TNFα and oxLDL Reduce Protein S-Nitrosylation in Endothelial Cells

Nitric oxide (NO) plays an important role in the regulation of the functional integrity of the endothelium. The intracellular reaction of NO with reactive cysteine groups leads to the formation of S-nitrosothiols. To investigate the regulation of S-nitrosothiols in endothelial cells, we first analyzed the composition of the S-nitrosylated molecules in endothelial cells. Gel filtration revealed that more than 95% of the detected S-nitrosothiols had a molecular mass of more than 5000 Da. Moreover, inhibition of de novosynthesis of glutathione using N-butyl-sulfoximine did not diminish the overall cellular S-NO content suggesting that S-nitrosylated glutathione quantitatively plays only a minor role in endothelial cells. Having demonstrated that most of the S-nitrosothiols are proteins, we determined the regulation of the S-nitrosylation by pro-inflammatory and pro-atherogenic factors, such as TNFα and mildly oxidized low density lipoprotein (oxLDL). TNFα and oxLDL induced denitrosylation of various proteins as assessed by Saville-Griess assay, by immunostaining with an anti-S-nitrosocysteine antibody, and by a Western blot approach. Furthermore, the caspase-3 p17 subunit, which has previously been shown to be S-nitrosylated and thereby inhibited, was denitrosylated by TNFα treatment suggesting thatS-nitrosylation and denitrosylation are important regulatory mechanisms in endothelial cells contributing to the integrity of the endothelial cell monolayer.

Nitric oxide (NO) plays an important role in the regulation of the functional integrity of the endothelium. The intracellular reaction of NO with reactive cysteine groups leads to the formation of S-nitrosothiols. To investigate the regulation of S-nitrosothiols in endothelial cells, we first analyzed the composition of the S-nitrosylated molecules in endothelial cells. Gel filtration revealed that more than 95% of the detected S-nitrosothiols had a molecular mass of more than 5000 Da. Moreover, inhibition of de novo synthesis of glutathione using N-butyl-sulfoximine did not diminish the overall cellular S-NO content suggesting that S-nitrosylated glutathione quantitatively plays only a minor role in endothelial cells. Having demonstrated that most of the S-nitrosothiols are proteins, we determined the regulation of the S-nitrosylation by pro-inflammatory and proatherogenic factors, such as TNF␣ and mildly oxidized low density lipoprotein (oxLDL). TNF␣ and oxLDL induced denitrosylation of various proteins as assessed by Saville-Griess assay, by immunostaining with an anti-Snitrosocysteine antibody, and by a Western blot approach. Furthermore, the caspase-3 p17 subunit, which has previously been shown to be S-nitrosylated and thereby inhibited, was denitrosylated by TNF␣ treatment suggesting that S-nitrosylation and denitrosylation are important regulatory mechanisms in endothelial cells contributing to the integrity of the endothelial cell monolayer.
Nitric oxide (NO) 1 plays an important role in the regulation of the functional integrity of the endothelium, which acts as a barrier between the circulating blood and the underlying tissue (1,2). In endothelial cells, nitric oxide is continuously synthesized by the endothelial NO synthase. NO is implicated in several physiological functions within the endothelium. Thus, NO regulates the vascular tone, provides anti-thrombotic and anti-inflammatory activity, and inhibits endothelial cell apoptosis (2)(3)(4).
NO can act via cGMP-dependent and independent pathways. During the last years, increasing evidence suggests that cGMPindependent processes are importantly contributing to the regulation of cellular signaling. Specifically, the S-nitrosylation of SH-groups by NO has been shown to contribute to cGMPindependent effects of NO (5-7). S-nitrosylation of caspases has been described to inhibit apoptosis signaling (8 -10). Caspase-3 has been shown to be S-nitrosylated at cysteine 163, which resulted in an inhibition of caspase-3 activity in vivo and in vitro (8,(11)(12)(13)(14). Furthermore, the small G-protein p21 ras and the MAP-kinase JNK are regulated by S-nitrosylation (15,16). Another example is the S-nitrosylation of transcription factors like NFB, c-jun, and c-fos, which regulates gene expression underscoring S-nitrosylation as a widespread regulatory mechanism (for review see Ref. 17). Beside the S-nitrosylation of high molecular weight proteins, the formation of S-nitrosylated glutathione, GSNO, has been proposed to be one of the important storage forms for NO in vivo (18,19).
Although various S-nitrosylated proteins and molecules have been identified, the regulation of the S-nitrosylation is not clear. A first hint for the potential reversibility of S-nitrosylation and thereby suggesting the process of denitrosylation as an important component of signal transduction cascades was reported by Mannick et al., who demonstrated that S-nitrosylation of caspase-3 is reversible (20). Stimulation of human T and B cells with Fas, a known pro-apoptotic stimulus, led to denitrosylation of overexpressed caspase-3 (20). Meanwhile, further studies revealed that the glutathione-dependent formaldehyde dehydrogenase, which is conserved from bacteria to humans, specifically metabolizes GSNO to glutathione (21). Therefore, we investigated the stability of GSNO and high molecular weight proteins in endothelial cells. The present study demonstrates that TNF␣ and mildly oxidized low density lipoprotein (oxLDL) reduce S-nitrosothiols in endothelial cells. The majority of S-nitrosothiols (95%) was associated with high molecular weight proteins suggesting a minor contribution of GSNO. TNF␣ and oxLDL induced denitrosylation of many proteins including the p17 subunit of caspase-3 suggesting that the mechanism of S-nitrosylation/denitrosylation plays an important regulatory role in endothelial cells.

MATERIALS AND METHODS
Cell Culture-Human umbilical vein endothelial cells were cultured in endothelial basal medium supplemented with hydrocortisone (1 g/ ml), bovine brain extract (12 g/ml), gentamicin (50 g/ml), amphotericin B (50 ng/ml), epidermal growth factor (10 ng/ml), and 10% fetal calf serum. After detachment with trypsin, cells were grown for at least 18 h. Cells were incubated with 10 g/ml oxLDL or 100 ng/ml TNF␣ as described previously (11,22). * This study was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 553, C2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Transfection-The plasmid encoding the caspase-3 p17 wt subunit and the caspase-3 p17 mutant type (C163S) were cloned as previously described (11). Human umbilical vein endothelial cells were transfected with 3 g of plasmid and 25 l of Superfect as described previously with a transfection efficiency of 40% (11).
S-NO Content-S-NO content was measured using the Saville-Griess assay as described (23). In brief, human umbilical vein endothelial cells were lysed in Griess lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM KCl, 1% Nonidet-P40, 1 mM phenylmethylsulfonyl fluoride, 1 mM bathocuproinedisulfonic acid, 1 mM diethylenetriaminepenta-acetic acid, 10 mM N-ethylmaleimide), and 80 g of cell lysate was incubated with 1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine in the presence or absence of 3.75 mM p-chloromercuribenzosulfonic acid for 20 min, and S-NO content was measured photometrically at 540 nm. The amount was calculated using defined GSNO concentrations as a standard.
Separation of Low Molecular Thiols and Protein S-NO-Cell lysates (500 g of total protein) were passed through G-25 Sephadex columns preequilibrated with Griess lysis buffer. Protein S-NO was separated from low molecular mass S-NO by desalting with 150 mM NaCl. S-NO content was detected by using the Saville-Griess assay as described above. Data were calculated as percent of protein loaded on the column.
Intracellular GSH Content-After the indicated times, cells were washed with PBS. After addition of 1% sulfosalicyclic acid cells were immediately shock frozen at Ϫ80°C for 2 min and the resulting lysate was centrifuged for 5 min 15,000 ϫ g at 4°C. The supernatant was incubated with Tris-EDTA buffer (20 mM Tris, pH 7.4, 1 mM EDTA) and dithiobis(2-nitrobenzoic acid) at room temperature for 10 min, and the optical density was determined with a photometer at 412 nm. The pellet was resuspended in 10 mM Tris, pH 8, 0.32 M sucrose, 5 mM EDTA, 1% Triton, 1 mM phenylmethylsulfonyl fluoride for 30 min on ice and then centrifuged for 10 min at 15,000 ϫ g at 4°C. The total protein content of the probe was determined.
Detection of S-Nitrosylated Caspase-3 p17 Subunit-Detection of Snitrosylated caspase-3 p17 subunit was performed as described (16). In brief, cells were lysed in 50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet-P-40, 0.5% deoxycholic acid, 0.1% SDS and subjected to immunoprecipitation. Immunoprecipitates were washed twice with lysis buffer and two times with PBS. The pellet was resuspended in 500 l of PBS. After addition of 100 M HgCl 2 and 100 M 2,3-diamino-naphthalene (DAN), samples were incubated in the dark at room temperature for 30 min, and 1 M NaOH was added. The generated fluorescent triazole from the reaction of 2,3-diamino-naphthalene with the NO released from caspase-3 p17 subunit was measured using an excitation wavelength of 375 nm and an emission wavelength of 450 nm. As negative control the Myc antibody alone in lysis buffer was immunoprecipitated. The resulting background fluorescence intensity was subtracted from each experiment.
Detection of S-Nitrosylated Proteins by Western Blotting-Detection of S-nitrosylated proteins was performed as described with slight modifications (24). In brief, cells were lysed in HENS buffer (25 mM HEPES, pH 7.7, 0.1 mM EDTA, 0.01 mM neocuproine and 1% SDS) for 20 min and centrifuged at 20,000 g for 15 min. 800 g of total protein was incubated with 20 mM methyl-methanthiosulfate (MMTS) for 20 min at 50°C and vortexed every 2 min for 5 s. MMTS was removed by protein precipitation with acetone. After resuspending the pellet in HENS buffer, 0.4 mM N- [6-(biotinamido)hexyl]-3Ј-(2Ј-pyridyldithio)propionamide (biotin-HPDP) and 1 mM sodium ascorbate were added and incubated for 1 h at room temperature in the dark. Gel electrophoresis and Western blotting were performed in the dark. Biotinylated proteins were detected by using a horseradish peroxidase-linked streptavidin according to the manufacturer's protocol.
Statistics-Statistical analysis was performed with analysis of variance or student's t test (SPSS-Software; Excel software).

TNF␣ and oxLDL Reduce S-Nitrosothiol Content in Endothe-
lial Cells-To address whether TNF␣ or oxLDL reduce the overall S-nitrosothiol content, endothelial cells were incubated with TNF␣ and oxLDL for 12 and 18 h. As shown in Fig. 1A, TNF␣ as well as oxLDL time dependently decreased S-nitrosothiols as measured by Saville-Griess assay. To confirm these findings, immunostainings were performed using an anti-Snitrosocysteine antibody. Endothelial cells treated with TNF␣ and oxLDL showed significantly less S-nitrosocysteine compared with control cells. The specificity of the immunostaining was demonstrated by preincubation with HgCl 2 (Fig. 1B and  data not shown). To exclude a possible involvement of TNF␣induced degradation of the eNOS mRNA and protein (25-27), which might interfere with de novo S-nitrosylation, we incubated endothelial cells with N G -monomethyl-L-arginine (L-NMMA), a competitive inhibitor of the eNOS, and measured the content of S-nitrosothiols. We could not detect any reduction of S-nitrosothiols after 18 h, but a significant decrease was determined after 48 h of incubation with L-NMMA (Fig. 1C) indicating a strong stability of the protein S-nitrosothiol adducts. Similar results were obtained using a different inhibitor of the eNOS, N G -nitro-L-arginine-methyl ester (L-NAME), for 18 h (control: 20.3 Ϯ 1.5 nmol S-NO/mg protein; L-NAME: 19.6 Ϯ 1.6 nmol S-NO/mg protein). Moreover, coincubation of endothelial cells with TNF␣ and L-NMMA revealed similar results as for TNF␣ alone (9.9 Ϯ 1.7 nmol S-NO/mg protein TNF␣ ϩ L-NMMA versus 10.1 Ϯ 1.4 nmol S-NO/mg protein TNF␣, n ϭ 4).
Characterization of Low and High Molecular Weight S-Nitrosothiols-To characterize the S-nitrosothiols involved in the S-nitrosylation/denitrosylation process in endothelial cells, we performed column chromatography using Sephadex G25 (cutoff: 5000 Da). As shown in Fig. 2A, the amount of low molecular weight S-nitrosothiols including GSNO was less than 5% of the overall S-NO content. These data indicate that proteins and not GSNO were denitrosylated upon stimulation with TNF␣ and oxLDL. To further underscore that GSNO is not the predominant form of NO in endothelial cells, cells were incubated with butyl-N-sulfoximine (BSO) to inhibit the de novo synthesis of GSH. BSO incubation did not reduce the overall S-NO content in endothelial cells (Fig. 2B), although the GSH content was markedly decreased in the cells (Fig. 2C).
Regulation of Protein S-Nitrosylation/Denitrosylation-Having demonstrated that more than 95% of the S-nitrosothiols in endothelial cells are high molecular weight proteins, we characterized the pattern of S-nitrosylation using a Western blot approach. For this purpose, S-nitrosylated proteins were labeled with biotin using biotin-HPDP and detected with horseradish peroxidase-linked streptavidin. Incubation with TNF␣ showed a significant reduction of S-nitrosylated proteins (Fig.  3A). Quantification revealed that TNF␣ and oxLDL reduced S-nitrosylated proteins as shown in Fig. 3B. Equal loading of the blots was confirmed by reprobing the membranes with antibodies against the well established S-nitrosylated proteins, Ras and JNK (Fig. 3A). Moreover, bands with the proposed molecular mass for Ras (21 kDa) and JNK (46 kDa) seemed to be S-nitrosylated (Fig. 3A).
TNF␣ Induces Denitrosylation of Caspase-3 p17 Subunit in Endothelial Cells-To get further insights into specific targets, which are denitrosylated by TNF␣, we investigated the caspase-3 p17 subunit. We and others showed previously that TNF␣ and oxLDL induced apoptosis in endothelial cells via activation of the cysteine protease family, the caspases, and, moreover, that S-nitrosylation of caspase-3 leads to its inactivation (8,(11)(12)(13)(14)28). Therefore, we hypothesized that TNF␣ induces denitrosylation of caspase-3 p17 subunit as an important mechanism for apoptosis induction in endothelial cells. To test this hypothesis, we transfected endothelial cells with Myctagged caspase-3 p17 wild type subunit (p17 wt) and the caspase 3 p17 mutant subunit, which contained a serine instead of a cysteine in its active center at position 163 (p17C163S). After stimulation with TNF␣, the caspase-3 p17 subunit was immunoprecipitated, and S-nitrosylation was detected using the DAN reagent. Under control conditions, p17 wt was S-nitrosylated in vivo, which was significantly reduced in cells treated with TNF␣ (Fig. 3C). The data obtained for p17 wt after TNF␣ treatment were similar as for p17C163S without TNF␣ (Fig. 3C). These results demonstrate that TNF␣ induced denitrosylation of proteins, importantly the caspase-3 p17 subunit.

DISCUSSION
The present study demonstrates that TNF␣ and oxLDL can induce the denitrosylation of proteins in endothelial cells. The denitrosylation was demonstrated using different techniques including immunohistochemistry and the detection of S-NO with the Saville-Griess Assay. Furthermore, the recently developed method by Jaffrey et al. was used, which allows the detection of S-nitrosylated proteins by Western blot (24). Thereby, TNF␣ and oxLDL were shown to reduce the S-nitrosylation of various proteins. Furthermore, TNF␣ specifically denitrosylates the caspase-3 p17 subunit as demonstrated by the detection of the S-nitrosylation of p17 immunoprecipitates with the DAN assay. These data provide evidence for the denitrosylation as an important regulatory mechanism in endothelial cells. The characterization of the S-nitrosothiols in endothelial cells revealed that most of the S-nitrosothiols were high molecular weight proteins as demonstrated by gel filtration. More-over, inhibition of glutathione biosynthesis did not significantly affect the overall S-NO levels, suggesting that GSNO quantitatively does play a minor role. This is in accordance with a study by Eu et al., who also demonstrated a minor contribution of GSNO to the overall S-NO levels in macrophages (23). A possible explanation of why GSNO did not accumulate in the cells could be a rapid metabolization. In support of this hypothesis, the thioredoxin system was shown to rapidly cleave NO out of GSNO in a cell-free system (29). In addition, a glutathionedependent formaldehyde dehydrogenase was identified in eukaryotes, which specifically denitrosylates GSNO (21).
We and others showed previously that apoptosis induction in endothelial cells was dependent on the caspase cascade, and that predominantly caspase-3 activity was inhibited by direct S-nitrosylation of the cysteine 163 in the active center of the caspase-3 p17 subunit (8,(11)(12)(13)(14)28). First evidence that this process is reversible came from Mannick et al., who reported a denitrosylation of the caspase-3 p17 subunit in iNOS-transfected B and T cells upon Fas-receptor stimulation (20). In line with these findings, the data of the present study revealed that the stimulation of endothelial cells with pro-inflammatory or pro-atherogenic stimuli reduced the S-nitrosylation of distinct proteins in endothelial cells and specifically reduced the Snitrosylation of the p17 subunit of caspase-3. The decline of S-nitrosylation could be due to an inhibition of eNOS expression and/or NO bioavailability induced by TNF␣-mediated eNOS mRNA destabilization (27) or increased oxidative stress (30,31). However, pharmacological inhibition of NO synthesis for 18 h with L-arginine-L-NAME or L-NMMA did not decrease the S-NO levels, although control experiments confirmed the inhibition of eNOS (data not shown). These data indicate that the intracellular S-nitrosylation under basal conditions is very stable, which is in accordance with a recent study by Mannick et al. (20). Furthermore, the reduction of S-nitrosylation by incubation with TNF␣ or oxLDL seems to be caused by an active denitrosylation process rather than a decline in de novo S-nitrosylation. The molecular mechanism underlying this denitrosylation requires further investigation. In bacteria, constitutive and inducible enzymes exist, which cleave S-NO to NO (32,33). Furthermore, a metabolic enzyme with GSNO reductase activity was identified in cytokine-stimulated macrophages, which also can reduce protein-S-NO (21). However, since GSNO is the preferred substrate (21), it is not clear whether this reductase can mediate TNF␣-or oxLDL-induced denitrosylation.
The regulation of S-nitrosylation may importantly contribute to cellular signaling and endothelial function. Specifically, the inhibition of endothelial cell apoptosis has at least in part been attributed to the blockade of caspase via S-nitrosylation. The reversal of the S-nitrosylation may allow for caspaseactivation, which is required for apoptosis signaling. Furthermore, S-nitrosylation of various other proteins was detected under basal conditions. The identity of these proteins needs to be defined in future studies. Given that small GTP-binding proteins, kinases, proteases and transcription factors are known to be S-nitrosylated, one may speculate that the reversal of the S-nitrosylation by pro-inflammatory or pro-atherosclerotic factors may play a crucial role in cellular signaling. FIG. 3. A, TNF␣ reduced S-nitrosylated protein content in endothelial cells. After incubation with TNF␣ (100 ng/ml) for 18 h, S-nitrosylated proteins were determined by Western blot as described under "Materials and Methods." To detect all proteins containing an SH group independent of S-nitrosylation, cell lysates were incubated without MMTS as a positive control. To demonstrate equal loading, blots were stripped and reprobed with anti-JNK and anti-Ras antibodies. Arrows indicate possible S-nitrosylated bands for JNK and Ras. A representative Western blot is shown (n ϭ 4). B, densitometric analysis of S-nitrosylated proteins. Extent of S-nitrosylation was quantified by scanning densitometry. Data are mean Ϯ S.E., n ϭ 4. C, denitrosylation of p17 wt by TNF␣. Endothelial cells were transfected with Myc-tagged p17 wt or p17C163S. After treatment with TNF␣ for 18 h, S-nitrosylation was determined as described under "Materials and Methods." Data are mean Ϯ S.E., n ϭ 4; *p Ͻ 0.05 versus p17 wt. Protein lysates were taken prior to immunoprecipitation to assure equal expression of p17 wt and p17C163S (inset).