Co-regulation of constitutive nitric oxide synthases and NADPH oxidase by the small GTPase Rac

Nitric oxide (NO), generated by NO synthases (NOSs), has multifarious roles in signal transduction. Reactive oxygen species (ROS), generated by ubiquitous NADPH oxidases (NOXs), also participate in cellular signaling. However, the coordination of signals conveyed by NO and ROS is poorly understood. We show that the small GTPase Rac, a component of some NOXs, also interacts with and regulates the constitutively-expressed NOSs. Cellular NO and O(2)(-) production increase or decrease together following activation or inhibition of Rac, and Rac inhibition reveals transduction mechanisms that depend upon NO (vasodilation), ROS (actin polymerization) or both (cytoskeletal organization). Thus, signaling by NO and ROS may be coordinated through a common control element.     

iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease.     

Human endogenous retrovirus W env increases nitric oxide production and enhances the migration ability of microglia by regulating the expression of inducible nitric oxide synthase

Human endogenous retrovirus W env(HERV-W env) plays a critical role in many neuropsychological diseases such as schizophrenia and multiple sclerosis(MS). These diseases are accompanied by immunological reactions in the central nervous system(CNS). Microglia are important immunocytes in brain inflammation that can produce a gasotransmitter – nitric oxide(NO). NO not only plays a role in the function of neuronal cells but also participates in the pathogenesis of various neuropsychological diseases. In this study, we reported increased NO production in CHME-5 microglia cells after they were transfected with HERV-W env. Moreover, HERV-W env increased the expression and function of human inducible nitric oxide synthase(hi NOS) and enhanced the promoter activity of hi NOS. Microglial migration was also enhanced. These data revealed that HERV-W env might contribute to increase NO production and microglial migration ability in neuropsychological disorders by regulating the expression of inducible NOS. Results from this study might lead to the identification of novel targets for the treatment of neuropsychological diseases, including neuroinflammatory diseases, stroke, and neurodegenerative diseases.     

Endothelial nitric oxide synthase is segregated from caveolin-1 and localizes to the leading edge of migrating cells

The enzyme endothelial Nitric Oxide Synthase (eNOS) is involved in key physiological and pathological processes, including cell motility and apoptosis. It is widely believed that at the cell surface eNOS is localized in caveolae, where caveolin-1 negatively regulates its activity, however, there are still uncertainties on its intracellular distribution. Here, we applied high resolution confocal microscopy to investigate the surface distribution of eNOS in transfected HeLa cells and in human umbilical vein endothelial cells (HUVEC) endogenously expressing the enzyme. In confluent and non-confluent HUVEC and HeLa cells, we failed to detect substantial colocalization between eNOS and caveolin-1 at the cell surface. Instead, in non-confluent cells, eNOS was concentrated in ruffles and at the leading edge of migrating cells, colocalizing with actin filaments and with the raft marker ganglioside G(M1), and well segregated from caveolin-1, which was restricted to the posterior region of the cells. Treatments that disrupted microfilaments caused loss of eNOS from the cell surface and decreased Ca(2+)-stimulated activity, suggesting a role of the cytoskeleton in the localization and function of the enzyme. Our results provide a morphological correlate for the role of eNOS in cell migration and raise questions on the site of interaction between eNOS and caveolin-1.     

Electrochemical study of the intracellular transduction of vascular endothelial growth factor induced nitric oxide synthase activity using a multi-channel biocompatible microelectrode array

Background

Nitric oxide (NO) plays a major role in physiology as a biological mediator. NO has been identified in nervous, immune and vascular systems and is a critical parameter in numerous pathologies, such as cancer. This article describes the electrochemical biomeasurements of NO synthase (NOS) activity from cultured endothelial cells using a multiple microelectrode array.

Methods

Firstly, the effect of biocompatible fibronectin coating on electrochemical measurements was investigated. Secondly, endothelial cells were deposited on the fibronectin coated sensor and NO release was triggered with vascular endothelial growth factor (VEGF). N^G-nitro-l-arginine methyl ester (L-NAME) was used as an inhibitor of NO production, and different kinase blockers were investigated. Change in NOS activity was quantified using differential pulse voltammetry before and after addition of VEGF.

Results

Our results show that carefully applied layers of fibronectin have a very limited effect on electrochemistry and that VEGF induces an increase in NOS activity that is mainly mediated through the phosphatidylinositol 3 kinase (PI-3), and not by the extracellular signal-regulated kinases 1/2. Results obtained using electrochemical sensors were supported by wound healing assay demonstrating the critical role of phosphatidylinositol 3 kinase and extracellular signal-regulated kinases 1/2 for angiogenesis.

Conclusion

Electrochemical study of the intracellular transduction of the VEGF signal leading to NO synthesis was achieved, showing the critical role of PI-3 kinase.

General significance

This study presents an electrochemical sensor allowing measurements of NOS activity in cell cultures and tissue samples.     

Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis

Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN = 116 ± 10 s⁻¹ at 1 μM NO, T = 37 °C). The activity is [O₂]-dependent and characterized by an apparent K_M,O2 = 22 ± 7 μM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with ?5 mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.     

Proteomic analysis of ribosomes: translational control of mRNA populations by glycogen synthase GYS1

Ribosomes exist as a heterogenous pool of macromolecular complexes composed of ribosomal RNA molecules, ribosomal proteins, and numerous associated “nonribosomal” proteins. To identify nonribosomal proteins that may modulate ribosome activity, we examined the composition of translationally active and inactive ribosomes using a proteomic multidimensional protein identification technology. Notably, the phosphorylated isoform of glycogen synthase, glycogen synthase 1 (GYS1), was preferentially associated with elongating ribosomes. Depletion of GYS1 affected the translation of a subset of cellular mRNAs, some of which encode proteins that modulate protein biosynthesis. These findings argue that GYS1 abundance, by virtue of its ribosomal association, provides a feedback loop between the energy state of the cells and the translation machinery.     

Lipopolysaccharide (LPS) Stimulates the Production of Tumor Necrosis Factor (TNF)-α and Expression of Inducible Nitric Oxide Synthase (iNOS) by Osteoclasts (OCL) in Murine Bone Marrow Cell Culture

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N^G-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.     

MicroRNA-326 suppresses iNOS expression and promotes autophagy of dopaminergic neurons through the JNK signaling by targeting XBP1 in a mouse model of Parkinson's disease

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons, and substantia nigra is primarily one of the damaged brain regions. Evidence indicates that microRNAs (miRNAs) is involved in the pathophysiology of this disease. The present study aimed to investigate the biological function of miR-326 in PD through the JNK signaling pathway by targeting X-box binding protein 1 (XBP1). After liposome complexes were prepared, healthy male C57BL/6 mice were selected to construct a mouse model of PD. The targeting relationship between miR-326 and XBP1 was confirmed. The expression of miR-326 and XBP1 was measured in PD mice, and gain- and loss-function assay was conducted to examine the regulatory effect of miR-326 and XBP1 on inducible nitric oxide synthase (iNOS) expression and autophagy of dopaminergic neurons of PD mice. Mice treated with miR-326 mimic and siRNA-XBP1 showed increased traction test scores, activation of autophagy, expression of LC3-II, c-Jun, and p-α-Syn, but diminished climbing time and expressions of iNOS, α-Syn, and p-c-Jun. The siRNA-XBP1 treatment could reverse the effect of miR-326 inhibitor on PD mice. Overexpression of miR-326 inhibits iNOS expression and promotes autophagy of dopaminergic neurons through JNK signaling by targeting XBP1.     

The dual behavior of heat shock protein 70 and asymmetric dimethylarginine in relation to serum CRP levels in type 2 diabetes

Background

Experimental evidence suggests that heat shock proteins (HSP) and asymmetric dimethylarginine (ADMA) are induced in the state of chronic inflammation and stress conditions. They are both inhibitors of nitric oxide synthase (NOS). The aim of this study was to evaluate the correlation between ADMA and HSP70, in patients with type 2 diabetes with respect to serum levels of C reactive protein (CRP).

Methods

We quantified serum HSP70, ADMA and CRP in 80 newly-diagnosed patients with type 2 diabetes plus 80 age-, sex and BMI-matched healthy controls. The patients and controls were also stratified into groups of high and low CRP levels (cut-point: 2.5 mg/ml).

Results

Patients with type 2 diabetes had significantly higher serum HSP70 (0.52 [0.51–0.66] vs. 0.27 [0.26–0.36], p < 0.001), ADMA (0.86 [0.81–0.92] vs. 0.72 [0.71–0.85], p < 0.05) and CRP (2.9 [1.7–3.4] vs. 1.6[1.2–2.3], p < 0.05) compared with healthy controls. Serum HSP70 and ADMA levels were significantly correlated in patients with high CRP levels (r = 0.89, p < 0.01), whereas there were no correlation in patients with low CRP (r = − 0.37, p = 0.07) and controls. This correlation was significant (r = 0.77, p < 0.001) in patients with high CRP and also in patients with low CRP levels (r = − 0.51, p < 0.05), after multiple adjustments for LDL and HDL levels.

Discussion

We showed that, in a state of high inflammation; serum levels of ADMA parallel the HSP70 levels. However in low inflammation, they are negatively correlated. The duality in HSP70 and ADMA correlation may be related to the duality of NOS function in low and high CRP levels.     

Cobalt protoporphyrin inhibition of lipopolysaccharide or lipoteichoic acid-induced nitric oxide production via blocking c-Jun N-terminal kinase activation and nitric oxide enzyme activity

In the present study, low doses (0.5, 1, and 2 μM) of cobalt protoporphyrin (CoPP), but not ferric protoporphyrin (FePP) or tin protoporphyrin (SnPP), significantly inhibited lipopolysaccharide (LPS) or lipoteichoic acid (LTA)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages under serum-free conditions. IC₅₀ values of CoPP inhibition of NO and iNOS protein individually induced by LPS and LTA were around 0.25 and 1.7 μM, respectively. This suggests that CoPP is more sensitive at inhibiting NO production than iNOS protein in response to separate LPS and LTA stimulation. NO inhibition and HO-1 induction by CoPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). Decreasing iNOS/NO production and increasing HO-1 protein by CoPP were observed with CoPP pretreatment, CoPP co-treatment, and CoPP post-treatment with LPS and LTA stimulation. LPS- and LTA-induced NOS/NO productions were significantly suppressed by the JNK inhibitor, SP600125, but not by the ERK inhibitor, PD98059, through a reduction in JNK protein phosphorylation. Transfection of a dominant negative JNK plasmid inhibited LPS- and LTA-induced iNOS/NO production and JNK protein phosphorylation, suggesting that JNK activation is involved in LPS- and LTA-induced iNOS/NO production. Additionally, CoPP inhibition of LPS- and LTA-induced JNK, but not ERK, protein phosphorylation was identified in RAW264.7 cells. Furthermore, CoPP significantly reduced NO production in a cell-mediated, but not cell-free, iNOS enzyme activity assay accompanied by HO-1 induction. However, attenuation of HO-1 protein stimulated by CoPP via transfection of HO-1 siRNA did not affect NO's inhibition of CoPP against LPS stimulation. CoPP effectively suppressing LPS- and LTA-induced iNOS/NO production through blocking JNK activation and iNOS enzyme activity via a HO-1 independent manner is first demonstrated herein.     

Group IIA and group V secretory phospholipase A2: quantitative analysis of expression and secretion and determination of the localization and routing in rat mesangial cells

Mesangial cells can be induced to express group IIA and group V secretory phospholipase A₂ (sPLA₂) at the mRNA level and at the protein level. In this report we quantitatively analyze the expression of both proteins in stimulated cells by Western blot techniques. We found that 75–80% of the total amount of synthesized group IIA sPLA₂ was secreted. The synthesized group V sPLA₂, however, was present almost exclusively intracellularly. The amount of group V present in the cell was comparable to the intracellular amount of group IIA sPLA₂. We furthermore studied the localization and routing of both proteins. Using fusion proteins of the group IIA or group V pre-sPLA₂ with green fluorescent protein it was established that both presequences are able to direct the proteins to the Golgi system. In immunofluorescence studies group V sPLA₂ expressed by rat mesangial cells was located in a punctate pattern in the cytosol with an enrichment near the nucleus. Immunofluorescent confocal laser scanning microscopy revealed that the group V and IIA sPLA₂ show partial colocalization in a Golgi-like structure in the inner part in the cell, but no colocalization was seen in the vesicles in the cytoplasm. The images also showed that group IIA sPLA₂ was located throughout the cell while group V was mainly present in the inner part of the cell. After treatment of the cells with brefeldin A or monensin the group IIA enzyme could no longer be detected, while group V sPLA₂ was still present although its localization was somewhat dependent on the treatment. Collectively, these results indicate that the two enzymes differ in both localization and routing in the cell, which underscores the hypothesis that the enzymes might have different functions.     

Acoustic overstimulation activates 5'-AMP-activated protein kinase through a temporary decrease in ATP level in the cochlear spiral ligament prior to permanent hearing loss in mice

Inner ear disorders are known to be elicited by mitochondrial dysfunction, which decreases the ATP level in the inner ear. 5′-AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by metabolic stress and by an increase in the AMP/ATP ratio. To elucidate the involvement of AMPK-derived signals in noise-induced hearing loss, we investigated whether in vivo acoustic overstimulation would activate AMPK in the cochlea of mice. Std-ddY mice were exposed to 8 kHz octave band noise at a 90-, 110- or 120-dB sound pressure level (SPL) for 2 h. Exposure to the noise at 110 or 120 dB SPL produced outer hair cell death in the organ of Corti and permanent hearing loss. Exposure to the noise at 120-dB SPL elevated the level of the phospho-AMPK α-subunit (p-AMPKα), without affecting the protein level of this subunit, immediately and at 12-h post-exposure in the lateral wall structures including the spiral ligament and stria vascularis. In the hair cells and spiral ganglion cells, no marked change in the level of p-AMPKα was observed at any time post-exposure. The level of phospho-c-Jun N-terminal kinase (p-JNK) was increased in the lateral wall structures at 2- to 4-h post-exposure at 120 dB SPL. Noise exposure significantly, but temporarily, decreased the ATP level in the spiral ligament, in an SPL-dependent manner at 110 dB and above. Likewise, elevation of p-AMPKα and p-JNK levels was also observed in the lateral wall structures post-exposure to noise at an SPL of 110 dB and above. Taken together, our data suggest that AMPK and JNK were activated by ATP depletion in the cochlear spiral ligament prior to permanent hearing loss induced by in vivo acoustic overstimulation.     

Leishmania donovani: inhibition of phagosomal maturation is rescued by nitric oxide in macrophages

Leishmania donovani promastigotes, the causative agent of visceral leishmaniasis, survive inside macrophages by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for parasite survival. LPG has several detrimental effects on macrophage function, including inhibition of periphagosomal filamentous actin (F-actin) breakdown during phagosomal maturation. However, in RAW 264.7 macrophages pre-stimulated with lipopolysaccharide (LPS) and interferon gamma (IFNgamma), known to up-regulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, L. donovani promastigotes are unable to inhibit periphagosomal F-actin breakdown and phagosomal maturation proceeds normally. Moreover, the iNOS inhibitor aminoguanidine, blocked the positive effects of LPS/IFNgamma suggesting that NO is a key player in F-actin remodeling. In conclusion, production of NO by stimulated macrophages seems to allow phagosomal maturation following uptake of L. donovani promastigotes, suggesting a novel mechanism whereby NO facilitates killing of an intracellular pathogen.     

Activation of cultured astrocytes by amphotericin B: Stimulation of NO and cytokines production and changes in neurotrophic factors production

Amphotericin B (AmB) is a polyene antibiotic and reported to be one of a few reagents having therapeutic effects on prion diseases, such as the delay in the appearing of the clinical signs and the prolongation of the survival time. In prion diseases, glial cells have been suggested to play important roles by proliferating and producing various factors such as nitric oxide, proinflammatory cytokines, and neurotrophic factors. However, the therapeutic mechanism of AmB on prion diseases remains elusive. We have previously reported that AmB changed the expression of neurotoxic and neurotrophic factors in microglia (Motoyoshi et al., 2008, Neurochem. Int. 52, 1290–1296). In the present study, we examined the effects of AmB on cellular functions of rat cultured astrocytes. We found that AmB could activate astrocytes to produce nitric oxide via inducible nitric oxide synthase induction. AmB also induced mRNA expression of interleukin-1β and tumor necrosis factor-α, and productions of their proteins in astrocytes. Moreover, AmB changed levels of neurotrophic factor mRNAs and proteins. Among three neurotrophic factors examined here, neurotrophin-3 mRNA expression and its protein production in the cells were down-regulated by AmB stimulation. On the other hand, AmB significantly enhanced the amounts of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor proteins in the cells and the medium. These results suggest that AmB might show therapeutic effects on prion diseases by controlling the expression and production of such mediators in astrocytes.     

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.     

Characterization of the Interaction between Diferric Transferrin and Transferrin Receptor 2 by Functional Assays and Atomic Force Microscopy

Transferrin receptor 2 (TfR2), a homologue of the classical transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein, but the β form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2α, we expressed the protein with FLAG tagging in transferrin-receptor-deficient Chinese hamster ovary cells. The association constant for the binding of diferric transferrin (Tf) to TfR2α is 5.6 × 10⁶ M^− 1, which is about 50 times lower than that for the binding of Tf to TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2α without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2α with Tf was further investigated using atomic force microscopy, a powerful tool used for investigating the interaction between a ligand and its receptor at the single-molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2α and TfR1, with Tf-TfR1 unbinding characterized by two energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2α by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2α may help account for the different physiological roles of the two receptors.