Distinct Protein Kinase C Isoforms Mediate Regulation of Vascular Endothelial Growth Factor Expression by A2A Adenosine Receptor Activation and Phorbol Esters in Pheochromocytoma PC12 Cells*

Alicia M. GardnerDagger and Mark E. Olah§

From the Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0575

Received for publication, August 15, 2002, and in revised form, January 23, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vascular endothelial growth factor (VEGF) stimulates angiogenesis during development and in disease. In pheochromocytoma (PC12) cells, VEGF expression is regulated by A2A adenosine receptor (A2AAR) activation. The present work examines the underlying signaling pathway. The adenylyl cyclase-protein kinase A cascade has no role in the down-regulation of VEGF mRNA induced by the A2AAR agonist, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Conversely, 6-h exposure of cells to either phorbol 12-myristate 13-acetate (PMA) or protein kinase C (PKC) inhibitors mimicked the CGS21680-induced down-regulation. PMA activated PKCalpha , PKCepsilon , and PKCzeta , and CGS21680 activated PKCepsilon and PKCzeta as assessed by cellular translocation. By 6 h, PMA but not CGS21680 decreased PKCalpha and PKCepsilon expression. Neither compound affected PKCzeta levels. Following prolonged PMA treatment to down-regulate susceptible PKC isoforms, CGS21680 but not PMA inhibited the cobalt chloride induction of VEGF mRNA. The proteasome inhibitor, MG-132, abolished PMA- but not CGS21680-induced down-regulation of VEGF mRNA. Phorbol 12,13-diacetate reduced VEGF mRNA levels while down-regulating PKCepsilon but not PKCalpha expression. In cells expressing a dominant negative PKCzeta construct, CGS21680 was unable to reduce VEGF mRNA. Together, the findings suggest that phorbol ester-induced down-regulation of VEGF mRNA occurs as a result of a reduction of PKCepsilon activity, whereas that mediated by the A2AAR occurs following deactivation of PKCzeta .

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vascular endothelial growth factor (VEGF)1 was described initially as a vascular permeability factor (1), and was characterized subsequently as an endothelial cell mitogen (2). VEGF is involved primarily in angiogenesis, the pruning and reorganization of pre-existing vasculature to create new vasculature, and it has a critical role in embryonic development (3). In the adult, VEGF is required for the development and maintenance of the female reproductive cycle (4) and may be cardioprotective during ischemia (5). However, elevated levels of VEGF have been associated with pathologies, such as diabetic retinopathy, endometriosis, rheumatoid arthritis, and tumorigenesis (3, 6-8). Many tumors demonstrate elevated levels of VEGF, which can be correlated to disease progression (9-12). This correlation reflects the requirement of an expanding vasculature for tumor growth, and disruption of VEGF signaling retards cancer progression (13-15).

Several factors, including hypoxia (16-19), various growth factors (20-22), and oncogenic mutations (23-25), up-regulate VEGF and the underlying mechanisms have been extensively examined. Less is known about the factors that down-regulate VEGF: natriuretic peptides (26), N-acetylcysteine (27), somatostatin (28), and certain anti-inflammatory drugs (29, 30). The pathways mediating the down-regulation of VEGF have not been elucidated.

Rat pheochromocytoma (PC12) cells are a frequently employed model for hypoxia-initiated responses and have been used to study VEGF gene regulation, as hypoxia is a potent stimulant of VEGF expression (16-19). Additionally, PC12 cells express A2A and A2B adenosine receptors (AR) (31) and have been employed to study AR signal transduction and physiologic activity. This laboratory has shown previously that activation of the A2AAR in PC12 cells results in a substantial reduction of VEGF, which is observed at both the mRNA and protein levels (32). Furthermore, this down-regulation of VEGF mRNA occurs because of an inhibition of VEGF gene transcription (32). The nonselective AR agonist, 5'-(N-ethylcarboxamido)adenosine, was also reported to down-regulate VEGF expression in PC12 cells (33). Other cell types have been shown to respond to AR agonists with either increases or decreases in VEGF expression (34-36). This differential regulation may exist because of the subtype specificity of various AR ligands, and because of cell-specific variations in the signal transduction cascade to which a distinct AR subtype may be linked.

The A2AAR is typically coupled via the Gs protein to the stimulation of adenylyl cyclase (AC) and activation of protein kinase A (PKA) (31, 37, 38). However, certain effects mediated by the A2AAR have been linked to protein kinase C (PKC) activation (39-42). Based on their requirements for activation, three PKC classes are defined: conventional (alpha , beta I, beta II, gamma ), which are activated by Ca2+ and diacylglycerol (DAG); novel (delta , epsilon , eta , theta ), which are Ca2+-independent; and atypical (zeta , lambda , tau ), which are activated independently of Ca2+ or DAG. PC12 cells have been reported to express PKC isoforms alpha , beta I, beta II, delta , epsilon , eta , theta , and zeta  (43-45). Two specific PKC isoforms, PKCzeta and PKCepsilon , have been demonstrated to regulate VEGF expression. For example, increases in PKCzeta activity up-regulate VEGF expression in glioblastoma U373 cells (46), and in HT1080 fibrosarcoma and 786-0 renal carcinoma cells (25). Activation of PKCzeta has also been implicated in stretch-induced up-regulation of VEGF in retinal capillary pericytes (47). In addition, it has been reported that ischemic preconditioning induces translocation of PKCepsilon to the nucleus in cardiomyocytes, which causes up-regulation of VEGF expression (48).

The goal of the present study was to elucidate the signal transduction cascade responsible for the down-regulation of VEGF mRNA that is induced by 2-[4-[(2-carboxyethyl)phenyl]-ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680), a selective agonist for the A2AAR. Our results indicate that stimulation of PKC activity by either CGS21680 or phorbol 12-myristate 13-acetate (PMA) produces an initial up-regulation of VEGF mRNA that is rapidly followed by a marked reduction in VEGF expression. The latter response appears to result from a decrease in PKC activity with specifically PKCepsilon and PKCzeta mediating the PMA- and CGS21680-induced response, respectively.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell Culture-- Rat pheochromocytoma (PC12) cells were grown in complete RPMI medium (RPMI medium 1640 supplemented with 10% fetal bovine serum, 10% equine serum, 1× penicillin-streptomycin-glutamine, and 0.25 µg/ml fungizone), and were maintained in a 5% CO2-humidified incubator at 37 °C as previously described (32). Cells were subcultured into collagen-coated six-well dishes, 100-mm dishes, or T-75 flasks for experiments 24 h prior to treatment. Culture medium was replaced with fresh complete RPMI medium or with RPMI 1640 medium, when noted, 30-60 min prior to treatment. The times of treatment with various agonists and inhibitors are provided under "Results." Control cells were treated with appropriate volumes of dimethyl sulfoxide when appropriate.

Radioimmunoassay of cAMP-- Intracellular cAMP levels in PC12 cells were determined with a cAMP 125I-Radioimmunoassay Kit (PerkinElmer Life Sciences). Cells in six-well dishes were treated for the indicated amount of time and washed twice with phosphate-buffered saline (PBS) (1.36 M NaCl, 27 mM KCl, 80.5 mM Na2HPO4, 14.7 mM KH2PO4), scraped, and suspended in 1 ml of EtOH. A 250-µl aliquot of the lysate was then dried at 60 °C for 3 h in a SpeedVac (Savant), and lysates were resuspended in 250 µl of sterile H2O. These solutions were diluted 1:50 in assay buffer, and 100-µl aliquots of the diluted solutions were used for the assay in duplicate. Samples were incubated overnight with 125I-labeled cAMP and antiserum complex prior to cAMP precipitation and centrifugation the following day. Gamma counts of the precipitated cAMP pellets were determined, and intracellular cAMP concentrations determined from a set of freshly prepared standards as instructed by the manufacturer.

Northern Blot Analysis-- Northern blot analysis was performed to determine VEGF mRNA content as previously described (32), with minor modifications. Total RNA was isolated from PC12 cells with TRIzol reagent (Invitrogen) and RNA samples were run on 1% agarose gels containing 2.2 M formaldehyde. RNA was then transferred to Zeta-Probe nylon membranes (Bio-Rad) and UV cross-linked with a Strata-linker (Stratagene, La Jolla, CA) prior to prehybridization at 42 °C for 2-5 h. Hybridization was conducted overnight with a 600-bp fragment of murine VEGF165 cDNA random prime-labeled with [32P]dCTP. The membrane was sequentially washed and subjected to autoradiography. To normalize total RNA levels, membranes were additionally hybridized with a 1,100-bp fragment of human glyceraldehyde-3-phosphate dehydrogenase (Clontech, Palo Alto, CA) random prime labeled with [32P]dCTP. Autoradiographic signals were quantitated by an AlphaImager 2000 (Alpha Innotech Corp.).

Western Blot Analysis-- Total protein from PC12 cells was isolated and analyzed to determine PKC protein levels. Cells were treated for the indicated amounts of time and then scraped in 250 µl of lysis buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 0.1 M dithiothreitol), boiled for 5 min, and microcentrifuged at 13,000 × g for 15 min at 4 °C. Total protein concentration of the resulting supernatant was determined with Bio-Rad Protein Assay, and equal amounts of protein were electrophoresed on 8% polyacrylamide gels. Protein was then transferred to nitrocellulose membranes and blocked for 1 h with Blotto (5% nonfat dry milk, 0.2% Triton X-100, 0.05% thiomerosal, in PBS), prior to being incubated overnight at 4 °C with the appropriate primary antibody at a 1:1000 dilution in Blotto. The following antibodies were employed: cPKCalpha (C-20), nPKCepsilon (C-15), and aPKCzeta (C-20)-G (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed three times for 5 min with Blotto and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at 1:10,000 dilution for 1 h at room temperature. Membranes were then washed three times for 5 min in Blotto and two times for 5 min with PBS prior to being developed with ECL Western blotting detection reagents (Amersham Biosciences) and being exposed to x-ray film. Signals were analyzed with an AlphaImager 2000.

Nuclear, Cytosolic, and Membrane PKC Analysis-- PC12 cells were treated with the appropriate agonists for 5 min. After treatment, subcellular fractions were isolated as previously described (49), with minor modifications. Briefly, cells from 100-mm dishes were scraped in 100 µl of extraction buffer (20 mM Tris, pH 7.6, 2 mM EDTA, 5 mM EGTA, 10 mM beta -mercaptoethanol (beta -ME), and protease inhibitors including 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.5 µg/ml aprotinin). Cells were then Dounce homogenized with 10 strokes, and lysates were microcentrifuged at 500 × g for 10 min. The supernatant (plasma membrane and cytosolic fractions) was removed and centrifuged at 100,000 × g for 60 min, and the resulting pellet (plasma membrane) was resuspended in 50 µl of suspension buffer (10 mM Tris, pH 7.6, 5 mM MgCl2, 5 mM beta -ME, and protease inhibitors as described above) by sonication. The supernatant was collected as the cytosolic fraction. The pellet from the initial 500 × g spin (nuclear fraction) was resuspended in 100 µl of nuclei buffer (5 mM Tris, pH 7.6, 10.5 mM MgCl2, 10 mM beta -ME, and protease inhibitors described above) supplemented with 0.1% Triton X-100 and then layered over 100 µl of nuclei buffer + 0.5 M sucrose and microcentrifuged for 10 min at 500 × g. The pellet (nuclear fraction) was resuspended in 25 µl of suspension buffer by sonication. All fractions were then resuspended in lysis buffer, boiled for 5 min, and microcentrifuged for 15 min at 13,000 × g prior to protein concentration determination by Bio-Rad protein assay. Equal amounts of protein were run on 8% polyacrylamide gels as described above. Nuclear sample purity was demonstrated with the histone H1 (AE-4) antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Adenoviral Vector Expression of PKCzeta -- PC12 cells were infected with previously characterized replication-deficient adenoviral vectors containing either wild-type or kinase-deficient mutant (dominant negative) forms of PKCzeta , or a vector containing beta -galactosidase as a control (50, 51). Each recombinant adenovirus was prepared as previously described (52). The vectors were expanded in HEK293 cells, and the amount of plaque-forming units for each was determined to infect at an equal multiplicity of infection (m.o.i.). The wild-type PKCzeta adenoviral vector was infected at an m.o.i. of 600, whereas the dominant negative PKCzeta adenoviral vector was infected at an m.o.i. of 800. PC12 cells were infected for 8 h in 4 ml of RPMI 1640 medium supplemented with 2% fetal bovine serum with gentle shaking in a 5% CO2-humidified 37 °C cell incubator. Complete RPMI medium was then added to the cells overnight, and cells were either treated for 24 h with 1 µM PMA prior to a 6-h treatment with the appropriate agonist in fresh complete medium, or cells were directly given fresh complete RPMI medium and treated for 6 h with the appropriate agonist. Cells were then scraped in 1 ml of PBS, and a 150-µl aliquot was microcentrifuged at 13,000 × g, and the resulting pellet was lysed in 100 µl of lysis buffer. PKC protein levels were assessed by Western blot as described above. The remaining cell suspension was spun at 200 × g, and the resulting pellet was used for RNA isolation. VEGF mRNA levels were assessed by Northern blot analysis as described above.

Data Analysis-- All experiments were performed a minimum of three times, and in duplicate when noted. Results are expressed as mean ± S.E. Statistical analysis was performed by one-way analysis of variance followed by a Newman-Keuls post-test. A p value < 0.05 was considered significant.

Reagents-- The following compounds were purchased from Calbiochem (La Jolla, CA): forskolin, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89), protein kinase A inhibitor 14-22 amide (PKI), PMA, MG-132, bisindolylmaleimide IX (Ro-31-8220), and bisindolylmaleimide I (GFX). Epidermal growth factor (EGF) and all cell culture reagents were obtained from Invitrogen. The following compounds were purchased from Sigma: 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), CGS21680, and phorbol 12, 13-diacetate (PDA).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Examination of the Involvement of PKA in the A2AAR-mediated Down-regulation of VEGF-- As stimulation of the A2AAR is linked to AC activation (31, 37, 38), the signaling pathway mediating the down-regulation of VEGF mRNA induced by CGS21680, a selective agonist for the A2AAR, was initially explored using activators and inhibitors of PKA (Fig. 1A). As previously described (32), treatment of PC12 cells for 6 h with 1 µM CGS21680 reduced the VEGF mRNA level to 27.0 ± 8.1% of that in control cells. Forskolin (5 µM), an activator of AC and 8-Br-cAMP (1 mM), a cell-permeable cAMP analogue, did not alter VEGF mRNA levels (Fig. 1A). The PKA inhibitors, H-89 (5 µM) and PKI (10 µM), had no effect on VEGF mRNA and were unable to reverse the CGS21680-induced down-regulation of VEGF mRNA (Fig. 1A). To further examine the role of cAMP in CGS21680-induced down-regulation of VEGF mRNA, whole cell accumulation of cAMP was assessed in response to forskolin (5 µM) or CGS21680 (1 µM) (Fig. 1B). Radioimmunoassay of cAMP content showed that forskolin elicited a greater increase in cAMP levels than CGS21680 at all time points examined during a 6-h time course. As forskolin increased cAMP levels significantly more than CGS21680, but had no effect on VEGF mRNA levels, the data strongly suggest that cAMP is not involved in the A2AAR-mediated down-regulation of VEGF. Overall, the sum of these data indicates that pathways involving cAMP and/or PKA are not involved in the observed down-regulation of VEGF following the stimulation of the A2AAR in PC12 cells.


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Fig. 1.   CGS21680-induced down-regulation of VEGF mRNA in PC12 cells is not mediated by the adenylyl cyclase-PKA pathway. A, effect of elevated intracellular cAMP and PKA inhibitors on VEGF mRNA in control and CGS21680-treated cells. PC12 cells were treated for 6 h with 5 µM forskolin, 1 mM 8-Br-cAMP, 5 µM H-89, 10 µM PKI, or 1 µM CGS21680 (CGS), or cells were pretreated for 1 h with a PKA inhibitor prior to 6 h of treatment with 1 µM CGS21680. Total RNA was collected, and Northern blot analysis was performed. Significant difference from control denoted by ** (p < 0.001). There was no significant difference between CGS21680-treated cells and cells treated with PKA inhibitors in combination with CGS21680 (p > 0.05). B, forskolin elevated intracellular cAMP levels to a greater degree than CGS21680. PC12 cells were treated with CGS21680 (1 µM) or forskolin (5 µM) for the indicated amount of time, and intracellular cAMP levels were quantitated by radioimmunoassay. All experiments were conducted in duplicate. Data are presented as percentage of cAMP in untreated cells. Significant difference between CGS21680- and forskolin-treated cells is denoted by * (p < 0.05), # (p < 0.01), or ** (p < 0.001).

PKC Activation in Response to Stimulation of the A2AAR-- As activation of the A2AAR has been linked to PKC stimulation in certain systems (39-41), including PC12 cells (42), the role of PKC in the regulation of VEGF mRNA was explored. For the initial analysis, cells were serum-starved for 14 h prior to a 6-h agonist treatment, as growth factors found in serum can stimulate PKC activity and/or up-regulate VEGF expression (21, 32). Under serum-free conditions, CGS21680 (1 µM), PMA (100 nM), and EGF (10 ng/ml), a growth factor known to activate PKC (21, 53), similarly regulated VEGF mRNA in a biphasic manner over a 6-h time period (Fig. 2A). At 1 h of treatment, all three compounds induced an initial increase in VEGF mRNA levels, followed by a down-regulation of VEGF mRNA by 6 h. The similarity of the PMA-, EGF-, and CGS21680-induced responses indicated that PKC may be involved in the A2AAR-mediated regulation of VEGF. To explore this possibility, the up-regulation of VEGF mRNA induced at 1 h by CGS21680 and PMA was examined for sensitivity to chemical inhibitors of PKC. As shown in Fig. 2B, administration of GFX (5 µM) and Ro-31-8220 (5 µM) blocked the increase in VEGF expression that was induced by CGS21680 and PMA. Under these conditions, the PKC inhibitors alone had no effect on VEGF mRNA.


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Fig. 2.   PKC regulation of VEGF expression in serum-starved PC12 cells. A, biphasic regulation of VEGF mRNA by CGS21680, PMA, and EGF. PC12 cells were serum-starved for 14 h prior to treatment for the indicated times with CGS21680 (1 µM), PMA (100 nM), or EGF (10 ng/ml). Total RNA was collected, and Northern blot analysis was performed. Significant difference from control denoted by * (p < 0.05), # (p < 0.01), or ** (p < 0.001). B, PKC inhibitors Ro-31-8220 and GFX blocked CGS21680- or PMA-induced up-regulation of VEGF mRNA in serum-starved PC12 cells. PC12 cells were serum-starved for 14 h prior to treatment with 1 µM CGS21680 (CGS) or 100 nM PMA, either alone or in combination with 5 µM Ro-31-8220 or 5 µM GFX. Total RNA was collected, and Northern blot analysis was performed. Significant difference from control is denoted by * (p < 0.05) or # (p < 0.01). Significant difference between CGS21680 or PMA treatments and CGS21680 or PMA treatments in combination with a PKC inhibitor is denoted by + (p < 0.05).

Based on the above findings, the ability of CGS21680 and PMA to activate various PKC isoforms was assessed by examining the translocation of PKC isoforms in the nuclear, cytosolic, and membrane fractions as translocation to the membrane or the nucleus is an indicator of enzyme activation (54-56). PKCalpha , PKCepsilon , and PKCzeta isoforms were studied, as each represents one of the three PKC classes: conventional, novel, and atypical, respectively. As shown in Fig. 3, PMA promoted the nuclear translocation of PKCalpha , PKCepsilon , and PKCzeta by 710.8 ± 181.8%, 520.5 ± 107.2%, and 397.7 ± 98.9%, respectively. CGS21680 induced the nuclear translocation of PKCepsilon and PKCzeta by 358.7 ± 86.3% and 416.0 ± 146.1%, respectively. This correlates with previous data indicating activation of the PKC pathway following stimulation of the A2AAR (39-42).


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Fig. 3.   PMA activated PKCalpha , PKCepsilon , and PKCzeta , whereas CGS21680 activated PKCepsilon and PKCzeta in PC12 cells. Cells were treated for 5 min with either 1 µM CGS21680 (CGS) or 100 nM PMA and nuclear, cytosolic, and membrane protein fractions were collected as described under "Experimental Procedures." Protein fractions were analyzed by Western blot. Significant difference from control is denoted by * (p < 0.05) or ** (p < 0.001).

Role of PKC Inhibition in the Down-regulation of VEGF mRNA-- To further explore the role of PKC activity in the down-regulation of VEGF, modulation of VEGF mRNA expression by a 6-h treatment with CGS21680 (1 µM) or PMA (100 nM) was examined for sensitivity to GFX (5 µM) and Ro-31-8220 (5 µM). These experiments were conducted in cells maintained in complete growth medium. As shown in Fig. 4A, the CGS21680- or PMA-induced down-regulation of VEGF mRNA was unaltered in the presence of GFX and Ro-31-8220. Interestingly, however, when these PKC inhibitors were administered alone, GFX and Ro-31-8220 lowered VEGF mRNA to 44.8 ± 8.7% and 40.7 ± 7.8% of control, respectively. To characterize this decrease in VEGF mRNA elicited by GFX and Ro-31-8220, time-course studies for these PKC inhibitors were performed. As shown in Fig. 4B, both compounds significantly reduced VEGF mRNA levels at 30 min of treatment (to 55.0 ± 17.2% by Ro-31-8220 and to 48.3 ± 0.7% by GFX) and this down-regulation remained throughout 6 h.


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Fig. 4.   PKC inhibitors Ro-31-8220 and GFX did not reverse the CGS21680-induced down-regulation of VEGF mRNA, but alone these inhibitors decreased basal VEGF mRNA levels in PC12 cells. A, cells were treated for 6 h with 1 µM CGS21680 (CGS), 100 nM PMA, 5 µM Ro-31-8220 (Ro), or 5 µM GFX; or cells were treated simultaneously with 5 µM Ro-31-8220 or 5 µM GFX and 1 µM CGS21680 or 100 nM PMA for 6 h. Total RNA was collected, and Northern blot analysis was performed. Significant difference from control is denoted by * (p < 0.001). There is no significant difference between CGS21680- or PMA-treated cells and cells treated with PKC inhibitors in combination with CGS21680 or PMA (p > 0.05). B, Ro-31-8220 and GFX promoted a rapid and sustained down-regulation of VEGF mRNA in PC12 cells. Cells were treated for the indicated time with either Ro-31-8220 (5 µM) or GFX (5 µM). Total RNA was collected, and Northern blot analysis was performed. Significant difference from control is denoted by * (p < 0.05), # (p < 0.01), or ** (p < 0.001).

The above data suggest that prolonged exposure of PC12 cells to CGS21680 or PMA may decrease VEGF mRNA content via an inhibition of PKC activity that may result from a down-regulation of PKC levels. Such a regulation of novel and conventional PKC isoforms is well documented in cells exposed for extended periods of time to PMA (57, 58). To examine this possibility, PC12 cells were treated with CGS21680 (1 µM) or PMA (100 nM) for 6 h and whole cell lysates were analyzed for PKC content (Fig. 5). Relative to control cells, PMA reduced PKCalpha and PKCepsilon expression by 55.7 ± 4.5% and 91.2 ± 1.7%, respectively. PMA did not significantly alter PKCzeta levels. Treatment of PC12 cells with CGS21680 for 6 h caused no significant change in the expression of any examined PKC isoform.


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Fig. 5.   Regulation of PKC isoform expression by 6-h treatment of PC12 cells with either CGS21680 or PMA. Cells were treated for 6 h with either 100 nM PMA or 1 µM CGS21680 (CGS). Total protein was collected and analyzed by Western blot. Significant difference from control denoted by * (p < 0.05) or ** (p < 0.001).

Identification of PKC Isoforms Involved in the CGS21680- and PMA-induced Down-regulation of VEGF mRNA-- To further define a role for PKC and to identify the specific PKC isoform(s) that may be involved in the CGS21680- or PMA-induced down-regulation of VEGF mRNA, PC12 cells were treated with 1 µM PMA for 24 h to remove conventional and novel PKCs. Cells were then treated with cobalt chloride (50 µM) with or without CGS21680 (1 µM) or PMA (100 nM) for 6 h. Application of cobalt chloride mimics hypoxia and has been demonstrated to elevate VEGF mRNA expression (16). As shown in Fig. 6, in control cells and cells treated for 24 h with PMA, cobalt chloride induced VEGF expression by 3.1 ± 0.5- and 4.9 ± 1.3-fold, respectively. In control cells, this cobalt chloride-induced up-regulation of VEGF mRNA was inhibited by 71.0 ± 9.5% by CGS21680 and 124.9 ± 13.8% by PMA, i.e. PMA reduced VEGF mRNA to a level lower than that observed in cells not treated with cobalt chloride. In cells treated for 24 h with PMA, CGS21680 similarly produced a 66.7 ± 7.5% inhibition of the cobalt chloride-induced response. Conversely, the ability of PMA to block the cobalt chloride-induced up-regulation of VEGF mRNA was nearly abolished, with an observed inhibition of 20.3 ± 9.1%. These data indicate that CGS21680-induced down-regulation of VEGF mRNA is not dependent on conventional and/or novel PKC isoforms, but that it may depend on PKCzeta , as CGS21680 can further down-regulate VEGF mRNA when PKCzeta remains available.


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Fig. 6.   CGS21680, but not PMA, inhibited cobalt chloride-induced VEGF mRNA up-regulation in PC12 cells pre-treated with PMA for 24 h. PC12 cells were treated with PMA (1 µM) for 24 h prior to a 6-h treatment with 50 µM cobalt chloride (Cobalt) alone or in combination with either 1 µM CGS21680 (CGS) or 100 nM PMA. Control cells did not receive 24 h PMA treatment. Total RNA was collected, and Northern blot analysis was performed. For control group, significant difference from cobalt chloride treatment is denoted by * (p < 0.05) or ** (p < 0.001); and for the 24-h PMA group, significant difference from cobalt chloride treatment is denoted by # (p < 0.01).

Use of a Proteasomal Inhibitor and a Selective Phorbol Ester to Identify the Specific PKC Isoform That Is Involved in the PMA-induced Response-- The present findings suggest that the PMA-induced reduction in VEGF mRNA occurs as a result of the ability of this phorbol ester to down-regulate susceptible PKC isoforms. The down-regulation of PKC isoforms frequently results from targeting of activated PKCs for degradation through proteasome pathways (58-60). Thus, PMA- and CGS21680-induced down-regulation of VEGF mRNA was analyzed for sensitivity to MG-132, a chemical inhibitor of proteasomal degradative activity. As shown in Fig. 7, treatment with MG-132 (500 µM) alone increased VEGF mRNA levels to 215.0 ± 29.0% of control. It may be speculated that MG-132 inhibits the degradation of HIF-1alpha , a transcription factor known to up-regulate VEGF mRNA and to be subject to constitutive proteasomal degradation (61). MG-132 abolished the ability of PMA to down-regulate VEGF mRNA while having no effect on the reduction of VEGF mRNA elicited by CGS21680. To confirm the ability of MG-132 to inhibit the PMA-induced down-regulation of PKC isoforms, whole cell lysates of PC12 cells were analyzed for PKC expression by Western blotting following treatment with PMA in the absence or presence of MG-132 (Fig. 7, inset). The PMA-induced down-regulation of both PKCalpha and PKCepsilon was completely reversed by MG-132. MG-132 itself had no effect on PKC expression. These data indicate a possible role for PKCalpha and/or PKCepsilon in PMA down-regulation of VEGF mRNA, while also supporting the above described findings (Fig. 6) that indicate these PKC isoforms are not involved in the mechanism by which CGS21680 down-regulates VEGF mRNA.


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Fig. 7.   MG-132 blocked PMA-induced degradation of PKCalpha and PKCepsilon and inhibited PMA-induced, but not CGS21680-induced, down-regulation of VEGF mRNA in PC12 cells. PC12 cells were treated with 1 µM CGS21680 (CGS), 100 nM PMA, or 500 nM MG-132, alone or in combination, for 6 h as indicated. Total RNA was collected, and Northern blot analysis was performed. Significant difference from control is denoted by * (p < 0.05), whereas significant difference between MG-132 and MG-132/CGS21680 is denoted by ** (p < 0.001). Inset, PC12 cells were treated for 6 h with either 100 nM PMA or 500 nM MG-132 alone or in combination as indicated. Total protein was collected, and PKC isoform expression was analyzed by Western blot. Significant difference from control denoted by # (p < 0.01) or ** (p < 0.001).

To further support the role of PKC down-regulation as the mechanism by which PMA reduces VEGF mRNA, PDA was explored for its ability to regulate PKC isoform expression and VEGF mRNA levels. PDA has been reported to activate but not promote the degradation of PKCalpha in rat brain cortical slices (62). As demonstrated in Fig. 8, a 6-h treatment of PC12 cells with 10 µM PDA promoted a down-regulation of VEGF mRNA nearly identical to that observed with 100 nM PMA. Although producing an 85.2 ± 4.3% reduction in PKCepsilon expression, 10 µM PDA had no significant effect on PKCalpha levels relative to untreated cells. As observed with PMA, PDA did not regulate PKCzeta expression. The sum of these findings indicates a role for PKCepsilon in the phorbol ester-mediated down-regulation of VEGF mRNA.


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Fig. 8.   PDA promoted specific degradation of PKCepsilon and down-regulated VEGF mRNA in PC12 cells. Cells were treated for 6 h with the indicated concentration of PDA or 100 nM PMA. Total RNA was collected, and Northern blot analysis was performed. Significant difference from control denoted by * (p < 0.05). Inset, in parallel, PC12 cells were treated under the same conditions, total protein was collected, and expression of PKC isoforms was analyzed by Western blot. Significant difference from control is denoted by * (p < 0.05) or ** (p < 0.001).

Application of Adenoviral Vectors Expressing Either Wild-type or Dominant Negative PKCzeta to Alter the CGS21680-induced Response-- To study the putative role of PKCzeta in constitutive VEGF expression in PC12 cells and more specifically the A2AAR-mediated down-regulation of VEGF mRNA, we employed adenoviral vectors that directed expression of either wild-type PKCzeta or a dominant negative form of PKCzeta (DNPKCzeta ). Control cells were infected at the same m.o.i. with a beta -galactosidase adenoviral construct. 24 h after infection, cells were treated with 1 µM PMA for 24 h prior to treatment with CGS21680. The addition of long term PMA allowed for the specific analysis of PKCzeta activity, as novel and conventional PKC isoforms were removed. As shown in Fig. 9A, 1 µM CGS21680 reduced VEGF mRNA to 45.0 ± 12.1% of that observed in untreated cells expressing beta -galactosidase. In cells in which there was a 3.9 ± 0.9-fold overexpression of wild-type PKCzeta (Fig. 9A, inset), CGS21680 was unable to induce a significant decrease in VEGF mRNA. In a separate set of experiments, cells were infected with the beta -galactosidase or DNPKCzeta adenoviral constructs at the same m.o.i. (Fig. 9B). The addition of CGS21680 to cells expressing the beta -galactosidase construct reduced the VEGF mRNA level to 40.7 ± 6.4% of that in untreated cells. In cells expressing DNPKCzeta (Fig. 9B, inset), VEGF mRNA levels were 43.7 ± 1.3% of that observed in beta -galactosidase-expressing cells. However, 1 µM CGS21680 did not further down-regulate VEGF mRNA in cells expressing DNPKCzeta .


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Fig. 9.   Involvement of PKCzeta in the CGS21680-induced down-regulation of VEGF mRNA in PC12 cells. A, overexpression of wild-type PKCzeta . Cells were infected for 8 h with adenovirus directing either the expression of beta -galactosidase or wild-type PKCzeta prior to exposure for 24 h to 1 µM PMA. Cells were then treated for 6 h with 1 µM CGS21680 (CGS) as indicated. Total RNA was collected, and Northern blot analysis was performed. Significant difference from control is denoted by * (p < 0.05). Inset, protein samples from cells treated as described above were analyzed for PKCzeta expression by Western blot. Representative blot is shown. B, expression of DNPKCzeta . Cells were infected for 8 h with adenovirus directing either the expression of beta -galactosidase or DNPKCzeta . After 24 h, cells were treated with 1 µM CGS21680 (CGS) as indicated. Total mRNA was collected, and Northern blot analysis was performed. Significant difference from control is denoted by ** (p < 0.001). Inset, protein samples from cells treated as described above were analyzed by Western blot with PKCzeta antibody. Representative blot is shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Research examining the signal transduction cascade linking A2AAR activation to physiologic responses has typically demonstrated a critical role for the AC-PKA pathway (31, 37, 38). Therefore, the initial focus of this study was the role of AC and PKA in the A2AAR-mediated down-regulation of VEGF mRNA. Forskolin and 8-Br-cAMP did not decrease VEGF mRNA, and PKA inhibitors did not modulate the CGS21680-induced down-regulation of VEGF. As there is evidence for PKA-independent, but cAMP-dependent events in signal transduction (63), changes in cellular cAMP were evaluated. Forskolin elevated cAMP levels to a greater degree than CGS21680 throughout a 6-h time course, indicating that CGS21680-induced VEGF down-regulation is not cAMP-dependent. Thus, multiple results strongly imply that AC and PKA are not involved in the A2AAR-mediated down-regulation of VEGF mRNA.

Subsequent focus was placed on the PKC pathway, as there have been reports linking A2AAR stimulation to PKC activation (39-42). Under serum-free conditions, CGS21680, PMA, and EGF initially up-regulated VEGF mRNA, and this was followed by a down-regulation at 6 h. The ability of two well described activators of PKC, PMA and EGF, to mimic the CGS21680-induced response suggested that the A2AAR may be linked to PKC stimulation. Indeed, the up-regulation of VEGF mRNA produced by CGS21680, PMA, and EGF was blocked by two different chemical inhibitors of PKC. Moreover, CGS21680 induced the translocation of PKCepsilon and PKCzeta to the nucleus whereas PMA directed nuclear translocation of PKCalpha , PKCepsilon , and PKCzeta . Recently, Huang and co-workers (42) demonstrated the rapid nuclear translocation of PKCzeta following A2AAR activation in PC12 cells and associated this response with the anti-apoptotic effects of CGS21680. It should be noted that, although insensitivity to direct activation by DAG has served to define atypical PKC isoforms, stimulation of PKCzeta by PMA in various cell types including PC12 cells has been observed (64, 65). Several studies have demonstrated that translocation of PKC to the nucleus is concurrent with activation (42, 44, 48, 55). This facet of PKC activity is particularly relevant to VEGF mRNA regulation, as PKC-mediated activation of nuclear transcription factors such as Sp-1 has a critical role in VEGF gene transcription (66, 67). The importance of PKC activity in VEGF mRNA regulation was further demonstrated, as GFX and Ro-31-8220 were found to rapidly down-regulate VEGF mRNA in PC12 cells maintained in complete medium. This suggests that VEGF mRNA is maintained at a high constitutive level in PC12 cells most likely because of high basal PKC activity resulting from growth factors present in the serum-supplemented medium. Indeed, VEGF mRNA and protein expression are markedly lower in serum-starved PC12 cells relative to cells maintained in complete medium (32).

The findings with the PKC inhibitors suggested that down-regulation of VEGF mRNA in response to CGS21680 and PMA over a prolonged period may occur as a result of a decrease in PKC activity, and several approaches were taken to examine this possibility. These studies demonstrated the differential regulation and roles of distinct PKC isoforms in the CGS21680- and PMA-induced responses. Treatment of PC12 cells with PMA for 6 h significantly decreased PKCalpha levels, and PKCepsilon expression was nearly abolished. It is well documented that long term (>= 24 h) treatment with PMA down-regulates conventional and novel PKC (59), but it was surprising to observe such a dramatic decrease at 6 h. In cells depleted of conventional and novel PKC isoforms by prolonged exposure to PMA, CGS21680, but not PMA, maintained the ability to inhibit the cobalt chloride-induced up-regulation of VEGF mRNA. This finding strongly implies a role for conventional/novel PKC isoforms in the PMA-induced VEGF down-regulation whereas PKCzeta may mediate the response observed with A2AAR activation.

The proteasome inhibitor MG-132 was employed to determine whether PKC degradation was central to the mechanism by which PMA down-regulates VEGF mRNA. MG-132 blocked PMA-induced down-regulation of VEGF mRNA, but had no effect on the CGS21680-induced response. PDA, a phorbol ester reported not to promote the degradation of PKCalpha (62), was administered to determine which PKC isoform may mediate the PMA-induced down-regulation of VEGF mRNA. PDA promoted the degradation of specifically PKCepsilon , and in a consistent temporal fashion down-regulated VEGF mRNA. These findings indicate that PMA-induced down-regulation of VEGF mRNA occurs because of the degradation of specifically PKCepsilon that follows the activation of this PKC isoform.

To further explore the putative role of PKCzeta in the A2AAR-mediated down-regulation of VEGF mRNA, replication-deficient adenoviruses containing either wild-type PKCzeta or a dominant negative PKCzeta construct were employed. In cells overexpressing wild-type PKCzeta and treated for 24 h with PMA, CGS21680 did not down-regulate VEGF mRNA. One possibility for this lack of responsiveness is that the mechanism by which prolonged stimulation of the A2AAR abrogates PKCzeta signaling is overwhelmed in the presence of excess PKCzeta levels. We did not observe up-regulation of VEGF mRNA levels upon overexpression of PKCzeta , and this was similarly reported for retinal capillary pericytes (47). Conversely, in other cell lines, an up-regulation of VEGF mRNA occurs upon overexpression of PKCzeta (25, 46). It is possible that, in PC12 cells, a relatively high basal activity of endogenous PKCzeta prevents any activity of experimentally introduced PKCzeta to be apparent. When a dominant negative PKCzeta isoform was expressed, VEGF mRNA levels were reduced relative to those in beta -galactosidase-expressing cells, an observation in agreement with the hypothesis regarding high constitutive activity of endogenous PKCzeta in PC12 cells. In these cells, CGS21680 did not down-regulate VEGF mRNA. Thus, the reduction in PKCzeta function induced via expression of the dominant negative construct may result in the loss of the signaling activity that is typically targeted for inhibition upon prolonged A2AAR stimulation.

Our findings implicating PKCepsilon and PKCzeta in VEGF regulation in PC12 cells are consistent with observations in other cell lines. For example, Kawata et al. (48) found that the specific translocation of PKCepsilon to the nucleus of cardiomyocytes 10 min after an ischemia/reperfusion protocol in rats up-regulated VEGF mRNA at 3 h. A PKC inhibitor blocked both the translocation of PKCepsilon and the up-regulation of VEGF mRNA. PKCzeta has also been shown to regulate VEGF mRNA expression in several cell types. In both human glioblastoma (46) and fibrosarcoma (25) cells, overexpression of PKCzeta resulted in constitutive up-regulation of VEGF mRNA. Similarly, expression of a dominant negative PKCzeta has been shown to abrogate VEGF up-regulation induced by various stimulants (25, 47). Particularly intriguing is the present finding that the reduction, and not the direct activation, of PKC signaling by a physiologically relevant stimulus, adenosine, underlies the VEGF mRNA down-regulation. The cellular effects occurring in response to acute PKC activation have been extensively explored. However, there have been reports of PKC deactivation/degradation upon prolonged agonist exposure as the mechanism underlying cellular response. For example, in an examination of the tumor-promoting effects of PMA in rat fibroblasts overexpressing c-Src, Lu and co-workers (68) found that phenotypic transformation of these cells corresponded temporally with phorbol ester-induced depletion of PKCdelta . Similar to results we describe, inhibitors of PKC and expression of a dominant negative PKCdelta also promoted phenotypic changes similar to those observed with prolonged PMA exposure (68). Additionally, Shizukuda et al. (69) reported that the VEGF-induced migration and proliferation of endothelial cells was mediated by a decrease in PKCdelta activity although PKCdelta expression remained unchanged. Overexpression of wild-type PKCdelta blocked the ability of VEGF to induce cell response (69), and this finding is similar to our observation that CGS21680 did not regulate VEGF in cells overexpressing PKCzeta . It is apparent that deactivation of PKCs, either through reduction in kinase activity or protein expression, can be as important or more important than their activation.

Our findings also raise questions regarding the mechanisms of differential feedback regulation of PKC isoforms. It is clear that PMA ultimately promotes the proteasomal degradation of PKCepsilon , but it is not known how A2AAR activation apparently decreases PKCzeta activity without modifying protein levels. Indeed, relatively little is known about the regulation of atypical PKC isoforms although it has been reported that PKCzeta may be processed by ubiquitinylation followed by proteasomal degradation (60). Additionally, of special interest in light of the lack of CGS21680-induced PKCzeta degradation is the prostate apoptosis response-4 (Par-4) gene product. Par-4 has been shown to directly and specifically associate with PKCzeta with a resulting dramatic reduction in PKCzeta kinase activity (70). Our PC12 cells express the Par-4 gene product (data not shown), and it is currently being examined whether A2AAR activation modulates Par-4 expression or promotes its complex formation with PKCzeta .

    ACKNOWLEDGEMENTS

We are very grateful to Dr. Jeff Molkentin (Children's Hospital Medical Center, Cincinnati, OH) for the generous gift of the PKCzeta adenoviral constructs. We thank Dr. Ron Millard (University of Cincinnati, Cincinnati, OH) for advice with statistical analysis. We very much appreciate the excellent technical assistance of John Meinken.

    FOOTNOTES

* This work was supported in part by National Institutes of Health NCI Grant R01 CA79531 (to M. E. O.).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.

Dagger Supported by National Institutes of Health Training Grant 5T32 HL07382.

§ To whom correspondence should be addressed. Tel.: 513-558-2361; Fax: 513-558-1169; E-mail: mark.olah@uc.edu.

Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M208366200

    ABBREVIATIONS

The abbreviations used are: VEGF, vascular endothelial growth factor; PC12, pheochromocytoma; AR, adenosine receptor; AC, adenylyl cyclase; PKA, protein kinase A; PKC, protein kinase C; DAG, diacylglycerol; CGS21680, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine; PMA, phorbol 12-myristate 13-acetate; beta -ME, beta -mercaptoethanol; DNPKCzeta , dominant negative PKCzeta ; m.o.i., multiplicity of infection; H-89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide; PKI, protein kinase A inhibitor 14-22 amide; Ro-31-8220, bisindolylmaleimide IX; GFX, bisindolylmaleimide I; EGF, epidermal growth factor; 8-Br-cAMP, 8-bromoadenosine 3',5'-cyclic monophosphate; PDA, phorbol 12, 13-diacetate; Par-4, prostate apoptosis response-4; PBS, phosphate-buffered saline.

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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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