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Ken-ichi Kodama, Yoshihiko Nishio, Osamu Sekine, Yoshinori Sato, Katsuya ..... changes in some critical amino acids in the DNA binding domain prevent it from.
Articles in PresS. Am J Physiol Cell Physiol (April 13, 2005). doi:10.1152/ajpcell.00558.2004

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Bidirectional regulation of monocyte chemoattractant protein-1 gene at distinct sites of its promoter by nitric oxide in vascular smooth muscle cells

Ken-ichi Kodama, Yoshihiko Nishio, Osamu Sekine, Yoshinori Sato, Katsuya Egawa, Hiroshi Maegawa, and Atsunori Kashiwagi

From the Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan

Corresponding

name: Yoshihiko Nishio telephone number: 81-77-548-2223

fax number: 81-77-543-3858

email address: [email protected]

Running title: Regulation of MCP-1 by NO through CHOP in VSMCs

Copyright © 2005 by the American Physiological Society.

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Abstract We reported that chronic activation of phosphatidylinositol 3-kinase (PI3K) by the overexpression of membrane-targeted p110CAAX induced proinflammatory gene expression in rat vascular smooth muscle cells (VSMCs) through the induction of CCAAT/enhancer binding protein-β (C/EBP-β) and C/EBP-δ. To examine the antiinflammatory effect of nitric oxide (NO) on the proinflammatory gene expression, we investigated the effects of sodium nitroprusside (SNP) on the monocyte chemoattractant protein-1 (MCP-1) gene expression in VSMCs under chronic activation of PI3K. At low concentration (0.05 mM) of SNP, but not at high concentration (0.5-1.0 mM), it significantly reduced MCP-1 mRNA and protein expression as well as its transcriptional activity. We found that SNP induced C/EBP homologous protein (CHOP) expression, which inhibited C/EBP binding activity and reduced the C/EBP activity induced by chronic activation of PI3K in a dose-dependent manner up to 1.0 mM. Consistently, the increase in CHOP expression significantly reduced the MCP-1 promoter activity induced by PI3K. However, the overexpression of CHOP alone upregulated MCP-1 promoter activity in a dose-dependent manner up to high concentration. Deletion analysis of MCP-1 promoter and electrophoretic mobility shift assay identified the CHOP-response element (CHOP-RE) at the region between -190 and -179 bp of MCP-1 promoter. By CHOP-RE used as a decoy, the increase in promoter activity of MCP-1 induced by either CHOP or SNP was suppressed significantly. Thus, CHOP induced by an NO-donor has bidirectional effects on MCP-1 gene expression: it decreases the gene expression by inhibition of C/EBPs, and it increases the gene expression through the CHOP-RE.

Key Words: insulin resistance, gene regulation, nitric oxide, monocyte chemoattractant protein-1, C/EBP homologous protein

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Introduction

We have reported that physiological concentration of insulin preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway rather than Ras/mitogen-activated protein kinase (MAPK) pathway in VSMCs (21, 32) and that when the PI3K is activated chronically with the recombinant adenovirus expressing membrane-targeted PI3K (p110CAAX) in VSMCs, the expression of proinflammatory genes including monocyte chemoattractant protein-1 (MCP-1) is stimulated through the induction of CCAAT/enhancer binding protein-β (C/EBP-β) and - δ (27). These results suggest the roles of insulin and chronic activation of PI3K in the induction of proinflammatory genes; however, other studies have revealed that insulin increases nitric oxide (NO) production through the induction of endothelial nitric oxide synthase (eNOS) by the activation of PI3K and Akt (8, 14, 39, 40). Since NO is known to have antiinflammatory actions (5, 33), insulin may have both pro- and antiinflammatory effects on VSMCs. Steinberg et al. reported that insulin stimulated endothelium-dependent vascular dilatation in human subjects (30) and that the dilatation was reduced in obese insulinresistant subjects, suggesting that production of NO in the endothelium is impaired in hyperinsulinemic

insulin-resistant

subjects

(31).

We

also

observed

that

hyperinsulinemic rats fed a diet with high-fructose have impairment of NO production (29), while equivalent hyperinsulinemic rats by implantion with insulin pellets exhibited increased NO production (29). Thus, hyperinsulinemia in the insulin-resistant states might stimulate the proinflammatory gene expression without protective effects of NO, suggesting supplement of NO reduces the PI3K-induced proinflammatory gene expression. However, high dose of NO induce oxidative stress (2) and apoptosis of VSMCs (10), it may be critical to determine the dose of NO to reduce the proinflammatory gene expression. Recently, it was reported that NO induces the expression of C/EBP homologous protein (CHOP) in several cell lines (9, 12, 22). Originally, CHOP was cloned as an inhibitory molecule of C/EBPs, to which it binds with its leucine zipper region, thus

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blocking the binding of C/EBPs to DNA (25). In addition, by forming a heterodimer, CHOP has been reported to act as a transcription factor inducing the gene expression (41). Thus, these various functions of CHOP might be related to the various functions of NO in VSMCs. In the present study, by assessing the promoter activity of MCP-1 as a target of gene regulation by chronic activation of PI3K in VSMCs, we investigated whether NO modifies the effects of chronic activation of PI3K on VSMCs through induction of CHOP expression. We have found that NO showed bidirectional effects on MCP-1 promoter activity at two distinct regions, dependent on its concentration and the function of CHOP. Here, we present a novel molecular mechanism in the regulation of the MCP-1 gene expression by NO mediated through the CHOP expression.

MATERIALS AND METHODS

Materials. Sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), DETA-NONOate, and pan-Akt antibody were purchased from Sigma (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were from Invitrogen (Carlsbad, CA). Phospho-specific Akt antibody was from New England BioLabs, Inc. (Beverly, MA). Antibody to CHOP was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). XAR-5 film was purchased from Eastman Kodak Co. (Rochester, NY). [γ-32P]ATP was purchased from PerkinElmer Life Sciences (Boston, MA). Expression vector for p110CAAX and Ad5-p110CAAX were generous gifts from Dr. Jerrold M. Olefsky (University of California, San Diego, CA) and expression vector for C/EBP-δ was generous gift from Dr. Steven L. McKnight.

Cell Culture. VSMCs were isolated from the aortas of male Sprague-Dawley rats (200300 g) by enzymatic digestion, and they were maintained in DMEM supplemented with 10% FCS, 80 units/ml penicillin G, and 80 µg/ml streptomycin, in 100-mm plates (5 × 6

10 cells per dish) as described previously (21). Culture media were changed every third

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day, and VSMCs were used between the 4th and 12th passages. Cell growth was arrested for 24 h in DMEM supplemented with 0.1% FCS before the experiments with Western blot analysis.

Cell Treatment. VSMCs were infected at 50 m.o.i. for 1 h with stocks of either a control recombinant adenovirus (Ad5-LacZ) containing the cytomegalovirus promoter, pUC 18 polylinker, and a fragment of the SV40 genome, or the recombinant adenovirus Ad5-p110CAAX (7). Infected cells were incubated for 48 h at 37°C in DMEM with 10% FCS, followed by incubation in the starvation medium required for assay. The efficiency of adenovirus-mediated gene transfer was ~90%, as measured by βgalactosidase staining. The survival of the VSMCs was unaffected by the incubation of cells with the different adenovirus constructs, because the total cell protein remained the same in infected or uninfected cells.

RNA Extraction and Real-Time RT-PCR. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify MCP-1 transcript levels. Briefly, RNA was extracted with TRIzol reagent (Invitrogen) and total RNA (1 µg) was reverse transcribed with Superscript II (Invitrogen). Real-time quantitative PCR was performed using fluorescence dye SYBR Green I (Roche Molecular Biochemicals, Mannheim, Germany) and LightCycler (Roche). The following PCR primers sets were designed: MCP-1 sense primer, 5’-TGTTGTTCACAGTTGCTGCCTG-3’, MCP-1 antisense primer, 5’-GTGCTGAAGTCCTTAGGGTTGAT-3’, GAPDH sesnse primer, 5’CCCTCAAGATTGTCAGCAATGC-3’, and GAPDH antisense primer, 5’-GTCCTC ATGTTAGCCCAGGAT-3’ for a two-step PCR protocol. In the first part, polymerase was activated for 10 min at 95°C. During the second part, the target region was amplified (40 cycles for MCP-1 or 35 cycles for GAPDH: 10 sec, 95°C; 10 sec, 55°C; 5 sec, 72°C). GAPDH was used as a house-keeping gene.

Enzyme-Linked Immunosorbent Assay (ELISA)

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MCP-1 concentrations were measured in undiluted supernatants from cultured VSMCs by using commercially available rat MCP-1 ELISA kits (Biosource International, Camarillo, CA).

Construction of Plasmids. Reporter vectors, pGL3-MCP1 (MCP1-3.6kb/Luc), MCP1C/EBPMAB/Luc which has mutated C/EBP binding sites and MCP1-NFκBMAB/Luc which has mutated NF-κB binding sites were constructed as described previously (27). The 619-bp rat MCP-1 promoter region between -565 and +54 bp was cloned by enzyme digestion with SacI (MCP1-565bp/Luc). The 5’-flanking region of rat MCP-1 from -189 to +54 bp was amplified using MCP1-3.6kb/Luc by PCR with the sense primer, 5’-GCGGTACCAAATTCCAATCCGCGGT-3’ , and antisense primer, 5’-TG CATAGTGGTGGAGGAAGAGAGATCTGG-3’. The resultant amplification was gelpurified, digested with KpnI/BglII, and inserted into KpnI/BglII-linearized pGL3-basic to form MCP1-189bp/Luc. In the same way, MCP1-124bp/Luc was cloned with the sense

primer,

5’-GCGGTACCAGCAGATTCAAACTTCCACT-3’,

and

MCP1-

32bp/Luc with the sense primer, 5’-GCGGTACCAATAAAAGGCTGAG GCAGA-3’. The reporter plasmid of the C/EBP binding sites (C/EBP/Luc) was made by ligating a double-stranded oligonucleotide containing three tandem copies of the consensus sequence of the C/EBP binding site (5’-TGCAGATTGCGCAATCTGCA-3’) into KpnI/MulI-linearized pGL3-basic, and the sequence of the adenovirus-TATA box (5’AGGGGGCTATAAAAGGGGGTGGGGGCGTTCGTCCTCACTCT-3’)

into

XbaI/HindIII-linearized pGL3-basic. The expression vector of CHOP was created as follows: an exactly 500-bp fragment from the rat CHOP gene was cloned by PCR with the sense primer, 5’ACACCTGAAAGCAGAACCTG-3’, and antisense primer, 5’-ATGCCCACTGTTC ATGCTTGGT-3’, and was then fused to murine sarcoma virus (MSV)-driven expression vector.

Western Blot Analysis. VSMCs were starved for 24 h in DMEM with 0.1% FCS. The

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cells were stimulated with each dose of SNP for 6 h at 37°C and lysed in a solubilizing buffer containing 20 mM Tris (pH7.5), 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 50 units/ml aprotinin, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 50 mM NaF for 10 min at 4°C. The cell lysates were centrifuged to remove insoluble materials. For analysis, whole-cell lysates (50 µg of protein per lane) were denatured by boiling in Laemmli sample buffer containing 100 mM DTT and resolved by SDS-PAGE. Gels were transferred to nitrocellulose by electroblotting in Towbin buffer containing 20% methanol. For immunoblotting, membranes were blocked and probed with the specific antibodies. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection, according to the manufacturer’s (PerkinElmer Life Sciences) instructions.

Cell Transfection and Luciferase Assay. Rat VSMCs were seeded on 12-well cell culture plates. After 24 h of incubation, the cells were 60-80% confluent. The VSMCs were cotransfected with each dose of luciferase expression plasmid, 0.05 µg of PRL-TK plasmid as normalization reference for transfection efficiency, and each dose of expression plasmid or empty plasmid using as control with LipofectAMINE plus (Invitrogen) following the instructions for the reagent. At 12 h before harvesting, each dose of SNP was added, and the cells were harvested 48 h after transfection. The Firefly and Renilla luciferase activities were determined by using a dual-luciferase reporter assay kit (Promega, Madison, WI) with a signal detection duration of 30 sec by a luminometer (Auto LUMI-counter Nu1422ES, Microtec, Tokyo, Japan).



Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared from VSMCs according to the method described by Dignam et al. (6). After protein concentrations were determined using Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA), the nuclear extracts were divided into small aliquots, quickly frozen in liquid nitrogen, and stored at -80°C. For electrophoretic mobility shift assay (EMSA), double-stranded oligodeoxynucleotide probes were generated. The double-stranded

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oligodeoxynucleotide probes were generated by annealing two complementary oligodeoxynucleotides corresponding to the nucleotide sequences as follows: each probe was end-labeled with [γ-32P]ATP using T4-polynucleotide kinase, and nuclear extracts (5 µg) were incubated with 1.0 × 105 cpm of the labeled probe for 30 min at room temperature in a 20-µl binding buffer containing 10 mM HEPES (pH 7.9), 50 mM NaCl, 1 mM DTT, 10% glycerol, and 0.5 mM EDTA. For competition, a 100-fold molar excess of unlabeled probe was added to the nuclear extracts. Then, all reaction mixtures were analyzed by 6% polyacrylamide gel electrophoresis in 0.25 TBE (45 mM Tris borate, 1 mM EDTA), and the gel was dried and visualized by autoradiography. Antibody competition assays used monoclonal antibodies against CHOP (Santa Cruz) or control β-galactosidase antibodies (1-2 µg) which were added to the nuclear extract and left for 30 min on ice before addition of the labelled oligonucleotides.

Oligonucleotides. The sequences of the oligonucleotides for the EMSA were as follows:

F1 (positions -192 to -173 bp): 5´-ACACCAAATTCCAATCCGCG-3´; F2

(positions -172 to -146 bp): 5´-GTTTCTCCCTTCTACTTCCTGGAAACA-3´; F3 (positions -145 to -126 bp): 5´-TCCAAGGGCTCGGCACTTAC-3´; and F4 (positions 135 to -116 bp): 5´-CGGCACTTACTCAGCAGATT-3´.

CHOP Decoy Experiments. Rat VSMCs were cotransfected with double-stranded oligonucleotides (0.1 µg) containing either the sequence of CHOP-response element (F1; described above under Oligonucleotides) or negative control (F4; described above), 0.1 µg of luciferase expression plasmid carrying the upstream 3.6 kb of the MCP-1 gene and 0.05 µg of PRL-TK plasmid as normalization reference for transfection efficiency using LipofectAMINE plus. 1 mM SNP was added 12 h before harvesting, and the cells were harvested 48 h after transfection.

Statistical Analysis. The values are expressed as mean ± S.E., unless otherwise stated. The Tukey-Welsch step-down multiple comparison test or Dunnet comparison test was

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used to determine the significance of any differences among four or more groups. p < 0.05 was considered significant.

RESULTS

NO-donor Reduces MCP-1 mRNA Expression Induced by PI3K Activation To determine whether SNP, an NO-donor, affects the MCP-1 expression in VSMCs induced by the overexpression of membrane-targeted p110CAAX, real-time reverse transcription-PCR was performed. VSMCs were infected with recombinant adenovirus expressing either the membrane-targeted p110CAAX or LacZ at 50 m.o.i. for 1 h. The mRNA expression of MCP-1 was significantly (p < 0.01) induced by the overexpression of membrane-targeted p110CAAX in VSMCs (Fig. 1A). SNP at 0.05 mM significantly (p < 0.01) reduced the p110CAAX-induced mRNA expression of MCP-1 (Fig. 1A). In contrast, SNP at 1.0 mM significantly increased the mRNA expression of MCP-1 in VSMCs infected with p110CAAX (Fig. 1A). We also investigated MCP-1 concentrations in the medium. According with the results of mRNA expression, SNP at 0.05 mM significantly reduced MCP-1 production by the VSMCs, and SNP at 1.0 mM increased the MCP-1 production (Fig. 1B). We next examined whether SNP affects the activity of PI3K. The level of phosphorylation of Akt, a serine/threonine kinase, downstream of the PI3K was analyzed by Western blotting analysis using p-Akt (Ser-473) antibody. The overexpression of p110CAAX increased the phosphorylation of Akt, and SNP at either 0.05 mM or 1.0 mM did not affect the phosphorylation of Akt (data not shown).

Effects of NO-donor on MCP-1 Transcription Induced by PI3K Activation To clarify whether SNP affects the p110CAAX-induced transcriptional activity of MCP-1 gene, functional promoter analysis was performed using a luciferase reporter plasmid carrying the 3.6-kb upstream region of the MCP-1 gene. The overexpresion of p110CAAX in VSMCs significantly increased the MCP-1 promoter-driven activity, and

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the addition of SNP at low doses (0.05-0.1 mM) decreased this effect (Fig. 2A). Similar to the results of mRNA levels, 0.5-1.0 mM SNP did not reduce the activity of the MCP1 promoter (Fig. 2A). To assess the possibility of contribution of C/EBP binding elements, we mutated two C/EBP binding sites and performed the promoter analysis. We have reported that p110CAAX induced MCP-1 transcriptional activity through two C/EBP binding elements located between 3.6 and 2.6kb upstream of the MCP-1 gene (27). As shown in Fig. 2B, the overexpression of p110CAAX did not increase MCP-1 promoter activity without C/EBP binding elements. In the absence of C/EBP response elements, the low dose of SNP (0.05 mM) did not affect the activity of

MCP-1 promoter, but high dose

(1.0 mM) enhanced the activity. Thus, 1.0 mM SNP increased the MCP-1 promoter activity independently of C/EBP binding elements. It has been reported that two NF-κB binding elements located between 2.3 and 2.6 kb upstream of the MCP-1 gene have a functional role (37). To assess the possibility of contribution of NF-κB, we mutated both the NF-κB binding sites and performed the promoter analysis. As shown in Fig. 2C, similar to the results obtained using the wildtype reporter vector, the induction of luciferase activity by the overexpression of p110CAAX in VSMCs was reduced by 0.05 mM SNP, but not by 1.0 mM SNP.

NO-donor Reduces C/EBPs Activity Induced by PI3K Activation To assess whether the increase of the promoter activity of MCP-1 caused by the overexpression of p110CAAX was inhibited by SNP through inhibiting the C/EBPs, we constructed a C/EBP-luciferase reporter plasmid containing three repeats of C/EBP binding sites in tandem. In contrast to the effects on MCP-1 promoter, low doses of SNP reduced the activity of C/EBPs caused by the overexpression of p110CAAX and higher doses enhanced this effect (Fig. 3).

NO-donor Induces CHOP Expression in VSMCs We investigated whether SNP induces CHOP expression in rat VSMCs, as in other cell

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types (9, 12, 22). As shown in Fig. 4A, low doses of SNP induced CHOP expression and higher doses enhanced this effect. SNAP and DETA-NONOate, other NO-donors, also induced CHOP expression in rat VSMCs (data not shown).

Effects of CHOP on the MCP-1 Transcription To determine the effects of CHOP expression on the activity of MCP-1 promoter, functional promoter analysis was performed by transfecting a luciferase reporter plasmid carrying the upstream 3.6-kb of the MCP-1 gene. As shown in Fig. 4B and C, transfection of 20 ng/well but not 500 ng/well of CHOP-expression vector reduced the MCP-1 promoter activity induced by C/EBP-δ or the activation of PI3K. These data suggested that NO-donor decrease MCP-1 expression by inhibiting C/EBPs through CHOP induction.

NO-donor Increase the MCP-1 Promoter Activity and Gene Expression in the Absence of Activation of PI3K To clarify the mechanism that high doses (0.5-1.0 mM) of SNP did not reduce MCP-1 promoter activity induced by the activation of PI3K, functional promoter analysis was performed using a luciferase reporter plasmid carrying the 3.6-kb upstream region of the MCP-1 gene. As shown in Fig. 5A and B, high amount of CHOP-expression vector and high doses of SNP increased MCP-1 promoter activity, respectively. In addition, high dose of SNP also induced MCP-1 mRNA expression (Fig. 5C).

Identifying the CHOP-Response Element As shown in Fig. 6A, the -565/+54-luciferase plasmid (MCP1-565b/Luc) and the 189/+54-luciferase plasmid (MCP1-189b/Luc) produced enhanced promoter activities by the overexpression of CHOP. However, the luciferase activity of the -124/+54luciferase plasmid (MCP1-124b/Luc) was not enhanced by the overexpression of CHOP, suggesting that the region between -189 and -124 bp is involved in the CHOPinduced transcriptional activation of the MCP-1 gene.

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To locate the CHOP-response element (CHOP-RE) at the 5' upstream region of MCP1 gene, EMSA experiments were performed. We divided the region between -192 and 116 bp into four fragments, i.e. F1, F2, F3, and F4, and we used these oligonucleotides as a probe for the EMSA experiments (Fig. 6B). As shown in Fig. 6C, when F1 fragment was used as a probe, the band induced by SNP was detected. This band was deleted by addition of antibody against CHOP, suggesting the involvement of CHOP in the band (Fig. 6C). Recently, it has been reported that some genes such as Cpn60, Cpn10, ClpP, and mtDnaJ have CHOP-RE as shown in Fig. 6D (41). To localize the CHOP-RE of rat MCP-1 promoter further, we mutated F1 fragments (M1-3), as shown in Fig. 6D, and used these oligonucleotides as competitor. As shown in Fig. 6E, the band was completely displaced by the wild-type or M3 oligonucleotide, but not by the M1 or M2 oligonucleotide, suggesting that both of the mutated regions of M1 and M2 oligonucleotides contain CHOP-RE. We further analyzed the location of CHOP-RE using luciferase reporter and mutation of the region between -186 and -184 bp completely disappeared the enhancement of the MCP-1 promoter activity induced by CHOP (data not shown).

SNP Up-regulates the Transcriptional Activity of MCP-1 Gene through CHOP-RE Finally, we investigated whether SNP up-regulates the transcriptional activity of MCP-1 through the CHOP-RE. We used F1-oligonucleotide (shown in Fig. 6B) as a decoy and F4-oligonucleotide (shown in Fig. 6B) as a negative control. With or without the decoy, functional promoter analysis was performed by transfecting a luciferase reporter plasmid carrying the MCP-1 promoter. The decoy significantly decreased the activities of MCP-1 promoter induced by the overexpression of CHOP (Fig. 7A). Likewise, the decoy also decreased the activities of MCP-1 promoter induced by SNP (Fig. 7B). These data strongly suggested that SNP up-regulates the transcriptional activity of rat

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MCP-1 gene through the CHOP induction.

DISCUSSION It has been reported anti-atherogenic or atherogenic effects of NO. In fact, Zeifer et al. or Tsao et al. reported that NO suppressed the MCP-1 expression (33, 38), suggesting that NO has anti-atherogenic effects. However, a long-term nitrate therapy in chronic coronary artery disease significantly increased mortality risk (20). In addition, analysis of transgenic mice that lack or overexpress NO synthases indicated that NO exerts both anti-atherogenic effects (13, 16) and atherogenic effects (15, 23, 28). In the present study, we found in the rat VSMCs with chronic activation of PI3K, low dose of SNP reduced MCP-1 expression, but high dose of SNP enhanced MCP-1 expression. These opposing effects of NO on MCP-1 expression might contribute to the interpretation of the discrepancy of the effects of NO on atherosclerosis. Although our results of high dose of

SNP on MCP-1 expression showed opposites effect reported Zeifer et al or

Tsao et al. cell types or condition of our study were different from their studies. Furthermore, it is noted that chronic activation of PI3K increased MCP-1 expression through C/EBP, but not NF-κB (27). Therefore NF-κB is not activated in the condition of the present study. Thus, in the condition with the activation of NF-κB, for example, with the stimulation of inflammatory cytokines, high dose of NO or much amount of NO produced by inducible type of NO synthase (iNOS) may suppress MCP-1 expression through decreasing the activity of NF-κB. In the present study, we found that SNP as well as SNAP and DETA-NONOate induced the expression of CHOP in VSMCs in a dose-dependent manner. CHOP has been known to be induced by stress of the endoplasmic reticulum (ER) (11, 19, 34) and to play a key role in the induction of apoptosis (3, 18, 36, 42). For example, Oyadomari et al. recently reported that NO depletes Ca2+ stored in the ER and causes ER stressdependent apoptosis in mouse-cell-derived MIN 6 cells (22). Although we did not investigate the precise mechanism of the CHOP induction by NO-donors in the present study, we have observed the induction of CHOP in VSMCs by NO-donors at the

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concentration similar to that reported in other cells (22) and we have also found that the cyclic-GMP-dependent kinase is involved in the induction of CHOP by NO-donors (data not shown). Further studies are clearly needed to elucidate in detail the mechanisms of CHOP induction by NO-donors. Aside from the role in apoptosis, CHOP has been reported to inhibit the function of C/EBPs (25). C/EBPs are members of a family of transcription factors containing a leucine zipper domain that is needed to form a heterodimer or a homodimer, a DNA binding domain, and a transcription activating domain in its molecule (17, 35). CHOP also has these domains, but the changes in some critical amino acids in the DNA binding domain prevent it from binding to C/EBP response elements. Forming a heterodimer with C/EBPs via its leucine zipper domain, CHOP blocks the binding of C/EBPs to DNA, resulting in inhibition of C/EBP activities (25). Recently, it has been reported that CHOP not only inhibits the C/EBP activities but also induces expression of genes such as chaperonin 60, chaperonin 10, ClpP, and mtDnaJ through the activation of cis-acting elements other than C/EBP response elements (41). Thus, by means of induction of CHOP, NO may regulate the gene expression of VSMCs. We demonstrated that 0.05-0.1 mM of SNP reduced the transcriptional activity of MCP-1 induced by chronic activation of PI3K. As a result, 0.05 mM SNP reduced the MCP-1 mRNA expression. It is not clear whether the levels of NO-donor used in this study produced physiological or pathophysiological range of NO levels in the cells. However, the range of NO-donor in this experiment was reported to inhibit the proliferation or TNF-α-induced inflammatory responsens (1, 33). Recent reports indicate the possibility that an NO-donor affects the activity of PI3K (24), but we found that the levels of phosphorylation of Akt, a downstream molecule of PI3K, were not changed by incubating the cells with SNP, suggesting that the decrease in the activity of MCP-1 promoter by SNP was not due to the reduction of the activities of PI3K. Tsao et al. reported that an NO-donor attenuates the lipopolysaccaride- or oxidized lipoproteininduced expression of MCP-1 in cultured VSMCs through inhibiting the transcription factor NF-κB (33). However, regarding the present results, it is reasonable to conclude

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that the inhibition of NF-κB is not involved in the mechanism by which NO inhibits the activity of MCP-1 promoter induced by PI3K. At first, as we reported previously, the induction of the transcriptional activity of MCP-1 by chronic activation of PI3K is dependent on the activation of C/EBP-β and -δ, but not on NF-κB (27). Secondly, the inhibitory effect of SNP on the activity of MCP-1 promoter was observed even after NF-κB binding sites were deleted from the MCP-1 promoter. Interestingly, when we examined the activities of C/EBPs using the reporter vector carrying three repeats of binding sites of C/EBPs in tandem, we found that SNP decreased the PI3K-induced C/EBP activities in a dose-dependent manner up to the higher doses such as 1.0 mM SNP. These results suggest that SNP may regulate the C/EBP activities at the level of protein-DNA interaction. As discussed above, CHOP is a possible candidate molecule for regulation of the binding activities of C/EBPs to DNA and is induced by NO-donors. In fact, the inhibitory effect of SNP on C/EBP activities and the induction of CHOP expression by SNP in VSMCs were observed in parallel. Furthermore, at adequate amount of the expression of CHOP using CHOP-expression vector in VSMCs decreased the activity of MCP-1 promoter induced by the activation of PI3K as well as the overexpression of C/EBP-δ. We found that the inhibitory effects of SNP on the activity of MCP-1 promoter were extinguished at the dose of 1.0 mM in the presence or the absence of NF-κB binding sites in the promoter region of MCP-1 gene, suggesting that NF-κB is not participated in the mechanisms involved in the loss of function of the NO-donor at higher doses. To assess whether the induction of CHOP by SNP is involved in the mechanism by which the inhibitory effect on the activity of MCP-1 promoter is lost at higher doses of SNP, we overexpressed CHOP in VSMCs; we found that the overexpression of CHOP increased the activities of MCP-1 promoter. Deletion analysis of MCP-1 promoter revealed that a functional response element of CHOP exists in the region between -189 and -124 bp from the starting point of MCP-1 transcription. Furthermore, EMSA experiments showed that the nuclear protein bindings were increased by 1.0 mM SNP at the region between -190 and -179 bp of MCP-1 promoter. According to this result, a

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search of the database of the MatInspector program (www.genomatix.de) for the sequence from -189 to -124 bp revealed a predicted CHOP-RE in this region. Finally, we observed that the introduction of double-stranded oligonucleotide from the region between -192 and -173 bp of the MCP-1 promoter but not one from the region between -135 and -116 bp of the MCP-1 promoter into VSMCs inhibited the SNP-induced as well as the CHOP-induced activities of MCP-1 promoter. These results clearly indicate that the induction of CHOP by SNP at higher doses is involved in the mechanisms participating in the loss of effect of NO-donor on the inhibition of the activity of MCP-1 promoter. Thus, we know of no other significance of the CHOP-dependent MCP-1 expression beside the loss of the inhibitory effects of NO-donor on MCP-1 at the higher doses examined in this study. Recently, Schaub et al. reported that apoptosis induces expression of proinflammatory genes including MCP-1 in VSMCs (26). They suggested that an interleukin-1-mediated pathway is involved in this induction of MCP-1. Since interleukin-1 is reported to increase iNOS in VSMCs (4), and iNOS produces much more amount of NO as compared with eNOS, it might be possible that the higher level of NO produced by iNOS induced MCP-1 through the CHOP expression, although they did not measure the expression of CHOP. In summary, we found that an NO-donor, SNP, showed bidirectional effects on PI3Kinduced transcriptional activities of MCP-1 promoter. As shown in Fig. 8, these bidirectional effects of NO-donor can be explained by the induction of CHOP in VSMCs. The inhibitory effect of NO-donor on the PI3K-induced activation of MCP-1 promoter may be caused by the blocking of the C/EBP binding to the C/EBP response elements at the promoter of the MCP-1 gene by CHOP. In contrast, the increased expression of CHOP at higher doses of NO-donor stimulates the activity of MCP-1 promoter directly through the CHOP-RE located between -190 and -179 bp of MCP-1 promoter.

Acknowledgments We thank Dr. Jerrold M. Olefsky for providing expression vector for p110CAAX and

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Ad5-p110CAAX and Dr. Steven L. McKnight for giving expression vector for C/EBPδ. Grants This work supported by research grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan.

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Figure legends

Fig. 1. NO-donor exhibits bidirectional effects on MCP-1 gene expression induced by PI3K. VSMCs were infected with a control recombinant adenovirus (Ad5-LacZ) or the

22

recombinant adenovirus Ad5-p110CAAX for 1 h at 50 m.o.i. and exposed to SNP for 12 h before harvesting. Then, the effect of SNP on MCP-1 expression induced by the overexpression of p110CAAX was investigated with real-time reverse transcriptionPCR (A, n=8) and ELISA (B, n=6). #, p < 0.01 versus LacZ; *, p < 0.01 versus p110CAAX without SNP; **, p < 0.05 versus p110CAAX without SNP.

Fig. 2. NO-donor exhibits bidirectional effects on MCP-1 promoter activity induced by PI3K. VSMCs were cotransfected with 0.2 µg of MCP1-3.6kb/Luc (A),

MCP1-

C/EBPMAB/Luc (B), or MCP1-NFκBMAB/Luc (C), PRL-TK, and 0.75 µg of control or p110CAAX-expression plasmid and exposed to SNP for 12 h (n=4 (A), 6 (B) and 5 (C), respectively). #, p < 0.01 versus control; *, p < 0.01 versus p110CAAX without SNP.

Fig. 3. NO-donor reduces C/EBP activities. VSMCs were cotransfected with 1.0 µg of C/EBP/Luc, PRL-TK, 0.05 µg of control or p110CAAX-expression plasmid and exposed to SNP for 12 h (n=5). #, p < 0.01 versus control; *, p < 0.01 versus p110CAAX without SNP; **, p < 0.001 versus p110CAAX without SNP.

Fig. 4. NO-donor induces CHOP expression. A: VSMCs were exposed to SNP for 6 h. The levels of CHOP were analyzed with Western blotting. The signal in the absence of SNP was expressed as 1 (n=4). B: VSMCs were cotransfected with 0.1 µg of MCP13.6kb/Luc, PRL-TK, 0.5 µg of control or C/EBP-δ-expression plasmid, and the indicated amounts of CHOP-expression plasmid (n=5). #, p < 0.01 versus control; *, p < 0.01 versus C/EBP-δ without CHOP. C: VSMCs were cotransfected with 0.1 µg of MCP1-3.6kb/Luc, PRL-TK, 0.5 µg of control or p110CAAX-expression plasmid, and the indicated amounts of CHOP-expression plasmid (n=4). #, p < 0.01 versus control; *, p < 0.05 versus p110CAAX without CHOP.

Fig. 5. SNP increases MCP-1 promoter activity and gene expression. A: VSMCs were

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cotransfected with 0.1 µg of MCP1-3.6kb/Luc, PRL-TK, and the indicated amounts of CHOP-expression plasmid (n=5). *, p < 0.01 versus without CHOP. B: VSMCs were cotransfected with 0.1 µg of MCP1-3.6kb/Luc and PRL-TK and exposed to SNP for 12 h (n=7). *, p < 0.05 versus SNP 0 mM; **, p < 0.01 versus SNP 0 mM. C: VSMCs were exposed to SNP for 12 h. MCP-1 gene expression was investigated with real-time reverse transcription-PCR (n=5). *, p < 0.01 versus SNP 0 mM.

Fig. 6. MCP-1 promoter contains CHOP-response element. A: VSMCs were cotransfected with 0.1 µg of reporter plasmid (MCP1-565b/Luc, MCP1-189b/Luc, MCP1-124b/Luc, or MCP1-32b/Luc), PRL-TK, and 0.5 µg of control or CHOPexpression plasmid (n=5). *, p < 0.05 versus MCP1-189b/Luc with control; #, p < 0.05 versus MCP1-565b/Luc with control. B: schematic structures of different fragments of 5'-flanking region from -192 to -116 bp of rat MCP-1 gene. C, E: VSMCs were stimulated with or without 1.0 mM SNP for 6 h. The binding activity of F1, F2, F3, or F4 (C) and F1 wild type (WT) or each mutant (M1-3) (E) was examined using EMSA. For competition, a 100-fold molar excess of unlabelled probe (cold) was added to nuclear extracts. For antibody competition, anti-CHOP antibody or control (βgalactosidase) IgG antibody was added to nuclear extracts. A representative result of three experiments is shown respectively. D: Nucleotide sequences of the consensus sequence of CHOP-response element (41), F1 fragment of rat MCP-1 promoter, and three forms of mutated F1 fragment (M1, 2, and 3) are described. The number refers to distance from transcription initiation site. The box indicates a putative key element for the action of CHOP.

Fig. 7. A CHOP decoy decreases MCP-1 promoter activity induced by CHOP or SNP. A: VSMCs were cotransfected with 0.1 µg of MCP1-3.6kb/Luc, PRL-TK, 0.5 µg of control or CHOP-expression plasmid, and double-stranded oligonucleotide of F1 or F4 as shown in Fig. 6B (n=5). *, p < 0.01 versus control without decoy; #, p < 0.01 versus control with decoy. B: VSMCs were cotransfected with 0.1 µg of MCP1-3.6kb/Luc,

24

PRL-TK, and double-stranded oligonucleotide of F1 or F4, and exposed to SNP (1.0 mM) for 12 h (n=5). *, p < 0.01 versus control without decoy; #, p < 0.01 versus control with decoy.

Fig. 8. Schematic presentation of bidirectional effects of NO on MCP-1 promoter by two distinct mechanisms.

Figure 1 A

B *

10

#

6 4 2 0

*

LacZ p110CAAX 0 0.01 05 0.1 0.5 1.0 SNP(mM) 0 0.

MCP-1 concentration (pg/ml)

MCP-1 mRNA expression (fold activation of LacZ)

12

8

**

800 700

#

600 500 400

*

300 200 100 0

LacZ SNP(mM) 0

p110CAAX 0 0.05

1.0

Figure 2 A

*

Relative luciferase activity (fold activation of control)

6 5 4

#

3

*

2

*

1

0 control p110CAAX

(+) (-) (-) (-) (-) (-) (-) (-) (+) (+) (+) (+) (+) (+) 0 0 . 0 1 . 0 5 0.1 0.5 .0 1 0 0 -2.3kb -2.6kb

-3.6kb

A

B

C/EBP

B Relative luciferase activity (fold activation of control)

5 4 3 2 1

0 control (+) p110CAAX (-) SNP(mM) 0 -3.6kb

NF-κB

*

6

(-) (+) 0

-2.6kb

A B C/EBP

(-) (+) 0.05

(-) (+) 1.0

-2.3kb

A B NF-κB

Luc

A B C

5 4

#

3 2

*

1

0 control (+) p110CAAX (-) SNP(mM) 0 -3.6kb

Luc

*

6

Relative luciferase activity (fold activation of control)

SNP(mM)

(-) (+) 0

-2.6kb

A B C/EBP

(-) (+) 0.05

(-) (+) 1.0

-2.3kb

A B

NF-κB

Luc

Relative luciferase activity (fold activation of p110CAAX only)

Figure 3

1.4 1.2

#

1

*

0.8

* **

0.6 0.4

0.2 0 control

**

(+)

(-)

(-)

(-)

(-)

(-)

(-)

p110CAAX (-)

(+)

(+)

(+)

(+)

(+)

(+)

SNP (mM) 0

0

0.01 0.05 0.1

0.5

1.0

Luc C/EBP repeat 

CHOP expression (arbitrary unit)

CHOP SNP (mM) 0

Relative luciferase activity (fold activation of control)

B

0.01

0.05

0.1

0.5

1.0

C 12

#

10 8 6

4 2 0 control (+) (-) (-) (-) (+) C/EBP δ (-) (+) (+) (+) (-) CHOP 0 0 10 20 500 (ng/well)

-3.6kb

*

-2.6kb

-2.3kb

C/EBP NF-κB

Luc

6 4

2 0 SNP (mM) 0 0.010.05 0.1 0.5 1.0 Relative luciferase activity (fold activation of control)

Figure 4 A

14 12 10 8

# 3 2

*

1

0 control (+) p110CAAX (-) CHOP 0 (ng/well) -3.6kb

(-) (+) 0 -2.6kb

(-) (+) 10

(-) (+) 20

(-) (+) 500

-2.3kb

C/EBP NF-κB

Luc

* 3 2 1

0 CHOP 0 (ng/well) -3.6kb

50 100 250 500 -2.6kb -2.3kb

C

MCP-1 mRNA expression (arbitrary unit)

C/EBP NF-κB

Luc

*

6 4 2

0 SNP (mM) 0

0.05

0.1

1.0

B

Relative luciferase activity (arbitrary unit)

A

Relative luciferase activity (arbitrary unit)

Figure 5

** 3

*

2 1 0

SNP (mM) 0 -3.6kb

1 .05 0.1 0.5 1.0 0 . 0 0 -2.6kb

-2.3kb

C/EBP NF-κB

Luc

Figure 6 (-565)

A

Relative luciferase activity   (fold activation of control-MCP1-32b) (-132) (+54) (-189) 0 10 20 30 40 50 Luc

(-565)

MCP1-565b

B (-192)

(-173/-172) (-146/-145) (-135) (-126)

#

(-189) MCP1-189b (-132)

*

F2 F1

MCP1-124b

(+54) MCP1-32b

control CHOP

C Probe F1 F2 SNP (-) (+) (+) (-) (+) (+) Cold (-) (-) (+) (-) (-) (+)

F3 F4 (-) (+) (+) (-) (+) (+) (-) (-) (+) (-) (-) (+)

Probe SNP (+) anti-CHOP(-) IgG (-)

F1 (+) (+) (+) (-) (-) (+)

F4 F3

(-116)

Figure 6 D

E

Probe

F1

SNP (+)(+)(+)(+)(+) Cold (-)WT M1M2M3 CHOP consensus MCP-1(WT) (-192) MCP-1(M1) (-192) MCP-1(M2) (-192) MCP-1(M3) (-192)

5' 5' 5' 5' 5'

TGCAAC TC ACACCAAATTCCAATCCGCG ACtggttcTTCCAATCCGCG ACACCAAAgagacgTCCGCG ACACCAAATTCCAAgggcgc

3' 3' 3' 3' 3'

B

*

3 2

#

1

0 control (+) CHOP (-) Decoy (-)

-3.6kb

(-) (+) (-)

-2.6kb

A

Relative luciferase activity (arbitary unit)

Relative luciferase activity (fold activation of control without decoy)

Figure 7 A

B

C/EBP

A

(+) (-) (+)

(-) (+) (+)

-2.3kb

B

NF-κB

*

3 2

#

1

0 SNP(1.0mM) (-) Decoy (-)

-3.6kb

Luc

(+) (-)

-2.6kb

A

B

C/EBP

A

(-) (+)

(+) (+)

-2.3kb

B

NF-κB

Luc

Figure 8

Insulin

(+): activation of MCP-1 promoter (-): inhibition of MCP-1 promoter

NO

Activation of PI3K

CHOP

C/EBPs (+) (+)

high affinity binding

(-) A

(+) low affinity binding

(-) B

C/EBP response elements

CHOP response element

MCP-1