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

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  • Additional Information
    • Source:
      Publisher: American Physiological Society Country of Publication: United States NLM ID: 100901228 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1522-1539 (Electronic) Linking ISSN: 03636135 NLM ISO Abbreviation: Am J Physiol Heart Circ Physiol Subsets: MEDLINE
    • Publication Information:
      Original Publication: Bethesda, Md. : American Physiological Society,
    • Subject Terms:
      Cell Adhesion*; Abdominal Muscles/*blood supply ; Endothelial Cells/*drug effects ; Leukocytes/*metabolism ; Nitric Oxide/*metabolism ; Nitric Oxide Synthase Type III/*metabolism ; Tumor Necrosis Factor-alpha/*pharmacology; Animals ; Cell Line ; Coculture Techniques ; Endothelial Cells/enzymology ; Enzyme Activation ; Humans ; Intercellular Adhesion Molecule-1/metabolism ; Male ; Mice, Inbred C57BL ; Phosphorylation ; Protein Kinase C/metabolism ; Protein Processing, Post-Translational ; Signal Transduction ; Time Factors ; Mice
    • 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.
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    • Grant Information:
      R01 HL076259 United States HL NHLBI NIH HHS; R01 GM122940 United States GM NIGMS NIH HHS; R01 HL146539 United States HL NHLBI NIH HHS; R56 HL134842 United States HL NHLBI NIH HHS; R01 HL146829 United States HL NHLBI NIH HHS; S10 OD025047 United States OD NIH HHS; P30 NS046593 United States NS NINDS NIH HHS
    • Contributed Indexing:
      Keywords: S-nitrosylation; leukocyte adhesion; nitric oxide; protein kinase
    • Accession Number:
      0 (ICAM1 protein, human)
      0 (Icam1 protein, mouse)
      0 (Tumor Necrosis Factor-alpha)
      126547-89-5 (Intercellular Adhesion Molecule-1)
      31C4KY9ESH (Nitric Oxide)
      EC 1.14.13.39 (Nitric Oxide Synthase Type III)
      EC 1.14.13.39 (Nos3 protein, mouse)
      EC 2.7.11.1 (protein kinase C zeta)
      EC 2.7.11.13 (Protein Kinase C)
    • Publication Date:
      Date Created: 20211015 Date Completed: 20211206 Latest Revision: 20240923
    • Publication Date:
      20240923
    • Accession Number:
      PMC8782658
    • Accession Number:
      10.1152/ajpheart.00065.2021
    • Accession Number:
      34652985