Inactivation of adenosine A2A receptor attenuates basal and angiotensin II-induced ROS production by Nox2 in endothelial cells

Endothelial cells (ECs) express a Nox2 enzyme, which, by generating reactive oxygen species (ROS), contributes to EC redox signaling and angiotensin II (AngII)-induced endothelial dysfunction. ECs also express abundantly an adenosine A(2A) receptor (A(2A)R), but its role in EC ROS production remains unknown. In this study, we investigated the role of A(2A)R in the regulation of Nox2 activity and signaling in ECs with or without acute AngII stimulation. In cultured ECs (SVEC4-10), AngII (100 nm, 30 min) significantly increased Nox2 membrane translocation and association with A(2A)R. These were accompanied by p47(phox), ERK1/2, p38 MAPK, and Akt phosphorylation and an increased ROS production (169 ± 0.04%). These AngII effects were inhibited back to the control levels by a specific A(2A)R antagonist (SCH58261), or adenosine deaminase, or by knockdown of A(2A)R or Nox2 using specific siRNAs. Knockdown of A(2A)R, as determined by Western blotting, decreased Nox2 and p47(phox) expression. In wild-type mouse aorta, SCH58261 significantly reduced acute AngII-induced ROS production and preserved endothelium-dependent vessel relaxation to acetylcholine. These results were further confirmed by using aortas from A(2A)R knock-out mice. In conclusion, A(2A)R is involved in the regulation of EC ROS production by Nox2. Inhibition or blockade of A(2A)R protects ECs from acute AngII-induced oxidative stress, MAPK activation, and endothelium dysfunction.     

Mitochondrial c-Jun N-terminal kinase (JNK) signaling initiates physiological changes resulting in amplification of reactive oxygen species generation

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.     

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Enhances Activation of p38 and ERK MAPKs,Increasing Interleukin-6 Synthesis in Airway Epithelial Cells Exposed to Pseudomonas aeruginosa

In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRΔF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRΔF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRΔF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRΔF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.     

Constitutive NADPH oxidase 4 activity resides in the composition of the B-loop and the penultimate C terminus

Redox regulation of signaling molecules contributes critically to propagation of intracellular signals. The main source providing reactive oxygen species (ROS) for these physiological processes are activated NADPH oxidases (Nox/Duox family). In a pathophysiological context, some NADPH oxidase complexes produce large amounts of ROS either as part of the antimicrobial immune defense or as pathologic oxidative stress in many chronic diseases. Thus, understanding the switch from a dormant, inactive conformation to the active state of these enzymes will aid the development of inhibitors. As exogenously expressed Nox4 represents the only constitutively active enzyme in this family, analysis of structural determinants that permit this active conformation was undertaken. Our focus was directed toward a cell-based analysis of the first intracellular loop, the B-loop, and the C-terminus, two regions of Nox family enzymes that are essential for electron transfer. Mutagenesis of the B-loop identified several unique residues and a polybasic motif that contribute to the catalytic activity of Nox4. By using a multifaceted approach, including Nox4-Nox2 chimeras, mutagenesis, and insertion of Nox2 domains, we show here that the penultimate 22 amino acids of Nox4 are involved in constitutive ROS generation. The appropriate spacing of the C-terminal Nox4 sequence may cooperate with a discrete arginine-based interaction site in the B-loop, providing an intrinsically active interface that could not be disrupted by peptides derived from the Nox4 C-terminus. These results indicate that accessibility for a Nox4-specific peptide inhibitor might be difficult to achieve in vivo.     

Monocarboxylate transporter-1 is required for cell death in mouse chondrocytic ATDC5 cells exposed to interleukin-1beta via late phase activation of nuclear factor kappaB and expression of phagocyte-type NADPH oxidase

Interleukin-1β (IL-1β) induces cell death in chondrocytes in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner. In this study, increased production of lactate was observed in IL-1β-treated mouse chondrocytic ATDC5 cells prior to the onset of their death. IL-1β-induced cell death in ATDC5 cells was suppressed by introducing an siRNA for monocarboxylate transporter-1 (MCT-1), a lactate transporter distributed in plasma and mitochondrial inner membranes. Mct-1 knockdown also prevented IL-1β-induced expression of phagocyte-type NADPH oxidase (NOX-2), an enzyme specialized for production of ROS, whereas it did not have an effect on inducible NO synthase. Suppression of IL-1β-induced cell death by Nox-2 siRNA indicated that NOX-2 is involved in cell death. Phosphorylation and degradation of inhibitor of κBα (IκBα) from 5 to 20 min after the addition of IL-1β was not affected by Mct-1 siRNA. In addition, IκBα was slightly decreased after 12 h of incubation with IL-1β, and the decrease was prominent after 36 h, whereas activation of p65/RelA was observed from 12 to 48 h after exposure to IL-1β. These changes were not seen in Mct-1-silenced cells. Forced expression of IκBα super repressor as well as treatment with the IκB kinase inhibitor BAY 11-7082 suppressed NOX-2 expression. Furthermore, Mct-1 siRNA lowered the level of ROS generated after 15-h exposure to IL-1β, whereas a ROS scavenger, N-acetylcysteine, suppressed both late phase degradation of IκBα and Nox-2 expression. These results suggest that MCT-1 contributes to NOX-2 expression via late phase activation of NF-κB in a ROS-dependent manner in ATDC5 cells exposed to IL-1β.     

Distinct roles for Nod2 protein and autocrine interleukin-1beta in muramyl dipeptide-induced mitogen-activated protein kinase activation and cytokine secretion in human macrophages

Elucidating factors regulating Crohn's disease-associated nucleotide-binding oligomerization domain 2 (Nod2) responses is critical to understanding the mechanisms of intestinal immune homeostasis. Stimulation of primary monocyte-derived macrophages by muramyl dipeptide (MDP), a component of bacterial peptidoglycan and specific Nod2 ligand, produces cytokines, including IL-1β. We found that IL-1β blockade profoundly inhibits MDP-induced cytokine production in human monocyte-derived macrophages, demonstrating a key role for IL-1β autocrine secretion in Nod2-mediated responses. Importantly, although MAPK activation has previously been attributed directly to Nod2 signaling, we determined that the IL-1β autocrine loop is responsible for the majority of MDP-induced MAPK activation. Because the critical effects of IL-1β autocrine secretion on MAPK activation are observed as early as 10 min after Nod2 stimulation, we hypothesized that secretion of IL-1β from preexisting intracellular pro-IL-1β stores is necessary for optimal MDP-mediated cytokine induction. Consistently, we detected IL-1β secretion within 10 min of MDP treatment. Moreover, caspase-1 inhibition significantly attenuates MDP-mediated early MAPK activation. Importantly, selective JNK/p38 activation is sufficient to rescue the decreased cytokine secretion during Nod2 stimulation in the absence of autocrine IL-1β. Finally, we found that the IL-1β autocrine loop significantly enhances responses by a broad range of pattern recognition receptors. Taken together, MDP stimulation activates Nod2 to process and release preexisting pro-IL-1β stores in a caspase-1-dependent fashion; this secreted IL-1β, in turn, contributes to the majority of MDP-initiated MAPK activation and leads to subsequent cytokine secretion. Our findings clarify mechanisms of IL-1β contributions to Nod2 responses and elucidate the dominant role of IL-1β in MDP-initiated MAPK and cytokine secretion.     

Up-regulation and sustained activation of Stat1 are essential for interferon-gamma (IFN-gamma)-induced dual oxidase 2 (Duox2) and dual oxidase A2 (DuoxA2) expression in human pancreatic cancer cell lines

Dual oxidase 2 is a member of the NADPH oxidase (Nox) gene family that plays a critical role in the biosynthesis of thyroid hormone as well as in the inflammatory response of the upper airway mucosa and in wound healing, presumably through its ability to generate reactive oxygen species, including H2O2. The recently discovered overexpression of Duox2 in gastrointestinal malignancies, as well as our limited understanding of the regulation of Duox2 expression, led us to examine the effect of cytokines and growth factors on Duox2 in human tumor cells. We found that exposure of human pancreatic cancer cells to IFN-γ (but not other agents) produced a profound up-regulation of the expression of Duox2, and its cognate maturation factor DuoxA2, but not other members of the Nox family. Furthermore, increased Duox2/DuoxA2 expression was closely associated with a significant increase in the production of both intracellular reactive oxygen species and extracellular H2O2. Examination of IFN-γ-mediated signaling events demonstrated that in addition to the canonical Jak-Stat1 pathway, IFN-γ activated the p38-MAPK pathway in pancreatic cancer cells, and both played an important role in the induction of Duox2 by IFN-γ. Duox2 up-regulation following IFN-γ exposure is also directly associated with the binding of Stat1 to elements of the Duox2 promoter. Our findings suggest that the pro-inflammatory cytokine IFN-γ initiates a Duox2-mediated reactive oxygen cascade in human pancreatic cancer cells; reactive oxygen species production in this setting could contribute to the pathophysiologic characteristics of these tumors.     

Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 μM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.     

Role of PKCζ‐NADPH oxidase signaling axis in PKCα‐mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C‐ζ (PKC‐ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC‐α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ, NADPH oxidase, and PKC‐α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC‐ζ and NADPH oxidase attenuate AngII induced increase in PKC‐α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ and NADPH oxidase activities. Overall, PKCζ‐NADPH oxidase‐PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII‐mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.