Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

NF-kappaB, but not p38 MAP kinase, is required for TNF-alpha-induced expression of cell adhesion molecules in endothelial cells

In response to inflammation stimuli, tumor necrosis factor-alpha (TNF-alpha) induces expression of cell adhesion molecules (CAMs) in endothelial cells (ECs). Studies have suggested that the nuclear factor-kappaB (NF-kappaB) and the p38 MAP kinase (p38) signaling pathways play central roles in this process, but conflicting results have been reported. The objective of this study is to determine the relative contributions of the two pathways to the effect of TNF-alpha. Our initial data indicated that blockade of p38 activity by chemical inhibitor SB203580 (SB) at 10 microM moderately inhibited TNF-alpha-induced expression of three types of CAMs; ICAM-1, VCAM-1 and E-selectin, indicating that p38 may be involved in the process. However, subsequent analysis revealed that neither 1 microM SB that could completely inhibit p38 nor specific knockdown of p38alpha and p38beta with small interference RNA (siRNA) had an apparent effect, indicating that p38 activity is not essential for TNF-alpha-induced CAMs. The most definitive evidence to support this conclusion was from the experiments using cells differentiated from p38alpha knockout embryonic stem cells. We could show that deletion of p38alpha gene did not affect TNF-alpha-induced ICAM-1 and VCAM-1 expression when compared with wild-type cells. We further demonstrated that inhibition of NF-kappaB completely blocked TNF-alpha-induced expression of ICAM-1, VCAM-1 and E-selectin. Taken together, our results clearly demonstrate that NF-kappaB, but not p38, is critical for TNF-alpha-induced CAM expression. The inhibition of SB at 10 microM on TNF-alpha-induced ICAM-1, VCAM-1 and E-selectin is likely due to the nonspecific effect of SB.     

Effects of antioxidants and nitric oxide on TNF-alpha-induced adhesion molecule expression and NF-kappaB activation in human dermal microvascular endothelial cells

Cell adhesion molecules expressed on endothelial cells in inflamed skin appear to be controlled by the actions of cytokines and reactive oxygen species. However, molecular mechanisms of the expression of adhesion molecules during skin inflammation are currently not well understood. To evaluate the role of antioxidants and nitric oxide in modulating inflammatory processes in the skin, we examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on adhesion molecule expression and nuclear factor kappa B (NF-kappaB) activation induced by TNF-alpha (10 ng/ml) in cultured human dermal microvascular endothelial cells (HDMEC). Treatment of cells with TNF-alpha for 4 h significantly induced the surface expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1). Treatment with TNF-alpha for 8 h significantly induced the surface expression of E-selectin, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1). The up-regulation of these adhesion molecules was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h. The mRNA expression of E-selectin, ICAM-1 and VCAM-1, and activation of NF-kappaB induced by TNF-alpha for 2 h were significantly decreased by the above two pretreatments. N-acetylcysteine (10 mM) and S-nitroso-N-acetylpenicillamine (1 mM) had no significant inhibitory effects on the cell surface and mRNA expression of these adhesion molecules stimulated by TNF-alpha. These findings indicate that both cell surface and mRNA expression of adhesion molecules in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly in part through blocking the activation of NF-kappaB. These results suggest a potential therapeutic approach using antioxidant agents or nitric oxide pathway modulators in the treatment of inflammatory skin diseases.     

p38 MAPK: A dual role in hepatocyte proliferation through reactive oxygen species

Abstract

p38 MAPKs are important mediators of signal transduction that respond to a wide range of extracellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines, and oxidative stress. The most abundant family member is p38α, which helps to couple cell proliferation and growth in response to certain damaging stimuli. In fact, increased proliferation and impaired differentiation are hallmarks of p38α-deficient cells. It has been reported that reactive oxygen species (ROS) play a critical role in cytokine-induced p38α activation. Under physiological conditions, p38α can function as a mediator of ROS signaling and either activate or suppress cell cycle progression depending on the activation stimulus. The interplay between cell proliferation, p38 MAPK activation, and ROS production plays an important role in hepatocytes. In fact, low levels of ROS seem to be needed to activate several signaling pathways in response to hepatectomy and to orchestrate liver regeneration. p38 MAPK works as a sensor of oxidative stress and cells that have developed mechanisms to uncouple p38 MAPK activation from oxidative stress are more likely to become tumorigenic. So far, p38α influences the redox balance, determining cell survival, terminal differentiation, proliferation, and senescence. Further studies would be necessary in order to clarify the precise role of p38 MAPK signaling as a redox therapeutical target.     

Angiotensin II and tumor necrosis factor-alpha synergistically promote monocyte chemoattractant protein-1 expression: roles of NF-kappaB, p38, and reactive oxygen species

We examined whether ANG II and TNF-alpha cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-alpha stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-kappaB-dependent pathway. In contrast, TNF-alpha-induced MCP-1 expression was potently suppressed by blockade of NF-kappaB activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-alpha-induced activation of NF-kappaB- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-alpha-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-alpha cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-alpha further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-alpha alone. These results suggested that ANG II and TNF-alpha synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NFkappaB-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-alpha cooperatively stimulate vascular inflammation in vivo as well as in vitro.     

Parthenolide and DMAPT induce cell death in primitive CML cells through reactive oxygen species

Tyrosine kinase inhibitors (TKI) have become a first‐line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34⁺lin^?) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF‐κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G₀ and G₂ phases. Furthermore, we found cell cycle inhibition to correlate with down‐regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF‐κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.     

Sulindac-derived reactive oxygen species induce apoptosis of human multiple myeloma cells via p38 mitogen activated protein kinase-induced mitochondrial dysfunction

Non-steroidal anti-inflammatory drugs are well known to induce apoptosis of cancer cells independent of their ability to inhibit cyclooxygenase-2, but the molecular mechanism for this effect has not yet been fully elucidated. The purpose of this study was to elucidate the potential signaling components underlying sulindac-induced apoptosis in human multiple myeloma (MM) cells. We found that sulindac induces apoptosis by promoting ROS generation, accompanied by opening of mitochondrial permeability transition pores, release of cytochrome c and apoptosis inducing factor from mitochondria, followed by caspase activation. Bcl-2 cleavage and down-regulation of the inhibitor of apoptosis proteins (IAPs) family including cIAP-1/2, XIAP, and survivin, occurred downstream of ROS production during sulindac-induced apoptosis. Forced expression of survivin and Bcl-2 blocked sulindac-induced apoptosis. Most importantly, sulindac-derived ROS activated p38 mitogen-activated protein kinase and p53. SB203580, a p38 mitogen-activated protein kinase inhibitor, and RNA inhibition of p53 inhibited the sulindac-induced apoptosis. Furthermore, p53, Bax, and Bak accumulated in mitochondria during sulindac-induced apoptosis. All of these events were significantly suppressed by SB203580. Our results demonstrate a novel mechanism of sulindac-induced apoptosis in human MM cells, namely, accumulation of p53, Bax, and Bak in mitochondria mediated by p38 MAPK activation downstream of ROS production.     

Sphingosylphosphorylcholine induces apoptosis of endothelial cells through reactive oxygen species-mediated activation of ERK

Sphingosylphosphorylcholine (SPC) produces reactive oxygen species (ROS) in MS1 pancreatic islet endothelial cells. In the present study, we explored the physiological significance of the SPC-induced ROS generation in endothelial cells. SPC induced cell death of MS1 cells at higher than 10 microM concentration through a caspase-3-dependent pathway. SPC treatment induced sustained activation of an extracellular signal-regulated kinase (ERK), in contrast to transient activation of ERK in response to platelet-derived growth factor (PDGF)-BB, which stimulated proliferation of MS1 cells. Both the SPC-induced cell death and ERK activation were abolished by pretreatment of the cells with the MEK inhibitor U0126 or by overexpression of a dominant negative mutant of MEK1 (DN-MEK1). Pretreatment of the cells with N-acetylcysteine, an antioxidant, completely prevented the SPC-induced ROS generation, apoptosis, and ERK activation, whereas the ROS generation was not abrogated by treatment with U0126. Consistent with these results, SPC induced cell death of human umbilical vein endothelial cells (HUVECs) through ROS-mediated activation of ERK. These results suggest that the SPC-induced generation of ROS plays a crucial role in the cell death of endothelial cells through ERK-dependent pathway.     

Carnosic acid, a rosemary phenolic compound, induces apoptosis through reactive oxygen species-mediated p38 activation in human neuroblastoma IMR-32 cells

Carnosic acid (CA), a rosemary phenolic compound, has been shown to display anti-cancer activity. We examined the apoptotic effect of CA in human neuroblastoma IMR-32 cells and elucidated the role of the reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) associated with carcinogenesis. The result indicated that CA decreased the cell viability in a dose-dependent manner. Further investigation in IMR-32 cells revealed that cell apoptosis following CA treatment is the mechanism as confirmed by flow cytometry, hoechst 33258, and caspase-3/-9 and poly(ADP-ribose) polymerase (PARP) activation. Immunoblotting suggested a down-regulation of anti-apoptotic Bcl-2 protein in the CA-treated cells. In flow cytometric analysis, CA caused the generation of reactive oxygen species (ROS); however, pretreatment with the antioxidant N-acetylcysteine (NAC) attenuated the CA-induced generation of ROS and apoptosis. This effect was accompanied by increased activation of p38 and by decreased activation of extracellular signal-regulated kinase (ERK) as well as activation of c-Jun NH₂-terminal kinase (JNK). Moreover, NAC attenuated the CA-induced phosphorylation of p38. Silencing of p38 by siRNA gene knockdown reduced the CA-induced activation of caspase-3. In conclusion, ROS-mediated p38 MAPK activation plays a critical role in CA-induced apoptosis in IMR-32 cells.     

Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-kappaB activation in endothelial cells

Visfatin has recently been identified as a novel visceral adipokine which may be involved in obesity-related vascular disorders. However, it is not known whether visfatin directly contributes to endothelial dysfunction. Here, we investigated the effect of visfatin on vascular inflammation, a key step in a variety of vascular diseases. Visfatin induced leukocyte adhesion to endothelial cells and the aortic endothelium by induction of the cell adhesion molecules, ICAM-1 and VCAM-1. Promoter analysis revealed that visfatin-mediated induction of CAMs is mainly regulated by nuclear factor-kappaB (NF-kappaB). Visfatin stimulated IkappaBalpha phosphorylation, nuclear translocation of the p65 subunit of NF-kappaB, and NF-kappaB DNA binding activity in HMECs. Furthermore, visfatin increased ROS generation, and visfatin-induced CAMs expression and NF-kappaB activation were abrogated in the presence of the direct scavenger of ROS. Taken together, our results demonstrate that visfatin is a vascular inflammatory molecule that increases expression of the inflammatory CAMs, ICAM-1 and VCAM-1, through ROS-dependent NF-kappaB activation in endothelial cells.     

Panaxydol induces apoptosis through an increased intracellular calcium level,activation of JNK and p38 MAPK and NADPH oxidase-dependent generation of reactive oxygen species

Panaxydol, a polyacetylenic compound derived from Panax ginseng roots, has been shown to inhibit the growth of cancer cells. In this study, we demonstrated that panaxydol induced apoptosis preferentially in transformed cells with a minimal effect on non-transformed cells. Furthermore, panaxydol was shown to induce apoptosis through an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), activation of JNK and p38 MAPK, and generation of reactive oxygen species (ROS) initially by NADPH oxidase and then by mitochondria. Panaxydol-induced apoptosis was caspase-dependent and occurred through a mitochondrial pathway. ROS generation by NADPH oxidase was critical for panaxydol-induced apoptosis. Mitochondrial ROS production was also required, however, it appeared to be secondary to the ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the membrane translocation of regulatory p47^phox and p67^phox subunits and shown to be necessary for ROS generation by panaxydol treatment. Panaxydol triggered a rapid and sustained increase of [Ca²⁺]_i, which resulted in activation of JNK and p38 MAPK. JNK and p38 MAPK play a key role in activation of NADPH oxidase, since inhibition of their expression or activity abrogated membrane translocation of p47^phox and p67^phox subunits and ROS generation. In summary, these data indicate that panaxydol induces apoptosis preferentially in cancer cells, and the signaling mechanisms involve a [Ca²⁺]_i increase, JNK and p38 MAPK activation, and ROS generation through NADPH oxidase and mitochondria.     

The endogenous reactive oxygen species promote NF-kappaB activation by targeting on activation of NF-kappaB-inducing kinase in oral squamous carcinoma cells

Reactive oxygen species (ROS) could stimulate or inhibit NF-κB pathways. However, most results have been obtained on the basis of the exogenous ROS and the molecular target of ROS in NF-κB signalling pathways has remained unclear. Here, the oral squamous carcinoma (OSC) cells, with a mild difference in the endogenous ROS level, were used to investigate how slight fluctuation of the endogenous ROS regulates NF-κB activation. This study demonstrates that NF-κB-inducing kinase (NIK) is a critical target of the endogenous ROS in NF-κB pathways. The results indicate that ROS may function as a physiological signalling modulator on NF-κB signalling cascades through its ability to facilitate the activity of NIK and subsequent NF-κB transactivation. In addition, the data are useful to explain why the altered intracellular microenvironment related to redox state may influence biological behaviours of cancer cells.     

Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta: involvement of reactive oxygen species

AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS.     

Ebselen inhibits tumor necrosis factor-alpha-induced c-Jun N-terminal kinase activation and adhesion molecule expression in endothelial cells

Tumor necrosis factor-alpha (TNF-alpha) stimulates expression of endothelial cell (EC) genes that may promote atherosclerosis in part by an activation of mitogen-activated protein (MAP) kinases. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a selenoorganic compound, is effective for acute ischemic stroke; however, its effect on EC has not yet been elucidated. We examined the effect of ebselen on TNF-alpha-induced MAP kinase activation and adhesion molecule expression in cultured human umbilical vein endothelial cells (HUVEC). Extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 were rapidly and significantly activated by TNF-alpha in HUVEC. TNF-alpha-induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 were not affected. Apoptosis signal-regulated kinase 1 (ASK1) was suggested to be involved in TNF-alpha-induced JNK activation because transfection of kinase-inactive ASK1 inhibited TNF-alpha-induced JNK activation. Ebselen inhibited TNF-alpha-induced TNF receptor-associated factor 2 (TRAF2)-ASK1 complex formation and phosphorylation of stress-activated protein kinase ERK kinase 1 (SEK1), which is an upstream signaling molecule of JNK. Finally, TNF-alpha-induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) activation and resultant intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions were inhibited by ebselen. Specific inhibitors for JNK and NF-kappaB also inhibited TNF-alpha-induced ICAM-1 and VCAM-1 expressions in HUVEC. These findings suggest that ebselen prevents TNF-alpha-induced EC activation through the inhibition of TRAF2-ASK1-SEK1 signaling pathway, which leads to JNK activation. Inhibition of JNK by ebselen may imply its usefulness for the prevention of atherosclerosis relevant to EC activation.     

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.