Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signalling. Here we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H₂O₂ originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppresses the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical to support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumour cell metabolism.

Endothelial nitric oxide synthase (eNOS)–mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1–mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca²⁺ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor l-N^G-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca²⁺-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1–Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition.

The NF-κB pathway is central to the regulation of inflammation. Here, we demonstrate that the low-output nitric oxide (NO) synthase 1 (NOS1 or nNOS) plays a critical role in the inflammatory response by promoting the activity of NF-κB. Specifically, NOS1-derived NO production in macrophages leads to proteolysis of suppressor of cytokine signaling 1 (SOCS1), alleviating its repression of NF-κB transcriptional activity. As a result, NOS1^−/− mice demonstrate reduced cytokine production, lung injury, and mortality when subjected to two different models of sepsis. Isolated NOS1^−/− macrophages demonstrate similar defects in proinflammatory transcription on challenge with Gram-negative bacterial LPS. Consistently, we found that activated NOS1^−/− macrophages contain increased SOCS1 protein and decreased levels of p65 protein compared with wild-type cells. NOS1-dependent S-nitrosation of SOCS1 impairs its binding to p65 and targets SOCS1 for proteolysis. Treatment of NOS1^−/− cells with exogenous NO rescues both SOCS1 degradation and stabilization of p65 protein. Point mutation analysis demonstrated that both Cys147 and Cys179 on SOCS1 are required for its NO-dependent degradation. These findings demonstrate a fundamental role for NOS1-derived NO in regulating TLR4-mediated inflammatory gene transcription, as well as the intensity and duration of the resulting host immune response.

Background: Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase–Akt–nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability. Methods: Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed. Results: Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC₅₀] = 8.611 × 10⁻¹⁰ M for ropivacaine; IC₅₀ = 5.864 × 10⁻¹⁰ M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC₅₀ = 7.572 × 10⁻¹⁰ M for ropivacaine; IC₅₀ = 6.377 × 10⁻¹⁰ M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated. Conclusions: Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory “side-effect” of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.

In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav‐1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav‐1 protein despite increased Cav‐1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary artery endothelial cells (PAECs) from patients with IPAH. In control human lung endothelial cell cultures, tumor necrosis factor α–induced nitric oxide (NO) production and S‐nitrosation (SNO) of Cav‐1 Cys‐156 were associated with Src displacement and activation, Cav‐1 Tyr‐14 phosphorylation, and destabilization of Cav‐1 oligomers within 5 minutes that could be blocked by eNOS or Src inhibition. Prolonged stimulation (72 hours) with NO donor DETANONOate reduced oligomerized and total Cav‐1 levels by 40%–80%, similar to that observed in IPAH patient–derived PAECs. NO donor stimulation of endothelial cells for >72 hours, which was associated with sustained Src activation and Cav‐1 phosphorylation, ubiquitination, and degradation, was blocked by NOS inhibitor L‐NAME, Src inhibitor PP2, and proteosomal inhibitor MG132. Thus, chronic inflammation, sustained eNOS and Src signaling, and Cav‐1 degradation may be important causal factors in the development of IPAH by promoting PAEC dysfunction/activation via sustained oxidative/nitrosative stress.