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. 2021 Dec 1;321(6):H1083-H1095.
doi: 10.1152/ajpheart.00065.2021. Epub 2021 Oct 15.

TNF-α-activated eNOS signaling increases leukocyte adhesion through the S-nitrosylation pathway

Gaynor Aguilar  1 , Francisco Córdova  1 , Tania Koning  1 , José Sarmiento  2 , Mauricio P Boric  3 , Konstantin Birukov  4 , Jorge Cancino  5 , Manuel Varas-Godoy  5 , Andrea Soza  5   6 , Natascha G Alves  7 , Patricio E Mujica  7   8 , Walter N Durán  7 , Pamela Ehrenfeld  9   10 , Fabiola A Sánchez  1   10
Affiliations

TNF-α-activated eNOS signaling increases leukocyte adhesion through the S-nitrosylation pathway

Gaynor Aguilar et al. Am J Physiol Heart Circ Physiol. .

Abstract

Nitric oxide (NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-α (TNF-α) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase Cζ (PKCζ) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-α, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α-induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCζ blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of purified PKCζ identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCζ may be an important regulatory step in early leukocyte adhesion in inflammation.NEW & NOTEWORTHY Contrary to the well-established inhibitory role of NO in leukocyte adhesion, we demonstrate a positive role of nitric oxide in this process. We demonstrate that NO induced by eNOS after TNF-α treatment induces early leukocyte adhesion activating the S-nitrosylation pathway. Our data suggest that PKCζ S-nitrosylation may be a key step in this process.

Keywords: S-nitrosylation; leukocyte adhesion; nitric oxide; protein kinase.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Figure 1.
Figure 1.
NO signaling regulates leukocyte adhesion in vivo. A: intravital microscopy of cremaster showing leukocyte adhesion at 5 and 10 min after treatment with 1 nM TNF-α. Pretreatment with l-NMA (50 mg/kg iv) inhibits TNF-α-induced leukocyte adhesion. B: statistical analysis of leukocyte adhesion. Two-way ANOVA and Tukey test, *P < 0.05 compared with time 0 of treatment with TNF-α; #P < 0.05 in comparison to the treatment with TNF-α. n = 3 different animals. l-NMA, NG-methyl-l-arginine; NO, nitric oxide; TNF-α, tumor necrosis factor-α.
Figure 2.
Figure 2.
S-nitrosylation regulates early PMN adhesion induced by TNF-α in vitro. A: confluent monolayers of EAhy926 cells were incubated with 1 nM TNF-α for 5 and 15 min. Then, isolated human preactivated PMNs were added to the monolayers and allowed to adhere for 30 min at 37°C. Nonadherent PMN were washed away and MPO activity was assayed in the cell lysates. One-way ANOVA and Tukey’s multiple comparison test. *P < 0.05 compared with time 0. n = 8 independent experiments. Confluent monolayers of EAhy926 cells were preincubated with 300 µM l-NMA for 45 min or 10 µM ODQ for 10 min before TNF-α treatment for 5 (B) and 15 min (C). Increase in leukocyte adhesion induced by TNF-α was blocked by inhibition of eNOS with l-NMA but not by inhibition of GC-1-PKG with ODQ. One-way ANOVA and Bonferroni’s multiple comparison test. *P < 0.05 compared with time 0; #P < 0.05 compared with TNF-α treatment. n = 6 independent experiments. D: confluent monolayers of EAhy926 cells were preincubated with 300 µM l-NMA for 45 min or 10 µM ODQ for 10 min. Neither agent caused leukocyte adhesion. One-way ANOVA and Bonferroni’s multiple comparison test. n = 8 independent experiments. All data were normalized to adhesion seen at time 0. E: TNF-α significantly increases phosphorylation of eNOS at Ser1177 at 5 and 15 min. One-way ANOVA and Bonferroni’s multiple comparison test. *P < 0.05; n = 3 independent experiments. F: real-time detection of NO induced by TNF-α in live EAHy926 cells. Paired t test. ****P < 0.0001; n = 5 independent experiments. G: representative image of one experiment showing the increases in NO levels in the time after TNF-α application. eNOS, endothelial nitric oxide synthase; l-NMA, NG-methyl-l-arginine; NO, nitric oxide; ODQ, 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one; PMN, polymorphonuclear leukocyte; TNF-α, tumor necrosis factor-α.
Figure 3.
Figure 3.
TNF-α induces ICAM-1 clustering dependent on NO signaling. A: confluent monolayers of EAhy926 cells were incubated with 1 nM TNF-α for 15 min and processed by immunofluorescence to detect ICAM-1 in the cell surface. The images are representative of 3 independent experiments. B: confluent monolayers of EAhy926 cells were incubated with 1 nM TNF-α for different times and membrane and cytosolic fractions were isolated, separated by PAGE-SDS, and probed with anti-ICAM antibodies. One-way ANOVA and Dunnett’s multiple comparison test, n = 3 independent experiments. C: confluent monolayers of EAhy926 cells were incubated with 1 nM TNF-α for different times in the presence or absence of l-NMA; ICAM-1 was immunoprecipitated and the interaction with cortactin evaluated by Western blot analysis. Two-way ANOVA and Tukey’s test, *P < 0.05 in comparison to time 0, #P < 0.05 in comparison to the treatment with TNF-α; n = 4 independent experiments. Data were normalized to band intensity seen at time 0. ICAM-1, intercellular adhesion molecule 1; l-NMA, NG-methyl-l-arginine; NO, nitric oxide; TNF-α, tumor necrosis factor-α.
Figure 4.
Figure 4.
TNF-α-induced NO increases ICAM-1 immobile fraction. A: ICAM-1-GFP (grayscale, inverted contrast) was expressed in EAhy926 cells, and FRAP was recorded. Arrow depicts the FRAP region. Top shows FRAP without treatment applied. Middle shows ICAM-1-GFP FRAP after treatment with 1 nM TNF-α for 5 min. Bottom shows ICAM-1-GFP FRAP after pretreatment of the cells with l-NMA before TNF-α application for 5 min. B: recovery ICAM-GFP fluorescence curves after different treatments. C: quantification of the FRAP data show increase in the immobile fraction of ICAM-1-GFP following TNF-α treatment and inhibition of this effect by application of l-NMA. Two-way ANOVA and Bonferroni t test. *P < 0.05 in comparison to time 0, #P < 0.05 in comparison to the treatment with TNF-α; n = 5 independent experiments. FRAP, fluorescence recovery after photobleaching; ICAM-1, intercellular adhesion molecule 1; l-NMA, NG-methyl-l-arginine; NO, nitric oxide; TNF-α, tumor necrosis factor-α.
Figure 5.
Figure 5.
TNF-α leads to PKCζ S-nitrosylation, PKCζ activation, association with ICAM-1, and ICAM-1 phosphorylation. A: confluent monolayers of EAhy926 cells were incubated with 1 nM TNF-α for different times. Cell lysates were obtained and processed by biotin switch to detect S-nitrosylated ICAM-1. One-way ANOVA and Tukey’s test, n = 3 independent experiments. B: confluent monolayers of EAhy926 cells were incubated with 1 nM TNF-α for 5 min in the presence or absence of 300 µM l-NMA for 1 h. Cell lysates were obtained and processed to detect S-nitrosylated PKCζ by biotin switch. C: depletion of eNOS in EAHy926 cells abrogates PKCζ S-nitrosylation induced by TNF-α. EAHy926 cells transfected with scrambled siRNA served as a control. Western blot for eNOS confirms protein depletion. One-way ANOVA and Dunnett’s multiple comparison test, n = 3 independent experiments; *P < 0.05 in comparison to control (C), #P < 0.05 in comparison to TNF-α treatment in the absence of eNOS siRNA. Data were normalized at band intensity seen at control without eNOS siRNA treatment. Confluent monolayers of EAhy926 cells were incubated with 1 nM TNF-α for 5 min in the presence or absence of 300 µM l-NMA for 1 h. Cell lysates were obtained and processed to detect phosphorylated p-PKCζ by Western blot analysis (D) and interaction between ICAM-1 and PKCζ by immunoprecipitation (E). Two-way ANOVA and Holm–Sidak test, *P < 0.05 in comparison to time 0, #P < 0.05 in comparison to the treatment with TNF-α; n = 3 independent experiments. All data were normalized at band intensity seen at time 0. F: ICAM-Y512 by Western blot analysis. Two-way ANOVA and Holm–Sidak test, *P < 0.05 in comparison to time 0, #P < 0.05 in comparison to the treatment with TNF-α; n = 4 independent experiments. G: ICAM-1 Thr phosphorylation by immunoprecipitation with anti ICAM-1 antibody, followed by Western blot analysis against p-Thr. Two-way ANOVA and Holm–Sidak test, *P < 0.05 in comparison to time 0, #P < 0.05 in comparison to the treatment with TNF-α; n = 3 independent experiments. Data were normalized at band intensity seen at time 0. eNOS, endothelial nitric oxide synthase; ICAM-1, intercellular adhesion molecule 1; l-NMA, NG-methyl-l-arginine; PKCζ, protein kinase Cζ; TNF-α, tumor necrosis factor-α.
Figure 6.
Figure 6.
PKCζ regulates leukocyte adhesion. Confluent monolayers of EAhy926 cells were preincubated with 1 µM inhibitor peptide for 20 min before TNF-α treatment for 5 and 15 min. Then, isolated human preactivated PMNs were added to the monolayers and allowed to adhere for 30 min at 37°C. Nonadherent PMNs were washed away and MPO activity was assayed in the cell lysates. Two-way ANOVA and Holm–Sidak test, *P < 0.05 in comparison to time 0, #P < 0.05 in comparison to the treatment with TNF-α; n = 3 independent experiments. Data were normalized at adhesion seen at time 0. MPO, myeloperoxidase; PKCζ, protein kinase Cζ; PMN, polymorphonuclear leukocyte; TNF-α, tumor necrosis factor-α.
Figure 7.
Figure 7.
Identification of S-nitrosylated Cys 503 in PKCζ. Protein sequence from human PKCζ. Purified PKCζ was S-nitrosylated with GSNO for 30 min, subjected to biotin switch assay followed by in-solution trypsin digestion. The kinase domain of PKCζ is shown in gray background. Cysteine 503 (red background) was the only S-nitrosylated cysteine according to mass spectrometry. GSNO, S-nitrosoglutathione; PKCζ, protein kinase Cζ.
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