Molecular Regulation of the Bovine Endothelial Cell Nitric Oxide Synthase by Transforming Growth Factor–β1
Abstract The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor–β1 (TGFβ1). TGFβ1 plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGFβ1 might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGFβ1 increased ecNOS mRNA in a dose-dependent manner. TGFβ1 also increased ecNOS protein content. The production of nitrogen oxides (NOx), assessed by chemiluminescence, and nitric oxide synthase activity, assessed by arginine/citrulline conversion, were increased in TGFβ1-treated cells. Transcriptional activity of the 5′-flanking promoter region of the ecNOS gene was increased by TGFβ1, as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGFβ1-response element was present between nucleotides −1269 and −935 from the first transcription start site, in which a putative nuclear factor–1 (NF-1) binding site existed. Gel shift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding site in a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGFβ1. In conclusion, TGFβ1 increases ecNOS expression associated with an increase in production of NO in BAEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter.
- Received September 9, 1994.
- Accepted May 8, 1995.
The endothelium regulates vascular tone by producing prostacyclin, EDRF, and endothelium-derived constricting factors. The EDRF is a labile potent vasodilator, which is NO or a related compound.1 2 Recently, ecNOS cDNA3 4 5 6 has been cloned and the 5′-flanking region of the gene sequenced.7 8 9 A variety of cis-regulatory sequences, such as potential AP-1 and AP-2 binding sites, sterol-regulatory elements, shear stress response elements, and a putative NF-1 binding site are present in the promoter region of the ecNOS gene.7 8 Although the ecNOS is considered a constitutive enzyme, recent evidence suggests that chronic shear stress, tumor necrosis factor, and proliferating state influence the level of ecNOS mRNA. Thus, various physical and humoral stimuli may regulate its expression.3 10 11
TGFβ1, a 25-kD homodimeric multifunctional peptide, plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy in hypertension, vascular response to injury after balloon angioplasty, and angiogenesis in wound healing.12 13 14 15 TGFβ1 modulates a wide variety of cellular activities through transcription of various genes. The precise molecular mechanism of gene expression induced by TGFβ1 is still unknown, although several mechanisms have been proposed. The TGFβ1-activating element of the promoter region of the mouse α2(I) collagen gene is very similar to a consensus sequence NF-1 binding site [TGG(N)6GCCAA], and NF-1 mediates expression of this gene.16 NF-1 is also reported to mediate the increases in PAI-1 gene expression induced by TGFβ1.17 In contrast, TGFβ1 autoinduction is mediated by a signaling pathway involving AP-1 regulatory elements.18 Both putative NF-1 and AP-1 binding sites are present in the 5′-flanking promoter region of the bovine ecNOS, −1014 and −441 nucleotides from the first transcription start site, respectively.8 On the basis of these considerations, we examined the effect of TGFβ1 on gene expression and protein induction of ecNOS, NO production, and the activity of NOS in cultured BAECs.
BAECs were grown in culture as previously described.19 The cells were cultured in Medium 199 supplemented with 10% fetal calf serum (Hyclone Laboratories). The cells used in the present study were from the fifth to the ninth passage, and were studied upon reaching confluency.
Northern Blot Analysis
Northern blot analysis was performed as previously described.3 11 Total RNA was isolated by phenol extraction, size-fractionated on a 1% agarose/3% formaldehyde gel, and transferred to a nitrocellulose membrane. Hybridizations were performed overnight at 42°C with a [32P]dCTP-labeled ecNOS cDNA fragment obtained by Sst I (GIBCO BRL) digestion corresponding to sequences from nucleotides 941 to 2099 of the full-length ecNOS cDNA. The membranes were then washed twice with 2× standard saline citrate and 1% SDS for 30 minutes at 55°C and subsequently once with 0.2× standard saline citrate and 0.1% SDS for 30 minutes at 55°C.
In all studies, the nitrocellulose membranes were stripped and subsequently hybridized with cDNA for GAPDH to serve as a control. The autoradiographs were quantified by densitometric scanning.
Western Blot Analysis
As previously described,3 11 Western blot analysis was performed with a rabbit antibody against BAEC NOS (antibody p459) and a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. The polyclonal antibody p459 was raised against the peptide sequence 628 to 638 of the bovine ecNOS linked to BSA and then immunopurified. This peptide sequence does not exist in the macrophage enzyme, and in preliminary studies p459 did not detect macrophage NOS by either Western blot analysis or immunohistochemistry. Signals were detected with the ECL detection system (Amersham Corp) on a standard x-ray system.
Measurement of Endothelial Cell NO Production
The production of NO from BAECs was assessed by measurement of nitrite and nitrate, the stable degradation products of NO, with a chemiluminescence technique, as previously described.20 The amounts of NO released were normalized to the number of cells in each culture dish.
Measurement of NOS Activity
The activity of NOS was assessed by conversion of [3H]arginine to [3H]citrulline, as previously described.11 Cell homogenates were incubated in the reaction buffer ([mmol/L] Tris HCl 50, EDTA 0.1, EGTA 0.1, pH 7.5) containing calmodulin 100 nmol/L, CaCl2 2.5 mmol/L, NADPH 1 mmol/L, tetrahydrobiopterin 3 μmol/L, and the substrate l-arginine 100 μmol/L combined with l-[2,3-3H]arginine (0.2 μCi; specific activity, 55 Ci/mmol) for 15 minutes at 37°C. After incubation, the reaction was quenched by the addition of 1 mL of the stop buffer ([mmol/L] EDTA 2, EGTA 2, HEPES 20, pH 5.5). The reaction mix was applied to a 1-mL column containing Dowex AG 50WX-8 (Na+ form) resin (Bio-Rad). l-[2,3-3H]Citrulline was eluted twice with 0.5 mL of the stop buffer and radioactivity was determined by liquid scintillation counting.
Transfection of BAECs With Promoter/Reporter Constructs
To determine whether the TGFβ1 effect on ecNOS gene expression was mediated by the 5′-flanking region of the gene, a series of bovine ecNOS promoter/luciferase reporter gene plasmids were constructed as recently described.8 Deletion fragments of the bovine endothelial gene promoter with 5′ end points at nucleotides −2835, −1548, −1269, −935, −614, and −416 and a 3′ end point at nucleotide +240 were created by PCR amplification of bovine genomic DNA. Fragments were then subcloned into the promoterless plasmid, poLuc, upstream from a luciferase reporter gene. Subconfluent BAECs were transfected with promoter/luciferase constructs, a poLuc (negative control) construct, or a pSV2Luc (positive control) construct containing the SV40 promoter/enhancer. BAECs were cotransfected with a pSV2CAT construct containing the CAT gene to normalize for variation in transfection efficiencies. Luciferase and CAT activities were determined 48 hours after transfection.8
Gel Mobility Shift Assay
Nuclear extracts from BAECs were prepared as previously described.21 Cell pellets were suspended in buffer A ([mmol/L] KCl 10, MgCl2 1.5, dithiothreitol 1, HEPES 10) containing the proteinase inhibitors PMSF 1 mmol/L, leupeptin 10 μg/mL, antipain 10 μg/mL, and aprotinin 0.023 trypsin inhibitor units/mL. After centrifugation, the cell pellets were resuspended in buffer A with 0.1% Triton X-100. After incubation for 10 minutes at 4°C, the mixture was centrifuged at 4°C for 5 minutes at 750g to obtain nuclear pellets. The nuclear pellets were resuspended in the extraction buffer ([mmol/L] NaCl 420, MgCl2 1.5, EDTA 0.2, dithiothreitol 1, and HEPES 20 and glycerol 25%, pH 7.5) containing the same proteinase inhibitors. After incubation for 30 minutes at 4°C, nuclear extracts were obtained by centrifugation at 20 000g for 10 minutes. Synthesized double-stranded oligonucleotides were labeled with [γ-32P]ATP at the 5′ end with T4 polynucleotide kinase (Promega Corp). The nuclear extracts were then incubated with approximately 100 000 cpm of radiolabeled double-stranded oligonucleotides for 30 minutes at 30°C in 20 μL of binding solution ([mmol/L] KCl 150, MgCl2 5, EDTA 1, dithiothreitol 1, PMSF 1, HEPES 12, and Tris HCl 4 and glycerol 12%, pH 7.9) in the presence of 2 μg poly dIdC and 4.5 μg BSA. In some experiments, before radiolabeled probes were added, cold competitors were preincubated with nuclear extracts for 15 minutes at room temperature. In other studies, a rabbit polyclonal antibody directed against CTF/NF-1 (generously provided by Dr Naoko Tanese and Dr Robert Tjian) or preimmune serum was preincubated with nuclear extracts for 45 minutes at 37°C. The incubated mixtures were separated on 4% polyacrylamide gels. The gels were dried under vacuum and exposed to x-ray film. Sequences of the oligonucleotides were as follows: ecNOS promoter containing a putative NF-1 binding site (corresponding to sequences from nucleotides −1006 to −1034 in the bovine ecNOS promoter), 5′-CACCTTCTTGGTGTGACCCCAGAACTCGC-3′; random oligonucleotide, 5′-GCTGACTATGACAGCTTCGGAACTTGCTAC-3′; consensus NF-1 oligonucleotide, 5′-CCTTTGGCATGCTGCCAATATG-3′ (Promega Corp); and NF-1 mutant NOS promoter fragment, 5′-CACCTTCTGTTTGTGA-CCAACGAACTCGC-3′. Complementary sequences were used to produce double-stranded DNA.
Site-Directed Mutagenesis of NF-1 Binding Site in ecNOS Promoter/Reporter Construct
A 6-bp substitution of the putative NF-1 binding site was introduced into the ecNOS −1548/+240/luciferase construct by the overlapping extension PCR technique.22 A synthetic oligonucleotide (corresponding to the sequence from nucleotides −1049 to −990 in the bovine ecNOS promoter) containing a 6-bp substitution of the NF-1 binding site was used as a mutagenic primer and the ecNOS −1548/+240/luciferase construct was used as a template. This mutated oligonucleotide produced by PCR was subcloned into the ecNOS −1548/+240/luciferase construct in the Tth111 and BamHI sites. By use of this approach the putative NF-1 binding site (5′-TGGTGTGACCCCA-3′) was mutated to 5′-GTTTGTGACCAAC-3′. Incorporation of the mutation was confirmed by dideoxy chain termination sequencing with Sequenase 2.0 (United States Biochemical).
DNA Synthesis and Cell Growth Study
DNA synthesis was assessed by measurement of the incorporation of [methyl-3H]thymidine into the acid-insoluble material as previously described.23 To achieve quiescence, BAECs on 12-well dishes were cultured in Medium 199 with 0.1% BSA for 36 hours. Quiescent cells were cultured for 24 hours in Medium 199 with 5% fetal calf serum containing or lacking TGFβ1. Finally, the cells were incubated for 24 hours in the same medium supplemented with 2 μCi [methyl-3H]thymidine. After precipitation with 2% trichloroacetic acid, incorporated radioactivity was measured by liquid scintillation counting. In separate experiments, for examination of the effect on cell growth, cell numbers were determined by counting with a hemacytometer.
TGFβ1 was used as the homodimer isolated from porcine platelets (R&D Systems). Radiochemicals were purchased from Amersham Corp. All other reagents were purchased from Sigma Chemical Co except where specified.
The data in this article are expressed as mean±SD. Comparisons of data between two groups were made by unpaired Student’s t test. Values of P<.05 were considered significant.
TGFβ1 Increased mRNA of ecNOS in BAECs
Levels of ecNOS transcripts were increased as early as 3 hours after exposure to 1 ng/mL TGFβ1 (Fig 1A⇓). Maximal induction, observed at 12 hours, was by approximately 2.3-fold (three different experiments). The level of mRNA began to decrease at 24 hours.
TGFβ1 (0.01 to 10 ng/mL for 6 hours) increased in ecNOS transcripts in a dose-dependent manner (Fig 1B⇑). The effect was detectable in the presence of 0.01 ng/mL TGFβ1.
To determine whether the increase in ecNOS mRNA was caused by enhanced transcription, BAECs were exposed to 10 μg/mL actinomycin D for 30 minutes before and during exposure to 1 ng/mL TGFβ1 for 6 hours. Actinomycin D slightly decreased the level of ecNOS mRNA in control cells, whereas it abolished the increase in ecNOS mRNA in TGFβ1-treated cells (Fig 1C⇑).
TGFβ1 Increased ecNOS Protein Expression in BAECs
Stimulation of BAECs with 1 ng/mL of TGFβ1 for 24 hours potentiated ecNOS protein expression 2.0-fold (in three different experiments assessed by densitometry) (Fig 2A⇓).
TGFβ1 Potentiated NO Production From BAECs
The release of NO from control cells was 1.48± 0.28×10−4 and 6.09±0.42×10−4 pmol/cell under basal conditions and in response to 1 μmol/L calcium ionophore A23187, respectively. After incubation with 1 ng/mL TGFβ1 for 24 hours, the release of NO under basal conditions and in the presence of calcium ionophore A23187 was increased to 3.11±0.27×10−4 and 9.22±0.36×10−4 pmol/cell, respectively (Fig 2B⇑).
TGFβ1 Potentiated NOS Activity in BAECs
As assessed by arginine/citrulline conversion, NOS activity in homogenates of TGFβ1-treated cells (1 ng/mL for 24 hours) was increased by 1.6 times that in control cells. Conversion of l-[3H]arginine to l-[3H]citrulline was inhibited by addition of 10 μmol/L NG-monomethyl-l-arginine, an inhibitor of NOS, in both control and TGFβ1-treated cells (Fig 2C⇑).
TGFβ1 Increased the Transcriptional Activity of the ecNOS Gene Promoter
Basal and TGFβ1-stimulated (1 ng/mL for 16 hours) promoter activity was assessed by luciferase assay. As shown in Fig 3⇓, ecNOS −2835/+240, ecNOS −1548/+240, and ecNOS −1269/+240 promoter activities were increased more than fourfold in response to TGFβ1 treatment. In contrast, TGFβ1 treatment did not alter transcriptional activities of poLuc, pSV2Luc, ecNOS −935/+240, ecNOS −614/+240, and ecNOS −416/+240 constructs. These results indicate that the TGFβ1-response element was likely present between nucleotides −935 and −1269 of the ecNOS promoter, in which a putative NF-1 binding site exists (nucleotide −1014).
Nuclear Extracts From BAECs Bound to ecNOS Promoter Fragment Containing the Putative NF-1 Binding Site in a Sequence-Specific Manner
The deletional analysis described above and the previous reports indicating a role of NF-1 in transcriptional regulation by TGFβ116 17 prompted us to further examine possible interactions of the putative NF-1 binding site of the ecNOS promoter with endothelial cell nuclear proteins. Gel mobility shift assays were performed with a 29-bp oligonucleotide corresponding to the sequence from nucleotide −1006 to nucleotide −1034 of the bovine ecNOS promoter (NOS promoter fragment) and a consensus NF-1 oligonucleotide. When nuclear extracts prepared from BAECs were incubated with radiolabeled NOS promoter fragments, several retarded bands were observed (lanes 1 and 9 in Fig 4⇓). Addition of excess unlabeled NOS promoter fragments competed formation of a major band, indicated by the arrow, but random oligonucleotides had no such effect (lanes 2, 4, and 10 in Fig 4⇓). This competition was dependent on the concentration of unlabeled NOS promoter fragments (lanes 1 to 5 in Fig 5⇓). These findings indicated that this band was a sequence-specific complex of the NOS promoter fragment with nuclear proteins. When the consensus NF-1 oligonucleotide was used as a radiolabeled probe, a major retarded band was observed (lanes 5 and 14 in Fig 4⇓). This major band was a sequence-specific complex of the consensus NF-1 oligonucleotide with nuclear proteins, because this complex was competitively inhibited by excess amounts of unlabeled NF-1 consensus oligonucleotides but not by random nonsense oligonucleotides (lanes 7, 8, and 15 in Fig 4⇓ and lanes 14 to 16 in Fig 5⇓). To further characterize the nuclear protein(s) involved in these interactions, a rabbit polyclonal antibody against CTF/NF-1 was used in a gel mobility shift assay. This antibody (designated 8199), which was raised against the N-terminal half of CTF/NF-1 that contains the DNA-binding domain, crossreacts with other proteins in the CTF/NF-1 family and other proteins related to CTF/NF-1 if they contain a similar DNA-binding domain. Antibody 8199 strongly reduced the electrophoretic mobility of the specific complexes of nuclear extracts with not only the consensus NF-1 oligonucleotide but also the NOS promoter fragment, whereas preimmune rabbit serum did not (lanes 11, 12, 16, and 17 in Fig 4⇓). These supershifted bands (indicated on the figure by an arrow) were competed by excess amount of unlabeled oligonucleotides (lanes 13 and 18 in Fig 4⇓). These results indicate that protein(s) bound to the NOS promoter fragment have a structure closely related to proteins of the CTF/NF-1 family. Interestingly, competition experiments indicated that protein(s) bound to the NOS promoter fragment had low affinity for the consensus NF-1 oligonucleotide. Excess amounts of unlabeled consensus NF-1 oligonucleotides only minimally competed the binding of nuclear protein(s) with the NOS promoter fragment (lane 3 in Fig 4⇓ and lanes 10 to 13 in Fig 5⇓). Conversely, unlabeled NOS promoter fragments had also little effect on the binding of nuclear extracts with the consensus NF-1 oligonucleotide (lane 6 in Fig 4⇓ and lanes 6 to 9 in Fig 5⇓).
The binding activity of nuclear extracts prepared from TGFβ1-treated cells (1 ng/mL for 16 hours) for the NOS promoter fragment was inconsistently increased compared with that of control cells (1.3-fold increase in three different experiments, as assessed by densitometry). TGFβ1 treatment had no effect on the binding activity to radiolabeled consensus NF-1 oligonucleotides.
Site-Directed Mutagenesis of the NF-1 Binding Site
To further explore the significance of the putative NF-1 binding site in transcriptional activation by TGFβ1, we introduced a 6-bp substitution into the core (nucleotides −1026 to −1014) of the putative NF-1 binding site of the ecNOS −1548/+240/luciferase construct (Fig 6A⇑). Gel mobility shift assay demonstrated that this substitution caused the loss of the specific binding affinity. As shown in Fig 6B⇑, excess amounts of unlabeled 6-bp–substituted NOS promoter fragments (NF-1 mutant NOS promoter fragment) did not compete the specific binding of the wild-type NOS promoter fragment with nuclear protein. In a transfection study with the NF-1 mutant ecNOS promoter/luciferase construct, mutation of the putative NF-1 binding site in the promoter/luciferase construct markedly decreased the responsiveness to TGFβ1, although even in the mutant ecNOS promoter/luciferase construct TGFβ1 increased promoter activity 1.9-fold (Fig 6C⇑).
TGFβ1 Inhibited DNA Synthesis and Cell Growth
Because ecNOS expression of proliferating cells is increased compared with quiescent cells,11 we sought to determine whether the effect of TGFβ1 on ecNOS was simply due to a stimulation of cell growth. TGFβ1 (0.1 to 10 ng/mL) inhibited both cell growth assessed by cell counting and DNA synthesis assessed by [methyl-3H]thymidine incorporation in a dose-dependent manner. TGFβ1 (1.0 ng/mL for 48 hours) reduced thymidine incorporation and cell number by 68.7% and 80.8%, respectively. The viability of TGFβ1-treated cells was greater than 97.3%, as assessed by trypan blue exclusion.
The present study demonstrates that TGFβ1 increases expression of ecNOS associated with an increased enzyme activity of NOS and production of NO in endothelial cells. Transcriptional activity of the 5′-flanking promoter of the ecNOS gene was increased by TGFβ1. Deletion analysis suggests that the TGFβ1 response element is present between nucleotides −1269 and −935 from the first transcription start site, in which a putative NF-1 binding site exists. Mutation of the putative NF-1 binding site significantly decreased the responsiveness to TGFβ1. Gel shift assays show that nuclear protein(s), immunologically similar to CTF/NF-1, bind to the putative NF-1 binding site in a sequence-specific manner.
The increase in ecNOS mRNA induced by TGFβ1 could be caused either by an increased transcriptional rate or by decreased degradation of mRNA. The results of the present study demonstrate that this effect is probably due at least in part to increased ecNOS transcription. First, inhibition of mRNA transcription with actinomycin D completely prevented the increase in ecNOS mRNA by TGFβ1. Second, promoter/luciferase constructs demonstrated a fourfold increase in ecNOS promoter activity in the presence of TGFβ1. Taken together, these data strongly suggest that the increase in ecNOS mRNA by TGFβ1 is mediated by increased mRNA transcription. The present study does not address the possibility that TGFβ1 might increase ecNOS stability, but this possibility seems unlikely given the reported stability of ecNOS mRNA10 and the relatively short period of time necessary for TGFβ1 to induce this effect.
Several mechanisms for gene transcription regulation by TGFβ1 have been proposed. TGFβ1 activates transcription of the mouse α2(I) collagen gene16 and the PAI-1 gene through NF-1.17 TGFβ1 is also reported to increase transcription of its own gene promoter by means of the AP-1 binding region.18 In studies of promoter/luciferase constructs we found that promoter activity was increased by TGFβ1 in constructs containing a putative NF-1 binding site (−1269/+240), whereas the −935/+240 luciferase construct (which lacked a putative NF-1 site but contained a putative AP-1 site) was not responsive to TGFβ1. Mutation of the putative NF-1 binding site markedly decreased TGFβ1 responsiveness. These results indicate that the NF-1 binding site in the promoter region plays an important role in transcriptional activation of the ecNOS induced by TGFβ1 treatment.
NF-1 interacts with the consensus sequence TGG(N)6-GCCAA, which is found in viral and cellular genomes.24 A similar sequence (TGGTGTGACCCCA) is found at nucleotide −1014 upstream from the first transcription start site of the ecNOS.8 Because the second half of the recognition sequence for NF-1 resembles the CCAAT box and NF-1 has been proposed to be identical to CTF,25 NF-1 is often referred to as CTF/NF-1. Recent data suggest that CTF/NF-1 is a heterogeneous family of nuclear proteins. The diversity of these proteins is in part due to alternative splicing of NF-1 gene transcripts.26 In gel mobility shift assays, a polyclonal antibody against CTF/NF-1 crossreacted with the protein(s) bound to the putative NF-1 binding site of the ecNOS promoter, whereas they had low affinity for consensus NF-1 oligonucleotides. These results suggest that nuclear protein(s) involved in the interaction of the putative NF-1 binding site of the ecNOS promoter have a structure closely related to the DNA-binding domain of proteins in the CTF/NF-1 family, but they are probably not identical to NF-1. These findings are similar to recent data indicating that the human α1(I) collagen gene promoter contains a TGFβ-activating element similar to the NF-1 binding region, which does not interact with NF-1.27 Whereas transcriptional activity of the ecNOS promoter by TGFβ1 treatment was increased fourfold as assessed by transfection of promoter/luciferase constructs, the binding activity of nuclear extracts in TGFβ1-treated cells was only modestly and inconsistently increased. From these findings, we speculate that a TGFβ1-responsive transcriptional factor(s) similar to CTF/NF-1 probably participates in activating the ecNOS gene transcription without enhancing the binding affinity. The present findings are entirely analogous to those of the PAI-1 promoter in which transcription is activated by TGFβ1 by means of a similar NF-1 site.17 In the cases of both PAI-1 and ecNOS, gel mobility shift assay failed to demonstrate enhancement of the binding affinity by TGFβ1 treatment. It is possible that TGFβ1 may activate gene transcription by means of NF-1–like factors already bound to the promoter. Alternatively, other regions of the ecNOS promoter may also participate in transcriptional activation by TGFβ1. This possibility is strengthened by the observation that even in the NF-1 mutant ecNOS/luciferase construct some responsiveness to TGFβ1 was preserved.
The present experiments also demonstrate an increase in ecNOS protein, NO production, and enzyme activity in endothelial cells treated with TGFβ1. The increases in protein and NO production were in general less than the increase in mRNA. This is not surprising because levels of mRNA and protein are not always directly correlated and factors other than the level of ecNOS protein may modulate cell NO production (eg, ecNOS cofactors, intracellular Ca2+). Although the increase in cellular NO release may seem modest, it is important to note that even small increases in NO production may markedly decrease the tone of intact vascular segments.2 28 Thus, small increases in NOS activity and NO production caused by TGFβ1 may importantly influence vascular function. Another consideration related to this issue is that TGFβ1 dramatically increases the production of extracellular matrix proteins, which were measured in our protein determinations and used to normalize enzyme activity and to calculate the amount of protein loaded onto gels for Western blot analysis. Although this was an unavoidable experimental condition, it is conceivable that if these data could have been normalized for intracellular proteins only, the amount of increases in ecNOS would have been greater.
Recently our laboratory demonstrated that expression of ecNOS of preconfluent growing cells is increased compared with that of postconfluent quiescent cells.11 Thus, one explanation for the present findings is that TGFβ1 might have a growth-stimulating effect. As previously observed,29 30 however, TGFβ1 decreased both endothelial cell number and [3H]thymidine incorporation. This finding excludes the possibility that the TGFβ1 effect was simply due to stimulation of cell growth. Furthermore, in preliminary experiments, TGFβ1 increased both ecNOS mRNA and the enzyme activity in quiescent cells several days after confluence was reached.
In previous experiments, we observed that vessels from animals with diet-induced atherosclerosis produced larger quantities of NO than normal animals.31 Because TGFβ1 is present in increased quantities in atherosclerotic lesions,13 our findings in the present study may in part explain the enhanced production of NO in atherosclerotic vessels.
It has previously been demonstrated that TGFβ1 decreases adhesiveness of neutrophils to endothelial cell monolayers.32 More recent studies have shown that NO also decreases neutrophil adhesion to endothelial cell monolayers, probably by means of scavenging of superoxide anions.33 34 It is interesting to speculate that an increase in NO production by TGFβ1 plays a role in the antiadhesive effect of TGFβ1. It has also been demonstrated that pretreatment with TGFβ1 preserves endothelial function and has a cardioprotective effect in rat hearts subjected to ischemia/reperfusion injury, possibly by means of inhibition of superoxide production.35 TGFβ1 might have beneficial effects in such pathophysiological situations by increasing endothelial cell NO production. A major source of TGFβ1 in vivo is α-granules in platelets. It is interesting to speculate that TGFβ1 released from platelets might serve to increase endothelial cell NO production through enhanced ecNOS expression. This could serve as a mechanism whereby platelet adhesion to endothelial cells could be modulated by enhanced endothelial NO release that was mediated by TGFβ1 released from platelets.
In conclusion, TGFβ1 upregulates the expression of ecNOS mRNA and protein as well as NO production and NOS activity in endothelial cells. This response is mediated by transcriptional activation of the ecNOS promoter. This study is the first report to demonstrate involvement of a growth factor in gene regulation of the “constitutively expressed” ecNOS.
Selected Abbreviations and Acronyms
|BAEC(s)||=||bovine aortic endothelial cell(s)|
|CTF||=||CCAAT transcriptional factor|
|ecNOS||=||endothelial cell NO synthase|
|EDRF||=||endothelium-derived relaxing factor|
|PAI-1||=||plasminogen activator inhibitor–1|
|PCR||=||polymerase chain reaction|
|TGFβ1||=||transforming growth factor–β1|
This work was supported by National Institutes of Health grants HL32717, HL39006, and HL48667 and a merit grant from the Veterans Administration. We are grateful to Dr Naoko Tanese (Department of Microbiology, New York University Medical Center) and Dr Robert Tjian (Howard Hughes Institute, Department of Biochemistry, University of California) for kindly providing us with antibody against CTF/NF-1. We appreciate the secretarial assistance of Cynthia M. Curry.
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