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

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-a (TNF-a) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase Cf (PKCf) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-a, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-a-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-a-induced NO also produced S-nitrosylation and phosphorylation of PKCf, association of PKCf with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCf blocked leukocyte adhesion induced by TNF-a. Mass spectrometry analysis of purified PKCf 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 PKCf may be an important regulatory step in early leukocyte adhesion in inflammation.