Shear Stress Regulates Endothelial Nitric-oxide Synthase Promoter Activity through Nuclear Factor κB Binding*

We have previously demonstrated that shear stress increases transcription of the endothelial nitric-oxide synthase (eNOS) by a pathway involving activation of the tyrosine kinase c-Src and extracellular signal-related kinase 1/2 (ERK1/2). In the present study sought to determine the events downstream of this pathway. Shear stress activated a human eNOS promoter chloramphenicol acetyl-CoA transferase chimeric construct in a time-dependent fashion, and this could be prevented by inhibition of the c-Src and MEK1/2. Studies using electromobility shift assays, promoter deletions, and promoter mutations revealed that shear activation of the eNOS promoter was due to binding of nuclear factor κB subunits p50 and p65 to a GAGACC sequence –990 to –984 base pairs upstream of the eNOS transcription start site. Shear induced nuclear translocation of p50 and p65, and activation of the eNOS promoter by shear could be prevented by co-transfection with a dominant negative I kappa Bα. Exposure of endothelial cells to shear resulted in Iκ kinase phosphorylation, and this was blocked by the MEK1/2 inhibitor PD98059 and the cSrc inhibitor PP1, suggesting these signaling molecules are upstream of NFκB activation. These experiments indicate that shear stress increases eNOS transcription by NFκB activation and p50/p65 binding to a GAGACC sequence present of the human eNOS promoter. While NFκB activation is generally viewed as a proinflammatory stimulus, the current data indicate that its transient activation by shear may increase expression of eNOS, which via production of nitric oxide could convey anti-inflammatory and anti-atherosclerotic properties.

We have previously demonstrated that shear stress increases transcription of the endothelial nitric-oxide synthase (eNOS) by a pathway involving activation of the tyrosine kinase c-Src and extracellular signal-related kinase 1/2 (ERK1/2). In the present study sought to determine the events downstream of this pathway. Shear stress activated a human eNOS promoter chloramphenicol acetyl-CoA transferase chimeric construct in a time-dependent fashion, and this could be prevented by inhibition of the c-Src and MEK1/2. Studies using electromobility shift assays, promoter deletions, and promoter mutations revealed that shear activation of the eNOS promoter was due to binding of nuclear factor B subunits p50 and p65 to a GAGACC sequence ؊990 to ؊984 base pairs upstream of the eNOS transcription start site. Shear induced nuclear translocation of p50 and p65, and activation of the eNOS promoter by shear could be prevented by co-transfection with a dominant negative I kappa B␣. Exposure of endothelial cells to shear resulted in I kinase phosphorylation, and this was blocked by the MEK1/2 inhibitor PD98059 and the cSrc inhibitor PP1, suggesting these signaling molecules are upstream of NFB activation. These experiments indicate that shear stress increases eNOS transcription by NFB activation and p50/p65 binding to a GAGACC sequence present of the human eNOS promoter. While NFB activation is generally viewed as a proinflammatory stimulus, the current data indicate that its transient activation by shear may increase expression of eNOS, which via production of nitric oxide could convey anti-inflammatory and anti-atherosclerotic properties.
Unidirectional laminar shear stress, the frictional force of blood over the surface of the endothelium, exerts atheroprotective effects by preventing adhesion molecule expression, reducing platelet aggregation, and inhibiting both smooth muscle cell proliferation and endothelial cell apoptosis (1). In contrast, areas of the vasculature exposed to low levels of shear stress are prone to atherosclerotic lesion formation (2,3). At least a portion of the beneficial effects of laminar shear stress is due to modulation of nitric oxide (NO ⅐ ) production via two mecha-nisms. Immediately after the onset of shear, there is an acute activation of the endothelial NO synthase (eNOS) leading to NO ⅐ release within seconds thereafter (4). Over several hours, shear stress stimulates an increase in eNOS mRNA and protein expression (5). Recent work from our laboratory has shown that this latter effect occurs by two distinct pathways regulating eNOS transcription and mRNA stability, with transcription peaking at 1 h and returning to baseline levels shortly thereafter. Both of these pathways share a need for activation of c-Src; however, eNOS transcription, as measured by nuclear run-on analysis, involves c-Src activation of the mitogen-activated protein kinases Raf, MEK1/2, 1 and extracellular-signal related kinase 1/2 (ERK1/2) (6).
The precise nuclear events that lead to an increase in eNOS transcription in response to shear stress remain poorly defined. Endothelial cells respond to increases in shear stress by activation of a variety of signaling molecules, including c-Src, PKA, PKB, PKC, and mitogen-activated protein kinases (7,8). ERK1/2 is phosphorylated shortly (within 5 min) following onset of shear stress and subsequently stimulates binding of a variety of transcription factors, including AP-1 and SP-1 (9). These could both potentially mediate the increase in eNOS transcription by shear stress, as potential binding sites for these are present in the eNOS promoter. Several early response genes are also induced by shear stress including c-myc, c-fos, and c-jun (10). At early time points shear stress increases transcription of several proatherogenic genes including platelet-derived growth factor B (PDGF-B) and endothelin-1 (11). Laminar shear stress activates the PDGF-B promoter through an NFB-like element with the core sequence GAGACC, which has been characterized as a shear stress response element (SSRE) (12). NFB is a heterodimer consisting of p50 and p65 subunits (13). Under basal conditions, the subunits are sequestered in the cytosol by binding to an inhibitory molecule IB. Following phosphorylation of IB, the p50 and p65 subunits are released and translocate to the nucleus where they can bind specific DNA sequences (13). Finally, shear stress has been shown to increase NFB/DNA binding during the first hour of flow stimulation (9,13).
While abundant data indicate that eNOS gene expression is increased by shear stress, the exact binding region in the eNOS promoter and the transcription factor(s) involved remain poorly defined. In this study, using a chimeric human eNOS promoter-chloramphenicol acetyltransferase (CAT) construct, we defined a 25-base pair region that is responsible for eNOS promoter activity in response to laminar shear stress. Transactivation of this element was found to be regulated by c-Src and ERK1/2 activity as PP1 and PD98059 completely inhibited shear-induced eNOS-CAT activity. Our studies indicate the NFB subunits p50 and p65 are translocated to the nucleus and bind to this site in response to shear stress. This phenomenon seems to be a critical mediator for eNOS transcription in response to laminar shear stress.
Shear Apparatus-A cone-in-plate viscometer with a 1°angle was used to shear cells (15). All shear studies used a shear rate of 15 dynes/cm 2 unless otherwise stated and were performed in an incubator at 37°C in 5% CO 2 . The culture medium was changed to 5% FCS the night before the experiments. Cells were pretreated with the indicated agent in media containing 5% FCS for 1 h prior to shear.
Cloning of the Human eNOS Promoter-The human eNOS promoter was cloned by polymerase chain reaction using Elongase (Invitrogen) with human genomic DNA (50 ng, Invitrogen) as a template. This approach used a forward primer from the 5Ј-upstream genomic sequence with a XhoI site at the 5Ј-end (underlined) (5Ј-TGCCTGTCAC-CTCGAGCCTGAGGAT-3Ј, nucleotide Ϫ1578) and a reverse primer from 3Ј-downstream prior to the initiator methionine codon with a HindIII site at the 5Ј-end (underlined) (5Ј-CACGCTCTTCAAGCTTC-CCAAGTTACTGTG-3Ј, nucleotide ϩ27). The resultant PCR product was sequenced by the dideoxy-chain termination method using the Sequenase 2.0 kit (United States Biochemical, Cleveland, OH). The sequence obtained was similar to that reported previously (16).
Human eNOS Promoter Deletion and Mutagenesis-A modified pGL2 vector was used as a CAT reporter gene vector. This was generated by replacement of the luciferase gene of pGL2 Basic vector (Promega, Madison, WI) by the CAT gene derived from pCAT Basic vector (Promega) using the BamH1/HindIII site. The full-length eNOS promoter (Ϫ1578 to ϩ27) was subcloned into the XhoI/HindIII sites of a modified pGL2 vector. This was done because in preliminary experiments we found that shear increased activity of a promoterless-luciferase construct.
Transfection Protocol-Cells were transfected when 50 -70% confluent using Effectene (Qiagen, Valencia, CA) as follows. When 50 -70% confluent, cells were washed with 1ϫ phosphate-buffered saline and then incubated for 18 h in medium containing Effectene (1:30), eNOS-CAT (1.5 g), and ␤-galactosidase DNA (0.5 g). Following incubation, transfection medium was replaced with media containing 10% FCS and cells were exposed to shear or quiescent conditions the following day. The cells were washed and scraped with 200 l of 1ϫ phosphatebuffered saline containing 5 mmol/liter EDTA. After subjecting the pellet to five freeze-thaw cycles, samples were centrifuged and divided for CAT and ␤-galactosidase assays. CAT activity was measured as described previously (17). Samples were counted in a Beckman scintillation counter in duplicate (50 l) following 1 h of incubation. CAT activity was normalized to ␤-galactosidase activity, which was assayed in duplicate (by absorption at 420 nm using a ␤-galactosidase assay kit (Promega).
Electrophoretic Mobility Shift Assay Protocol-Following shear, nuclear protein was extracted as described previously (18). For gel shift studies, a 25-base pair oligonucleotide shown to be responsive to shear stress plus 6-flanking bases on each end was used (5Ј-CAGGGGTC-GAAGCCTCGGGATTTCGAGACCTCAGAGA-3Ј and complement). Biotinylated DNA (5Ј-) was used for all gel shift studies. Four g of nuclear protein was incubated at room temperature for 5 min with 2ϫ binding buffer (Pierce). Biotinylated DNA was then added, and samples were incubated at room temperature for an additional 25 min. Complexes were then separated on a 6% acrylamide-GTG non-denaturing gel and transferred to a nylon membrane for detection using the Lightshift electrophoretic mobility shift assay (EMSA) kit (Pierce). In some studies, antibodies to specific DNA-binding proteins were incubated for 30 min prior to addition of biotinylated DNA.
Materials-PD98059, UO126, and PPM-18 were obtained from Calbiochem. PP1 was obtained from Biomol (Plymouth Meeting, PA). All drugs were dissolved in Me 2 SO, and the resulting stock solutions were filtered (0.2 M) before use. All antibodies used in EMSA experiments were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Effect of Shear on eNOS Promoter Activity and Identification of the Region Responsive to
Shear-BAECs transfected with the full-length eNOS-CAT promoter construct were sheared for 0, 1, 2, 4, 6, 12, and 24 h and CAT activity measured. We found significant increases in shear-induced eNOS promoter activity at 12 and 24 h of shear stress ( Fig. 2A).
We have previously shown that shear-induced eNOS transcription, as measured by nuclear run-on analysis, occurs

FIG. 2. Chimeric eNOS-CAT promoter activity in BAECs.
A, BAECs were transfected with constructs containing the full-length (F1) promoter and ␤-galactosidase and sheared at 15 dynes/cm 2 for the indicated time points. CAT activity was determined using scintillation counting and normalized to ␤-galactosidase activity and expressed as fold over non-sheared controls. Mean Ϯ S.E. from five to seven separate experiments is shown. Asterisks indicate significant differences from static control (**, p Ͻ 0.01; *, p Ͻ 0.05; Tukey-Kramer test following one-way ANOVA). B, BAECs were sheared or held static for 24 h following transfection of the indicated construct (PO ϭ promoterless). Data are expressed as fold increase over non-sheared cells. Asterisks indicate significant differences from non-sheared cells (**, p Ͻ 0.01, Tukey-Kramer test following one-way ANOVA). through c-Src activation of the MEK/ERK pathway (6). In keeping with these findings, pretreatment of BAECs with the Src inhibitor PP1 (10 M) completely prevented eNOS-CAT activity in response to shear stress (Fig. 3). Furthermore, pretreatment of BAECs with the MEK1/2 inhibitors PD98059 (50 M) and UO126 (50 M) also abolished shear induction of eNOS promoter activity. Taken together, these data indicated that the activity of the chimeric eNOS promoter/CAT construct is regulated by shear stress in response to signaling events that also modulate activation of the of the endogenous eNOS gene by shear.
Identification of the Region of the Human eNOS Promoter Responsive to Shear Stress-To examine the region of the eNOS promoter sensitive to shear, several truncation mutants were generated (Fig. 1). Deletion of the first 600 bases resulted in no significant reduction in eNOS-CAT activity (2.7-fold). Deletion of the next 25 bases (Ϫ1000 to Ϫ975) completely inhibited shear stress activation of the eNOS promoter. Deletion of the following 25-75 bases maintained the inhibition of shear-induced eNOS-CAT activity (Fig. 2B). In keeping with prior studies, there was no difference in the basal activities of these various constructs (p ϭ 0.4, ANOVA, data not shown). These data define a 25-base pair region in the eNOS promoter between Ϫ1000 and Ϫ975 as the region responsible for shear responsiveness. Using the Transfac sequence analysis, we found that this region contained potential binding sites for several transcription factors, including NFB, Sp-1, Elk-1, and GATA (19).
Determination of Transcription Factor Binding to the Shearresponsive Promoter Sequence-To determine the factors binding to the shear responsive element, EMSAs were performed. Using the 25-base pair sequence and flanking 6 bases on either side as a probe, we found that shear stress resulted in the binding of several complexes in a time-dependent manner (Fig.  4) correlating with the time course we have previously reported for eNOS mRNA transcription by shear stress (6).
To determine the composition of the complex induced by shear stress that binds to Ϫ1000 to Ϫ975 base pairs, we used cold competitor oligonucleotides and antibodies for supershift analysis. As seen in Fig. 5A, preincubation of the binding reaction with 50ϫ excess of cold probe completely prevented binding of the complex. Furthermore, preincubation with an NFB unlabeled consensus probe abrogated binding (Fig. 5A). In contrast, binding was unaffected by AP-1, SP-1, and CREB cold consensus oligonucleotides (data not shown). Additionally, the antibodies against p65 and p50 diminished binding of both complexes, suggesting that they are both comprised of p65 and p50 subunits (Fig. 5B). Finally, pretreatment of BAECs with the NFB inhibitor PPM-18 completely prevented binding to the probe (Fig. 6A) and also preventing nuclear translocation of p50 and p65 in response to shear (Fig. 6B). These data implicate a role for NFB binding to the 25 base pair segment that regulates shear stress induction of eNOS transcription.
Determination of the Importance of Binding-regulating eNOS Promoter Activity-Transfac analysis indicated that a consensus NFB site exists in the human eNOS promoter at nucleotides Ϫ994 to Ϫ988 and that this is adjacent to an SSRE site between nucleotides Ϫ987 to Ϫ981. Because both of these sites could potentially bind NFB and regulate eNOS promoter activity in response to shear stress, we generated mutations of the NFB site alone, the SSRE site alone, as well as both sites and performed EMSAs (Fig. 7A). Mutation of the consensus NFB site had no effect on complex binding, whereas mutation of the GAGACC abolished binding of the lower band. Mutation of both sites completely abolished all complex binding (Fig. 7B).
The above data indicate that either the putative NFB con-sensus site or the GAGACC sequence might be responsible for increasing eNOS transcription in response to shear stress. To determine which of these are involved, mutations identical to those describe above were made in the full-length eNOS promoter and shear induction of eNOS promoter activity was examined following 24 h of shear stress. Mutation of the NFB consensus site had no effect on eNOS promoter activity; however, mutation of the GAGACC sequence completely prevented eNOS promoter activity in response to shear stress (Fig. 7C). These data clearly demonstrate the importance of NFB binding to the GAGACC sequence, rather than to the putative NFB site, as being the critical event for initiation of eNOS transcription in response to shear stress FIG. 7. A, sequences of mutations used for EMSA and promoter activity studies. Bold sequences are sequences of interest (underline ϭ consensus NFB; italics ϭ SSRE; NFBm ϭ NFB mutant; SS-REm ϭ SSRE mutant; DBLm ϭ double mutant). B, representative EMSA showing loss of complex binding with SSRE mutation and not NFB mutation. C, BAECs were transfected with either the F1 or mutant NFBm, SSREm, or DBLm promoter constructs, as well as ␤-galactosidase and sheared for 24 h. CAT activity was determined using scintillation counting and normalized to ␤-galactosidase activity and expressed as fold over static control. Mean Ϯ S.E. from four separate experiments is shown. Asterisks indicate significant differences from F1 and NFBm (**, p Ͻ 0.01; Tukey-Kramer test following one-way ANOVA).

FIG. 8. Chimeric eNOS-CAT promoter activity in BAECs treated with dominant negative IB␣ (IB-DN).
BAECs were transfected with constructs containing the full-length human eNOS promoter as well as ␤-galactosidase and/or IB-DN and sheared at 15 dynes/cm 2 for 24 h. Transfected cells were also treated with lysophosphatidylcholine (LPC; 100 M) for 12 h. CAT activity was determined using scintillation counting and normalized to ␤-galactosidase activity and expressed as fold over static control. Mean Ϯ S.E. from three separate experiments is shown. FIG. 9. A, Western analysis showing the time course of IK induction by unidirectional shear stress. BAECs were sheared for the indicated time and 20 g of protein were loaded on a 12.5% SDS-polyacrylamide gel. Membranes were probed with antibodies against phospho-and total IK␣. The top panel is a representative blot for phospho-IK␣, and the lower panel is a representative blot for total IK␣ (n ϭ 4). B, Western analysis showing inhibition of IK phosphorylation by c-Src and MEK1/2 inhibitors. BAECs were pretreated with PD98059 (50 M) or PP1 (10 M) for 1 h prior to shear, and 20 g of protein were loaded on a 12.5% SDS-polyacrylamide gel. Membranes were probed with antibodies against phospho and total IK␣. The top panel is a representative blot for phospho-IK␣, and the lower panel is a representative blot for total IK␣ from three separate experiments.
Determination of NFB Activation in Regulating eNOS Promoter Activity-To determine whether NFB binding is critical for eNOS promoter activation in response to unidirectional shear stress, BAECs were transfected with full-length eNOS/ CAT chimeric construct and a dominant negative IB␣ (20). In resting cells, p50 and p65 are sequestered to IB␣ and can only be released by phosphorylation of IB␣ on serine 32 (21). Mutation of the serine to lysine prevents phosphorylation and subsequent dissociation (20). Co-transfection of F1-CAT and IB␣ dominant negative resulted in complete inhibition of eNOS promoter activity in response to shear stress (Fig. 8). In contrast, dominant negative IB␣ had no effect on eNOS promoter activity in response to lysophosphatidylcholine, which increases eNOS expression by enhancing Sp-1 binding to the eNOS promoter (22). These data clearly show NFB binding regulates eNOS promoter activity following unidirectional shear stress.
Determination of NFB Pathway Activation by Laminar Shear Stress-To determine whether shear stress could activate the NFB pathway, phosphorylation of IK, the major kinase involved in IB phosphorylation, was examined using Western analysis. Shear stress caused a time-dependent phosphorylation of IK, with no effect on the total levels of the protein (Fig. 9A). Furthermore, IK phosphorylation by shear stress was prevented by preincubation with the Src inhibitor PP1 and the MEK1/2 inhibitor PD98059 (Fig. 9B). These data demonstrate NFB pathway activation in response to shear stress and that this pathway is regulated through c-Src, MEK1/2 and ERK1/2.

DISCUSSION
One of the most important physiological stimuli for regulation of the eNOS expression is laminar shear stress. Previously, we have shown that this occurs by a transient increase in transcription and a prolonged increase in eNOS mRNA stability (6). The increase in eNOS transcription, while transient, is likely important as it could underlie the increase in eNOS caused by bouts of exercise training, which via increasing cardiac output enhance eNOS expression (23,24). In the present study, we defined a specific region of the eNOS promoter that regulates induction of eNOS transcription by shear. We demonstrated that shear stress stimulates binding of the NFB subunits p50 and p65 to this region and that this phenomenon is crucial for increasing promoter activity.
Our present findings our in keeping with previous studies in which deletion of the eNOS promoter Ϫ1600 to Ϫ779 base pairs upstream from the transcription start site inhibited induction by laminar shear stress (25,26). There are numerous potential binding sites for a variety of transcription factors within this large region of DNA (19). Using eNOS-CAT promoter constructs, we identified a more precise region, specifically between Ϫ1000 and Ϫ975 base pairs, which confers eNOS transcriptional activation by shear stress. Even within this 25-base pair region, there are several putative binding sites for transcription factors such as Sp-1, Elk-1, and GATA. Our studies, however, suggest that these are not involved in modulation of eNOS promoter activity in response to shear but strongly support a role of NFB in transactivation of the eNOS reporter in response to shear stress. Studies using cold competitor oligonucleotides, antibodies against p50 and p65, and pharmacological inhibition with PPM-18 (27) indicated that the proteins responsible for the complexes formed in the electrophoretic mobility shift assays were p50 and p65. Studies with the eNOS promoter further supported a role of NFB as co-transfection with the dominant negative IB prevented transactivation of eNOS by shear stress.
Analysis of the 25-base pair segment indicated that two potential binding sites for NFB are present within this region, both present on the antisense strand. Interestingly, in electrophoretic mobility assays, both of these regions were capable of binding p50 and p65, as mutation of either alone did not completely abolish binding while mutation of both prevented complex formation. In studies of the full-length eNOS promoter, however, only the GAGACC sequence seemed important, as mutation of the potential NFB consensus site had no effect on transactivation by shear stress, while mutation of the GAGACC site completely prevented the effect of shear. These seemingly disparate results might be due to differences in secondary structure between the full-length promoter and the simple 37-base pair oligonucleotide used in the EMSAs. Our data further provide some insight into how shear activates NFB and eNOS transcription. We have previously found that eNOS transcription is signaled by a pathway involving cSrc, Ras, Raf, MEK1/2, and ERK1/2. In keeping with these findings, pharmacological inhibition of either cSrc (with PP1) or MEK1/2 (with PD98059 or UO126) prevented activation of the eNOS promoter. Likewise, phosphorylation of IK␣ was prevented by PP1 and PD98059. These data indicate that cSrc and ERK1/2 are upstream of IK␣ activation in response to shear stress. This finding is similar to the situation described recently by Dhawan and Richmond (28) who demonstrated that MEK1/2 and ERK1/2 activation are upstream of NFB activation in melanoma cells.
Historically, NFB activation has been implicated in regulation of several proatherogenic related genes including VCAM-1, MCP-1, and PDGF (29). Indeed, in lesion prone areas of the circulation there is increased expression of p65 as well as IK␣ and IK␤ (30). Interestingly, NO ⅐ has been shown to inhibit NFB activation via at least two mechanisms. One involves increased expression of the inhibitor subunit IB (31), while a second involves nitrosylation of p50, which diminishes its nuclear translocation (32). One can therefore envision that this represents a classical negative feedback loop in which shear stress activates NFB, which in turn increases eNOS expression. The increase in eNOS expression would allow for an increase NO ⅐ production, which would ultimately inhibit NFB activation. This is potentially important, because several common cardiovascular diseases, such as hypercholesterolemia, hypertension, and diabetes, are associated with oxidative inactivation of NO⅐ or oxidation of critical NOS co-factors. In these situations the reduction in the ambient levels of biologically active NO ⅐ shear stress could lead to an unmitigated activation of NFB. Such an interruption of a normal negative feedback situation might dramatically predispose to endothelial inflammation and atherosclerosis.