Sp1 Transcription Factor as a Molecular Target for Nitric Oxide– and Cyclic Nucleotide–Mediated Suppression of cGMP-Dependent Protein Kinase-Iα Expression in Vascular Smooth Muscle Cells
cGMP-dependent protein kinase (PKG) expression is highly variable and decreases in cultured vascular smooth muscle cells (VSMCs), exposure of cells to nitric oxide (NO), or in response to balloon catheter injury in vivo. In this study, the mechanisms of human type I PKG-α (PKG-Iα) gene expression were examined. Three structurally unrelated NO donors decreased PKG-Iα promoter activity after transfection of a promoter/luciferase construct in VSMCs. Promoter deletion analysis demonstrated that (1) a 120-bp promoter containing tandem Sp1 sites was sufficient to drive basal PKG-Iα promoter activity, and (2) NO was inhibitory at this site. Cyclic nucleotide analogues also suppressed PKG-Iα promoter activity with cAMP being more potent than cGMP. The effects of cyclic nucleotides to suppress PKG-Iα promoter activity were attenuated by a specific cAMP-dependent protein kinase (PKA) inhibitor. Single or double mutation of Sp1 binding sites abolished PKG-Iα expression. Moreover, Sp1 binding activity on the PKG-Iα promoter was detected in A7r5 cells, and this binding was inhibited by NO and cyclic nucleotides. These results indicate that PKG-Iα gene expression is driven by an Sp1 transcription mechanism, and that NO and cAMP inhibit Sp1-mediated PKG-Iα gene expression through separate mechanisms.
Cyclic GMP and cGMP-dependent protein kinase (PKG) regulate smooth muscle relaxation, platelet aggregation, cell growth, and differentiation.1–4⇓⇓⇓ Two genes encoding mammalian PKG exist.5,6⇓ Type I PKG is highly expressed in vascular smooth muscle cells (VSMCs) and exists as 2 isoforms, type Iα and type Iβ. These isoforms differ only in the initial coding region encoded by the first exon and therefore contain identical catalytic domains.7 The type II PKG is most abundant in brain and intestinal epithelium.6,8⇓ The expression of PKG-I, and particularly the Iα isoform, predominates in VSMCs9 and decreases as VSMCs are passaged in vitro.10 Exposure of bovine aortic VSMCs to NO or cyclic nucleotide analogues decreases PKG gene expression,11 and recently, it was shown that PKG-I expression is suppressed in coronary artery VSMCs after balloon catheter injury in vivo.12
Accumulating evidence suggests that NO modulates gene expression.13,14⇓ This involves activation or inhibition of different transcription factors such as NF-κB, OxyR, c-Myc, AP-1, and Sp1.15–18⇓⇓⇓ As mentioned, there are 2 reports demonstrating that high concentrations of NO suppress PKG-I gene expression in VSMCs.11,19⇓ The human PKG-I promoter20 for either the Iα or Iβ isoform lacks a typical TATA or CCAAT box, although several potential Sp1 binding sites have been detected. In many genes lacking a TATA box, a proximally positioned Sp1 site serves as the critical determinant of promoter activity and dictates the start site of transcription.21,22⇓ In this study, we have examined the effects of NO and cyclic nucleotide analogues on the modulation of PKG-Iα expression at the transcriptional level. For this purpose, embryonic vascular smooth muscle cells (A7r5) were transfected with a human PKG-Iα promoter (500 bp) and then treated with NO donors or cyclic nucleotide analogues. The data suggest that the inactivation of zinc finger factor Sp1 by NO and cyclic nucleotide analogues appears to be at least one mechanism involved in the downregulation of PKG-Iα gene expression in A7r5 cells.
Materials and Methods
Smooth Muscle Cell Culture
Bovine aortic SMCs in passage 2 (BASMCs) were cultured as described previously.11 Embryonic rat aortic SMCs (A7r5) were purchased from American Type Culture Collection (Rockville, Md). Cells were maintained in DMEM (Gibco) containing 10% calf serum and 50 μg/mL gentamicin. The routine subculturing procedure was to remove the cells using buffered trypsin and to split the cells 1:4. Viability was assessed after each experiment using trypan blue exclusion. A7r5 cells express PKG-Iα, unlike rat aortic SMCs, which lose PKG-Iα after few passages in culture.
Transfection and Reporter Gene Assay
A7r5 cells and BASMCs were seeded in 12-well plates at 60% to 80% confluence. After 16 to 20 hours, cells were transfected in the absence of serum, with 500 ng of PKG-Iα promoter (500 bp)/luciferase reporter gene construct using 3 μL/mL of transfecting reagent Tfx-20 (Promega, Madison, WI). One hour later, cells were incubated in the presence of serum (1%) with different NO donors or cyclic nucleotide analogues as indicated in Figures. Forty-eight hours later, luciferase assays were performed and expressed as relative luciferase light units (RLU). Firefly luciferase activity was normalized to Renilla luciferase by cotransfecting with pRL-null Vector (Promega). Dual luciferase (Dual-Luciferase Reporter Assay System, Promega) was quantified using a luminometer. Luciferase reporter gene assays were confirmed using the β-galactosidase reporter assay.
Generation of PKG Promoter Deletions
The human PKG-Iα promoter was cloned into pGL3 basic vector (Promega) at the KpnI/HindIII site. Polymerase chain reaction (PCR) and restriction enzyme digestions were used to generate deletion constructs pGL3 to 430 bp, pGL3 to 260 bp, and pGL3 to 120 bp. The truncated 260-bp fragment was created using PCR, with primers designed from the published sequence of the human PKG-Iα.20 The fragments 430 bp and 120 bp were generated by digestion of pGL3 containing PKG-Iα 500 bp with NheI/HindIII and XhoI/HindIII, respectively, and cloned in pGL3 basic vector. The integrity of promoter/reporter constructs was assessed by DNA sequence analysis.
Point mutations in Sp1 binding sites were introduced into the 500-bp promoter in pGL3 using Quick Change Site-Directed Mutagenesis Kit (Strategene) following the protocol described by the manufacturer. Oligonucleotides used to mutate the first Sp1 site within the 500-bp promoter were 5′-CGAAACTTTTTCTCGTTTGGCGGCGGCGGCGG-3′ and 3′-CTTTGAAAAAGAGCAAACCGCCGCCGCCGCC-5′; oligonucleotides used to mutate the second Sp1 site were 5′-TTTCACTGAGCC-TTTCCGCGAAACTTTTTC3′ and 3′-AAAAAGTGACTCGGAAAG- GCGCTTTGAAAAAG-5′ with the underlined case letters indicating the mutation sites. These oligonucleotides were synthesized by Research Genetic, Inc (Huntsville, Ala). The double-site mutant was made by performing a second round of PCR using the single-site mutant as a template and the appropriate pairs of oligonucleotides. Mutated Sp1 binding sites in the 500-bp PKG promoter as well as the wild type were used for transfection of A7r5 cells as described.
Nuclear Extract Preparation and Electrophoretic Mobility Shift Assays
Nuclear extracts from A7r5 cells were prepared as described elsewhere,23 with the addition of fresh 0.5 mmol/L phenylmethylsulfonyl fluoride and 0.2 mmol/L dithiothreitol (DTT) to all solutions. Oligonucleotides representing 2 putative Sp1 binding sites corresponding to −26 to −47 (5′-CCGCCGCCGCCGCCCGAGAAAA-3′, Sp1[a]) and −7 to −27 (5′-AGTTTCGCGGAGGGGCTCAG- TG-3′, Sp1) from the transcription start site of PKG-Iα were synthesized and used in the electrophoretic mobility shift assays (EMSAs). The bold letters in the sequences represent the Sp1 binding sites. The sequences were annealed, end-labeled with γ-[32P] ATP and T4 oligonucleotide kinase, and purified. Nuclear extracts (5 μg) from control, NO-, and cyclic nucleotide analogue-treated cells were incubated with the 32P-labeled Sp1 (a or b) oligonucleotides (50 000 cpm) in a buffer containing 2 μg of poly (dI-dC), 10 mg/mL BSA, 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 1 mmol/L DTT, 1 mmol/L EDTA, and 5% glycerol (total volume 20 μL). DNA-protein complexes were resolved by 4% nondenaturing polyacrylamide gel electrophoresis at 12 V/cm for 3 hours in low ionic strength buffer (0.5×TBE) at room temperature. A double-stranded Sp1 consensus oligonucleotide (5′-ATTCGATCGGGGCGGG-GCGAGC-3′) was used for competition to confirm Sp1 binding sites identified in the EMSA experiment. In experiments involving competitive EMSA, the unlabeled consensus oligonucleotides (Sp1, 50-fold excess) were added in the preincubation buffer. In some experiments, nuclear extracts from control A7r5 cells were prepared and directly treated with NO donors in the presence and absence of DTT.
A7r5 cells and adult rat aortic SMCs (as control) at passage 6 were treated for 3 minutes with different concentrations of the NO donor DEA-NONOate (1, 5, 10, and 50 μmol/L). At the appropriate time points, media were aspirated, and the cells were washed twice with ice-cold PBS and extracted with acidified methanol. Extracts were then lyophilized and samples were processed for cGMP measurement by radioimmunoassay. The protein concentrations were determined by the Bradford assay.
All experiments were performed in at least quadruplicate and repeated a minimum of 3 times. Results are expressed as mean±standard deviation (SD). Significant differences between means were identified using student’s t test as *P<0.05 and **P<0.01.
NO Donors and Inflammatory Mediators Inhibit PKG-Iα Promoter Activity in SMCs
Treatment of A7r5 cells or BASMCs with DETA-NONOate (DetaNO) resulted in decreased PKG-Iα promoter activity (Figure 1). To assess whether the levels of NO generated by DetaNO could be physiologically significant, the effects of lipopolysaccharide (LPS) and TNF-α, which activate inducible NO synthase (iNOS) expression,were also examined. Both LPS and TNF-α also suppressed PKG-Iα promoter activity in these two SMC lines. To further study the mechanisms involved, the effects of NO donors on PKG-Iα promoter activity in A7r5 cells were examined more extensively. Treatment of A7r5 cells with 3 structurally unrelated NO donors, DetaNO, S-nitroso-N-acetyl penicillamine (SNAP), and S-nitroso-l-glutathione (GSNO), resulted in decreased PKG promoter activity (Figure 2A). Inhibition of PKG-Iα promoter expression by NO donors was concentration dependent, and the order of potency was DetaNO>SNAP>GSNO. Decomposed NO donors had no effect on PKG-Iα promoter expression (data not shown). To exclude possible cytotoxic effects of the NO donor drugs, cell viability was examined using trypan blue as well as effects on cell morphology. NO donors, at the concentrations used, did not affect either cell viability or morphology (data not shown). Control studies demonstrated that NO by itself, at the concentrations used in these experiments, had no effect on luciferase activity (data not shown).
Sin-1 Induces PKG-Iα Promoter Activity
The addition of 3-morpholinosydnonimine (Sin-1) to transfected cells resulted in an increase in PKG-Iα promoter activity (Figure 2B). Because Sin-1 generates both superoxide and NO leading to peroxynitrite formation,24 the effects of superoxide dismutase (SOD) on PKG promoter activity were examined. The results in Figure 3A show that SOD abolished the increase in luciferase activity induced by Sin-1, resulting only in Sin-1–induced inhibition of PKG-Iα promoter activity. The inhibition was concentration dependent and the maximum effect was obtained with 1000 U/mL SOD. At this concentration of SOD, Sin-1 completely suppressed PKG-Iα promoter activity. Heat-inactivated SOD had no effect on the Sin-1–induced inhibition of PKG-Iα promoter activity (data not shown). In addition, catalase, which eliminated hydrogen peroxide (H2O2) generated by O2·− dismutation, did not inhibit the NO effect (data not shown). To confirm that NO mediates PKG-Iα promoter suppression and that this effect was predominant over activation of the promoter by Sin-1, cells were simultaneously incubated with Sin-1 alone, DetaNO alone, or both. The results in Figure 3B show that NO abolished the stimulatory effect of Sin-1 on PKG-Iα promoter activity. Taken together, these results suggest that NO alone accounts for the suppression of PKG-Iα promoter activity, although other species generated by Sin-1 could be responsible for increasing PKG-Iα promoter activity.
Soluble Guanylyl Cyclase Is Not Necessary for Inhibition of PKG-Iα Promoter Activity
To determine whether NO acts through soluble guanylyl cyclase (sGC) activation, A7r5 cells were transfected and treated with the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). ODQ did not restore PKG-Iα promoter activity in the presence of NO (Figure 4A). To assess whether A7r5 cells synthesize cGMP in response to NO, cells were incubated with DetaNO, and cGMP levels were determined. Under these conditions, A7r5 cells did not generate cGMP in response to DetaNO. As a control, adult rat aortic SMCs were examined under the same conditions. Figure 4B shows that these cells generated up to 50-fold increase in cGMP in response to DEA NO. These results indicate that the NO-induced PKG-Iα promoter suppression was independent of sGC activation and cGMP elevation.
Cyclic Nucleotide Analogues Suppress PKG-Iα Promoter Activity
To determine whether the PKG-Iα promoter might be sensitive to cyclic nucleotides, A7r5 cells were transfected and treated with cyclic nucleotide analogues and assayed for PKG-Iα promoter activity. The results shown in Figure 5 A and B demonstrate that the 8-parachlorophenylthio (pCPT) analogues of cAMP and cGMP inhibited PKG promoter activity. The effects were more potent with 8pCPT-cAMP (Figure 5A) compared with 8pCPT-cGMP (Figure 5B).
Suppression of PKG-Iα Promoter Activity Is Mediated by cAMP-Dependent Protein Kinase Activation
To determine whether PKA mediates the effects of 8pCPT-cAMP and 8pCPT-cGMP on PKG promoter activity, selective inhibitors of PKA and PKG were examined. As shown in Figure 5C, A7r5 cells preincubated with the PKA inhibitor KT5720 partially reversed the effects of both cyclic nucleotide analogues on PKG-Iα promoter suppression. Two structurally unrelated PKG-selective inhibitors, Rp-8Br-cGMPs and KT5823, did not reverse the suppression of PKG-Iα promoter (data not shown). These results suggest that cyclic nucleotide analogues inhibited PKG promoter activity primarily through PKA activation.
Sp1 Elements Are Responsible for NO-Mediated Suppression of PKG-Iα Promoter Activity
To localize the putative NO and cyclic nucleotide regulatory site(s) on the PKG-Iα promoter, deletions of the human 500-bp PKG-Iα promoter were generated. The sequence of the human PKG-Iα promoter used in these studies is shown in Figure 6A. After transfection with different deletion constructs, cells were incubated in the presence of DetaNO or 8pCTP-cAMP. The results in Figure 6B show that the shortest promoter (120 bp) preserved expression of the PKG-Iα promoter. In addition, this 120-bp promoter fragment was still sensitive to DetaNO and cyclic nucleotide inhibition, indicating that it contained the NO-responsive element(s). Analysis of the 120-bp fragment revealed the existence of 2 Sp1 binding sites located from −36 bp to −42 bp and from −13 bp to −19 bp upstream of the transcription start site.
To confirm that these putative Sp1 binding sites were responsible for the NO-suppression of PKG-Iα promoter activity, site-directed mutagenesis was performed. Plasmids carrying single or double mutations, pGL3 to 500ΔSp1(a), pGL3 to 500ΔSp1(b), and pGL3 to 500ΔSp1(a)Sp1(b), respectively, were generated. Results in Figure 6C show that either single or double mutation of Sp1 binding site abolished the basal PKG promoter activity in transfected A7r5 cells.
Effects of NO and Cyclic Nucleotides on Sp1 Binding to the PKG-Iα Promoter
The binding activity of Sp1 from A7r5 nuclear extracts was analyzed by EMSA using labeled Sp1(a) as a probe. To determine whether NO and cyclic nucleotide analogues affected Sp1 protein binding, nuclear extracts were prepared from A7r5 cells treated with NO donors and cyclic nucleotide analogues. The results in Figure 7A show that DetaNO, SNAP, and GSNO reduced the binding of Sp1 to its probe (Figure 7, lanes 3, 4, and 5, respectively) compared with untreated cells (lane 2). The band with the lower mobility was determined to be Sp1 binding, whereas Sp3 binding was included in the band having higher mobility based on supershift assays using specific antibodies (data not shown). Similarly, cyclic nucleotide analogues diminished the binding of Sp1 to the probe (Figure 7B, lane 3 and 4, respectively). The addition of a 50-fold excess of nonlabeled consensus Sp1 oligonucleotide abolished Sp1 binding (Figure 7A, lane 6, and Figure 7B, lane 5), although an unrelated oligonucleotide did not alter Sp1 binding (Figure 7B, lane 6). As the 120-bp promoter fragment contains 2 Sp1 binding sites, similar results were obtained when Sp1(b) was used as a probe (data not shown). It was interesting to note that only Sp1 appeared to be modulated by NO, whereas both Sp1 and Sp3 binding was decreased by cyclic nucleotides.
Additional experiments were performed to determine whether NO directly affects the DNA binding activity of Sp1. Nuclear extracts were prepared from A7r5 cells in the absence of DTT and then incubated in the presence of NO donors. Figure 7C shows the untreated nuclear extract contains a retarded band (lane 2) and this was abolished by incubation with 50-fold excess of unlabeled Sp1 consensus oligonucleotide (lane 6). Sp1 binding was reduced when nuclear extracts were directly treated with DetaNO, SNAP, and GSNO. All experiments were performed in the absence of a sulfhydryl reducing agent and Zn (as Sp1 is a zinc finger protein) to explore the effects of NO on the Sp1 binding activity.
An intriguing new function ascribed to NO is the modulation of gene expression in cells. In this study, we have demonstrated that cytokines, NO, and cyclic nucleotides suppress PKG gene expression in vascular smooth cells. Previously, we reported that the treatment of primary cultures of bovine aortic SMCs with NO donor drugs and cyclic nucleotide analogues suppressed PKG-I mRNA and protein expression, and the effects on mRNA expression were verified using nuclear run-on assays.11 In the present study, we have extended these observations using the human PKG-Iα promoter in an in vitro transfection assay system to elucidate the mechanism by which NO and cyclic nucleotides suppress PKG-Iα gene expression. Three structurally unrelated NO donors resulted in the inhibition of PKG-Iα promoter activity in a cyclic nucleotide-independent manner in A7r5 cells and primary cultures of BASMCs, but not in cells deficient in PKG expression, ie, repetitively passaged rat aortic SMCs or 3T3 fibroblasts (Sellak and Lincoln, unpublished observation, 2002). These effects of NO are mimicked by inflammatory mediators that are known to increase the expression of iNOS, suggesting that the suppression of expression of PKG-Iα mRNA may have pathophysiological relevance. The effects of NO and cyclic nucleotides on PKG-Iα promoter suppression appear to be due to different mechanisms. Not only did the sGC inhibitor fail to block the suppression of PKG-Iα gene expression, but NO donor drugs failed to increase cGMP levels in the A7r5 cells. We have since determined that A7r5 cells are deficient in the expression of the β-subunit of sGC (N.B. Browner and T.M. Lincoln, unpublished observations, 2002). Nevertheless, cyclic nucleotide analogues also suppressed PKG promoter activity, suggesting that NO and cyclic nucleotides act independently of each other in mediating PKG-Iα gene suppression. Studies using selective kinase inhibitors demonstrate that activation of PKA by either cAMP or cGMP analogues mediates the inhibitory effect of cyclic nucleotide analogues on PKG-Iα promoter activity.
The effects of NO donor drugs on PKG-Iα promoter activity appear to be due to the generation of authentic NO because Sin-1 increased PKG-Iα promoter activity in the absence of SOD. It is known that Sin-1 generates both NO and O2·− on decomposition, and the reaction of NO and O2·− leads to peroxynitrite production. SOD, by dismutating O2·−, abolishes the stimulatory effect of Sin-1 on PKG-Iα promoter activity. In this situation, the available NO suppresses PKG-Iα promoter activity. Furthermore, the combination of DetaNO with Sin-1 also leads to the suppression of PKG promoter activity, suggesting that NO is the inhibitory factor in modulating PKG-Iα gene expression. The possible relevance of the stimulatory action of Sin-1 and the role of peroxynitrite effect on PKG-1α gene are still unclear and under investigation.
The effective NO donor concentrations used in this study are relatively high but typical of the concentrations of NO likely to be available to cells expressing iNOS when considering the NO bioavailability and the NO donor half-life. Thus, these studies may be relevant to pathophysiological situations such as inflammation where iNOS is produced in response to cytokine release.
Modulation of gene expression by NO has been reported to occur in numerous cell types by either cGMP-dependent or cGMP-independent mechanisms and may result in either upregulation or downregulation of gene transcription.13–18,25–30⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ Several transcription factors, including NF-κB,16 AP-1,13–15⇓⇓ heat shock factor,26 and Oct-1,27 were reported to be modulated by NO. Recently, Wang et al17 reported that NO decreases the binding of Sp1 to its site on TNF-α promoter. Furthermore, mutation within the Sp1 binding site abolished the TNF-α promoter response to NO donors. In addition, NO has been shown to abrogate the DNA binding activities of Sp1 derived from NO-treated nuclear extracts as well as from NO-treated lymphocytes.28–30⇓⇓ The PKG-Iα promoter contains 2 Sp1 sequences located from −13 bp to −19 bp and from −34 bp to −40 bp upstream of the transcription start site, raising the possibility that NO may affect Sp1 activity and thus downregulate PKG-Iα promoter activity. The importance of Sp1 for PKG gene expression may be related to studies showing that genes lacking TATA or CCAAT elements require Sp1 binding for transcription.21,22⇓ PKG-Iα is one such gene that does not contain the putative TATA or CCAAT boxes. The mechanism by which NO directly modulates Sp1 transcriptional activity may be related to its ability to interact with Zn. NO was reported to inhibit the DNA binding activity of the yeast zinc finger transcription factor lac929 and it has been suggested that zinc finger proteins may be primary targets of NO-derived stress.30 In VSMCs, NO may suppress PKG expression by directly inhibiting Sp1 transcription factor binding activity to the PKG promoter, an observation supported by the direct effect of NO on Sp1 binding in nuclear extracts of cells pretreated with NO donors.
A second mechanism for the suppression of PKG-Iα promoter activity involves the generation of cyclic nucleotides by NO or other signaling agents leading to the activation of PKA. Exposure of PKG promoter–transfected cells to cyclic nucleotide analogues decreases PKG-Iα promoter activity. The finding that selective inhibitors for PKA, but not for PKG, reverse cyclic nucleotide–induced PKG gene suppression suggest that PKA activation may downregulate PKG-Iα gene expression. These data are also consistent with previous studies demonstrating that cyclic nucleotide–dependent cross-activation of protein kinases occurs in cells, including VSMCs.31,32⇓ Under pathophysiological conditions, inflammatory cytokines induce robust increases in cGMP levels sufficient to cross-activate PKA.31 The 500-bp promoter used in this study does not contain a cAMP-response element (CRE), and there are no consensus CREs that are found in the human PKG-Iα promoter. Recently, PKA activation was reported to regulate Sp1 activity,33 whereas other studies support the notion that Sp1 binding activity is influenced by phosphorylation.34,35⇓ Our data show that a decrease in Sp1 binding activity to the PKG-Iα promoter by cyclic nucleotide analogues are dependent on activation of PKA consistent with the role of phosphorylation in modifying Sp1 binding activity. Because both Sp1 and Sp3 protein binding are decreased on exposure of SMCs to cyclic nucleotides, the phosphorylation of these transcription factors (or most likely, SMC-specific accessory proteins that modulate the activity of these proteins) may affect their DNA binding activity. In VSMCs, exactly how Sp1 specifically drives PKG-Iα promoter expression and how its activity is modulated by phosphorylation is unknown. The Sp1 may interact with a VSMC-specific protein that could be the target of PKA. We are presently investigating the contribution of other VSMC nuclear factors on Sp1 function, and it will be of interest to determine how these factors are involved in the altered expression of PKG-Iα.
The studies reported here have implications in the pathophysiology of vascular disease. In several pathophysiological situations, including atherosclerosis, restenosis, and aging, the VSMCs modulate phenotype from contractile to a synthetic phenotype. It is the synthetic phenotype of VSMCs that predominates in vascular lesions where proliferation and excessive extracellular matrix protein production are observed. Our laboratory has shown that transfection of PKG-Iα cDNA into cultured VSMCs deficient in PKG results in the restoration of the contractile phenotype.36,37⇓ Likewise, the expression of PKG in coronary arterial SMCs in response to injury decreases as the phenotype of the cells modulates from contractile to synthetic. In addition to the inhibition of PKG-Iα promoter activity produced by LPS and TNF-α in BASMCs observed in this study, we have also observed that exposure of BASMCs to IL-1β, TNF-α, or LPS causes the suppression of PKG-Iα protein expression (N.B. Browner and T.M. Lincoln, unpublished observations, 2002). Inflammatory cytokines are present in vascular lesions and induce the expression of iNOS and cyclooxygenase-2 (COX-2). The high output production of NO and eicosanoids in these lesions would be predicted, based on the result of this study, to increase cyclic nucleotides levels, activate PKA, and suppress PKG expression. Hence, modulation of the VSMCs from the more contractile phenotype to a more fibroproliferative phenotype as a result of the suppression of PKG expression may underlie in part the development of vascular lesions.
This work was supported by grants HL53426 and HL66164 from the NIH. We are grateful to Drs Victor Darley-Usmar and Hanjoong Jo, Department of Pathology, and Natasha Browner, Department of Pharmacology, for helpful discussions.
Original received August 23, 2001; revision received January 17, 2002; accepted January 17, 2002.
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