective in blocking activation of the JNKs in vitro (51) and in vivo (49). Positive recombinant adenoviruses were isolated by plaque purification and propagated in H293 cells that had been transformed with E1 genes (26). The recombinant viral cell lysates were purified by double CsCl gradient. The integrity of transgene expression was confirmed by PCR and Southern blotting.

Transfection of Cardiac Cells with Adenoviruses

In all groups, 10 plaque-forming units (pfu) of recombinant adenovirus per cardiac myocyte were used for transfection. Transfection efficiency was assessed with the use of recombinant adenovirus encoding the green fluorescence protein (GFP). We found that 10 pfu/cell consistently yielded 85-90% transfection efficiency in adult rabbit cardiac myocytes. The following experimental groups were studied: a control group, in which cardiac myocytes received recombinant adenovirus encoding a null transgene; a FL-PKCepsilon group, in which cardiac myocytes received recombinant adenovirus encoding rabbit full-length active PKCepsilon (30); and a DN-PKCepsilon group, in which cardiac myocytes received recombinant adenovirus encoding a dominant negative mutant of rabbit PKCepsilon (30).

To block PKCepsilon -induced activation of p44/p42 MAPKs, one group of cardiac myocytes received the MAP or extracellular signal-regulated kinase (ERK) kinase (MEK)-1/2 inhibitor PD98059 (10 µM) 4 h before incubation with recombinant adenovirus encoding FL-PKCepsilon . To block PKCepsilon -induced activation of p54/p46 JNKs, another group of myocytes received both the recombinant adenovirus encoding FL-PKCepsilon and that encoding DN-MKK4.

The expression of the PKCepsilon transgene in cardiac cells (and thus the activity of this enzyme) is also dependent on the time interval after transfection. In pilot experiments, cardiac cells were collected 8, 12, 16, 18, and 24 h after receiving 10 pfu/cell of FL-PKCepsilon . We found that 18 h was the optimal expression time, because it produced an increase in PKCepsilon phosphorylation activity [208 ± 5% of control (null vector)], similar to that observed in conscious rabbits after ischemic PC in our previous studies (32, 33). Thus, in all experiments related to PKCepsilon , 18 h after transfection with PKCepsilon adenoviruses, cells were washed twice with warm PBS, harvested, frozen immediately in liquid nitrogen, and stored at -80°C. Our pilot experiments also showed that activation of PKCeta after ischemic PC in vivo was best mimicked after a 16-h transfection with PKCeta adenovirus. Specifically, cardiac cells that received FL-PKCeta exhibited a level of PKCeta phosphorylation activity (228 ± 17% of control) 12 h posttransfection that was similar to that induced by ischemic PC in conscious rabbits (in separate studies in three rabbits, we found that cardiac PKCeta activity 30 min after an ischemic PC protocol consisting of six 4-min coronary occlusions and reperfusions was 198 ±23% of that in control rabbits; Refs. 32-34). Thus, in all experiments related to PKCeta , cells were collected 16 h after transfection with PKCeta adenovirus, washed twice with warm PBS, harvested, frozen immediately in liquid nitrogen, and stored at -80°C.

Preparation of Nuclear Extracts

Nuclear extracts of myocytes were prepared by a modified detergent treatment method as previously described (48). Briefly, the myocytes were homogenized in ice-cold buffer A [in mmol/l: 10 HEPES with pH 7.9, 10 KCl, 0.1 EDTA, 0.1 EGTA, 1.0 dithiothreitol (DTT), 0.5 PMSF, 1 NaF, and 1 Na3VO4] and incubated on ice for 15 min, followed by centrifugation at 3,800 rpm at 4°C for 10 min. A value of 0.5% Nonidet NP-40 was added to the reaction, followed by brief vigorous vortexing and incubation on ice for an additional 10 min. The isolated nuclear pellets were resuspended in ice-cold buffer B (in mmol/l: 20 HEPES with pH 7.9, 400 NaCl, 1.0 EDTA, 1.0 EGTA, 1.0 DTT, 1.0 PMSF, 1 NaF, and 1 Na3VO4), homogenized with a glass-glass homogenizer at 4°C, and incubated on ice for an additional 30 min. The nuclear proteins were extracted by collecting the supernatant of an 8,000-rpm spin of the nuclear homogenates. The Bradford system (Bio-Rad, Hercules, CA) was used to determine the protein content of the nuclear extracts.

Electrophoretic Mobility Shift Assay

Double-stranded synthetic oligonucleotides were 5' end labeled with [gamma -32P]ATP and T4 polynucleotide kinase. The oligonucleotide sequences used for labeling the probes were as follows: AP-1, 5'-CGCTTGATGACTCAGCCGGAA-3'; NF-kappa B, 5'-AGTTGAGGGGACTTTCCC-AGGC-3'; and Elk-1, 5'-GGGGTCCTTGAGGAAGTATAAGAAGAAT-3'.

Standard DNA-binding reactions were carried out in 20 µl of mixture containing (in mmol/l) 25 HEPES with pH 7.6, 50 KCl, 1 EDTA, 1 DTT, 0.5 spermidine, 0.5 PMSF, 10% glycerol, 0.1 mg · mmol-1 · l-1 poly (dI-dC), and 6 µg of the extracted nuclear protein. The DNA probe (40,000-60,000 counts/min) was added, and the reaction was carried out on ice for 20 min. The reaction samples were then loaded onto 4% native polyacrylamide gels made in a 0.5× TBE buffer, and electrophoresis was performed at a constant voltage of 140 V for 90 min. After electrophoresis, the gels were vacuum dried and autoradiographed by use of an intensifying screen at -80°C.

To verify the specificity of the DNA-binding activity, competition assays were performed to identify the specific binding signal for AP-1 and NF-kappa B by addition of a 100-fold molar excess of unlabeled double-stranded oligonucleotides. Supershift assays were performed by incubation of the nuclear extracts with the corresponding antibodies.

Western Blotting Analysis

Standard Western immunoblotting techniques (32, 48) were used to assess the protein expression of AP-1, NF-kappa B, and MAPKs. Briefly, the protein content was measured by the Bradford assay. A quantity of 100 µg of either total cellular proteins or nuclear proteins was separated with a 12% SDS-PAGE, and standard ECL methods were used to visualize the protein signal.

Statistical Analysis

Data are expressed as means ± SE of five or six experiments, each from a different rabbit heart. To facilitate comparisons, measurements of nuclear DNA-binding activity and protein expression in each experiment were expressed as a percentage of the average value for the control group. Differences among groups were tested by one-way ANOVA. If the F test showed an overall significance, comparisons between two groups were performed by unpaired Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of PKCepsilon Enhances AP-1 and NF-kappa B DNA-Binding Activity

We found that 18 h after transfection with FL-PKCepsilon adenovirus, PKCepsilon phosphorylation activity increased to 208 ± 5% of control (null vector), a level similar to that observed in conscious rabbits after ischemic PC in our previous studies (32, 33). This level of activation has also been previously shown to protect adult rabbit cardiac myocytes against hypoxic injury (30). To determine whether PKCepsilon modulates the transcription factors AP-1, NF-kappa B, and Elk-1 in cardiac myocytes, recombinant adenovirus encoding active PKCepsilon (FL-PKCepsilon ) was used (Figs. 1, 2, and 3). Cardiac cells transfected with the active PKCepsilon adenovirus exhibited increased DNA-binding activity for both AP-1 [227 ± 19% of control (null vector); Figs. 1A and 2] and NF-kappa B [264 ± 21% of control (null vector); Figs. 1B and 2] but not for Elk-1 [127 ± 13.2% of control (null vector); Fig. 1C]. To determine whether the increased DNA-binding activity was dependent on the activation of PKCepsilon , cardiac cells were either pretreated with GF109203 (1 µM), a selective PKC inhibitor, or received recombinant adenovirus encoding the dominant negative mutant of PKCepsilon (DN-PKCepsilon ) in conjunction with that encoding the FL-PKCepsilon . Separate experiments showed that the concentration of GF109203 used (1 µM) effectively blocked the increase in PKCepsilon -selective phosphorylation activity induced by transfection with FL-PKCepsilon adenovirus in adult rabbit cardiac myocytes (107 ± 5% of control, n = 5). The ability of DN-PKCepsilon adenovirus transfection to inhibit PKCepsilon activation after transfection with FL-PKCepsilon has been previously demonstrated (30). Both GF109203 and DN-PKCepsilon completely abrogated PKCepsilon -induced activation of AP-1 (Figs. 1A and 2) and NF-kappa B (Figs. 1B and 2).


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  		Fig. 1.   Representative gels demonstrating the effect of protein kinase C (PKC)-epsilon activation on the nuclear factor (NF)-kappa B, activator protein (AP)-1, and Elk-1 DNA-binding activity in adult rabbit cardiac myocytes. A: a representative electrophoretic mobility shift assay (EMSA) for AP-1. B: a representative EMSA for NF-kappa B. C: a representative EMSA for Elk-1. Lane 1 is a negative control (reactions without nuclear proteins). In lanes 2-7, cardiac myocytes were treated with (+) or without (-) recombinant adenovirus encoding rabbit full-length active PKCepsilon (FL-PKCepsilon ; lanes 5-7), recombinant adenovirus encoding a null transgene (control; lanes 2-4), the PKC inhibitor GF109203 (lanes 3 and 6), or recombinant adenovirus encoding the dominant negative mutant of rabbit PKCepsilon (DN-PKCepsilon ; lanes 5 and 7). Activation of PKCepsilon resulted a robust increase in the DNA-binding activity of AP-1 (A, lane 5) and NF-kappa B (B, lane 5). In contrast, activation of PKCepsilon did not affect the DNA-binding activity of Elk-1 (C).



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  		Fig. 2.   Effect of the PKC inhibitors GF109203 and DN-PKCepsilon on PKCepsilon -induced DNA-binding activity of NF-kappa B and AP-1. Four groups of adult rabbit cardiac myocytes received recombinant adenovirus encoding a null transgene (control), recombinant adenovirus encoding active PKCepsilon (FL-PKCepsilon ), GF109203 and FL-PKCepsilon , or DN-PKCepsilon and FL-PKCepsilon . Each group included six experiments, each from a different rabbit heart. EMSAs were performed to determine the DNA-binding activity. Data are expressed as a percentage of the null vector (means ± SE); n = no. of rabbits.



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  		Fig. 3.   Representative gels demonstrating the specificity of EMSAs. The proteins bound to the AP-1 and NF-kappa B probes were identified by supershift assays. The specificity of DNA binding was determined by competition assays. A: nuclear proteins were preincubated with antibodies against c-Jun (lane 3) or Jun-D (lane 4) or with a 100-fold molar excess of unlabeled cold AP-1 oligonucleotides (100×oligo; lane 5) before EMSA. B: nuclear proteins were preincubated with antibodies against p65 (lane 3) or p50 (lane 4) or with a 100-fold molar excess of unlabeled cold NF-kappa B oligonucleotides (lane 5) before EMSA.

The identity of the proteins bound to either the AP-1 or the NF-kappa B consensus probes was verified by supershift analysis and competition assays. Antibodies to c-Jun and Jun-D or to the p50 and p65 subunits of NF-kappa B were used to characterize these protein-probe complexes. Figure 3, A and B, shows that the c-Jun and Jun-D antibodies supershifted the protein-probe complex of AP-1 and that the p50 and p65 antibodies supershifted the protein-probe complex of NF-kappa B, indicating that these complexes were specific for AP-1 and NF-kappa B, respectively. Furthermore, competition assays performed by adding a 100-fold molar excess of unlabeled double-stranded AP-1 or NF-kappa B consensus probes completely blocked the binding, further confirming the specificity of these signals (Fig. 3, A and B).

In contrast, cardiac cells transfected with recombinant adenovirus encoding active PKCeta exhibited increased PKCeta phosphorylation activity (228 ± 17% of control), an activation of PKCeta that is similar to that evoked by ischemic PC in vivo, as indicated above, but no significant change in the DNA-binding activity of AP-1 (112 ± 8% of control), NF-kappa B (121 ± 13% of control), or Elk-1 (109 ± 9% of control), indicating that activation of PKCeta does not lead to AP-1-, NF-kappa B-, or Elk-1-dependent transcriptional regulation in cardiac cells.

Overexpression of PKCepsilon Results in Nuclear Translocation of NF-kappa B and c-Jun Proteins

To determine whether the increased DNA-binding activity of AP-1 and NF-kappa B was due to nuclear translocation of AP-1 or NF-kappa B subunits, we analyzed the nuclear protein content of AP-1 and NF-kappa B with the use of Western immunoblotting. Five experiments (each from a different rabbit heart) were used to assess c-Jun and Jun-D, and five were used to assess p65. As shown in Fig. 4, there was a significant increase in the nuclear expression of c-Jun [241 ± 16% of null vector (control)] and Jun-D [176 ± 9% of null vector (control)]. Cardiac cells transfected with PKCepsilon also exhibited increased nuclear p65 protein expression [204 ± 13% of null vector (control); Fig. 5]. The expression of AP-1 (data not shown) and NF-kappa B (Fig. 5) in the total cellular lysates was unaltered. These data indicate that activation of PKCepsilon induces nuclear translocation of the p65 subunit of NF-kappa B as well as the c-Jun and Jun-D subunits of AP-1, which results in enhanced DNA-binding activity of NF-kappa B and AP-1.


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  		Fig. 4.   Representative Western blots for the analysis of AP-1. Lanes 1-3 are protein extracts from myocytes transfected with null transgene (control), whereas lanes 4-6 are protein extracts from myocytes transfected with FL-PKCepsilon recombinant adenovirus. In addition to recombinant adenovirus, cardiac cells in lanes 2 and 5 also received GF109203. Lanes 3 and 6 were cotransfected with DN-PKCepsilon . Protein extracts were probed with antibodies against c-Jun (top) or Jun-D (bottom). Activation of PKCepsilon significantly increased the expression of c-Jun (lane 4) and Jun-D (lane 4) proteins in the nuclei. Both increases were abolished by inhibition of PKCepsilon (lanes 5 and 6).



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  		Fig. 5.   Representative Western blots for the analysis of NF-kappa B expression in total cell lysates and in nuclear extracts. Lanes 1-3 are from cardiac cells transfected with a null transgene (control), whereas lanes 4-6 are from myocytes transfected with FL-PKCepsilon recombinant adenovirus. In addition to recombinant adenovirus, cardiac cells in lanes 2 and 5 also received GF109203. Lanes 3 and 6 were cotransfected with DN-PKCepsilon . Total cell lysates and the nuclear protein extracts were probed with antibodies against the p65 subunit of NF-kappa B. Activation of PKCepsilon significantly increased the expression of p65 (lane 4) protein in the nuclear fraction, indicating nuclear translocation of the p65 subunit of NF-kappa B. Inhibition of PKCepsilon completely abolished the nuclear translocation of the p65 subunit of NF-kappa B (lanes 5 and 6).

To verify whether the effect of PKCepsilon transfection with FL-PKCepsilon on the nuclear translocation of these transcription factors is dependent on the activation of PKCepsilon , we treated cardiac myocytes with GF109203 or DN-PKCepsilon . Western immunoblotting showed that GF109203 (1 µM) and DN-PKCepsilon abrogated the nuclear translocation of both c-Jun/Jun-D (Fig. 4) and the p65 subunits of NF-kappa B (Fig. 5), confirming that such translocation was caused by the activation of PKCepsilon .

Activation of MAPKs Mediates PKCepsilon -Dependent Activation of NF-kappa B and AP-1

After establishing the molecular link between PKCepsilon and AP-1 and NF-kappa B, we investigated the role of MAPKs in mediating this signaling pathway. To address this question, we performed three experiments. 1) We determined which of the three MAPK pathways was activated by PKCepsilon with the use of antibodies against phosphorylated p44/p42 MAPKs, p54/p46 JNKs, and the p38 MAPKs. 2) To confirm that phosphorylation of MAPKs was induced by PKC, we tested whether the PKC inhibitor GF109203 abolished the phosphorylation of MAPKs. 3) To identify which MAPK signaling pathway(s) is necessary for PKCepsilon -induced activation of these two transcription factors, we selectively blocked the p44/p42 MAPK pathway with PD98059 and the p54/p46 JNK pathway with a DN-MKK4.

PKCepsilon -induced activation of MAPKs. Using in-gel kinase assays, we have previously shown that overexpression of PKCepsilon leads to increased phosphorylation activities of the p44/p42 MAPKs (30) and the p54/p46 JNKs (31). To confirm PKCepsilon -mediated activation of these MAPKs, we used specific phosphor-antibodies against the p44/p42 MAPKs, the p54/p46 JNKs, and the p38 MAPKs. Increased phosphorylation of p44/p42 MAPKs and p54/p46 JNKs was detected in cardiac myocytes transfected with PKCepsilon (Fig. 6A). In contrast, the signal for phosphorylated p38 MAPKs was unaffected (Fig. 6A), although these antibodies do not differentiate the individual subtypes within the p38 MAPK subfamily. Western immunoblotting analysis confirmed no change in total protein expression for all MAPKs (Fig. 6A). These results corroborate our previous findings (30, 31) that both p44/p42 MAPKs and p54/p46 JNKs are downstream signaling targets of PKCepsilon in rabbit cardiac myocytes.


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  		Fig. 6.   Representative Western blots demonstrating the effect of PKCepsilon on mitogen-activated protein (MAP) kinase (MAPK) phosphorylation. A: cardiac myocytes received recombinant adenovirus encoding a null transgene (control), recombinant adenovirus encoding active PKCepsilon (FL-PKCepsilon ), GF109203 before FL-PKCepsilon , or recombinant adenoviruses encoding both DN-PKCepsilon and FL-PKCepsilon . Total cell lysates were analyzed by Western blotting by use of antibodies against nonphosphorylated or phosphorylated p44/p42 MAPKs (left), p54/p46 c-Jun NH2-terminal kinases (JNKs; middle), and p38 MAPKs (right). Activation of PKCepsilon increased phosphorylation of the p44/p42 MAPKs and p54/p46 JNKs but had no effect on the phosphorylation activity of total p38 MAPKs. Inhibition of PKCepsilon with either the inhibitor GF109203 or a dominant negative mutant of PKCepsilon completely abolished the enhanced phosphorylation signals. Total protein expression of all MAPKs detected by nonphosphor antibodies was not altered by PKCepsilon activation. B, left: cardiac myocytes were transfected with recombinant adenovirus encoding either a null transgene (control) or active PKCepsilon (FL-PKCepsilon ), were pretreated with PD98059 and transfected with active PKCepsilon , or were cotransfected with both active PKCepsilon and a dominant negative mutant of MAPK kinase (MKK)-4 (DN-MKK4). Phosphor-antibodies to p44/p42 MAPKs were used to determined the activation of these kinases. Activation of PKCepsilon increased phosphorylation of the p44/p42 MAPKs, which was completely abolished by inhibition of MAP or extracellular signal-regulated kinase (ERK) kinase (MEK)-1/2 with PD98059. B, right: cardiac myocytes were transfected with recombinant adenovirus encoding either a null transgene (control) or active PKCepsilon (FL-PKCepsilon ), were cotransfected with active PKCepsilon and DN-MKK4, or were pretreated with PD98059 and transfected with active PKCepsilon . Phosphor-antibodies to p54/p46 JNKs were used to determined the activation of these kinases. Activation of PKCepsilon increased phosphorylation of the p54/p46 JNKs, which was completely abolished by inhibition of MKK4 with DN-MKK4. Total protein expression of all MAPKs detected by nonphosphor antibodies was not altered by PKCepsilon activation.

To determine whether the phosphorylation of p44/p42 MAPKs and p54/p46 JNKs in myocytes was caused by PKCepsilon activation, we pretreated myocytes with GF109203 (1 µM) or with DN-PKCepsilon together with the active PKCepsilon (FL-PKCepsilon ). The results (Fig. 6A) demonstrate that GF109203, a specific inhibitor of PKC, blocked the enhanced phosphorylation of p44/p42 MAPKs and p54/p46 JNKs. Similar results were observed when cells were pretreated with DN-PKCepsilon (Fig. 6A). These data demonstrate that the enhanced phosphorylation of p44/p42 MAPKs and p54/p46 JNKs was dependent on the activation of PKCepsilon .

To elucidate the role of MEK1/2 and MEK4 in the PKCepsilon -induced phosphorylation of p44/p42 MAPKs and p54/46 JNKs, we pretreated myocytes with PD98059, a specific inhibitor of the p44/p42 MAPK signaling pathway, or with DN-MKK4, which selectively blocks the p54/p46 JNK signaling pathway, before transfection with FL-PKCepsilon . Both PD98059 and DN-MKK4, at the doses used in this study, inhibited basal phosphorylation of p44/p42 MAPKs and JNKs, respectively (data not shown). As expected, PD98059 and DN-MKK4 completely blocked PKCepsilon -induced phosphorylation of p44/p42 MAPKs and p54/p46 JNKs, respectively (Fig. 6B). Thus, in adult rabbit cardiac myocytes, PKCepsilon induces the activation of p44/p42 MAPKs via a MEK1/2 signaling pathway and the activation of p54/p46 JNKs via a MKK4 signaling pathway.

Involvement of p44/p42 MAPKs and p54/p46 JNKs in PKCepsilon -induced activation of NF-kappa B and AP-1. To test whether PKCepsilon -induced activation of AP-1 and NF-kappa B involves the p44/p42 MAPK signaling pathway, the p54/p46 JNK signaling pathway, or both, we treated cardiac cells with PD98059 or with recombinant adenovirus encoding DN-MKK4.

Pretreatment of cells with either PD98059 or DN-MKK4 abolished PKCepsilon -induced activation of AP-1 (FL-PKCepsilon , 295 ± 32% of null vector control; PD98059, 120 ± 14%; DN-MKK4, 105 ± 7%) (Fig. 7A). Combined pretreatment with both PD98059 and DN-MKK4 did not result in inhibition greater than with either agent alone (Figs. 7A and 8). These results indicate that PKCepsilon utilizes both the p44/p42 MAPK and the p54/p46 JNK signaling pathways to activate AP-1 and that neither pathway is sufficient for PKCepsilon -induced activation of AP-1 to occur. Thus both the p44/p42 MAPKs and the p54/p46 JNKs are necessary downstream signaling elements in PKCepsilon -dependent regulation of AP-1 induction.


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  		Fig. 7.   Representative EMSAs for the effects of PD98059 and DN-MKK4 on DNA-binding activity of AP-1 (A) and NF-kappa B (B). Lane 1 is nuclear protein extracts from cardiac cells transfected with a null transgene (control). Lane 2 is with active PKCepsilon (FL-PKCepsilon ). Lane 3 was pretreated with PD98059 before receiving active PKCepsilon (FL-PKCepsilon ). Lane 4 was cotransfected with both DN-MKK4 and PKCepsilon (FL-PKCepsilon ). Lane 5 was pretreated with PD98059 and cotransfected with DN-MKK4 and PKCepsilon (FL-PKCepsilon ). Activation of PKCepsilon (lane 2) increased the DNA-binding activity of AP-1 (A) and NF-kappa B (B). Activation of AP-1 and NF-kappa B was abolished by inhibition of the p44/p42 MAPK signaling pathway with PD98059, by inhibition of the p54/46 JNK signaling pathway with DN-MKK4, and by inhibition of both pathways with combined treatment with PD98059 and DN-MKK4.



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  		Fig. 8.   Role of p44/p42 MAPKs and p54/p46 JNKs in PKCepsilon -induced activation of NF-kappa B and AP-1. Five groups of adult rabbit cardiac myocytes received recombinant adenovirus encoding a null transgene (control), recombinant adenovirus encoding active PKCepsilon (FL-PKCepsilon ), PD98059 before receiving FL-PKCepsilon , recombinant adenovirus encoding DN-MKK4 in conjunction with FL-PKCepsilon , or PD98059 combined with DN-MKK4 and FL-PKCepsilon . Each group included four experiments, each from a different rabbit heart. EMSAs were performed to determine the DNA-binding activity. Activation of AP-1 and NF-kappa B was abolished by inhibition of the p44/p42 MAPK signaling pathway with PD98059, by inhibition of the p54/46 JNK signaling pathway with DN-MKK4, and by inhibition of both pathways with combined treatment of PD98059 and DN-MKK4. Data are expressed as a percentage of control (null transgene) (means ± SE); n = no. of rabbits.

Similar to AP-1, pretreatment of cells with either PD98059 or DN-MKK4 completely blocked PKCepsilon -induced activation of NF-kappa B (FL-PKCepsilon , 210 ± 25% of null vector control; PD98059, 33 ± 4.1%; DN-MKK4, 22 ± 3.2%) (Figs. 7B and 8). These data support the concept that both the p44/p42 MAPK and the p54/p46 JNK signaling pathways are necessary for PKCepsilon -induced activation of NF-kappa B and that neither pathway is sufficient to mediate this response.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There are two important findings in this study. First, we found that selective activation of PKCepsilon induces activation of the transcription factors AP-1 and NF-kappa B, demonstrating that these two factors serve as downstream signaling targets of the epsilon -isoform of PKC in adult rabbit cardiac myocytes. Second, we found that activation of the p44/p42 MAPK and the p54/p46 JNK pathways is a critical intermediate signaling step during PKCepsilon -induced activation of AP-1 and NF-kappa B and that both of these pathways are necessary to produce the activation of the transcription factors. Taken together with our previous results demonstrating the role of the PKCepsilon isoform in the development of the late phase of PC (32, 33), these data indicate that activation of PKCepsilon not only can contribute to the posttranslational modulation of intracellular signaling molecules but also can participate in the AP-1- and NF-kappa B-dependent transcriptional regulation required for the delayed phase of cardioprotection. To our knowledge, this is the first report that PKCepsilon governs the activity of NF-kappa B and AP-1 in the heart, further underscoring the role of this PKC isoform in cardiovascular pathophysiology.

PKC comprises a large family of kinases with 11 known isoforms. Increasing evidence suggests that each individual PKC isoform possesses a unique biological function (29). The molecular structure of PKC isoforms dictates the regulatory mechanism for activation. Both the PKCepsilon and the PKCeta isoforms belong to the novel PKC subfamily and share very similar molecular structure domains (29). We have previously reported that both of these isoforms are translocated by ischemic PC (32) but appear to play distinct roles during the development of the cardioprotection: activation of PKCepsilon is essential for late PC (33), whereas translocation of PKCeta is not (33). In the present study, we found that PKCepsilon and PKCeta appear to be coupled to distinct downstream signaling pathways. Activation of PKCepsilon resulted in activation of the transcription factors NF-kappa B and AP-1, consistent with a role of this isoform of PKC in PC. In contrast, activation of PKCeta did not have any significant effect on any of the three transcription factors examined (AP-1, NF-kappa B, and Elk-1). Taken together with our previous finding that activation of NF-kappa B is necessary for the late phase of ischemic PC to occur (48), these results provide a molecular basis for the differential roles of PKCepsilon and PKCeta in late PC by indicating that the eta -isoform of PKC may not participate in the transcriptional regulation of new genes involved in this cardioprotective phenomenon.

MAPKs have been shown to be important mediators of signal transduction from the cell surface to the nucleus. Transcriptional regulation by MAPKs has been implicated in many cellular processes such as proliferation, differentiation, and apoptotic death (8, 18, 36, 42). In mammals, MAPKs are divided into at least three subfamilies: the ERKs, also known as the p44/p42 MAPKs; the JNKs, which include the p54/p46 JNKs; and the p38 family of MAPKs (11, 41). MAPKs are involved in multiple intracellular signaling cascades and are activated by the stimulation of a variety of cell surface receptors such as receptor tyrosine kinases, G protein-linked receptors, cytokine receptors, and so forth (11, 18, 41). Mounting evidence implicates PKC in signaling pathways leading to the activation of various MAPKs (2, 6, 14, 36). On activation, MAPKs can phosphorylate and activate their target proteins and transcription factors (12, 37), thereby initiating the transcription of new genes and the expression of new proteins. In noncardiac cells, it has been well documented that p44/p42 MAPKs can activate c-Fos and Elk-1 and that p54/p46 JNKs can activate c-Jun (18, 36). Recently, it has also been reported that MAPKs are involved in the activation of NF-kappa B and AP-1 (12, 37). However, the role of MAPKs in PKC-dependent AP-1 and NF-kappa B activation in the heart is unknown. Our data demonstrate for the first time that both p44/p42 MAPKs and p54/p46 JNKs are necessary for PKCepsilon -dependent modulation of these transcription factors in the myocardium.

Interestingly, the effect of p44/p42 MAPKs and p54/p46 JNKs on the basal activity of NF-kappa B and AP-1 appears to be very different. Neither PD98059 nor DN-MKK4 had an appreciable effect on the basal activity of AP-1 (Figs. 7A and 8). In contrast, inhibition of p44/p42 MAPKs with PD98059 or inhibition of p54/p46 JNKs with DN-MKK4 in the presence of PKCepsilon activation not only abolished the PKCepsilon -induced activation of NF-kappa B but also resulted in a reduced NF-kappa B activity that was lower than that observed under control conditions (Figs. 7B and 8). These data suggest that both p44/p42 MAPKs and p54/p46 JNKs may be important in determining the basal activity of NF-kappa B in cardiac myocytes, independent of PKCepsilon activation, whereas basal AP-1 activity does not receive input from these MAPK signaling pathways.

The precise molecular mechanisms for MAPK-mediated activation of AP-1 and NF-kappa B are unknown. NF-kappa B is activated by phosphorylation of inhibitory kappa B-alpha (Ikappa B-alpha ), either on serine residues 32 and 36 or on tyrosine residue 42 (1, 22, 51). Such phosphorylation leads to dissociation of Ikappa B-alpha from NF-kappa B and subsequent translocation of NF-kappa B to the nucleus (1). The NF-kappa B family consists of five different members, p50, p52, p65, c-Rel, and RelB, which can form various homodimers and heterodimers. The active form of NF-kappa B usually consists of heterodimers composed primarily of p50/p65 (1, 2). The AP-1 family is subdivided into three main subgroups: the Jun proteins (such as c-Jun and Jun-D), the Fos proteins, and the activating transcription factors. AP-1 activity is regulated at two levels: abundance of AP-1 proteins and posttranslational modification that dictates the DNA-binding activity of AP-1 (46). Because serine 32 and 36 and tyrosine 42 of Ikappa B-alpha do not appear to be direct phosphorylation sites for p44/p42 MAPKs or JNKs, it seems likely that these kinases activate NF-kappa B indirectly via other, as yet unidentified, downstream kinases. Further studies will be necessary to decipher the signaling pathway that links MAPKs to NF-kappa B and AP-1. Regardless of the precise mechanism, our data show that the activity of MAPKs results in nuclear translocation of NF-kappa B and AP-1, because activation of PKCepsilon enhanced the DNA-binding activities of these two transcription factors (Figs. 1 and 2) and, at the same time, caused translocation to the nucleus of the p65 subunit and the c-Jun and Jun-D subunits (Figs. 4 and 5).

The information provided by this investigation expands our understanding of the signaling mechanisms that underlie late PC. Previous in vivo studies have shown that both ischemia-induced and nitric oxide (NO)-induced late PC are associated with activation of PKCepsilon (32) and NF-kappa B (48) and that ischemia-induced activation of NF-kappa B can be blocked with inhibitors of PKC (48). Although these results imply that recruitment of NF-kappa B during late PC is PKC dependent, they cannot discern whether the epsilon -isoform of PKC (rather than PKCeta , which is also recruited during late PC, or possibly other PKC isotypes; Ref. 32) is specifically involved in this phenomenon, because chelerythrine is a general PKC inhibitor. The present finding that increased PKCepsilon activity is sufficient to recruit NF-kappa B in cardiac myocytes supports the concept that epsilon  is the PKC isoform responsible for activation of NF-kappa B after ischemia-induced or NO donor-induced late PC. However, further in vivo studies using isoform-specific approaches to inhibit PKCepsilon (e.g., transgenesis of dominant negative mutants) will be necessary to firmly establish an obligatory role of PKCepsilon in NF-kappa B activation during late PC. The role of AP-1 in this process will also require further investigation, because the ability of PKCepsilon to recruit this transcription factor does not, in itself, signify that AP-1 contributes to the delayed cardioprotection. Nevertheless, the notion that both NF-kappa B and AP-1 are downstream targets of PKCepsilon in cardiac myocytes has broad implications that transcend the specific process of late PC and may involve a variety of cardiovascular phenomena. In addition, our finding that p44/p42 MAPKs and p54/p46 JNKs are required for PKCepsilon -dependent activation of NF-kappa B and AP-1 in cardiac myocytes is consistent with the hypothesis that these subfamilies of MAPKs participate in the genesis of late PC. Thus the present study provides a rationale for future investigations aimed at assessing the effect of in vivo inhibition of p44/p42 MAPKs and p54/p46 JNKs on the acquisition of delayed cardioprotection.

In conclusion, although PKC has been implicated in the activation of NF-kappa B (3, 4, 7, 10, 19) and AP-1 (8, 39, 43) in a variety of cells, the signaling pathways linking PKC to the activation of transcription factors are poorly characterized, especially in cardiac myocytes. In the present study, we demonstrate that PKCepsilon activates the transcription factors AP-1 and NF-kappa B via the p44/p42 MAPK and the p54/p46 JNK signaling pathways. Recruitment of both of these signaling pathways is necessary for the activation of AP-1 and NF-kappa B to occur. These findings provide new insights into the role of PKCepsilon in the development of ischemic PC and, more generally, in the modulation of AP-1- and NF-kappa B-dependent genes. The identification of the PKCepsilon -MAPK-AP-1/NF-kappa B signal transduction pathways in the heart may have significant implications for our understanding of the signaling mechanisms underlying the delayed response of the heart to brief ischemic stresses as well as other pathophysiological events dependent on PKC signaling.


    ACKNOWLEDGEMENTS

This study was supported in part by American Heart Association (AHA) Kentucky Affiliate Research Fellowship 9804503 and AHA Ohio Valley Affiliate Research Fellowship 9920591V (both to R. C. X. Li); by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-58166 and AHA National Center Grant-in-Aid 9750721N (both to P. Ping); by NHLBI Grants HL-43151 and HL-55757 (both to R. Bolli); and by the Jewish Hospital Research Foundation, Louisville, KY.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Ping, Dept. of Physiology and Biophysics, Baxter Building Suite 122, 570 S. Preston St., Louisville, KY 40202 (E-mail: ping{at}ntr.net).

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 12 March 2000; accepted in final form 4 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baeuerle, PA, and Baltimore D. NF-kappa B: ten years after. Cell 87: 13-20, 1996[Web of Science][Medline].

2.   Berra, E, Diaz-Meco MT, Lozano J, Frutos S, Municio MM, Sanchez P, Sanz L, and Moscat J. Evidence for a role of MEK and MAPK during signal transduction by protein kinase Czeta . EMBO J 14: 6157-6163, 1995[Web of Science][Medline].

3.   Bomsztyk, K, Rooney JW, Iwasaki T, Rachie NA, Dower SK, and Sibley CH. Evidence that interleukin-1 and phorbol esters activate NF-kappa B by different pathways: role of protein kinase C. Cell Regul 2: 329-335, 1991[Web of Science][Medline].

4.   Bonizzi, G, Piette J, Schoonbroodt S, Merville MP, and Bours V. Role of the protein kinase C lambda /iota isoform in nuclear factor-kappa B activation by interleukin-1beta or tumor necrosis factor-alpha : cell type specificities. Biochem Pharmacol 57: 713-720, 1999[Web of Science][Medline].

5.   Chandrasekar, B, and Freeman GL. Induction of nuclear factor-kappa B and activation of protein 1 in postischemic myocardium. FEBS Lett 401: 30-34, 1997[Web of Science][Medline].

6.   Chang, YY, Kim SJ, Park TK, Kang SS, Ha MJ, Mushinski JF, and Chun JS. Modulation of MAP kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells. Biochem Mol Biol Int 45: 1139-1148, 1998[Web of Science][Medline].

7.   Cruise, L, Ho LK, Veitch K, Fuller G, and Morris BJ. Kainate receptors activate NF-kappa B via MAP kinase in striatal neurones. Neuroreport 11: 395-398, 2000[Web of Science][Medline].

8.   Dieckgraefe, BK, and Weems DM. Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK. Am J Physiol Gastrointest Liver Physiol 276: G322-G330, 1999[Abstract/Free Full Text].

9.   Eicher, DM, Tan TH, Rice NR, O'Shea JJ, and Kennedy ICS Expression of v-src in T cells correlates with nuclear expression of NF-kappa B. J Immunol 152: 2710-2719, 1994[Abstract].

10.   Folgueira, L, McElhinny JA, Bren GD, MacMorran WS, Diaz-Meco MT, Moscat J, and Paya CV. Protein kinase Czeta mediates NF-kappa B activation in human immunodeficiency virus-infected monocytes. J Virol 70: 223-231, 1996[Abstract].

11.   Force, T, Pombo CM, Avruch JA, Bonventre JV, and Kyriakis JM. Stress-activated protein kinases in cardiovascular disease. Circ Res 78: 947-953, 1996[Free Full Text].

12.   Garcia, J, Lemercier B, Roman-Roman S, and Rawadi G. A Mycoplasma fermentans-derived synthetic lipopeptide induces AP-1 and NF-kappa B activity and cytokine secretion in macrophages via the activation of mitogen-activated protein kinase pathways. J Biol Chem 273: 34391-34398, 1998[Abstract/Free Full Text].

13.   Genot, EM, Parker PJ, and Cantrell DA. Analysis of the role of protein kinase C-alpha , -epsilon , and -zeta in T cell activation. J Biol Chem 270: 9833-9839, 1995[Abstract/Free Full Text].

14.   Ghaffari-Tabrizi, N, Bauer B, Villunger A, Baier-Bitterlich G, Altman A, Utermann G, Uberall F, and Baier G. Protein kinase Ctheta , a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells. Eur J Immunol 29: 132-142, 1999[Web of Science][Medline].

15.   Gray, MO, Karliner JS, and Mochly-Rosen D. A selective epsilon-protein C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem 272: 30945-30951, 1997[Abstract/Free Full Text].

16.   Guo, Y, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, and Bolli R. The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci USA 96: 11507-11512, 1999[Abstract/Free Full Text].

17.   Haddad, J, Decker ML, Hsieh LC, Lesch M, Samarel AM, and Decker RS. Attachment and maintenance of adult rabbit cardiac myocytes in primary cell culture. Am J Physiol Cell Physiol 255: C19-C27, 1988[Abstract/Free Full Text].

18.   Hill, CS, and Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80: 199-211, 1995[Web of Science][Medline].

19.   Hirano, M, Hirai S, Mizuno K, Osada S, Hosaka M, and Ohno S. A protein kinase C isozyme, nPKC epsilon , is involved in the activation of NF-kappa B by 12-O-tetradecanoylphorbol-13-acetate (TPA) in rat 3Y1 fibroblasts. Biochem Biophys Res Commun 206: 429-436, 1995[Web of Science][Medline].

20.   Jones, WK, Flaherty MP, Tang XL, Takano H, Qiu Y, Banerjee S, Smith T, and Bolli R. Ischemic preconditioning increases iNOS transcript levels in conscious rabbits via a nitric oxide-dependent mechanism. J Mol Cell Cardiol 31: 1469-1481, 1999[Web of Science][Medline].

21.   Kawasaki, H, Moriguchi T, Matsuda S, Li HZ, Nakamura S, Shimohama S, Kimura J, Gotoh Y, and Nishida E. Ras-dependent and Ras-independent activation pathways for the stress-activated-protein-kinase cascade. Eur J Biochem 241: 315-321, 1996[Web of Science][Medline].

22.   La Porta, CA, and Comolli R. PKC-dependent modulation of Ikappa B alpha -NFkappa B pathway in low metastatic B16F1 murine melanoma cells and in highly metastatic BL6 cells. Anticancer Res 18: 2591-2597, 1998[Web of Science][Medline].

23.   Liu, GS, Cohen MV, Mochly-Rosen D, and Downey JM. Protein kinase C-epsilon is responsible for the protection of preconditioning in rabbit cardiomyocytes. J Mol Cell Cardiol 31: 1937-1948, 1999[Web of Science][Medline].

24.   Liu, Y, Ytrehus K, and Downey JM. Evidence that translocation of protein kinase C is a key event during ischemic preconditioning of rabbit myocardium. J Mol Cell Cardiol 26: 661-668, 1994[Web of Science][Medline].

25.   Maulik, N, Sato M, Price BD, and Das DK. An essential role of NFkappa B in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett 429: 365-369, 1998[Web of Science][Medline].

26.   McGrory, WJ, Bautista DS, and Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology 163: 614-617, 1988[Web of Science][Medline].

27.   Michell, MB, Meng X, Ao L, Brown JM, Harken AH, and Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res 76: 73-81, 1995[Abstract/Free Full Text].

28.   Nakano, A, Baines CP, Kim SO, Pelech SL, Downey JM, Cohen MV, and Critiz SD. Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK. Circ Res 86: 144-151, 1999[Abstract/Free Full Text].

29.   Newton, AC. Regulation of protein kinase C. Curr Opin Cell Biol 9: 161-167, 1997[Web of Science][Medline].

30.   Ping, P, Zhang J, Cao X, Li RCX, Kong D, Tang XL, Qiu Y, Manchikalapudi S, Auchampach JA, Black RG, and Bolli R. PKC-dependent activation of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious rabbits. Am J Physiol Heart Circ Physiol 276: H1468-H1481, 1999[Abstract/Free Full Text].

31.   Ping, P, Zhang J, Huang S, Cao X, Tang X-L, Li RCX, Zheng Y-T, Qiu Y, Clerk A, Sugden P, Han J, and Bolli R. PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits. Am J Physiol Heart Circ Physiol 277: H1771-H1785, 1999[Abstract/Free Full Text].

32.   Ping, P, Zhang J, Qiu Y, Tang XL, Manchikalapudi S, Cao X, and Bolli R. Ischemic preconditioning induces selective translocation of protein kinase C isoforms epsilon and eta in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res 81: 404-414, 1997[Abstract/Free Full Text].

33.   Qiu, Y, Ping P, Tang XL, Manchikalapudi S, Rizvi A, Zhang J, Takano H, Wu WJ, Teschner S, and Bolli R. Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved. J Clin Invest 101: 2182-2198, 1998[Web of Science][Medline].

34.   Rizvi, A, Tang X-L, Qiu Y, Xuan YT, Takano H., Jadoon AK, and Bolli R. Increased protein synthesis is necessary for the development of late preconditioning against myocardial stunning. Am J Physiol Heart Circ Physiol 277: H874-H884, 1999[Abstract/Free Full Text].

35.   Rybin, VO, and Steinberg SF. Protein kinase C isoform expression and regulation in the developing rat heart. Circ Res 74: 299-309, 1994[Abstract/Free Full Text].

36.   Schonwasser, DC, Marais RM, Marshall CJ, and Parker PJ. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol 18: 790-798, 1998[Abstract/Free Full Text].

37.   Schulze-Osthoff, K, Ferrari D, Riehemann K, and Wesselborg S. Regulation of NF-kappa B activation by MAP kinase cascades. Immunobiology 198: 35-49, 1997[Web of Science][Medline].

38.   Sharif, TR, and Sharif M. A high throughput system for the evaluation of protein kinase C inhibitors based on Elk1 transcriptional activation in human astrocytoma cells. Int J Oncol 14: 327-335, 1999[Web of Science][Medline].

39.   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 element. Mol Cell Biol 19: 1313-1324, 1999[Abstract/Free Full Text].

40.   Steffan, NM, Bren GD, Frantz B, Tocci MJ, O'Neill EA, and Paya CV. Regulation of Ikappa B alpha  phosphorylation by PKC- and Ca++-dependent signal transduction pathways. J Immunol 155: 4685-4691, 1995[Abstract].

41.   Sugden, PH, and Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res 83: 345-352, 1998[Free Full Text].

42.   Sugiyama, S, Kugiyama K, Ogata N, Doi H, Ota Y, Ohgushi M, Matsumura T, Oka H, and Yasue H. Biphasic regulation of transcription factor nuclear factor-kappa B activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway. Arterioscler Thromb Vasc Biol 18: 568-576, 1998[Abstract/Free Full Text].

43.   White, BR, Duval DL, Mulvaney JM, Roberson MS, and Clay CM. Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element. Mol Endocrinol 13: 566-577, 1999[Abstract/Free Full Text].

44.   Whitmarsh, AJ, and Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74: 589-607, 1996[Web of Science][Medline].

45.   Wilson, L, Szabo C, and Salzman LA. Protein kinase C-dependent activation of NF-kappa B in enterocytes is independent of Ikappa B degradation. Gastroenterology 117: 106-114, 1999[Web of Science][Medline].

46.   Wisdom, R. AP-1: one switch for many signals. Exp Cell Res 253: 180-185, 1999[Web of Science][Medline].

47.   Xiao, L, and Lang W. A dominant role for the c-Jun NH2-terminal kinase in oncogenic ras-induced morphologic transformation of human lung carcinoma cells. Cancer Res 60: 400-408, 2000[Abstract/Free Full Text].

48.   Xuan, YT, Tang XL, Banerjee S, Takano H, Li RC, Han H, Qiu Y, Li JJ, and Bolli R. Nuclear factor-kappa B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res 84: 1095-1109, 1999[Abstract/Free Full Text].

49.   Yang, D, Tournier C, Wysk M, Lu HT, Xu J, Davis RJ, and Flavell RA. Targeted disruption of the MKK4 gene causes embryonic death, inhibition of c-Jun NH2-terminal kinase activation, and defects in AP-1 transcriptional activity. Proc Natl Acad Sci USA 94: 3004-3009, 1997[Abstract/Free Full Text].

50.   Yao, A, Takahashi T, Aoyagi T, Kinugawa K, Kohmoto O, Sugiura S, and Serizawa T. Immediate-early gene induction and MAP kinase activation during recovery from metabolic inhibition in cultured cardiac myocytes. J Clin Invest 96: 69-77, 1995.

51.   Zhang, Y, Neo SY, Wang X, Han J, and Lin SC. Axin forms a complex with MEKK1 and activates c-Jun NH2-terminal kinase/stress-activated protein kinase through domains distinct from Wnt signaling. J Biol Chem 274: 35247-35254, 1999[Abstract/Free Full Text].

52.   Zhao, J, Renner O, Wightman L, Sugden PH, Stewart L, Miller AD, Latchman DS, and Marber MS. The expression of constitutively active isotypes of protein kinase C to investigate preconditioning. J Biol Chem 273: 23072-23079, 1998[Abstract/Free Full Text].

53.   Zhao, Q, and Lee FS. Mitogen-activated protein kinase/ERK kinase kinases 2 and 3 activate nuclear factor-kappa B through Ikappa B kinase-alpha and Ikappa B kinase-beta . J Biol Chem 274: 8355-8358, 1999[Abstract/Free Full Text].

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Protein kinase C-{varepsilon} coimmunoprecipitates with cytochrome oxidase subunit IV and is associated with improved cytochrome-c oxidase activity and cardioprotection
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[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
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Reperfusion kinase phosphorylation is essential but not sufficient in the mediation of pharmacological preconditioning: Characterisation in the bi-phasic profile of early and late protection
Cardiovasc Res, January 1, 2007; 73(1): 153 - 163.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. Inagaki, E. Churchill, and D. Mochly-Rosen
Epsilon protein kinase C as a potential therapeutic target for the ischemic heart
Cardiovasc Res, May 1, 2006; 70(2): 222 - 230.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
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Survival kinases in ischemic preconditioning and postconditioning
Cardiovasc Res, May 1, 2006; 70(2): 240 - 253.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
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Studying ischemic preconditioning in isolated cardiomyocyte models
Cardiovasc Res, May 1, 2006; 70(2): 286 - 296.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
E. McGregor and M. J. Dunn
Proteomics of the Heart: Unraveling Disease
Circ. Res., February 17, 2006; 98(3): 309 - 321.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
A. Denys, A. Hichami, and N. A. Khan
n-3 PUFAs modulate T-cell activation via protein kinase C-{alpha} and -{varepsilon} and the NF-{kappa}B signaling pathway
J. Lipid Res., April 1, 2005; 46(4): 752 - 758.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
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Expression of constitutively stable hybrid hypoxia-inducible factor-1{alpha} protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury
Am J Physiol Cell Physiol, February 1, 2005; 288(2): C314 - C320.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
M.-C. Beauchamp, S.-E. Michaud, L. Li, M. R. Sartippour, and G. Renier
Advanced glycation end products potentiate the stimulatory effect of glucose on macrophage lipoprotein lipase expression
J. Lipid Res., September 1, 2004; 45(9): 1749 - 1757.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
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Circ. Res., May 28, 2004; 94(10): 1375 - 1382.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
L. Li, T. Sawamura, and G. Renier
Glucose Enhances Human Macrophage LOX-1 Expression: Role for LOX-1 in Glucose-Induced Macrophage Foam Cell Formation
Circ. Res., April 16, 2004; 94(7): 892 - 901.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
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Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits
Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1455 - H1460.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
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Cardioprotection involves activation of NF-{kappa}B via PKC-dependent tyrosine and serine phosphorylation of I{kappa}B-{alpha}
Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1753 - H1758.
[Abstract] [Full Text] [PDF]

Home page
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Glucose Enhances Endothelial LOX-1 Expression: Role for LOX-1 in Glucose-Induced Human Monocyte Adhesion to Endothelium
Diabetes, July 1, 2003; 52(7): 1843 - 1850.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
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Role of the NF-{kappa}B Signaling Pathway and {kappa}B cis-Regulatory Elements on the IRF-1 and iNOS Promoter Regions in Mycobacterial Lipoarabinomannan Induction of Nitric Oxide
Infect. Immun., March 1, 2003; 71(3): 1442 - 1452.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
A. Castrillo, D. J. Pennington, F. Otto, P. J. Parker, M. J. Owen, and L. Bosca
Protein Kinase C{epsilon} Is Required for Macrophage Activation and Defense Against Bacterial Infection
J. Exp. Med., October 29, 2001; 194(9): 1231 - 1242.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. M. Vondriska, J. B. Klein, and P. Ping
Use of functional proteomics to investigate PKC{epsilon}-mediated cardioprotection: the signaling module hypothesis
Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1434 - H1441.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. M. Pass, Y. Zheng, W. B. Wead, J. Zhang, R. C. X. Li, R. Bolli, and P. Ping
PKC{epsilon} activation induces dichotomous cardiac phenotypes and modulates PKC{epsilon}-RACK interactions and RACK expression
Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H946 - H955.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. Bolli
The Late Phase of Preconditioning
Circ. Res., November 24, 2000; 87(11): 972 - 983.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
T. M. Vondriska, J. Zhang, C. Song, X.-L. Tang, X. Cao, C. P. Baines, J. M. Pass, S. Wang, R. Bolli, and P. Ping
Protein Kinase C {epsilon}-Src Modules Direct Signal Transduction in Nitric Oxide-Induced Cardioprotection : Complex Formation as a Means for Cardioprotective Signaling
Circ. Res., June 22, 2001; 88(12): 1306 - 1313.
[Abstract] [Full Text] [PDF]

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