Role of Endothelial Nitric Oxide Synthase in the Regulationof SREBP Activation by Oxidized Phospholipids

Nima M. Gharavi, Nancy A. Baker, Kevin P. Mouillesseaux, Winnie Yeung, Henry M. Honda,Xavier Hsieh, Michael Yeh, Eric J. Smart, Judith A. Berliner

Abstract—Oxidized-1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), found in atherosclerotic le-sions and other sites of chronic inflammation, activates endothelial cells (EC) to synthesize chemotactic factors, suchas interleukin (IL)-8. Previously, we demonstrated that the sustained induction of IL-8 transcription by Ox-PAPC wasmediated through the activation of sterol regulatory element–binding protein (SREBP). We now present evidence forthe role of endothelial nitric oxide synthase (eNOS) in the activation of SREBP by Ox-PAPC. Ox-PAPC treatment ofEC induced a dose- and time-dependent activation of eNOS, as measured by phosphorylation of serine 1177,dephosphorylation of threonine 495, and the conversion of L-arginine to L-citrulline. Activation of eNOS by Ox-PAPCwas regulated through a phosphatidylinositol-3-kinase/Akt-mediated mechanism. These studies also demonstrated thatpretreatment of EC with NOS inhibitor, N�-nitro-L-arginine-methyl ester (L-NAME), significantly inhibited Ox-PAPC–induced IL-8 synthesis. Because SREBP activation had been previously shown to regulate IL-8 transcription byOx-PAPC, we examined the effects of L-NAME on Ox-PAPC–induced SREBP activation. Our data demonstrated thatOx-PAPC–induced SREBP activation and expression of SREBP target genes were significantly reduced by pretreatmentwith L-NAME. Interestingly, treatment of EC with NO donor, S-nitroso-N-acetylpenicillamine, did not activate SREBP,suggesting that NO alone was not sufficient for SREBP activation. Rather, our findings indicated that superoxide (O2

��),in combination with NO, regulated SREBP activation by Ox-PAPC. We found that Ox-PAPC treatment generated O2

��

through an eNOS-mediated mechanism and that mercaptoethylguanidine, a peroxynitrite scavenger, reduced SREBPactivation by Ox-PAPC. Taken together, these findings propose a novel role for eNOS in the activation of SREBP andSREBP-mediated inflammatory processes. (Circ Res. 2006;98:768-776.)

Key Words: oxidized phospholipids � interleukin-8 � endothelium � nitric oxide � cholesterol

Atherosclerosis is a chronic inflammatory condition char-acterized by lipid accumulation and monocyte entry into

the subendothelium. Previous work by our laboratory hasdemonstrated that phospholipid oxidation products of1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine(PAPC) accumulate in atherosclerotic lesions and other sitesof chronic inflammation1 and activate endothelial cells (EC),leading to enhanced monocyte/EC interactions.2,3 OxidizedPAPC (Ox-PAPC) and its component phospholipids,1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoryl-choline (POVPC) and 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC), also increase transcrip-tion of the atherogenic chemotactic factor, interleukin (IL)-8,in a sustained manner through an nuclear factor (NF)-�B–independent pathway.4 In a recent article, we demonstratedthat the sustained induction of IL-8 transcription by Ox-PAPC and its component phospholipids was mediatedthrough a novel pathway, involving the activation of sterolregulatory element–binding protein (SREBP).5

SREBPs are a family of transcription factors that regulatecellular cholesterol homeostasis.6 These proteins are locatedin the endoplasmic reticulum in their inactive state, which onsterol depletion become activated and enzymatically cleavedinto smaller forms that translocate into the nucleus andincrease transcription of genes regulating cholesterol andfatty acid metabolism.7 Previously, we demonstrated thatOx-PAPC treatment of EC depleted caveolar cholesterol andactivated both SREBP 1 and 2 within 30 minutes.5 SREBPactivation by Ox-PAPC was maintained for at least 8 hours,and Ox-PAPC treatment also increased binding of SREBP toa novel SRE sequence in the IL-8 promoter.5 The currentstudies address the mechanism by which Ox-PAPC regulatesSREBP activation. We provide evidence supporting a role forendothelial nitric oxide synthase (eNOS) in the regulation ofSREBP activation and IL-8 transcription by Ox-PAPC.

eNOS is a member of the family of nitric oxide synthases(NOSs), which are responsible for the synthesis of NO fromthe metabolism of L-arginine to L-citrulline. In the normal

Original received July 27, 2005; revision received February 6, 2006; accepted February 15, 2006.From the Division of Cardiology, Department of Medicine (N.M.G., K.P.M., W.Y., H.M.H., X.H., M.Y., J.A.B.); Department of Pathology (N.M.G.,

N.A.B., M.Y., J.A.B.); and Department of Physiological Science (N.A.B.), University of California, Los Angeles; and Department of Pediatrics (E.J.S.),University of Kentucky, Lexington.

Correspondence to Nima M. Gharavi, 13-239 CHS-Pathology, 650 Charles E. Young Dr S, Los Angeles, CA 90095. E-mail ngharavi@ucla.edu© 2006 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000215343.89308.93

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Molecular Medicine

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endothelium, eNOS activity can be regulated by chemicalagonists, including bradykinin8 and acetylcholine,9 and phys-ical stimuli, such as shear stress.10 The subcellular localiza-tion of eNOS also plays an important role in its activity.Recent findings have demonstrated eNOS localization in theGolgi apparatus11 and in caveolar membranes12; the latterappears to be an important compartment regulating eNOS-mediated NO production. Furthermore, phosphorylation ofeNOS at serine residue 1177 (S1177) and dephosphorylationat threonine residue 495 (T495) have been associated withincreased eNOS activity.13 Under certain pathological condi-tions, eNOS can also independently generate superoxide(O2

��).14 In the presence of O2�� generated by eNOS or other

enzymes, NO can react to form peroxynitrite (ONOO�).ONOO� is a potent oxidizing agent that has been implicated toplay a role in several inflammatory conditions, includingatherosclerosis.15

In the current studies, we demonstrate that Ox-PAPCtreatment of EC activates eNOS in a dose- and time-dependent manner, while also inducing a concomitant in-crease in O2

�� production. Furthermore, our findings demon-strate for the first time that eNOS activation regulates theactivation of SREBP and the induction of SREBP targetgenes by oxidized phospholipids.

Materials and Methods

Material and ReagentsMedium 199 for Human Aortic EC (HAEC) and DMEM medium forbovine aortic EC (BAEC) were purchased from Irvine Scientific.FBS was obtained from Hyclone. Oxidized phospholipids wereprepared as previously described.2 Rabbit polyclonal antibodiesagainst eNOS S1177, eNOS T495, Akt S473, total Akt, and histoneH2A were purchased from Cell Signaling. Rabbit polyclonal anti-body against total eNOS, SREBP-1, and SREBP-2 were purchasedfrom Santa Cruz Biotechnology. N�-Nitro-L-arginine-methyl ester(L-NAME), N�-nitro-D-arginine methyl ester (D-NAME), methyl-�-cyclodextrin (M�CD), mercaptoethylguanidine sulfate (MEG), andsuperoxide dismutase (SOD), were purchased from Sigma.S-Nitroso-N-acetylpenicillamine (SNAP), and N-acetyl-Leu-Leu-norleucinal (ALLN) were purchased from Calbiochem. 2�,7�-Dichlo-rodihydrofluorescein diacetate bis-acetomethyl ester (DCFH-DA)was purchased from Molecular Probes.

Cell Culture and TreatmentHAEC (passages 4 to 8) were isolated and cultured as previouslydescribed.16 BAEC (passages 5 to 17) were purchased from VECTechnologies and cultured as previously described.5 Treatment withlipids and other activating agents was performed in media containing1% to 2% FBS. For all inhibitor studies, cells were pretreated withinhibitor and subsequently cotreated with lipid and inhibitor for theindicated times.

Enzyme-Linked Immunosorbent AssaySecreted IL-8 levels in cell media were measured using the IL-8ELISA kit (R&D Systems) according to the protocol of themanufacturer.4

Western Blot AnalysisWestern blot analyses were performed as previously described.5

Blots were developed using enhanced chemiluminescence (ECL)plus (Amersham International) and levels were detected using theBio-Rad VersaDoc Imaging System (Model 5000).

Real-Time RT-PCR Using SYBRRT-PCR was performed as previously described.5 Primer sequenceswere: IL-8, 5�-tctcttggcagccttcctgatttc-3�, 5�-aactgtcgccaccagaa-agctgag-3�; LDLR, 5�-cgtgcttgtctgtcacctgcaaat-3�, 5�-agaactga-ggaatgcagcggttga-3�; Insig-1, 5�-ggacgacagttagctatgggtgtt-3�, 5�-gagtcatttgtacagtcagcccga-3�; and GAPDH, 5�-cattgccctcaacgac-cactttgt-3�, 5�-accaccctgttgctgtagccaaat-3�.

Measurement of eNOS ActivationNO synthesis was determined in intact cells as previously de-scribed.17 Cells were incubated with 0.75 mCi/mL 3H-L-arginine for30 minutes and then treated with lipids at the indicated concentra-tions for 30 minutes. To ensure that treatment did not affect theloading of the cells with 3H-L-arginine, the amount of 3H-L-arginineassociated with the cells was determined. The cells contained212 957�9128 DPM/well of 3H-L-arginine independent of the treat-ment and time.

Measurement of Intracellular Oxidative StressOxidation of DCFH, a nonfluorescent probe, to a green fluorescentproduct, 2�,7�-dichlorofluorescein (DCF) was used to monitor intra-cellular oxidative stress. Following treatment, HAEC were rinsedwith 1xPBS and DCFH-DA was added at a final concentration of10 �mol/L and incubated for up to 30 minutes. Cells were rinsedagain with 1� PBS and maintained in culture medium. Fluorescencewas monitored using a Nikon fluorescence microscope (excitation,488 nm; emission, 610 nm) equipped with a FITC filter.

Measurement of Extracellular O2�� Production

O2�� production was measured using the cytochrome C reduction

assay as previously described.18 HAEC were exposed to lipids for upto 4 hours using media containing 100 �mol/L acetylated-ferricytrochrome C (Sigma). Supernatants were taken for absorbancemeasurements at 550 nm (Beckman 640 spectrophotometer). Thespecificity of reduction by O2

�� was established by comparingreduction rates in the presence and absence of SOD (60 �g/mL). Thecorrected rates of O2

�� release were calculated from SOD-inhibitableferricytochrome C absorbance at 550 nm using the molar extinctioncoefficient (��21 000 mol/L�1 cm�1).

Statistical AnalysisData for Western blots and RT-PCR were normalized to total proteinor housekeeping genes, and fold induction was calculated withrespect to normalized control, set at the baseline of 1. Computer-assisted statistical analyses were performed using Microsoft Excel2000. All probability values were calculated using the Student t test.A value of P�0.05 was considered significant.

ResultsOx-PAPC and Its Component Phospholipid,POVPC, Activate eNOSActivation of eNOS correlates positively with phosphoryla-tion at S1177 and dephosphorylation at T495.13 To determinewhether Ox-PAPC treatment of HAEC alters eNOS phos-phorylation, phospho-specific antibodies to eNOS residuesS1177 and T495 were used. Ox-PAPC treatment increasedS1177 phosphorylation within 10 minutes, and this phosphor-ylation was sustained for up to 4 hours (Figure 1A, top). Totallevels of eNOS, as measured by the eNOS antibody at all timepoints following Ox-PAPC treatment, remained unchanged(Figure 1A, middle). Using densitometry to normalize eNOSS1177 phosphorylation to total eNOS levels, Ox-PAPCtreatment was found to induce a 4-fold increase in eNOSphosphorylation at the 1-hour peak (Figure 1A, bottom). Ourdata demonstrated that phosphorylation of eNOS S1177 byOx-PAPC was also dose dependent (Figure 1B). Further-

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more, treatment of HAEC with POVPC induced eNOS S1177phosphorylation, whereas unoxidized PAPC had no effect(Figure 1C). In contrast, phosphorylation of eNOS at T495was reduced following treatment with Ox-PAPC; this effectwas observed within 10 minutes and was sustained for at least4 hours (Figure 1D).

An important measure of eNOS activity is the conversionof L-arginine to L-citrulline. Our data demonstrated thattreatment of HAEC with Ox-PAPC (25 �g/mL) for 30minutes induced a significant increase in L-arginine toL-citrulline conversion (Figure 1E). We also observed asignificant increase in L-arginine to L-citrulline conversion inBAEC, following treatment with both Ox-PAPC and POVPC(Figure 1F).

Ox-PAPC Treatment Activates eNOS Through aPhosphatidylinositol 3-Kinase/Akt-MediatedMechanism, Independent of c-Src Kinase and PKAPhosphorylation of eNOS S1177 can be mediated through aphosphatidylinositol 3-kinase (PI3K) pathway,19 in whichPI3K phosphorylates and activates Akt kinase, which in turnphosphorylates eNOS.10 To determine the role of the PI3K/Akt pathway in eNOS activation by Ox-PAPC, we firstexamined the activation state of Akt following Ox-PAPC

treatment. Activation of Akt correlates positively with phos-phorylation at S473.20 Using the phospho-specific antibody toAkt residue S473, we demonstrated that Ox-PAPC treatmentof HAEC activated Akt within 5 minutes and that thisactivation was maintained for at least 1 hour (Figure 2A, top).Total levels of Akt, as measured by the total Akt antibody atall time points following Ox-PAPC treatment, remainedunchanged (Figure 2A, middle). Following normalization tototal Akt levels, Ox-PAPC treatment was found to induce a5-fold increase in Akt phosphorylation at the 1-hour peak(Figure 2A, bottom).

We next examined the role of PI3K/Akt in eNOS activa-tion by Ox-PAPC, using the specific PI3K/Akt inhibitorLY294002.21 Pretreatment of HAEC with LY294002 signif-icantly reduced Ox-PAPC–induced eNOS S1177 phosphory-lation (Figure 2B, top), without having any significant affectson total eNOS levels (Figure 2B, middle). Following normal-ization, LY294002 pretreatment was found to cause a nearcomplete inhibition of Ox-PAPC–induced eNOS phosphory-lation (Figure 2B, bottom).

In addition to Akt, protein kinases such as c-Src kinase22

and protein kinase A (PKA)23 have also been demonstrated toregulate eNOS S1177 phosphorylation. Furthermore, wepreviously reported that Ox-PAPC treatment of HAEC acti-

Figure 1. Ox-PAPC treatment of EC acti-vates eNOS. HAEC were either untreated(control [C]) or treated with Ox-PAPC(Ox, 50 �g/mL) for the indicated times(A), at increasing concentrations for 1hour (B), or with PAPC (50 �g/mL),Ox-PAPC (Ox, 50 �g/mL), and POVPC(20 �g/mL) for 1 hour (C). Cell lysateswere collected, and Western blot analy-sis was performed analyzing eNOSS1177 phosphorylation (top). D, HAECwere either untreated (control [C]) ortreated with Ox-PAPC (Ox, 50 �g/mL) forthe indicated times, and phosphorylationof eNOS at T495 was analyzed by West-ern blot analysis (top). For A through D,total eNOS levels were also examined byWestern blot analysis (middle), and den-sitometry was performed to normalizephospho-eNOS levels to total eNOS lev-els (bottom). eNOS activation, as mea-sured by the conversion of 3H-arginine to3H-citrulline, was also analyzed in HAECuntreated (Control) or treated withOx-PAPC (25 �g/mL) (E) and in BAECuntreated (Control) or treated withOx-PAPC (20 �g/mL) or POVPC (20�g/mL) (F) for 30 minutes. For E and F,data represent mean�SD (n�3). *P�0.05and **P�0.01, compared with untreatedcells. All experiments were repeated atleast 3 times.

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vates both c-Src kinase24 and PKA.25 These studies, however,demonstrated that neither PP2 (c-Src inhibitor) nor RP-cAMP(PKA inhibitor) had any significant effects on Ox-PAPC–induced eNOS S1177 phosphorylation (Figure 2C).

Role of eNOS Activation in Ox-PAPC–InducedIL-8 SynthesisTo examine the role of eNOS activation in the induction ofIL-8 expression by Ox-PAPC, we used the NOS chemicalinhibitor L-NAME and its inactive D analog (D-NAME). Asa competitive inhibitor for L-arginine binding to the NOSactive site, L-NAME serves as a general inhibitor for all NOSfamily members.26 Through PCR analysis, however, we havedetermined that HAEC express neither inducible NOS norneuronal NOS (data not shown). Our data demonstrated thatpretreatment of HAEC with L-NAME reduced Ox-PAPC–induced IL-8 protein synthesis in a dose-dependent manner,whereas pretreatment of HAEC with D-NAME at similarconcentrations had no effect (Figure 3A). Pretreatment ofHAEC with L-NAME also significantly reduced the levels ofIL-8 mRNA induced by Ox-PAPC (Figure 3B). Furthermore,the inhibitory effects of L-NAME on IL-8 synthesis werespecific to Ox-PAPC, as L-NAME had no significant effectson LPS-induced IL-8 synthesis (data not shown).

To determine whether NO itself could induce IL-8 synthe-sis in HAEC, we used the NO donor (�)-SNAP. Thiscompound emits NO immediately after solubilization inaqueous buffers. Treatment of HAEC with SNAP induced adose-dependent increase in IL-8 protein (Figure 3C) andmRNA (Figure 3D) synthesis.

Role of eNOS in Ox-PAPC–InducedSREBP ActivationPreviously, we demonstrated that the sustained induction ofIL-8 by Ox-PAPC was mediated through the activation ofSREBP.5 We, therefore, examined the role of eNOS inOx-PAPC–induced SREBP activation. Activation of SREBPis mediated by a posttranslational cleavage in which theimmature (125-kDa) protein is enzymatically truncated into asmaller mature isoform that enters the nucleus. SREBPactivation, therefore, can be measured as a decrease in thelevels of the immature isoform, as well as an increase in thenuclear translocation of the mature isoform. Our findingsdemonstrated that pretreatment of HAEC with 2.0 mmol/LL-NAME for 1 to 2 hours significantly inhibited the activa-tion of SREBP in the cytoplasm by Ox-PAPC but not by thecholesterol-sequestering agent M�CD (Figure 4A). Similarresults were observed in HAEC following pretreatment with1.0 mmol/L L-NAME for 8 hours and 0.25 mmol/L L-NAMEfor 16 hours (data not shown). Furthermore, we observed thatpretreatment of HAEC with 2.0 mmol/L L-NAME for 1 to 2hours significantly reduced the nuclear translocation ofSREBP induced by Ox-PAPC but not by M�CD (Figure 4B).

To confirm the effects of L-NAME on Ox-PAPC–inducedSREBP activation, we next examined the effects of L-NAMEon the induction of SREBP target genes low-density lipopro-tein receptor (LDLR) and Insig-1 by Ox-PAPC. WhereasOx-PAPC treatment of HAEC induced an approximate 12-fold increase in LDLR mRNA levels in the absence ofinhibitor, pretreatment of HAEC with L-NAME, but not

Figure 2. Ox-PAPC–induced eNOS acti-vation is mediated through a PI3K/Aktmechanism, independent of c-Src kinaseand PKA. A, Cell lysates from HAEC thatwere either untreated (control [C]) ortreated with Ox-PAPC (Ox, 50 �g/mL) atthe indicated times were examined byWestern blot analysis using thephospho-specific antibody to Akt (S473)(top) and phosphorylation state-independent antibody to Akt (middle).Densitometry was performed to normal-ize phospho-Akt levels to total Akt levels(bottom). HAEC were pretreated for 1hour with vehicle or LY294002 (LY)(10 �mol/L) (B) or with vehicle, PP2(5 �mol/L), or RP-cAMP (10 �mol/L) (C)and stimulated for 1 hour with mediaalone (control [C]) or Ox-PAPC (Ox, 50�g/mL) as indicated. Cell lysates werecollected and eNOS activation wasexamined by Western blot analysis usingphospho-specific antibody to eNOSS1177 (top) and phosphorylation state-independent antibody to eNOS (middle).Densitometry was performed to normal-ize phospho-eNOS levels to total eNOSlevels (bottom). All experiments wererepeated at least 3 times.

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D-NAME, significantly reduced Ox-PAPC–induced LDLRmRNA levels (Figure 4C). L-NAME pretreatment did not,however, reduce the expression of LDLR mRNA induced byM�CD (data not shown). Furthermore, L-NAME pretreat-ment had similar effects on the induction of Insig-1 byOx-PAPC (Figure 4D).

To determine whether NO itself could activate SREBP, wetreated HAEC with SNAP and examined the expression ofSREBP target genes. Surprisingly, treatment of HAEC withSNAP induced a dose-dependent decrease in LDLR mRNAlevels (Figure 4E).

Role of eNOS-Induced O2�� Production in SREBP

Activation by Ox-PAPCWe next examined whether Ox-PAPC treatment of HAECwould induce oxidative stress and increase the levels ofreactive oxygen species (ROS). Our data demonstrated thattreatment of HAEC with Ox-PAPC induced a significantincrease in DCF fluorescence, a marker of intracellularoxidative stress, within 5 minutes, that was sustained for atleast 30 minutes following treatment (Figure 5A); unoxidizedPAPC had no significant effect on DCF fluorescence. Fur-thermore, treatment of HAEC with Ox-PAPC and its componentPEIPC significantly increased the rate of O2

�� production, ascompared with treatment with unoxidized PAPC (Figure 5B).

Because eNOS may generate O2�� under conditions in

which is activity state is “uncoupled,” we examined the effect

of L-NAME on Ox-PAPC–induced O2�� production. Our

findings demonstrated that pretreatment of HAEC withL-NAME significantly reduced the rate of O2

�� productioninduced by Ox-PAPC (Figure 5C). These findings suggestedthat O2

�� production, in combination with NO, might regulatethe activation of SREBP by Ox-PAPC. In the presence of O2

��,NO will react to form ONOO�, a highly reactive oxidizing agentwith potent inflammatory properties.15 We, therefore, examinedthe role of ONOO� in Ox-PAPC–induced SREBP activationusing the ONOO� scavenger, mercaptoethylguanidine (MEG).Our data demonstrated that pretreatment of EC with MEGsignificantly inhibited the activation of SREBP (Figure 5D) andthe induction of LDLR transcription by Ox-PAPC (Figure 5E).

DiscussionIn these studies, we have reported the following findings. (1)Ox-PAPC treatment of EC activates eNOS in a dose- andtime-dependent manner. (2) eNOS activation by Ox-PAPC ismediated through a PI3K/Akt-dependent mechanism, andOx-PAPC treatment activates Akt within 5 minutes. (3)Furthermore, eNOS activation plays a role in mediating theactivation of SREBP and the induction of IL-8 by Ox-PAPC.(4) Finally, we present evidence that eNOS activation gener-ates O2

�� and that O2��, in combination with NO, is responsi-

ble for the activation of SREBP by Ox-PAPC.

Figure 3. eNOS activation and NO regulate the induction of IL-8 by Ox-PAPC. HAEC were pretreated for 1 hour with the indicated con-centrations of L-NAME or D-NAME and then stimulated with control (C) media or Ox-PAPC (Ox, 50 �g/mL) for 4 hours. IL-8 proteinsynthesis, as measured by ELISA (A), and IL-8 mRNA synthesis, as measured by quantitative RT-PCR (B), were analyzed. HAEC weretreated with the indicated doses of SNAP for 4 hours and IL-8 protein synthesis, as measured by ELISA (C), and IL-8 mRNA synthesis,as measured by quantitative RT-PCR (D), were analyzed. Data represent mean�SD (n�3). *P�0.05 and **P�0.01, compared withOx-PAPC treated cells in the absence of inhibitor in A and B and compared with untreated cells in C and D. All experiments wererepeated at least 3 times.

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Whereas previous findings have demonstrated that oxi-dized lipids inhibit the activation of eNOS by agonists, suchas acetylcholine,27 our studies have examined the effect ofOx-PAPC treatment on the basal activity state of eNOS. Wehave demonstrated that Ox-PAPC treatment activates eNOSdirectly by increasing the conversion of L-arginine toL-citrulline (Figure 1). In addition, whereas activation ofeNOS by some agonists requires an increase in intracellularcalcium,28 we have obtained evidence that Ox-PAPC treat-ment of HAEC does not increase intracellular calcium levels(data not shown). Rather, activation of eNOS by Ox-PAPC ismediated through phosphorylation of S1177 (Figure 1),which has been reported to regulate eNOS activation in theabsence of detectable calcium fluxes.29 S1177 phosphoryla-tion by Ox-PAPC is mediated through the activation ofPI3K/Akt (Figure 2). Interestingly, we previously demon-strated an important role for PI3K activation in Ox-PAPC–

induced monocyte binding to EC.25 Taken together, thesefindings suggest an important role for PI3K in the initiation ofinflammatory signaling events mediated by Ox-PAPC.

Importantly, these studies have established a novel role foreNOS in the activation of SREBP by Ox-PAPC. In a recentarticle, we demonstrated that treatment of EC with Ox-PAPCactivates SREBP and increases transcription of SREBP targetgenes.5 We now demonstrate that pretreatment of EC withL-NAME significantly reduces the activation and nucleartranslocation of SREBP, as well as the expression of SREBPtarget genes LDLR and Insig-1 induced by Ox-PAPC (Figure4). Treatment of EC with NO alone (in response to SNAP),however, did not activate SREBP (data not shown) and, infact, inhibited the expression of SREBP target genes (Figure4). Rather, our findings indicated that regulation of SREBPactivation by eNOS was mediated through eNOS-induced O2

��

production (Figure 5). Because Ox-PAPC treatment induced

Figure 4. eNOS activation regulates Ox-PAPC–induced SREBPactivation. HAEC were pretreated for 2 hours with or withoutL-NAME (LN) (2.0 mmol/L) as indicated and then stimulated for 4hours with media alone (control [C]), Ox-PAPC (Ox, 50 �g/mL), orM�CD (M�, 0.3% m/v). Western blot analysis was performed forSREBP activation as measured by: loss of the immature (125-kDa)molecule in the cytoplasm (A, top), normalized to GAPDH (A, bot-tom); and nuclear translocation of the mature (68-kDa) molecule (B,top), normalized to histone H2A (B, bottom). C and D, HAEC werepretreated for 1 hour with or without L-NAME (2.0 mmol/L) orD-NAME (2.0 mmol/L) as indicated and then stimulated for 4 hourswith media alone (control [C]) or Ox-PAPC (Ox, 50 �g/mL). Quanti-tative RT-PCR was performed measuring the relative mRNA levelsof SREBP target genes LDLR (C) and Insig-1 (D). E, HAEC weretreated with the indicated doses of SNAP for 4 hours and LDLR

mRNA synthesis was analyzed using quantitative RT-PCR. For C, D, and E, data represent mean�SD (n�3). *P�0.05 and **P�0.01, com-pared with Ox-PAPC treated cells in the absence of inhibitor in C and D and compared with untreated cells in E. All experiments wererepeated at least 4 times.

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phosphorylation of eNOS at S1177 and dephosphorylation atT495, as well as an increase in L-NAME-inhibitable O2

��

production, we propose that Ox-PAPC treatment concurrentlyactivates and “uncouples” eNOS. Sessa and colleagues havereported that dephosphorylation at T495 may be an intrinsicswitch mechanism controlling the production of O2

��, rather thanNO, by eNOS.30 Their studies demonstrated maximal O2

��

production in cells where S1177 was constitutively phosphory-lated and T495 was dephosphorylated.30 Previously, it has also

been reported that eNOS uncoupling may occur during states inwhich eNOS is displaced from caveolae.14 We recently reportedthat Ox-PAPC treatment of EC disrupts caveolae and redistrib-utes caveolin-1, a major structural protein present in caveolae.5

O2�� can readily combine with NO to form ONOO� and it may

also catalyze the formation of other hydroperoxides.31 Thecurrent studies have demonstrated that MEG, a scavenger ofONOO�, inhibits the activation of SREBP by Ox-PAPC (Figure5). Taken together, these findings suggest that both NO and O2

��

Figure 5. ROS regulate Ox-PAPC–induced SREBP activation. A, HAEC were treated with PAPC (50 �g/mL, top) or Ox-PAPC (50�g/mL, bottom), and oxidative stress was measured at the indicated times using the DCF assay. B, HAEC were treated with PAPC (50�g/mL), Ox-PAPC (50 �g/mL), and PEIPC (2 �g/mL), and O2

�� production was analyzed using the cytochrome C reduction assay. C,HAEC were pretreated with vehicle or L-NAME (2.0 mmol/L) for 1 hour and then stimulated with PAPC (50 �g/mL) or Ox-PAPC (50�g/mL) for 4 hours. O2

�� production was assessed using the cytochrome C reduction assay. D, HAEC were pretreated for 1 hour withvehicle or MEG (100 �mol/L) and subsequently stimulated for 4 hours with media alone (control [C]) or Ox-PAPC (Ox, 50 �g/mL), andWestern blot analysis was performed measuring SREBP activation. E, Quantitative RT-PCR was performed measuring LDLR mRNA lev-els. For B, C, and E, data represent mean�SD (n�3). *P�0.05 and **P�0.01, compared with untreated cells in B and compared withOx-PAPC–treated cells in the absence of inhibitor in C and E. All experiments were repeated at least 3 times.

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production play an important role in regulating SREBP activa-tion by Ox-PAPC.

We propose the following potential mechanisms for therole of eNOS in SREBP activation by Ox-PAPC. First, theformation of ROS generated by eNOS, including O2

�� and/orONOO�, may alter caveolar membranes and regulate thedepletion of cellular cholesterol induced by Ox-PAPC. Radiet al have demonstrated that ROS can alter the integrity of cellmembranes via lipid peroxidation.31 Peterson et al havedemonstrated that ROS can decrease caveolae number andoxidize cholesterol in caveolar membranes, which may resultin cholesterol efflux from caveolae.32 Cholesterol oxidationhas also been shown to redistribute caveolin from caveolarcompartments to internal stores.33 Second, ROS may modifyenzymes that regulate SREBP activation, such as SREBPcleavage-activating protein (SCAP) and/or the S1/S2 pro-teases. Several investigators have demonstrated that ROS,including ONOO�, can regulate the activation of proteases34

via nitrosylation of cysteine and/or tyrosine residues.35 One orboth of the mechanisms may be important in mediatingSREBP activation by Ox-PAPC.

In the current studies, we have also established a role foreNOS activation in the induction of IL-8 transcription byOx-PAPC. We have demonstrated that pretreatment of ECwith L-NAME inhibits Ox-PAPC–induced IL-8 expression(Figure 3). Although the effect of L-NAME on IL-8 expres-sion may be attributable to the inhibitory action of L-NAMEon Ox-PAPC–induced SREBP activation, it is important tonote that eNOS can induce IL-8 expression via SREBP-independent mechanisms. Pae et al have demonstrated thattreatment of EC with NO donor SNAP induces IL-8 synthesisvia expression of vascular endothelial growth factor(VEGF).36 Furthermore, we have presented evidence thattreatment with SNAP induces IL-8 transcription in HAEC(Figure 3). Therefore, we hypothesize that eNOS activationby Ox-PAPC may regulate IL-8 transcription throughSREBP-dependent and SREBP-independent mechanisms.Because we previously reported a role for SREBP in thesustained induction of IL-8 transcription by Ox-PAPC, wepropose that eNOS also regulates the sustained induction ofIL-8 transcription by Ox-PAPC. This is in contrast with thec-Src/signal transducer and activator of transcription (STAT)3 pathway, which we previously reported to regulate the earlyinduction of IL-8 transcription by Ox-PAPC.24 In summary,our current model for the sustained activation of SREBP andinduction of IL-8 transcription by Ox-PAPC suggests thatOx-PAPC treatment activates Akt, which promotes the acti-vation of eNOS and the production of NO and O2

��. Subse-quently, O2

�� and/or ONOO�, through a mechanism mediatedby either cholesterol oxidation and depletion or enzymaticactivation, activate SREBP, which translocates to the nucleus,binds to the SRE in the IL-8 promoter, and increasestranscription of IL-8 (Figure 6).

Atherosclerosis is a chronic inflammatory condition char-acterized by endothelial dysfunction and lipid accumulationduring its early stages.37 Endothelial dysfunction is 1 of theearliest markers of atherosclerosis, detected even beforeangiographic and/or ultrasonic evidence of plaque forma-tion.38,39 Although others have clearly demonstrated that lipid

accumulation and subsequent oxidation can cause endothelialdysfunction,40 we present evidence supporting the hypothesisthat endothelial dysfunction can also promote lipid accumu-lation, thus perpetuating the cycle. In summary, these studiespresent evidence that dysregulation of NO synthesis, espe-cially in the presence of oxidative stress, can lead to SREBPactivation and expression of SREBP target genes, such asLDLR, which may increase accumulation of lipids in thesubendothelial space and aggravate the ensuing inflammatoryresponse.

AcknowledgmentsThis research was supported by NIH grants HL30568 (to J.A.B.),HL064731 (to J.A.B.), HL62844 (to E.J.S.), HL54056 (to E.J.S.),and GM008042 (NMG); and American Heart Association Grant0415020Y (to N.M.G.).

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Xavier Hsieh, Michael Yeh, Eric J. Smart and Judith A. BerlinerNima M. Gharavi, Nancy A. Baker, Kevin P. Mouillesseaux, Winnie Yeung, Henry M. Honda,

Oxidized PhospholipidsRole of Endothelial Nitric Oxide Synthase in the Regulation of SREBP Activation by

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