Synergy of aldosterone and high salt induces vascular smooth muscle hypertrophy through up-regulation of NOX1

Aldosterone and excessive salt intake are obviously implicated in human arteriosclerosis. Aldosterone activates NADPH oxidase that induces superoxide production and cardiovascular cell hypertrophy. The activity of NADPH oxidase is influenced by the expression of its subunit, through which, vasoactive agents activate in the enzyme. Here, we show that aldosterone elicited overexpression of the NOX1 catalytic subunit of NADPH oxidase in the presence of high salt in A7r5 vascular smooth muscle cells. We also showed that NOX1 is a key subunit involved in physiological aldosterone-induced NADPH oxidase activation. Aldosterone dose-dependently increased NOX1 expression and NADPH activity, which subsequently caused superoxide over-production and A7r5 cell hypertrophy. However, aldosterone had little effect on any of NOX1, superoxide over-production and cell hypertrophy in NOX1 knock-down A7r5 cells. These results suggest that the aldosterone-induced effects are mainly generated through NOX1. Aldosterone-induced NOX1 over-expression was augmented by 145 mM sodium chloride, as compared with control medium containing 135 mM NaCl. However, NOX1 over-expression was not induced in the absence of aldosterone, even in the presence of 185 mM NaCl. The mineralocorticoid receptor antagonist, eplerenone, completely abolished NOX1 over-expression, indicating that aldosterone is essential for this process.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species

Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS). This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H(2)O(2), because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H(2)O(2), acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.     

NADPH oxidase is involved in prostaglandin F2alpha-induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF2alpha

Prostaglandin (PG) F(2alpha), one of the primary prostanoids generated in vascular tissue, is known to cause hypertrophy in vascular smooth muscle cells. To clarify the molecular mechanisms underlying PGF(2alpha)-induced hypertrophy, the involvement of reactive oxygen species was examined in a rat vascular smooth muscle cell line, A7r5. PGF(2alpha) and (+)-fluprostenol, a selective agonist of the PGF receptor, significantly increased intracellular O(2)(-) in A7r5. The PGF(2alpha)-induced O(2)(-) increase was suppressed by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase that has been reported to be the major source of O(2)(-) in vascular cells. The augmented synthesis of the protein induced by PGF(2alpha) or (+)-fluprostenol was suppressed in the presence of DPI. In PGF(2alpha) or (+)-fluprostenol-treated cells, a dose-dependent increase in the expression of NOX1, a homolog of the catalytic subunit of the phagocyte NADPH oxidase gp91(phox), was demonstrated by Northern blot analysis. Finally, depletion of NOX1 mRNA in the cells transfected with ribozymes targeted for three independent cleavage sites on the mRNA sequence significantly reduced the PGF(2alpha)-induced increase in protein synthesis. Taken together, these results suggest that hypertrophy of vascular smooth muscle cells caused by PGF(2alpha) is mediated by NOX1 induction and the resultant overproduction of O(2)(-) by NADPH oxidase.     

A novel superoxide-producing NAD(P)H oxidase in kidney

During phagocytosis, gp91(phox), the catalytic subunit of the phagocyte NADPH oxidase, becomes activated to produce superoxide, a precursor of microbicidal oxidants. Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events such as cell proliferation and oxygen sensing for erythropoiesis. Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4. The NOX4 protein of 578 amino acids exhibits 39% identity to gp91(phox) with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase. The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADH- and NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA. The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys. In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells. In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth. The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney.     

Bidirectional interactions between NOX2‐type NADPH oxidase and the F‐actin cytoskeleton in neuronal growth cones

NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)‐type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F‐actin content, retrograde F‐actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91^phox localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40^phox. p40^phox itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91^phox and p40^phox with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40^phox with NOX2/gp91^phox at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth.

     

NOX4 as an oxygen sensor to regulate TASK-1 activity

When oxygen sensing cells are excited by hypoxia, background K+ currents are inhibited. TASK-1, which is commonly expressed in oxygen sensing cells and makes a background K+ current, is inactivated by hypoxia. Thus TASK-1 is a candidate molecule responsible for hypoxic excitation. However, TASK-1 per se cannot sense oxygen and may require a regulatory protein that can. In the present study, we propose that the NADPH oxidase NOX4 functions as an oxygen-sensing partner and that it modulates the oxygen sensitivity of TASK-1. Confocal imaging revealed the co-localization of TASK-1 and NOX4 in the plasma membrane. In HEK293 cells expressing NOX4 endogenously, the activity of expressed TASK-1 was moderately inhibited by hypoxia, and this oxygen response was significantly augmented by NOX4. Moreover, the oxygen sensitivity of TASK-1 was abolished by NOX4 siRNA and NADPH oxidase inhibitors. These results suggest a novel function for NOX4 in the oxygen-dependent regulation of TASK-1 activity.     

Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells

The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.     

Essential role of NKCC1 in NGF-induced neurite outgrowth

The Na(+)/K(+)/2Cl(-) cotransporter (NKCC) mediates electroneutral transport of 2Cl(-) coupled with Na(+) and K(+) across the plasma membrane, and plays crucial roles in Cl(-) uptake into the cells, homeostasis of cellular Cl(-), and cell volume regulation. However, we have very limited information on the roles of ion transporters in neurite outgrowth in neuronal cells. In the present study, we report the role of NKCC1 (an isoform of NKCC) in NGF-induced neurite outgrowth of rat pheochromocytoma PC12D cells. The expression level of NKCC1 protein was increased by NGF treatment. Knock-down of NKCC1 by RNA interference (RNAi) drastically diminished the NGF-induced neurite outgrowth. Transfection of enhanced green fluorescent protein (EGFP)-tagged rat NKCC1 into cells for clarification of intracellular localization of NKCC1 revealed that the EGFP-rNKCC1 was mainly localized in the plasma membrane at growth cone during neurite outgrowth. These observations suggest that NKCC1 plays a fundamental role in NGF-induced neurite outgrowth of PC12D cells.     

CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation.     

PVN adenovirus-siRNA injections silencing either NOX2 or NOX4 attenuate aldosterone/NaCl-induced hypertension in mice

Mineralocorticoid excess increases superoxide production by activating NADPH oxidase (NOX), and intracerebroventricular infusions of NADPH oxidase inhibitors attenuate aldosterone (Aldo)/salt-induced hypertension. It has been hypothesized that increased reactive oxygen species (ROS) in the brain may be a key mechanism in the development of hypertension. The present study investigated the brain regional specificity of NADPH oxidase and the role of NOX2 and NOX4 NADPH oxidase subunits in the hypothalamic paraventricular nucleus (PVN) in Aldo/salt-induced hypertension. PVN injections of adenoviral vectors expressing small interfering (si)RNA targeting NOX2 (AdsiRNA-NOX2) or NOX4 (AdsiRNA-NOX4) mRNAs were used to knock down NOX2 and NOX4 proteins. Three days later, delivery of Aldo (0.2 mg·kg(-1)·day(-1) sc) via osmotic pump commenced and 1% NaCl was provided in place of water. PVN injections of either AdsiRNA-NOX2 or AdsiRNA-NOX4 significantly attenuated the development of Aldo/NaCl-induced hypertension. In an additional study, Aldo/salt-induced hypertension was also significantly attenuated in NOX2 (genomic) knockout mice compared with wild-type controls. When animals from both functional studies underwent ganglionic blockade, there was a reduced fall in blood pressure in the NOX2 and NOX4 knockdown/knockout mice. Western blot analyses of the PVN of siRNA-NOX2- or siRNA-NOX4-injected mice confirmed a marked reduction in the expression of NOX2 or NOX4 protein. In cultured PVN neurons, silencing either NOX2 or NOX4 protein production by culturing PVN cells with siRNA-NOX2 or siRNA-NOX4 attenuated Aldo-induced ROS. These data indicate that both NOX2 and NOX4 in the PVN contribute to elevated sympathetic activity and the hypertensivogenic actions induced by mineralocorticoid excess.     

NOX3, a superoxide-generating NADPH oxidase of the inner ear

Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase (NOX) 3, a member of the NOX/dual domain oxidase family of NADPH oxidases, is highly expressed in specific portions of the inner ear. As assessed by real-time PCR, NOX3 mRNA expression in the inner ear is at least 50-fold higher than in any other tissues where its expression has been observed (e.g. fetal kidney, brain, skull). Microdissection and in situ hybridization studies demonstrated that NOX3 is localized to the vestibular and cochlear sensory epithelia and to the spiral ganglions. Transfection of human embryonic kidney 293 cells with NOX3 revealed that it generates low levels of ROS on its own but produces high levels of ROS upon co-expression with cytoplasmic NOX subunits. NOX3-dependent superoxide production required a stimulus in the absence of subunits and upon co-expression with phagocyte NADPH oxidase subunits p47(phox) and p67(phox), but it was stimulus-independent upon co-expression with colon NADPH oxidase subunits NOX organizer 1 and NOX activator 1. Pre-incubation of NOX3-transfected human embryonic kidney 293 cells with the ototoxic drug cisplatin markedly enhanced superoxide production, in both the presence and the absence of subunits. Our data suggest that NOX3 is a relevant source of ROS generation in the cochlear and vestibular systems and that NOX3-dependent ROS generation might contribute to hearing loss and balance problems in response to ototoxic drugs.     

Involvement of H-Ras and reactive oxygen species in proinflammatory cytokine-induced matrix metalloproteinase-13 expression in human articular chondrocytes

Proinflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) enhance degradation of cartilage-specific, type II collagen by matrix metalloproteinase-13 (MMP-13). We investigated the previously unknown role of H-Ras and reactive oxygen species (ROS) in the cytokine induction of MMP-13 gene expression in human articular chondrocytes by using pharmacological inhibitors, RNA interference (RNAi) and antioxidants. Manumycin A, an inhibitor of H-Ras farnesylation by farnesyltransferase, suppressed IL-1β- and TNF-α-induced MMP-13 mRNA and protein expression. Small interfering RNA (siRNA)-mediated H-Ras silencing down-regulated MMP-13 mRNA and protein induction by IL-1β and TNF-α. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase/NOX) inhibitor, diphenyleneiodonium (DPI) suppressed cytokine-induced MMP-13 expression and superoxide production. Apocynin, another NOX inhibitor, also diminished MMP-13 induction. Deoxyglucose an antimetabolite of glucose metabolism reduced MMP-13 increase. Role of NOX-mediated ROS production was reaffirmed by the observation that the antioxidants, trolox, nordihydroguaiaretic acid (NDGA), quercetin and resveratrol downregulated cytokine-induced MMP-13 mRNA and protein expression. These results provide strong pharmacological and genetic evidence for the implication of H-Ras and NADPH oxidase-generated superoxide production in MMP-13 gene regulation by IL-1β and TNF-α. These proteins could be potentially targeted for therapeutic inhibition of MMP-13-driven cartilage erosion by using H-Ras and NOX inhibitors and antioxidants.     

Inhibition of nerve growth factor-induced B-50/GAP-43 expression by antisense oligomers interferes with neurite outgrowth of PC12 cells.

Substantial evidence has now been gathered for the involvement of B-50/GAP-43 in neuronal development and regeneration. The precise role of this protein, however, is still debated. In an earlier study, a linear correlation between NGF-induced neurite outgrowth and B-50/GAP-43 levels was observed in PC12 cells. To establish the involvement of B-50/GAP-43 expression in neurite outgrowth in these cells, we interfered with the expression by antisense oligomers and measured the outgrowth. In the present study, a B-50/GAP-43 antisense 5'-oligomer interfered both with the NGF-induced increase in B-50/GAP-43 and with neurite outgrowth, whereas an antisense 3'-oligomer was ineffective. We conclude, that in PC12 cells under normal conditions B-50/GAP-43 expression and neurite outgrowth are or become coupled upon NGF-induction, in contrast to the situation in PC12 clones with no or very low B-50/GAP-43 expression.     

Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels

Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPCs changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1-TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75(NTR)-IKK(2)-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK(2) dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent "molecular damper" maintaining a submaximal velocity of neurite extension.     

A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes

Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.