Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Shiga, Japan
Submitted 18 November 2004 ; accepted in final form 11 April 2005
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ABSTRACT |
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insulin resistance; gene regulation; CCAAT/enhancer binding protein homologous protein
Steinberg et al. (30) reported that insulin stimulated endothelium-dependent vascular dilatation in humans and that the dilatation was reduced in individuals who were obese and insulin-resistant, suggesting that production of NO in the endothelium is impaired in hyperinsulinemic insulin-resistant patients (31). We also observed that hyperinsulinemic rats fed a diet with high fructose have impairment of NO production (29), while equivalent rats rendered hyperinsulinemic by undergoing implantation with insulin pellets exhibited increased NO production (29). Thus hyperinsulinemia in the insulin-resistant states might stimulate the proinflammatory gene expression without the protective effects of NO, suggesting that supplementation with NO reduces the PI3-kinase-induced proinflammatory gene expression. However, because high doses of NO induce oxidative stress (2) and apoptosis of VSMCs (10), it may be critical to determine the dose of NO required 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 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 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 PI3-kinase in VSMCs, we investigated whether NO modifies the effects of chronic activation of PI3-kinase on VSMCs through induction of CHOP expression. We found that NO showed bidirectional effects on MCP-1 promoter activity at two distinct regions, depending on its concentration and the function of CHOP. Herein we present a novel molecular mechanism in the regulation of MCP-1 gene expression by NO mediated by CHOP expression.
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MATERIALS AND METHODS |
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Cell culture. VSMCs were isolated from the aortas of male Sprague-Dawley rats (200300 g) using enzymatic digestion, and they were maintained in DMEM supplemented with 10% FCS, 80 U/ml penicillin G, and 80 µg/ml streptomycin in 100-mm plates (5 x 106 cells/dish) as described previously (21). Culture media were changed every third day, and VSMCs between passages 4 and 12 were used. Cell growth was arrested for 24 h in DMEM supplemented with 0.1% FCS before we performed the experiments using Western blot analysis.
Cell treatment.
VSMCs were infected at 50 multiplicities of infection (MOI) 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
-galactosidase 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 transcriptase-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). For a two-step PCR protocol, the following PCR primers sets were designed: MCP-1 sense primer, 5'-TGTTGTTCACAGTTGCTGCCTG-3'; MCP-1 antisense primer, 5'-GTGCTGAAGTCCTTAGGGTTGAT-3'; GAPDH sense primer, 5'-CCCTCAAGATTGTCAGCAATGC-3'; and GAPDH antisense primer, 5'-GTCCTCATGTTAGCCCAGGAT-3'. 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 s, 95°C; 10 s, 55°C; and 5 s, 72°C). GAPDH was used as a housekeeping gene.
Enzyme-linked immunosorbent assay. MCP-1 concentrations were measured in undiluted supernatants from cultured VSMCs using commercially available rat MCP-1 enzyme-linked immunosorbent assay (ELISA) kits (Biosource International, Camarillo, CA).
Construction of plasmids.
Reporter vectors pGL3-MCP1 (MCP13.6kb/Luc), MCP1-C/EBPMAB/Luc, which has mutated C/EBP binding sites, and MCP1-NFBMAB/Luc, which has mutated NF-
B binding sites, were constructed as described previously (27). The 619-bp rat MCP-1 promoter region between 565 and +54 bp was cloned using enzyme digestion with SacI (MCP1565bp/Luc). The 5'-flanking region of rat MCP-1 from 189 to +54 bp was amplified using MCP13.6kb/Luc by performing PCR with the sense primer 5'-GCGGTACCAAATTCCAATCCGCGGT-3' and the antisense primer 5'-TGCATAGTGGTGGAGGAAGAGAGATCTGG-3'. The resultant amplification was gel purified, digested with KpnI/BglII, and inserted into KpnI/BglII-linearized pGL3-basic to form MCP1189bp/Luc. In the same way, MCP1124bp/Luc was cloned with the sense primer, 5'-GCGGTACCAGCAGATTCAAACTTCCACT-3', and MCP132bp/Luc was cloned 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 by ligating 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 performing PCR with the sense primer, 5'-ACACCTGAAAGCAGAACCTG-3', and the antisense primer, 5'-ATGCCCACTGTTCATGCTTGGT-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 cells were stimulated with each dose of SNP for 6 h at 37°C and lysed in a solubilizing buffer containing 20 mM Tris (pH 7.5), 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 50 U/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 them in Laemmli sample buffer containing 100 mM DTT, and they were resolved using 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 secondary antibody followed by chemiluminescence detection, according to the manufacturers instructions (PerkinElmer Life Sciences).
Cell transfection and luciferase assay. Rat VSMCs were seeded onto 12-well cell culture plates. After 24 h of incubation, the cells were 6080% confluent. The VSMCs were cotransfected with each dose of luciferase expression plasmid, 0.05 µg of herpes simplex virus thymidine kinase promoter-linked Renilla luciferase vector (pRL-TK) plasmid (Promega, Madison, WI) as a normalization reference for transfection efficiency, and each dose of expression plasmid or empty plasmid was used as a control with LipofectAMINE Plus (Invitrogen) according to 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 using a dual luciferase reporter assay kit (Promega, Madison, WI) with a signal detection duration of 30 s measured using 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 with the use of 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 oligodeoxynucleotide probes were generated by annealing two complementary oligodeoxynucleotides corresponding to the nucleotide sequences as follows. Each probe was end labeled with [-32P]ATP using T4-polynucleotide kinase, and nuclear extracts (5 µg) were incubated with 1.0 x 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. Subsequently, all reaction mixtures were analyzed using 6% polyacrylamide gel electrophoresis in 0.25 TBE (45 mM Tris-borate, 1 mM EDTA), and the gel was dried and visualized using autoradiography.
Antibody competition assays used monoclonal antibodies against CHOP (Santa Cruz Biotechnology) or control -galactosidase antibodies (12 µg), which were added to the nuclear extract and left for 30 min on ice before addition of the labeled 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 in 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. SNP (1 mM) was added 12 h before the cells were harvested, and the cells were harvested 48 h after transfection.
Statistical analysis. Values are expressed as means ± SE unless otherwise stated. The Tukey-Welsch step-down multiple-comparison test or the Dunnett comparison test was used to determine the significance of differences among four or more groups. P < 0.05 was considered statistically significant.
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RESULTS |
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Effects of NO donor on MCP-1 transcription induced by PI3-kinase 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 overexpression of p110CAAX in VSMCs significantly increased the MCP-1 promoter-driven activity, and the addition of SNP at low doses (0.050.1 mM) decreased this effect (Fig. 2A). Similar to the results of mRNA levels, 0.51.0 mM SNP did not reduce the activity of the MCP-1 promoter (Fig. 2A).
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It has been reported that two NF-B 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-
B, we mutated both the NF-
B binding sites and performed promoter analysis. As shown in Fig. 2C, similar to the results obtained using the wild-type 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/EBP activity induced by PI3-kinase 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).
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NO donor increases the MCP-1 promoter activity and gene expression in the absence of the activation of PI3-kinase. To clarify the mechanism that high (0.51.0 mM) doses of SNP did not reduce MCP-1 promoter activity induced by the activation of PI3-kinase, 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. 5, A and B, high amounts of CHOP expression vector and high doses of SNP increased MCP-1 promoter activity, respectively. In addition, a high dose of SNP also induced MCP-1 mRNA expression (Fig. 5C).
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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 (M1M3) as shown in Fig. 6D and used these oligonucleotides as competitors. 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 the luciferase reporter, and mutation of the region between 186 and 184 bp caused the enhancement of the MCP-1 promoter activity induced by CHOP to disappear completely (data not shown).
SNP upregulates the transcriptional activity of MCP-1 gene through CHOP-RE. Finally, we investigated whether SNP upregulates 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 suggest that SNP upregulates the transcriptional activity of the rat MCP-1 gene through the CHOP induction.
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DISCUSSION |
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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. (22) recently reported that NO depletes Ca2+ stored in the ER and causes ER stress-dependent apoptosis in mouse cell-derived MIN6 cells. Although we did not investigate the precise mechanism of the CHOP induction by NO donors in the present study, we observed the induction of CHOP in VSMCs by NO donors at a concentration similar to that reported in other cells (22), and we also have 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 its 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. By 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 have demonstrated that 0.050.1 mM of SNP reduced the transcriptional activity of MCP-1 induced by chronic activation of PI3-kinase. 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 the present study produced a physiological or a 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--induced inflammatory responses (1, 33). Recent reports have described the possibility that an NO donor might affect the activity of PI3-kinase (24), but we found that the levels of phosphorylation of Akt, a downstream molecule of PI3-kinase, 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 PI3-kinase. Tsao et al. (33) reported that an NO donor attenuates the lipopolysaccharide- or oxidized lipoprotein-induced expression of MCP-1 in cultured VSMCs through inhibiting the transcription factor NF-
B. However, regarding the present results, it is reasonable to conclude that the inhibition of NF-
B is not involved in the mechanism by which NO inhibits the activity of the MCP-1 promoter induced by PI3-kinase. At first, as we reported previously, the induction of the transcriptional activity of MCP-1 by chronic activation of PI3-kinase is dependent on the activation of C/EBP-
and C/EBP-
, but not on NF-
B (27). Second, the inhibitory effect of SNP on the activity of the MCP-1 promoter was observed even after NF-
B 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 PI3-kinase-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 amounts of the expression of CHOP, use of the CHOP expression vector in VSMCs decreased the activity of MCP-1 promoter induced by the activation of PI3-kinase as well as the overexpression of C/EBP-
.
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 absence of NF-B binding sites in the promoter region of the MCP-1 gene, suggesting that NF-
B does not participate 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, and we found that the overexpression of CHOP increased the activities of the 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 nuclear protein binding was increased by 1.0 mM SNP at the region between 190 and 179 bp of the MCP-1 promoter. According to this result, a search of the database of the MatInspector program (available at: http://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 a 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 the 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 besides the loss of the inhibitory effects of NO donor on MCP-1 at the higher doses examined in this study. Recently, Schaub et al. (26) reported that apoptosis induces the expression of proinflammatory genes, including MCP-1, in VSMCs. They suggested that an interleukin-1-mediated pathway is involved in this induction of MCP-1. Because interleukin-1 is reported to increase iNOS in VSMCs (4) and because iNOS produces much more NO compared with eNOS, it might be possible that the higher level of NO produced by iNOS induces MCP-1 through CHOP expression, although Schaub et al. did not measure the expression of CHOP.
In summary, we have found in the present study that an NO donor, SNP, showed bidirectional effects on PI3-kinase-induced transcriptional activities of the MCP-1 promoter. As shown in Fig. 8, these bidirectional effects of the NO donor can be explained by the induction of CHOP in VSMCs. The inhibitory effect of NO donor on the PI3-kinase-induced activation of MCP-1 promoter may be caused by 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 the NO donor stimulates the activity of the MCP-1 promoter directly through the CHOP-RE located between 190 and 179 bp of MCP-1 promoter.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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