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Biochemistry and Biophysics |
Division of Molecular Biology, Department of Surgery, Medical Faculty Charité, Humboldt University [C. P., X. H., J. M., D. B-O., W. D.], and Department of Molecular Biology, Max-Planck-Institute of Infection Biology [M. N.], 10117 Berlin, Germany
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ABSTRACT |
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c-Jun(1–226), inhibits VEGF production in HeLa cells. The transcription factors AP-2 and SP-1 act independently of the CSN. They contribute less than a quarter to basal VEGF production. Under our experimental conditions, hypoxia-inducible factor 1
protein was not detected. Overexpression of the tumor suppressor p53 reduces VEGF production in HeLa cells. p53 competes with c-Jun for CSN-specific phosphorylation with the consequence of c-Jun destabilization. We conclude that CSN-directed c-Jun signaling mediates high VEGF production in HeLa and HL-60 cells. The data provide an explanation for the known antiangiogenic and antitumorigenic activities of curcumin. Because the CSN regulates the major part of VEGF production in the tested tumor cells, it constitutes a potentially important target for tumor therapy. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Angiogenesis is regulated by a variety of positively and negatively acting effectors. The VEGF,3
the sole endothelial cell-specific mitogen (5
, 6)
, is one of the most prominent proangiogenic regulators. It is expressed by a wide range of tumor cells (7
, 8) , and abrogation of VEGF with a monoclonal antibody inhibits tumor growth in vivo (9)
. Promoter analysis of the VEGF gene revealed AP-1, AP-2, SP-1, and HIF-1 binding sites (10
, 11)
. Hypoxia induces VEGF expression through activation of HIF-1 (11)
and AP-1 (12
, 13)
. AP-1-mediated transcription is also involved in the signaling cascade prompted by transforming growth factor ß1 (14)
and lipopolysaccharide (15)
. AP-2 mediates transcriptional activation by stimulators that act through the p42/p44 mitogen-activated protein kinase module (16)
such as transforming growth factor (17)
and UV A radiation (18)
. SP-1 is responsible for the stimulation of VEGF expression by TNF- (12
, 19)
. The drug mithramycin inhibits SP-1 activity by specifically binding to the GC box motif in SP-1 binding sites (20)
. Another transcription factor involved in the control of VEGF expression is p53, which down-regulates VEGF in human tumor cells (21)
. At least two levels of p53 action might be responsible for the observed effect. The region of the VEGF promoter that, as defined by deletion analysis, is necessary for the inhibitory action of p53 indicates interference with SP-1 activity (22)
. On the other hand, p53 induces Mdm2, which causes ubiquitination and subsequent degradation of HIF-1 by the Ub/26S proteasome system (23)
. The exact mechanisms, however, are not clear at the moment.
The CSN is a highly conserved multimeric protein complex with kinase activity (24)
. It is comprised of eight subunits named CSN1–8 (25)
that exhibit significant sequence homologies to the eight subunits of the 26S proteasome lid complex (26
, 27) . Although the exact function of the CSN has yet to be established, there is no doubt about its involvement in signal transduction. The CSN phosphorylates transcriptional regulators such as p105, I
ß, c-Jun (24)
, and p53 (28)
. CSN-specific phosphorylation of c-Jun leads to stabilization of the transcription factor in HeLa cells, resulting in increased AP-1 transcriptional activity (29)
. Overexpression of CSN2 stimulates CSN-dependent c-Jun activation, which is independent of the JNK signaling pathway (29)
. The most effective inhibitor of the CSN kinase activity is curcumin (diferuloylmethane; Ref. 27
), a major active component of the food flavoring turmeric (30)
, which is known to be antitumorigenic (31)
and antiangiogenic (32)
. A more specific inhibitor of the CSN-dependent c-Jun phosphorylation is the NH2-terminal c-Jun fragment c-Jun(1–226) (29)
.
There is increasing evidence for a functional cooperation of the CSN with the Ub/26S proteasome system in regulation of the stability of important cellular proteins. CSN-specific phosphorylation either stabilizes regulatory proteins against degradation or targets them for degradation by the Ub/26S proteasome pathway. CSN phosphorylates c-Jun at its NH2 terminus, including residue Ser-63 and Ser-73 (24) , which is known to prevent ubiquitination and subsequent degradation of the transcription factor (33) . In contrast to c-Jun, the CSN-specific phosphorylation of p53 targets the tumor suppressor to degradation mediated by the 26S proteasome (28) . The fate of the cyclin-dependent kinase inhibitor p27Kip1 might also be determined by the CSN in combination with the Ub/26S proteasome system. Its abundance is regulated predominantly by degradation via the 26S proteasome (34) . Jab1, CSN5, specifically binds p27Kip1 and promotes its degradation via the Ub/26S proteasome pathway (35) .
Here we show that the CSN controls the major part of VEGF production in HeLa and HL-60 cells. The CSN-directed VEGF production is mediated mostly through c-Jun signaling and can be reduced by elevated intracellular levels of the tumor suppressor p53.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was purchased from PharMingen (San Diego, CA). VEGF was quantified using the Quantikine human VEGF ELISA kit from R&D Systems (Minneapolis, MN). Recombinant human SP-1 protein was obtained from Promega (Madison, WI). An expression construct encoding human SP-1 was a gift from R. Tijan (University of California, Berkeley, CA). Wild-type p53 cDNA was obtained by reverse transcription-PCR and cloned into a pcDNA3.1 vector encoding an NH2-terminal Flag tag (28)
. Plasmids encoding human CSN2, CSN5, and c-Jun(1–226) possessing an NH2-terminal Flag tag have been described previously (29)
.
Rabbit polyclonal anti-AP-2 and anti-c-Jun/AP-1 antibodies were from Geneka (Montreal, Canada) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively; mouse monoclonal anti-HIF-1 (clone H167), anti-Flag, and anti-p53 antibodies were from Neomarkers (Fremont, CA), Stratagene (La Jolla, CA) and IC Chemikalien (Ismaning, Germany), respectively. Mouse polyclonal anti-CSN5 was from GeneTex (San Antonio, TX). Rabbit polyclonal anti-CSN2 antibody has been described previously, and human CSN complex was prepared from RBCs as outlined previously (24)
.
Quantification of VEGF Production.
The VEGF production is expressed as VEGF concentration determined after the indicated times in cell culture supernatants (pg VEGF/ml supernatant). VEGF concentration in cell culture supernatant was measured using the Quantikine human VEGF ELISA kit from R&D Systems, which has been calibrated against a highly purified recombinant human VEGF165. In short, 1 ml of media was centrifuged at 15,000 x g for 5 min at 4°C, and the supernatant was stored at -70°C for up to 72 h. Thawed supernatant (200 µl) was used in the ELISA according to manufacturer’s instructions. The absorbance of the samples was determined using a Dynatech MR5000 microplate reader. A serial dilution of human recombinant VEGF was included in each assay to obtain a standard curve from which sample VEGF concentrations were obtained.
Cell Culture.
HL-60 and HeLa cells were grown in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. With the exception of transient transfections, HeLa cells were plated at 105 cells/well in a 6-well plate 24 h before an experiment. HL-60 cells were used at 106 cells/ml.
To test the VEGF production and its inhibition by curcumin, HeLa and HL-60 cells were incubated in the presence of 50 µM curcumin or 0.25% DMSO as a control. At the indicated time points, cell culture supernatants were collected, and VEGF concentrations were determined.
To investigate the inhibitory effect of mithramycin, HeLa cells were incubated with varying concentrations of the drug as indicated. TNF--stimulated cells were incubated in the presence of 50 ng TNF-/ml media. After 2 h, cell culture supernatants were collected, and VEGF concentrations were determined.
To test whether mithramycin and curcumin had additive effects on VEGF production, HeLa cells were treated with 50 µM curcumin and 50 µM mithramycin alone and in combination for 2 h.
The effect of curcumin on TNF- stimulation was tested by incubating HeLa cells in the presence or absence of 50 ng TNF-/ml media with and without 50 µM curcumin for 4 h.
Transient Transfections and Luciferase Reporter Assays.
HeLa cells were harvested and resuspended to 1.7 x 107 cells/ml. Electroporation was performed with 300 µl of cell suspension and 10 µg of CSN2, CSN5, c-Jun(1–226) (29)
, or p53 cDNA (28)
in pcDNA3.1 encoding for an NH2-terminal Flag tag in the presence of 1.25% DMSO (36)
. If not otherwise specified, cell culture supernatants were collected after 6 h, and VEGF concentrations were determined.
Transactivating activity of AP-1 was measured as described previously (29) . In brief, HeLa cells were cotransfected with a luciferase expression plasmid containing three repeats of the AP-1 binding site and other expression constructs described above using cationic liposomes (DAC-30; Eurogentec, Sart Tilman, Belgium). Sixteen h after transfection, cells were either treated for 3 h with the indicated concentrations of curcumin or left untreated. Luciferase assays were performed as recommended by using the manufacturer’s instructions (Promega).
Western Blots.
For Western blot analysis, cells were harvested and lysed in radioimmunoprecipitation assay buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 5% glycerol, 1 mM EGTA, 100 µM phenylmethylsulfonyl fluoride, and 4 µM aprotinin. Membranes were disrupted by passing the cell suspension through a 26-gauge needle. After centrifugation, aliquots of the supernatants were separated by 12.5% SDS-PAGE. Proteins were blotted to nitrocellulose, and Western blots were developed using the enhanced chemiluminescence method (Amersham Pharmacia Biotech).
Densitometry was performed using Aida 2.11 software.
Kinase Assay.
Kinase assays were performed as described by Seeger et al. (24)
. In short, recombinant p53 and c-Jun were incubated with purified CSN and [
-32P]ATP for 30 min at 37°C. Reactions were stopped by adding 4x SDS sample buffer. Samples were separated by SDS-PAGE, and gels were stained with Coomassie Blue, dried, and exposed to X-ray film.
Statistics.
VEGF concentrations are reported as means ± SE. To compare VEGF concentrations, data from one experiment were treated as paired observations, and the Wilcoxon matched pairs signed rank-sum test was used. Ps < 0.05 were considered statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To test whether the CSN had any effect on VEGF production, HeLa and HL-60 cells were incubated with curcumin, an effective inhibitor of the CSN kinase (27)
. Treatment of HeLa and HL-60 cells with 50 µM curcumin for 2 h reduced VEGF production to 27 ± 3% and 27 ± 2% of the control value, respectively (Fig. 1A)
. Because the VEGF promoter contains an AP-1 binding site, we studied the influence of curcumin on basal AP-1 transactivating activity using the luciferase reporter assay. As shown in Fig. 1B
, curcumin inhibited AP-1 activity in a dose-dependent manner. It is important to note that under these conditions, the JNK is inactive (29)
. The CSN-specific phosphorylation stabilizes c-Jun in HeLa cells, resulting in elevated AP-1 activity (29)
. Therefore, we tested the impact of CSN inhibition on VEGF production and cellular c-Jun levels simultaneously. This is demonstrated in Fig. 1C
. The incubation of HeLa cells with curcumin for 6 h inhibited VEGF production and reduced the cellular c-Jun concentration to approximately 25% of control value. After the removal of curcumin, supernatant VEGF concentration and cellular c-Jun levels increased concomitantly. The same holds true for HL-60 cells. As shown in Fig. 1D
, the reduction of cellular c-Jun concentration by curcumin in HL-60 cells is also accompanied by decreased VEGF production. In HL-60 cell lysates, the anti-c-Jun antibody detected two c-Jun isoforms that might be explained by different phosphorylation grades of the transcription factor. The data indicate that high c-Jun levels in HeLa and HL-60 cells are due to CSN-directed stabilization of the transcription factor that is associated with elevated VEGF production.
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, overexpression of CSN2 elevated cellular VEGF production by 37%. In contrast, overexpression of subunit CSN5, which does not increase CSN activity (29)
, had no effect on VEGF production (Fig. 2A)
. As demonstrated by Western blotting and illustrated in Fig. 2B
, similar amounts of CSN2 and CSN5 proteins were expressed after transfection with the cDNA constructs.
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, left panel). Under this condition, the JNK was inactive (29)
. Interestingly, as shown in Fig. 2C
, right panel, AP-1 activity induced by CSN2 overexpression can be completely inhibited by curcumin in a concentration-dependent manner, indicating that it is dependent on the CSN kinase. As shown earlier, the CSN-mediated c-Jun phosphorylation can be inhibited by an NH2-terminal c-Jun fragment that competes with full-length c-Jun for binding to the CSN complex (29)
. If the VEGF production by HeLa cells depends on CSN-directed c-Jun signaling, then transfection with c-Jun(1–226) should reduce the amount of VEGF in the supernatant. Six h after transfection with c-Jun(1–226), the VEGF concentration was measured. As shown in Fig. 2A
, overexpression of c-Jun(1–226) inhibited VEGF production by 25%. The data provide additional evidence for the involvement of the CSN in the regulation of VEGF expression.
SP-1 and AP-2 Affect VEGF Production Independently of CSN.
The portion of VEGF production sensitive to curcumin amounts to approximately 75% and might be due to a combination of AP-1, AP-2, SP-1, and HIF-1 activity. Therefore, we tested whether, in addition to c-Jun, other transcription factors involved in the regulation of VEGF expression are regulated by the CSN.
Inhibition of basal VEGF production with 10 nM mithramycin, a specific inhibitor of SP-1 signaling, led to a decrease of VEGF protein by <10%, which was not statistically significant (Fig. 3A)
. As expected, mithramycin at 10 nM reduced TNF--stimulated VEGF production in HeLa cells to control level because TNF- stimulates VEGF expression, most likely via SP-1 (12
, 19)
. The application of higher concentrations of mithramycin resulted in a dose-dependent reduction of basal VEGF production. As shown in Fig. 3B
, a combination of curcumin and high concentrations of mithramycin did not have an additive effect on VEGF, indicating that large amounts of mithramycin nonspecifically inhibited the curcumin-sensitive, CSN-directed VEGF production. To further investigate the role of SP-1 in CSN-mediated regulation of VEGF production, HeLa cells were stimulated with 50 ng/ml TNF- for 4 h in the presence or absence of curcumin (Fig. 3C)
. TNF- elevated VEGF production by approximately 10% compared with the control value. In the presence of curcumin, TNF- enhanced VEGF protein concentration by 33%. However, the absolute values of approximately 30 pg/ml VEGF formed in response to TNF- were the same with and without curcumin. Thus, inhibition of CSN activity by curcumin did not compromise the TNF-/SP-1-dependent stimulation of VEGF production. In addition, preliminary experiments confirmed that SP-1 is not phosphorylated by the CSN (data not shown).
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indicate that there is no CSN-dependent modification of AP-2 stability.
HIF-1, a subunit of the transcription factor HIF-1, was not detectable in the lysate of HeLa cells by Western blotting (data not shown). Because cells grew under normal oxygen conditions, a contribution of HIF-1 to the regulation of VEGF expression was not expected and is rather unlikely.
p53 Is a Negative Regulator of CSN-directed VEGF Production.
Both p53 and c-Jun are phosphorylated by the CSN, suggesting competition between the two transcription factors. To test this possibility, kinase assays with purified CSN in the presence of constant amounts of c-Jun and increasing p53 concentrations were performed (Fig. 4A)
. At high p53 concentrations, CSN-dependent phosphorylation of c-Jun was almost completely diminished. This is in agreement with our in vivo data shown in Fig. 4B
. Overexpression of p53 in HeLa cells led to a reduction of VEGF production. To test the impact of CSN inhibition on endogenous p53 levels, HeLa cells were incubated with 50 µM curcumin for 6 h. After treatment, cells were lysed, and lysates were tested with anti-p53 and anti-c-Jun antibodies by Western blotting. As shown in Fig. 4C
, p53 was up-regulated in curcumin-treated cells, whereas endogenous c-Jun was reduced at the same time.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Under these conditions, the c-Jun stabilization has been explained by a de novo assembly of the CSN complex (29)
. Third, c-Jun(1–226), a competitor of full-length c-Jun for binding to the CSN complex, reduces AP-1 activity (29)
and inhibits basal VEGF production (Fig. 2A)
. Because the JNK pathway is not active, c-Jun(1–226) specifically blocks the CSN-directed c-Jun signaling pathway. Assuming usual efficiency of transfection, the obtained c-Jun(1–226) effect is highly significant. Finally, overexpression of wild-type p53 reduces VEGF production in HeLa cells, which can be explained by a competition between p53 and c-Jun for phosphorylation by the CSN (see below).
Our data show that in HeLa and HL-60 cells, most signaling that leads to VEGF expression is mediated by the AP-1 factor, c-Jun. The transcription factor stability against the Ub/26S proteasome system is regulated by CSN-specific phosphorylation (24
, 29)
. The relationship between intracellular c-Jun levels and VEGF production is shown by CSN2 and c-Jun(1–226) overexpressions and by curcumin treatment. No other transcription factor potentially involved in the regulation of VEGF expression is influenced by curcumin. Stimulation of VEGF production by TNF-, presumably mediated via SP-1 (12
, 19)
, was not affected by curcumin. This is in agreement with findings of Singh and Aggarwal (38)
, who reported that the treatment of a human myelomonoblastic leukemia cell line with curcumin resulted in a down-modulation of AP-1, whereas SP-1 remained unaffected. The fact that SP-1 is not phosphorylated by the CSN in vitro is another indication for CSN-independent SP-1 signaling. As shown by inhibition with 10 nM mithramycin, the portion of SP-1-mediated signaling responsible for basal VEGF production in HeLa cells amounts to <10%. The effects of mithramycin at high concentrations were not additive to curcumin inhibition. Therefore, at higher concentrations, mithramycin seems to exhibit nonspecific inhibitory effects on curcumin-sensitive signaling by mechanisms that are presently unknown. This is in agreement with results demonstrating that mithramycin at 100 nM inhibits VEGF gene activation by signaling that is not mediated via SP-1 (12)
.
Because HIF-1 was not detectable in HeLa cells, we assume that the transcription factor HIF-1 does not contribute to basal VEGF production under the normoxic conditions used. Therefore, the remaining 15–20% of basal VEGF production can be attributed to AP-2 activity. The abundance of AP-2 protein was not influenced by curcumin (Fig. 3D)
, indicating that it acts independently of the CSN. It is concluded, therefore, that curcumin-sensitive VEGF production is solely due to CSN-directed c-Jun signaling that predominantly contributes to high VEGF production in HeLa and HL-60 cells.
VEGF Production Depends on the Balance between c-Jun and p53, Which Is Determined by the CSN and the Ub/26S Proteasome System.
Our data confirm that overexpression of p53 (Fig. 4B)
leads to reduced VEGF production. Similar data were obtained with adenovirus-mediated p53 gene transfer in colon cancer cells, which down-regulated VEGF expression and inhibited angiogenesis (21)
. The amount of c-Jun and p53 protein is controlled by different mechanisms. For example, high levels of p53 expression in jun-/- fibroblasts have been explained by the loss of a negative feedback of c-Jun via direct binding to a variant AP-1 site in the p53 promoter (39)
. On the protein level, JNK signaling has been implicated in the stabilization and activation of both c-Jun and p53 (40)
, which, as mentioned above, cannot be relevant under our conditions. Here we show that p53 competes with c-Jun for CSN-specific phosphorylation. The consequence is the ubiquitination and subsequent degradation of c-Jun by the 26S proteasome. On the other hand, very low levels of p53 in HeLa cells and the lack of p53 expression in HL-60 cells might be reasons for the elevated c-Jun concentration with the consequence of increased VEGF expression in these tumor cells. The loss of the competitor p53 leads to an increased CSN-specific phosphorylation of c-Jun on Ser-63 and Ser-73 (24)
, stabilizing the protein against the Ub/26S proteasome system (33)
. Curcumin inhibits the CSN kinase, fixing a new equilibrium with low c-Jun and elevated p53 levels (Fig. 4C)
. After the removal of the inhibitor, the c-Jun levels recover in parallel with increasing VEGF production and decreasing p53 levels.
In summary, the basal VEGF production in HeLa and HL-60 cells is due mostly to the activity of c-Jun, which is stabilized by CSN-specific phosphorylation that is sensitive to curcumin. As outlined in Fig. 5
, the contribution of SP-1 and AP-2 amounted to <10% and 15–20% of basal VEGF production, respectively. The tumor suppressor p53 inhibits VEGF production, possibly by competition with c-Jun for CSN-specific phosphorylation. We conclude that the CSN controls most VEGF production, at least in HeLa and HL-60 cells. Therefore, it is a potentially important target for future antiangiogenic and antitumor therapy. In addition, our data provide an explanation for the action of curcumin as an antiangiogenic and antitumorigenic substance. The application of specific inhibitors of the CSN kinase in tumor therapy and the interference with signaling pathways upstream of VEGF might be advantageous compared with approaches that directly target VEGF (9)
. The inhibition of the CSN kinase not only reduces VEGF production and presumably tumor angiogenesis, but at the same time, it increases intracellular p53 levels (28)
that might drive tumor cells into apoptosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by Deutsche Forschungsgemeinschaft Grants Du 229/5-2 (to W. D.) and Na 292/5-2 (M. N.). 
2 To whom requests for reprints should be addressed, at Division of Molecular Biology, Department of Surgery, Medical Faculty Charité, Humboldt University, Monbijoustrasse 2, 10117 Berlin, Germany. Phone: 4930-450528190; Fax: 4930-450528918; E-mail: christianpollman{at}hotmail.com
3 The abbreviations used are: VEGF, vascular endothelial growth factor; AP, activator protein; COP9, constitutive photomorphogenesis 9; CSN, COP9 signalosome; HIF-1, hypoxia-inducible factor 1; JNK, c-Jun-NH2-terminal kinase; Ub, ubiquitin; TNF, tumor necrosis factor.
Received 2/14/01. Accepted 10/ 2/01.
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