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J Biol Chem, Vol. 275, Issue 5, 3075-3080, February 4, 2000

Transcriptional Induction of Endothelial Nitric Oxide Gene by Cyclosporine A
A ROLE FOR ACTIVATOR PROTEIN-1^*

Javier Navarro-Antolín^§¶, Javier Rey-Campos, and Santiago Lamas^§

From the  Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas and the ^§ Instituto Reina Sofía de Investigaciones Nefrológicas, 28006 Madrid, Spain

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have previously shown that the immunosuppressant cyclosporine A (CsA) increases the activity, the protein level, and the steady-state levels of the mRNA of the endothelial nitric-oxide synthase (eNOS) gene in bovine aortic endothelial cells (BAEC). We have now investigated the mechanisms responsible for these effects. Preincubation with an inhibitor of RNA polymerase II abolished CsA-induced eNOS up-regulation. Nuclear run-on experiments demonstrated a 1.6-fold increase in the induction of eNOS gene by CsA. In agreement with these results, transient transfections showed that CsA augmented the transactivation of the eNOS promoter. Electrophoretic mobility shift assays showed an increase in the activator protein-1 (AP-1) DNA binding activity in BAEC treated with CsA. An increase in the level of c-fos mRNA and in the nuclear content of c-Fos protein was detected in BAEC treated with CsA. Site-directed mutagenesis of the AP-1 cis-regulatory element in the context of the human eNOS promoter resulted in the abrogation of the induction mediated by CsA. Hence, up-regulation of eNOS mRNA by CsA is a transcriptional phenomenon involving the proximal AP-1 site in the 5'-regulatory region of the human eNOS gene. Furthermore, our data exemplify how immunosuppressive drugs may result in the regulation of specific genes involved in the homeostasis of endothelial function, such as eNOS.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Whereas cyclosporine A (CsA)¹ still stands as a cornerstone therapy for immunosuppression, its use encompasses serious side effects among which nephrotoxicity and hypertension are often encountered. The pathogenesis of their appearance has been investigated in clinical studies and animal models, but a complete understanding is still elusive. The partial reversibility of these effects suggests that the underlying disturbance has a functional component.

Acute nephrotoxicity is associated with renal vasoconstriction and a reduced glomerular filtration rate. A significant amount of data support that the vasoconstrictor peptide, endothelin-1, is involved in this response (1, 2). Furthermore, transforming growth factor  (TGF-) has been proposed as a link between CsA-enhanced endothelin synthesis and many of the pathological fibrotic lesions observed with chronic CsA toxicity (3).

Although endothelin and other paracrine vasoconstrictors have been identified as mediators of the hypertensive response (1, 4, 5) associated with CsA toxicity, a less clear picture has emerged regarding the potential dysfunction of the L-arginine-NO-cGMP pathway. Several reports have described that there is a defect in the relaxation of blood vessels in response to endothelial agonists, but the precise step of diminished NO synthesis, enhanced degradation, or receptor abnormality has not been established (6-8). In previous work, using endothelial cells in culture treated with CsA, we found that NO synthesis is not only not reduced but moderately enhanced (9). These observations have been also confirmed in rats (10), in healthy volunteers, in whom venous antecubital infusion of CsA significantly decreased forearm blood flow (11), and recently in patients treated with CsA after heart transplantation (12). These data indicate that nitric oxide may constitute an important regulatory mechanism that protects against CsA-associated vasoconstriction in vivo. In support of this hypothesis we found that the expression of the eNOS gene is increased in bovine aortic endothelial cells (BAEC) treated with CsA (12-14). We have now investigated the mechanisms by which eNOS is up-regulated by CsA.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Reagents-- BAEC were isolated from thoracic aortas using previously described methods (15). Phenotype characterization was based on their typical cobblestone appearance and uniform uptake of fluorescent acetylated low density lipoprotein. Cells were maintained in RPMI 1640 supplemented with 10% calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in an atmosphere of 95% O₂ and 5% CO₂. Experiments were performed on confluent monolayers (unless for transfection assays) at passages 3-6 and were made quiescent by serum deprivation. Specifically, cells were kept in serum-free medium for 24 h before starting the procedure and during incubations. Human umbilical vein endothelial cells were obtained and cultured as described elsewhere (16). The following reagents were obtained from the indicated sources: cell culture media, calf serum, glutamine, and penicillin/streptomycin were purchased from Bio-Whittaker (Servier, Belgium); cell culture plates were from Falcon (Beckton Dickinson, France); the Plasmid Maxi kit purification system was from Qiagen (Germany); restriction endonucleases, Tfx-50 reagent, Dual-Luciferase^TM Reporter assay system, and pGL3 basic plasmid were from Promega (Madison, WI); Opti-minimum Eagle's medium and T4 DNA ligase were from Life Technologies, Inc; Klenow enzyme, Taq DNA polymerase, and Taq extender^TM polymerase chain reaction (PCR) additive were from Stratagene (Cupertino, CA). Deoxycytidine 5'-triphosphate, [-³²P]- (3000 Ci/mmol), and [-³²P]UTP were from Amersham Pharmacia Biotech; X-OMAT S x-ray film was from Eastman Kodak Co. Cyclosporine A was a gift from Dr. D. Rodríguez-Puyol (Madrid, Spain). Reagents for electrophoretic mobility shift assay (EMSA) have been described elsewhere (14), rabbit IgG (100 µg/ml), rabbit polyclonal c-Jun/AP-1, and mouse monoclonal c-Fos antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); phorbol 12-myristate 13-acetate (PMA) and all other reagents were purchased from Sigma.

Plasmid Constructs-- A fragment corresponding to the 5'-flanking regulatory region of the human eNOS gene at nucleotide positions from 1910 to +48 designated 1.9peNOS was obtained by PCR, including Taq extender^TM PCR additive, using a human genomic clone (17) as template. Primers were: upper, 5'-GGGGTACCTCACCTGAGGCTAGGAGT-3' and lower, 5'-CCGGTACCTGGGCCACGCTCTTCAAG-3'. The PCR product was cloned into the KpnI site of the promoterless luciferase reporter vector pGL3-Basic and sequenced to confirm correct orientation. Constructs with progressive deletions of the 5'-region at nucleotide positions from 1514 to +22, 728 to +22, and 314 to +22, designated 1.5peNOS, 0.7peNOS, and 0.3peNOS, respectively, were derived from plasmids pUA7, pUA8, and pUA4 (18) and recloned into the KpnI/HindIII sites of the pGL3-Basic vector. To obtain a construct of the human eNOS promoter with a mutation of the proximal AP-1 cis-regulatory element (positions 662 to 656), site-directed mutagenesis was performed by PCR using the 1.5peNOS plasmid as a template. A primer derived from the sequence of pGL3-Basic vector (RVprimer3, Promega) and the primer AP-1 antisense (5'-CCCCATAGATCTAGTGGGGG-3'; mutated positions 662, 659, 658, and 657 are underlined, these mutations created a BglII site) were used to generate the upstream PCR fragment. Primers AP-1 sense (5'-CCACTAGATCTATGGGGGTG-3', complementary of AP-1 antisense) and GLprimer2 (from pGL3-Basic) antisense were used to generate the downstream PCR fragment. After appropriate restriction enzyme digestions, the PCR fragments were ligated into the KpnI/HindIII sites of the pGL3-Basic vector. This AP-1 mutant construct, named 1.5peNOSAP-1M, was confirmed by sequencing. The luciferase reporter plasmids containing AP-1-driven (-73pColLuc) and AP-1-unresponsive (-60pColLuc) promoters of the human collagenase gene have been described elsewhere (19). A HindIII-BamHI fragment (nucleotide positions 517 to +63) of the 5'-flanking region of the human collagenase gene (20) was subcloned into pGL3-Basic and designated -517pColLuc. This construct was sequenced to confirm correct orientation. To standardize Northern and run-on experiments, a 650-base pair PCR fragment of the human GAPDH gene (GenBank^TM accession number M33197) was generated by PCR and subcloned into the PCR-Script vector (Stratagene, La Jolla, CA).

RNA Isolation, Northern Blotting, and Hybridization-- Total RNA was isolated following the guanidinium thiocyanate-phenol-chloroform method (21), separated by electrophoresis in 1% agarose gels containing 0.66 M formaldehyde, transferred to nylon membranes (Hybond, Amersham Pharmacia Biotech), UV cross-linked (UV stratalinker 1800 from Cultek, Stratagene, La Jolla, CA), and hybridized as described previously (22). A bovine eNOS cDNA (23) (GenBank^TM accession number M89952) or a murine c-fos cDNA was used as a probe. Blots were washed at final stringency conditions of 42 °C, 1X SSC, 0.5% SDS and exposed on X-OMAT S film, using intensifying screens, at 80 °C. The amount of RNA loaded was estimated by ethidium bromide staining of the ribosomal RNAs. The density of autoradiographic signals or the band intensities of 28 S ribosomal RNA were quantitated with a Studiostar Agfa image scanner using the public domain software package NIH Image 1.55. Levels of mRNA were normalized to GAPDH transcript levels and expressed in relative densitometric units with respect to control value. For experiments of inhibition of RNA synthesis with the RNA polymerase II inhibitor, 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB), BAEC were preincubated with 10 µM DRB for 45 min, and then CsA (1 µM final concentration) was added and the incubation continued for 12 h more.

Nuclear Run-on Experiments-- Isolation of nuclei (by Dounce homogenization) and the nuclear run-on transcription experiments were performed according to the protocols described elsewhere (24) with minor modifications: 12 µl of 10 mCi/ml [-³²P]UTP were used for each condition, proteinase K incubation was for 40 min, and after hybridization membranes were washed with 2× SSC for 15 min.

Transfection Experiments-- For transfection experiments BAEC were grown in RPMI, 10% calf serum at 37 °C in 6-well plates. At 90% confluence medium was replaced, and 4 h later the transfection was initiated. Transfection mixtures were as following: (per well) 1.5 µg of plasmid of interest (1.5 µg), 3 ng of a cytomegalovirus-driven Renilla luciferase plasmid (used for normalization of transfection efficiency), 4.5 µl of Tfx-50, and 1 ml of Opti-minimum Eagle's medium. Mixtures were incubated for 20 min at room temperature. Cells were washed with serum-free RPMI and incubated with transfection mixtures for 3 h at 37 °C. Cells were washed again and maintained in RPMI with 10% calf serum for 24 h. Then fresh medium devoid of serum was added, and the incubation was continued for another 24 h. Following treatment with the specified agents, cells were harvested and lysed with Passive Lysis Buffer (Promega) and two freeze/thaw cycles. The extracts were centrifuged for 30 s at 15,000 rpm at 4 °C and then used for assaying of luciferase reporters following the Dual-Luciferase^TM Reporter Assay System (Promega). Luciferase activity was corrected for transfection efficiency by cotransfection of a cytomegalovirus promoter-driven Renilla luciferase gene plasmid and indicated as Firefly/Renilla luminescence ratio. Triplicates of each condition for the indicated number of independent experiments were considered for analysis.

Preparation of Nuclear Extracts and EMSA-- For preparation of nuclear extracts, a modification of the method of Schreiber et al. (25) was used. At the end of each experimental period, BAEC were washed twice with phosphate-buffered saline, scraped, and transferred to microcentrifuge tubes. Cell pellets were resuspended in 400 µl of 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml of leupeptin, antipain, trypsin inhibitor, and pepstatin A. After 15 min at 4 °C, 25 µl of 10% Nonidet P-40 was added. Tubes were vigorously vortexed for 10 s, and nuclei were sedimented for 30 s at 23,000 × g. Nuclear pellets were resuspended in 50 µl of 20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors at 1 µg/ml and vigorously rocked at 4 °C for 15 min. They were then centrifuged for 5 min, 23,000 × g at 4 °C, and the supernatants were aliquoted at 80 °C. Before use, their protein content was determined by the Bio-Rad protein assay (Bio-Rad). Oligonucleotides were synthesized in a Beckman Oligo 100 M DNA synthesizer. The oligonucleotide sequence corresponding to the AP-1 site in the bovine eNOS promoter was: 5'- GGCCCCCAACTTGAGTCACAGGGGGTG-3' and 3'-GGGGGTTGAACTCAGTGTCCCCCACGG-5'. Equal molar amounts of each oligonucleotide were mixed and annealed by incubation for 5 min at 85 °C in 50 mM Tris-HCl, pH 7.5, 1 mM spermidine, 10 mM MgCl₂, and 5 mM dithiothreitol and slowly cooled down to room temperature. Two hundred nanograms were end-labeled using Klenow DNA polymerase (Amersham Pharmacia Biotech) in the presence of 20 µCi of [-³²P]dCTP. For binding reactions, approximately 6000 cpm of labeled oligonucleotide probes were incubated with 7 µg of nuclear extract and 1 µg of poly(dI-dC) in 10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 4% glycerol, 1 mM dithiothreitol at 4 °C for 30 min. Where indicated c-Jun or IgG antibodies were added. Protein-DNA complexes were separated by electrophoresis, in 6% nondenaturing polyacrylamide gels in 0.25 × Tris-borate EDTA buffer at 20 mA, and visualized by autoradiography. For competition experiments 125-fold molar excess of competitor DNA was coincubated with the labeled oligonucleotide probe.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-- For SDS-polyacrylamide gel electrophoresis and immunoblotting, 15 µg/lane of total nuclear protein extract were electrophoresed on 10% polyacrylamide gels and transferred to Immobilon-P membranes using a semidry electroblotting system (Trans-Blot SD, Bio-Rad). Blots were probed with 1:500 mouse monoclonal c-Fos antibody, and the c-Fos protein was visualized using an enhanced chemiluminescence (ECL) detection system from Amersham Pharmacia Biotech.

Statistics-- Unless otherwise specified, data are expressed as means ± S.E. with the number (n) of experiments in parenthesis. Statistical analysis was performed by unpaired Student's t test in the case of normal distribution of data or using nonparametric tests as appropriate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CsA Up-regulated eNOSGene Expression at the Transcriptional Level-- The long half-life of eNOS mRNA, more than 24 h in human umbilical vein endothelial cells (26), is consistent with a low transcriptional rate of the eNOS gene in basal conditions. Thus, the relatively early increase (at 4 h) of the eNOS mRNA levels upon incubation with CsA (9) suggested that this immunosuppressant could be affecting the transcriptional rate of eNOS, rather than its mRNA stability. To confirm this, we first evaluated whether the inhibition of RNA synthesis was able to abrogate the CsA-induced increase in eNOS mRNA levels. To this end, we used a long half-life RNA polymerase II inhibitor, DRB. As shown in Fig. 1, A and B, in BAEC, the CsA-dependent up-regulation of the eNOS transcript was significantly reduced by DRB (CsA: 73.9 ± 0.9; control: 7 ± 6, 8% of reduction; n = 3, p < 0.05). Further nuclear run-on experiments showed that CsA induction of the eNOS mRNA occurred at the transcriptional level. Nuclei from CsA-treated BAEC showed an enhanced rate of eNOS mRNA synthesis (161.3 ± 5.2% of control; n = 3, p < 0.05) (Fig. 2, A and B). This provides direct evidence for transcriptional activation of the eNOS gene by CsA.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Effect of an RNA polymerase II inhibitor on the up-regulation of eNOS expression by CsA. A, Northern blot analysis (10 µg of total RNA/lane) of the eNOS transcript levels of BAEC after a 12-h treatment with 1 µM CsA. Where indicated BAEC were treated with 10 µM DRB and with 1 µM CsA or vehicle alone (ethanol 0.01%). A representative Northern blot is shown. B, densitometric band intensities were normalized to GAPDH (relative densitometric units) as described under "Experimental Procedures." Data are mean ± S.E. of three independent experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Effect of CsA on the transcription rate of the eNOS gene in cultured bovine aortic endothelial cells. A, nuclear run-on analysis of transcription rates of eNOS and GAPDH genes in BAEC treated with vehicle (Control) or 1 µM CsA for 16 h. B, densitometric analysis of the autoradiograph shown in A. Intensities of GAPDH bands were assigned an arbitrary densitometric unit of 100. This graph shows a 1.7-fold increase of the eNOS band intensity in the blot hybridized with RNA synthesized by nuclei isolated from CsA-treated BAEC. In two additional experiments 1.5- and 1.6-fold increases were observed. pBS, pBluescript plasmid.

The Promoter of the Human eNOSGene Is Inducible by CsA-- To determine whether the promoter region of the eNOS gene was responsive to the stimulatory effect of CsA, a 1.9-kilobase fragment of the 5'-regulatory region of the human eNOS gene was transiently transfected into BAEC. A 2-fold increase (200.7% ± 13.8 of vehicle; n = 5, p < 0.05) of the promoter activity was detected after a 24-h treatment with 1 µM CsA (Fig. 3). To further delineate which region of the promoter could be responsible for this induction, we transfected a series of 5'-deletion mutants of the 1.9-kb fragment of the human eNOS gene and treated the transfected cells with 1 µM CsA or vehicle alone. As shown in Fig. 3, the degree of inducibility by CsA correlated positively with the length of the transfected construct (a 1.7-, 1.4- and 1.1-fold increase with the 1.5-, 0.7-, and 0.3-kb constructs, respectively) (170 ± 14.7, 137.8 ± 8.1, and 108.7 ± 12.7% of vehicle, respectively; n = 5, p < 0.05 for the 1.5- and 0.7-kb constructs). Similarly, transfections of human umbilical vein endothelial cells with the 1.9-, 1.5-, and 0.3-kb constructs showed comparable results (202%, 154%, and 123% of vehicle, respectively). Therefore, a unique specific region responsible for this inducibility is not immediately apparent.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of CsA on the activity of different 5'-deletion mutants of the human eNOS promoter. BAEC, transiently transfected with 1.9, 1.5, 0.7, or 0.3 kb of the human eNOS promoter-luciferase gene constructs, were treated with 1 µM CsA or vehicle alone for 24 h. Luciferase activity was corrected for transfection efficiency by cotransfection of a cytomegalovirus promoter-driven Renilla luciferase gene plasmid. Firefly and Renilla luciferase activities were determined as described under "Experimental Procedures." Results are representative of five experiments (mean ± S.E.).

CsA Increased the Binding Activity of AP-1-- We had previously observed that the expression of the eNOS mRNA is a redox-sensitive process. Three reactive oxygen species-generating systems, glucose oxidase, hypoxanthine-xanthine oxidase, and CsA, have been shown to increase the abundance of the eNOS transcript in BAEC. This process was abolished by catalase and horseradish peroxidase. In addition, we have observed that pyrrolidinedithiocarbamate, an agent with a known capacity to increase AP-1 activity (27, 28), and PMA (data not shown) were able to increase eNOS mRNA levels in BAEC (14). In view of these results and taking into account that both human (1532 and 662) and bovine (442) eNOS promoters include putative AP-1 sites, we analyzed the DNA binding activity of AP-1-related proteins to the AP-1 site of the bovine eNOS promoter. We carried out EMSA with [-³²P]dCTP-labeled double-stranded oligonucleotide, which contained the 442 AP-1 site of the eNOS bovine promoter, and nuclear extracts of BAEC treated with CsA or vehicle. An increase in a retarded predominant band was observed in extracts of cells treated with 1 µM CsA (Fig. 4A) with a maximal binding activity at 60 min. This band was competed out by a 125-fold molar excess of the same nonlabeled AP-1 oligonucleotide, thus suggesting the specificity of an AP-1-related complex. Furthermore, the addition of an antibody directed against the DNA binding domain of the c-Jun subunit of AP-1 resulted in a marked reduction of the binding in the EMSA (Fig. 4B). Incubation with an unrelated antibody (IgG) was not associated with any competition of the CsA-mediated binding of AP-1 to DNA.

View larger version (68K): [in this window] [in a new window]   Fig. 4.   Time course of the AP-1 complex DNA binding in BAEC treated with CsA. BAEC were incubated with 1 µM CsA for the indicated periods of time. Nuclear protein extracts were analyzed by EMSA as described under "Experimental Procedures," using an oligonucleotide including the AP-1 site of the eNOS promoter. -Jun is an antibody directed against the DNA binding domain of the c-Jun subunit of AP-1.

CsA Increased the Expression of c-fos Gene and the Nuclear Content of c-Fos Protein in BAEC-- An increased stability of the AP-1 dimers results in higher levels of AP-1 DNA binding activity. As an increased synthesis of c-Fos and its translocation to the nucleus to combine with pre-existing Jun protein forms AP-1 dimers that are more stable than those formed by Jun protein alone, we investigated the potential effect of CsA on the expression of c-fos gene and on the nuclear content of c-Fos in BAEC. As shown in Fig. 5, A and B an increase in c-Fos mRNA was detected, with a peak at 30-60 min, in BAEC treated with 1 µM CsA. An enhanced detection of c-Fos in nuclear protein extracts of BAEC treated for 30 or 60 min with 1 µM CsA was observed (192 ± 36 and 239 ± 73% of control, respectively; n = 3) (Fig. 5C).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of CsA on the expression of c-fos gene and in the nuclear content of c-Fos protein in BAEC. A, Northern blot analysis (10 µg of total RNA/lane) of the c-Fos transcript levels of BAEC treated with 1 µM CsA. A representative Northern blot is shown. B, densitometric band intensities were normalized to GAPDH (relative densitometric units) as described under "Experimental Procedures." Data are mean ± S.E. of three independent experiments. C, Western blot analysis (15 µg of protein/lane) of the nuclear extracts of BAEC after treatment with 1 µM CsA for the indicated periods of time and incubated with an anti-c-Fos antibody. Shown is a representative figure of three independent experiments.

CsA-activated Transcription of an AP-1-driven Reporter Gene in BAEC-- To assess if the increase on the AP-1 binding activity observed in CsA-treated BAEC correlated with an increase in the AP-1-dependent transcription, we transfected BAEC with an AP-1-driven luciferase gene construct (73pColLuc) (containing one AP-1 site). The transcriptional response of this construct to 1 µM CsA for 24 h was compared with that of a similar construct lacking the AP-1 site, 60pColLuc. BAEC transfected in parallel with the same constructs were treated with 100 nM PMA for 24 h as a positive control. As shown in Fig. 6, both PMA and to a lesser extent CsA augmented the promoter activity of 73pColLuc (percentage of vehicle: 238% ± 7 and 150% ± 18, respectively). In transfections with a full promoter of the collagenase gene, containing this AP-1 site and 517 base pairs upstream from the transcription start site, similar results were obtained with CsA and PMA (194.8% ± 18.4 and 152.8% ± 16.1 of increase over control, respectively), and an increase of 232% was observed with 100 µM pyrrolidinedithiocarbamate (data not shown). Neither CsA nor PMA had an effect on the activity of the 60pColLuc construct.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of CsA and PMA on the activity of an AP-1-driven promoter transfected into BAEC. BAEC transiently transfected with -73pColLuc (with an AP-1 site) or -60pColLuc (without the AP-1 site) promoter-firefly luciferase gene constructs were treated with 1 µM CsA or 100 nM PMA for 24 h. Luciferase activity was corrected and determined as in Fig. 3. Results represent data from three experiments (mean ± S.E.).

AP-1 Participates in the Transcriptional Up-regulation of eNOS Gene by CsA-- To evaluate the functional contribution of the AP-1 site to the CsA-mediated transcriptional up-regulation of eNOS, we generated a mutant version of the human eNOS promoter in which the proximal AP-1 site was modified by substituting four of the seven nucleotides of the AP-1 sequence in the context of the wild type 1.5-kb construct. As shown in Fig. 7, BAEC transiently transfected with this construct no longer responded to CsA treatment (wild type: 138% ± 8.7; AP-1M: 89% ± 7.4 of vehicle; n = 4, p < 0.05), and its induction by PMA was markedly diminished (wild type: 172.3% ± 11; AP-1M: 123.3% ± 10.6 of vehicle; n = 4, p < 0.05). Altogether these results suggest that this AP-1 site within the human eNOS promoter is important in order to elicit an inductive response by CsA and PMA.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of CsA and PMA on the activity of the human eNOS promoter mutated at its proximal AP-1 site. BAEC transiently transfected with the 1.5-kb human eNOS promoter-firefly luciferase gene construct, wild type (WT) or proximal AP-1 mutant (AP-1M), were treated with 1 µM CsA or 100 nM PMA for 24 h. Luciferase activity was corrected and determined as in Fig. 3. Results represent data from four experiments (mean ± S.E.).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The regulation of NO synthesis in endothelial cells involves a complex pathway starting with the coupling of agonists to their receptors and ending in coordinated post-translational changes of eNOS, which allow initiation, development, and termination of its catalytic function. Accumulated evidence suggests that the endothelial content of eNOS enzyme is also subject to modest degrees of transcriptional and post-transcriptional regulation, which may have important physiological and pathophysiological implications. These add other points for the control of NO production (29, 30). Studies in cultured cells and in vivo models indicate that the expression of the eNOS gene is regulated at the transcriptional level by several stimuli, including shear stress (31-33), lysophosphatidylcholine (34), transforming growth factor- (35), and estrogens (36). Other agents or pathophysiological perturbations such as tumor necrosis factor- (26, 37, 38), hypoxia (18, 39), entry into the cell cycle (40), status of cell growth (29, 41), oxidized low density lipoprotein (42), and hydroxymethylglutaryl-CoA reductase inhibitors (43) modify the eNOS mRNA half-life. Basic fibroblast growth factor has been shown to enhance eNOS mRNA in BAEC (44) and to restore eNOS protein to normal levels in vessels of genetically hypertensive rats (45), although no mechanistic information for these changes is available at the moment. In this work, we provide data consistent with an induction of the transcription of eNOS gene in vascular endothelial cells exposed to the immunosuppressant cyclosporine A. In this induction, a role for the proximal AP-1 site in the 5'-regulatory region in the human eNOS gene is proposed.

Our results support the idea that transcriptional induction is the main mechanism by which CsA enhances eNOS mRNA expression. However, a minor effect on mRNA stabilization cannot be ruled out, according to data obtained with RNA polymerase inhibitors. The first 1.9 kb of the promoter region of the eNOS gene appeared to contain sequence elements responsive to CsA induction. The fact that a progressive decline of the induction is observed with sequential deletions of this 5'-regulatory region suggested that several regions in the eNOS promoter were participating in the response to CsA. Among these, we have been able to identify the participation of an AP-1 site, which according to our knowledge, had not been assigned to a specific functional response of the eNOS gene. Stimulation with phorbol esters has been shown to increase the promoter activity of the eNOS gene (46), a result consistent with our observations with PMA treatment. However, even though AP-1 is the classical target of phorbol esters, its presence in the eNOS promoter has not been related to PMA induction.² In fact, when we mutated the AP-1 site a partial, but not complete, loss of PMA induction was obtained, thus suggesting alternative sites for PMA activation. Among these, cis-regulatory sequences for the AP-2 sites present in the eNOS promoter (47-49) or cooperation with other transactivating proteins such as nuclear factor-1 (35) are possibilities that remain unexplored.

Previous data from our group suggest that CsA promotes the formation of reactive oxygen species in BAEC (13, 14) together with a moderate increase in the binding capacity to AP-1. Reactive oxygen species have been shown to induce AP-1 activation by increasing c-fos and c-jun expression and Jun kinase activity (50, 51). On the other hand AP-1 is considered a redox-sensitive transcription factor (52-54). Thus, it seemed reasonable to hypothesize that reactive oxygen species could serve as an intermediate transducer of the effects of CsA in BAEC. However, in T lymphocytes (55) and nonstimulated BAEC,³ CsA does not seem to modify c-Jun NH₂-terminal kinase activity. CsA has been shown to increase AP-1 binding activity in rat C6 glioma cells (56) and to increase c-Fos mRNA expression in human lymphoid cells (57) and in murine erythroleukaemia cells (58). Our own studies in BAEC treated for 30-60 min with 1 µM CsA resulted in an increase in c-fos mRNA expression and an increase in the nuclear content of c-Fos. This suggests that CsA-induced c-fos mRNA up-regulation may constitute an important mechanism by which CsA may regulate AP-1-driven responses.

The intermediate steps triggered by CsA in endothelial cells, which could result in transcriptional activation of eNOS, remain to be elucidated. Among these, TGF- is an obvious candidate, as its expression is positively modulated by CsA in other cells, and TGF- has been shown to up-regulate eNOS expression, possibly through the activation of the nuclear factor-1 transcription factor. Besides, TGF- has been recently implicated in the pathway leading to malignant transformation triggered by CsA in some cells (59). Very recently the requirements for eNOS promoter basal activation have been analyzed in detail (60), demonstrating the necessity for protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1, MAZ, and YY1. Even when our results are specifically related to CsA-mediated enhancement of eNOS transcription, we cannot exclude the possibility of underlying routes whereby CsA could positively interact with one or more of these trans-acting factors.

From a biochemical standpoint, the best characterized effect of CsA is its capacity to inhibit the phosphatase activity of calcineurin, after the CsA-cyclophillin complex has been formed (61). As a result of this inhibition, the transcription factor NF-AT remains phosphorylated and is unable to translocate to the nucleus and hence to activate transcription of its cognate-dependent genes. A putative NF-AT site (bases 1539 to 1532) adjacent to a distal AP-1 site (bases 1532 to 1526) is present in the human eNOS promoter. However, experiments of DNA mobility shift using an oligonucleotide containing the sequence of the eNOS promoter with these two putative sites (NF-AT and AP-1: 1543 to 1520) performed with nuclear extracts from nonstimulated BAEC (in which NF-AT activation is not expected) suggest that this consensus NF-AT site does not show NF-AT binding activity that could be interfering (cooperation or competition) with the adjacent AP-1 site (data not shown, n = 3). An increasing interest in the calcineurin-NF-AT pathway is substantiated by the importance of knock-out models for NF-AT, where severe defects in cardiac valve formation have been observed (62, 63). Noteworthily, calcineurin has also been shown to play a role in cardiac hypertrophy, and hence calcineurin inhibitors may continue to gain new therapeutic indications. In fact CsA has already been employed to prevent myocardial ischemia, thus rendering a potential link with enhanced eNOS expression a tempting speculation.

In the context of CsA-related endothelial toxicity, many aspects remain to be clarified, including immediate cellular targets and generators of toxic mediators within endothelial cells. Nevertheless we believe our data exemplify how the exposure of endothelial cells to CsA may result in the regulation of specific genes, such as eNOS. Whether this represents an adaptive response to injury at the physiological level or further contributes to the generation of damage remains to be investigated.

	ACKNOWLEDGEMENTS

We are indebted to Rafael Sánchez-Pascuala and Estrella Soria for technical assistance, Dr. Diego Rodríguez-Puyol for supplying us with CsA, Dr. Tom Michel, Dr. Charles Lowenstein, and Dr. Carlos López-Otín for supplying us with plasmids, and Dr. Juan M. Redondo for comments and for supplying us with plasmids. We also thank Dr. Dolores Pérez-Sala for assistance with the immunoblots and helpful discussion and Dr. Peter Klatt and all the members of the laboratory for helpful comments.

	FOOTNOTES

^* This work was supported by Grant SAF-97-0035 from the Comisión Interministerial de Ciencia y Tecnologia, Grant BMH4-CT96-0979 from European Union Program BIOMED-2, and Grant 08.4/0032/1998 from Comunidad Autonoma de Madrid.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.

^¶ Fellow from the Fondo de Investigación Sanitaria, Spain.

To whom correspondence should be addressed: Centro de Investigaciones Biológicas, c/Velázquez 144, 28006 Madrid, Spain. Tel.: 34-91-5644562 (ext. 4302); Fax: 34-91-5627518; E-mail: slamas@cib.csic.es.

² H. Li and U. Förstermann, personal communication.

³ J. Navarro-Antolín, J. Rey-Campos, and S. Lamas, unpublished data.

	ABBREVIATIONS

The abbreviations used are: CsA, cyclosporine A; TGF-, transforming growth factor ; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; AP, activator protein; BAEC, bovine aortic endothelial cells; EMSA, electrophoretic mobility shift assay; DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole; PMA, phorbol 12-myristate 13-acetate; NF-AT, nuclear factor of activated T cells; PCR, polymerase chain reaction; kb, kilobase(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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