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

Abstract

Nitric oxide (NO) is a key factor in inflammation produced by endothelial nitric oxide synthase (eNOS) in endothelium, whose activity increases after stimulation with pro-inflammatory cytokines. NO activates the soluble guanylate cyclase-protein kinase G and S-nitrosylation (NO modification of free-thiol cysteines in proteins) pathways. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce fast leukocyte adhesion suggesting 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 using tumor necrosis factor alpha (TNF-α) as pro-inflammatory agonist. ICAM-1 changes were evaluated by biochemical and imaging techniques. Protein kinase C zeta (PKCζ) activity and S-nitrosylation were evaluated by western-blot and biotin switch methods, 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 PKCz blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of PKCζ identified cysteine 503 as the S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS-S-nitrosylation mechanism that induces leukocyte adhesion suggesting that PKCζ--S-nitrosylation might be an important regulatory step in early leukocyte adhesion.

S-Nitrosylation in endothelial cells contributes to tumor cell adhesion and extravasation during breast cancer metastasis

“…Our results showing NO as a signal that promotes cell adhesion to the endothelium are in strong contrast to studies showing an inhibitory role of NO in this process [ 16 , 24 , 38 , 73 ]. Besides the differences in the cell and animal models used, we propose that these discrepancies can be explained by the fact that the physiological effects of NO strictly depend on its concentration, duration of exposure, location, and activity of NOS isoforms [ 2 , 3 ]. In non-stimulated cells, basal NO production could maintain an anti-adhesive phenotype [ 23 , 44 ].…”

“…We use a modification of the protocol previously described by us [ 2 ]. Briefly, confluent EAHy926 cells were loaded with DAF-FM Diacetate (5 mM, Invitrogen, Cat.…”

3β-Hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (SEC-B) Induces Proinflammatory Activation of Human Endothelial Cells Associated with Nitric Oxide Production and Endothelial Nitric Oxide Synthase/Caveolin-1 Dysregulation

“…Since NO is classically described as a negative regulator of leukocyte adhesion, upregulation of ICAM-1 and increased U937 recruitment on HMEC-1 cells was a quite unexpected result. However, a recent study by Aguilar et al demonstrated that TNF-α-activated eNOS signaling can increase leukocyte adhesion through ICAM-1 and the S-nitrosylation pathway [ 47 , 48 , 49 ]. In line with these data, our findings also demonstrated that overproduction of NO plays a key role in U937 cellular adhesion.…”

Connexin and Pannexin Large-Pore Channels in Microcirculation and Neurovascular Coupling Function

“…There is evidence of connexin contribution to the hyperpermeability response, inasmuch as agonist-induced eNOS translocation from the plasma membrane to cytosol and subsequent cytosolic NO production is crucial to agonist-induced hyperpermeability [126,[201][202][203][204][205], influencing S-nitrosylation and the disassembly of AJs proteins and VASP [206][207][208][209][210][211][212][213]. The participation of GJ channels or Cxs-hemichannels in hyperpermeability may be related to a NO-cGMP-PKG pathway.…”