Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis

Background Hyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes. Methods H9c2 cells (rat cardiomyoblast cells) and adult rat cardiomyocytes were incubated with Hcy and were analyzed for cell viability. Furthermore, we determined the effects of Hcy on intracellular mediators related to cell viability in cardiomyocytes, namely NOX2, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨ _m) and ATP concentrations. Results We found that incubation of H9c2 cells with 0.1 mM D,L-Hcy (= 60 μM l-Hcy) resulted in an increase of ΔΨ _m as well as ATP concentrations. 1.1 mM d,l-Hcy (= 460 μM l-Hcy) induced reversible flip-flop of the plasma membrane phospholipids, but not apoptosis. Incubation with 2.73 mM d,l-Hcy (= 1.18 mM l-Hcy) induced apoptosis and necrosis. This loss of cell viability was accompanied by a thread-to-grain transition of the mitochondrial reticulum, ATP depletion and nuclear NOX2 expression coinciding with ROS production as evident from the presence of nitrotyrosin residues. Notably, only at this concentration we found a significant increase in S-adenosylhomocysteine which is considered the primary culprit in HHC. Conclusion We found concentration-dependent effects of Hcy in cardiomyocytes, varying from induction of reversible flip-flop of the plasma membrane phospholipids, to apoptosis and necrosis.     

Homocysteine-Induced Apoptosis in Endothelial Cells Coincides With Nuclear NOX2 and Peri-nuclear NOX4 Activity

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01–2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47^phox expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ _m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ _m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47^phox, and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47^phox in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47^phox was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.     

Cdc42-dependent activation of NADPH oxidase is involved in ethanol-induced neuronal oxidative stress

It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47(phox) and p67(phox), the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47(phox) and p67(phox), which were prerequisites for NOX assembly and activation. Knocking down p47(phox) with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons.     

S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.     

Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate fluorescence] is detectable after a reduction in the extracellular osmolarity from 335 mosM (isotonic) to 300 mosM and reaches a maximal value after a reduction to 260 mosM. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (25 microM) but is unaffected by the nitric oxide synthase inhibitor N omega-nitro-l-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22 phox, a NOX4 isotype; p47 phox; and p67 phox (real-time PCR). Exposure to the Ca2+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22 phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca2+.     

NADPH oxidase-derived superoxide anion-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose

Hyperglycemia-induced generation of reactive oxygen species (ROS) can lead to cardiomyocyte apoptosis and cardiac dysfunction. However, the mechanism by which high glucose causes cardiomyocyte apoptosis is not clear. In this study, we investigated the signaling pathways involved in NADPH oxidase-derived ROS-induced apoptosis in cardiomyocytes under hyperglycemic conditions. H9c2 cells were treated with 5.5 or 33 mM glucose for 36 h. We found that 33 mM glucose resulted in a time-dependent increase in ROS generation as well as a time-dependent increase in protein expression of p22(phox), p47(phox), gp91(phox), phosphorylated IκB, c-Jun N-terminal kinase (JNK) and p38, as well as the nuclear translocation of NF-kB. Treatment with apocynin or diphenylene iodonium (DPI), NADPH oxidase inhibitors, resulted in reduced expression of p22(phox), p47(phox), gp91(phox), phosphorylated IκB, c-Jun N-terminal kinase (JNK) and p38. In addition, treatment with JNK and NF-kB siRNAs blocked the activity of caspase-3. Furthermore, treatment with JNK, but not p38, siRNA inhibited the glucose-induced activation of NF-κB. Similar results were obtained in neonatal cardiomyocytes exposed to high glucose concentrations. Therefore, we propose that NADPH oxidase-derived ROS-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose.     

Taurine prevents cardiomyocyte death by inhibiting NADPH oxidase-mediated calpain activation

Taurine has been shown to prevent cardiomyocyte apoptosis. This study investigated the effects of taurine on NADPH oxidase and calpain activation in mediating apoptosis in cardiomyocytes. Apoptosis was induced by norepinephrine (NE) in cultured adult rat ventricular cardiomyocytes. NE (5 microM) increased NADPH oxidase activation and reactive oxygen species (ROS) production and induced apoptosis. These effects of NE on cardiomyocytes were diminished by taurine (0.5 mg/kg) but not beta-alanine. Inhibition of gp91(phox)-NADPH oxidase or ROS production protected cardiomyocytes from apoptosis. NE also induced calpain-1 activation in cardiomyocytes. This effect of NE on calpain was abrogated by gp91(phox)-NADPH oxidase inhibition or ROS scavengers and was mimicked by H(2)O(2) (25 microM) in cardiomyocytes. Pharmacological inhibitors of calpain or overexpression of calpastatin, a specific calpain inhibitor, blocked calpain activation and prevented cardiomyocyte apoptosis during NE stimulation. Furthermore, taurine treatment inhibited NE- or H(2)O(2)-induced calpain activation in cardiomyocytes. In conclusion, NADPH oxidase induces calpain activation, leading to apoptosis in NE-induced cardiomyocytes. Taurine inhibits NADPH oxidase and calpain activation. Thus, inhibition of NADPH oxidase-mediated calpain activation may be an important mechanism for taurine's antiapoptotic action in cardiomyocytes.     

Angiopoietin-1 Requires Oxidant Signaling through p47phox to Promote Endothelial Barrier Defense

Background

Reactive oxygen species (ROS) are largely considered to be pathogenic to normal endothelial function in disease states such as sepsis. We hypothesized that Angiopoietin-1 (Angpt-1), an endogenous agonist of the endothelial-specific receptor, Tie-2, promotes barrier defense by activating NADPH oxidase (NOX) signaling.

Methods and Findings

Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied. As a result, there was attenuated ROS activity, disrupted junctional contacts, enhanced actin stress fiber accumulation, and induced gap formation between confluent HMVECs. All of these changes were associated with weakened barrier function. The ability of Angpt-1 to prevent identical changes induced by inflammatory permeability mediators, thrombin and lipopolysaccharides (LPS), was abrogated by p47phox knockdown. P47phox was required for Angpt-1 to activate Rac1 and inhibit mediator-induced activation of the small GTPase RhoA. Finally, Angpt-1 gene transfer prevented vascular leakage in wildtype mice exposed to systemically administered LPS, but not in p47phox knock out (p47^−/−) littermates.

Conclusions

These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation. More broadly, oxidants generated for signal transduction may have a barrier-promoting role in vascular endothelium.     

Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis

CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91phox (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47phox. gp91phox (Nox 2) and p47phox were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47phox, which was sensitive to inhibitors of sphingomyelinase and protein kinase Czeta (PKCzeta). These inhibitors and p47phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKCzeta are upstream events of the CD95L-induced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N-acetylcysteine, inhibitors of NADPH oxidase, PKCzeta, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor (EGFR), which became phosphorylated at Tyr845 and Tyr1173 but not at Tyr1045. Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fas-associated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKCzeta, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47phox, Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase- and PKCzeta-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation.     

Cordycepin inhibits albumin-induced epithelial-mesenchymal transition of renal tubular epithelial cells by reducing reactive oxygen species production

Albumin induced epithelial-mesenchymal transition (EMT) of renal tubular cells through reactive oxygen species (ROS) pathway plays an important role in tubulointerstitial fibrosis. Cordycepin (3 -deoxyadenosine), a potential antioxidant, was demonstrated to have various pharmacological effects and could inhibit EMT of some cells. However, the role of cordycepin on albumin-induced EMT in renal tubular cells (HK2) is unclear. In this study, we investigated the effect of cordycepin on albumin-induced EMT of HK2 cells and its mechanisms. HK-2 cells were exposed to bovine serum albumin with or without pretreatment with cordycepin. Results showed that albumin significantly induced EMT formation of HK-2 which associated with NADPH oxidase activation and intracellular ROS overproduction through increased Rac1 activity and expression of NOX4, p22phox and p47phox, while these effects were abolished in that pretreated with cordycepin. In conclusion, cordycepin could ameliorate albumin-induced EMT of HK2 cells by decreasing NADPH oxidase activity and inhibiting ROS production.     

质膜上的活性氧制造者--NOX家族

NADPH氧化酶特异存在于吞噬细胞质膜,能生成用于清除病原微生物的活性氧（reactive oxygen species,ROS）。NOX是NADPH氧化酶催化亚基gp91^phox的同源物,存在于多种非吞噬细胞。目前发现的NOX有NOX1、NOX3、NOX4及NOX5,虽然它们有一定的组织特异性,但与NADPH氧化酶一样均有催化生成ROS的能力。与吞噬细胞中NADPH氧化酶所制造的ROS不同,NOX所产生的ROS并不主要起细胞防御功能,而是作为第二信使,参与细胞增殖、分化、凋亡的调节。此外,NOX对血管生成及骨吸收也有一定的影响,同时还可作为氧感受器调节促红细胞生成素（EPO）的产生。     

Diazoxide Pretreatment Prevents Aβ1–42 Induced Oxidative Stress in Cholinergic Neurons Via Alleviating NOX2 Expression

The aggregation and accumulation of amyloid-β (Aβ) plays a significant role in the pathogenesis of Alzheimer’s disease. Aβ is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Diazoxide (DZ), a highly selective drug capable of opening mitochondrial ATP-sensitive potassium channels, has neuroprotective effects against neuronal cell death. However, the mechanism through which DZ protects cholinergic neurons against Aβ-induced oxidative injury is still unclear. The present study was designed to investigate the effects of DZ pretreatment against Aβ_1–42 induced oxidative damage and cytotoxicity. Through measures of DZ effects on Aβ_1–42 induced cellular damage, reactive oxygen species (ROS) and MDA generation and expressions of gp91phox and p47phox in cholinergic neurons, new insights into the neuroprotective mechanisms can be derived. Aβ_1–42 significantly decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide levels and increased ROS and MDA production; all effects were attenuated by pretreatment with DZ or diphenyleneiodonium chloride (a NOX2 inhibitor). Pretreatment with DZ also attenuated the upregulation of NOX2 subunits (gp91phox and p47phox) induced by Aβ_1–42. Since NOX2 is one of the main sources of free radicals, these results suggest that DZ can counteract Aβ_1–42 induced oxidative stress and associated cell death by reducing the level of ROS and MDA, in part, by alleviating NOX2 expression.     

Reactive oxygen species cause endothelial dysfunction in chronic flow overload

Although elevation of shear stress increases production of vascular reactive oxygen species (ROS), the role of ROS in chronic flow overload (CFO) has not been well investigated. We hypothesize that CFO increases ROS production mediated in part by NADPH oxidase, which leads to endothelial dysfunction. In six swine, CFO in carotid arteries was induced by contralateral ligation for 1 wk. In an additional group, six swine received apocynin (NADPH oxidase blocker and anti-oxidant) treatment in conjunction with CFO for 1 wk. The blood flow in carotid arteries increased from 189.2 ± 25.3 ml/min (control) to 369.6 ± 61.9 ml/min (CFO), and the arterial diameter increased by 8.6%. The expressions of endothelial nitric oxide synthase (eNOS), p22/p47(phox), and NOX2/NOX4 were upregulated. ROS production increased threefold in response to CFO. The endothelium-dependent vasorelaxation was compromised in the CFO group. Treatment with apocynin significantly reduced ROS production in the vessel wall, preserved endothelial function, and inhibited expressions of p22/p47phox and NOX2/NOX4. Although the process of CFO remodeling to restore the wall shear stress has been thought of as a physiological response, the present data implicate NADPH oxidase-produced ROS and eNOS uncoupling in endothelial dysfunction at 1 wk of CFO.     

NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells

Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca(2+) oscillation, the expression of apoptotic genes p53 and bax, and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a Ca(2+) chelator, BAPTA-AM, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22(phox) and p 47(phox) inhibited cerulein-induced ROS production, translocation of NADPH oxidase cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and the expression of apoptotic genes and apoptotic indices, as compared to the cells without treatment and those transfected with the corresponding sense oligonucleotides. These results indicate that NADPH oxidase may mediate ROS-induced apoptosis in pancreatic acinar cells in a Ca(2+)-dependent manner.     

Cooperative interaction between reactive oxygen species and Ca2+ signals contributes to angiotensin II-induced hypertrophy in adult rat cardiomyocytes

Reactive oxygen species (ROS) and Ca(2+) signals are closely associated with the pathogenesis of cardiac hypertrophy. However, the cause and effect of the two signals in cardiac hypertrophy remain to be clarified. We extend our recent report by investigating a potential interaction between ROS and Ca(2+) signals utilizing in vitro and in vivo angiotensin II (ANG II)-induced cardiac hypertrophy models. ANG II-induced initial Ca(2+) transients mediated by inositol trisphosphate (IP(3)) triggered initial ROS production in adult rat cardiomyocytes. The ROS generated by activation of the NAD(P)H oxidase complex via Rac1 in concert with Ca(2+) activates ADP-ribosyl cyclase to generate cyclic ADP-ribose (cADPR). This messenger-mediated Ca(2+) signal further augments ROS production, since 2,2'-dihydroxyazobenzene, an ADP-ribosyl cyclase inhibitor, or 8-Br-cADPR, an antagonistic analog of cADPR, abolished further ROS production. Data from short hairpin RNA (shRNA)-mediated knockdown of Akt1 and p47(phox) demonstrated that Akt1 is the upstream key molecule responsible for the initiation of Ca(2+) signal that activates p47(phox) to generate ROS in cardiomyocytes. Nuclear translocation of nuclear factor of activated T-cell in cardiomyocytes was significantly suppressed by treatment with NAD(P)H oxidase inhibitors as well as by shRNA against Akt1 and p47(phox). Our results suggest that in cardiomyocytes Ca(2+) and ROS messengers generated by ANG II amplify the initial signals in a cooperative manner, thereby leading to cardiac hypertrophy.     

Role of NADPH oxidase in the apoptotic death of cultured cerebellar granule neurons

Cerebellar granule neurons (CGN) cultured in a medium containing 25 mM KCl and treated with staurosporine (ST) or transferred to a medium with 5 mM KCl (K5) die apoptotically. CGN death is mediated by an increase in reactive oxygen species (ROS) production. When CGN are treated with antioxidants all apoptotic parameters and cell death are markedly diminished, showing a central role for ROS in this process. Recently, it has been suggested that a possible ROS source involved in cell death is a NADPH oxidase. In that regard, we found expression in CGN of the components of NADPH proteins, p40phox, p47phox and p67phox, and p22phox, as well as three homologues of the catalytic subunit of this complex, NOX1, 2, and 4. The inhibition of NADPH oxidase with diphenylene iodonium or 4-(2-aminoethyl)benzenesulfonyl fluoride significantly reduced ROS production, NADPH oxidase activity, all the apoptotic events, and cell death induced by both K5 and ST. We conclude that ROS could be an early signal of apoptotic neuronal death and that NADPH oxidase, including NOX1, 2, and/or 4, could have a central role in apoptotic death induced by different conditions in these neurons.     

NAD(P)H oxidase isoform Nox2 plays a prosurvival role in human leukaemia cells

The mechanism involved in the prosurvival effect of interleukin-3 on the human acute myeloid leukaemia cell line M07e is investigated. A decrease in intracellular reactive oxygen species (ROS) content, glucose transport activity and cell survival was observed in the presence of inhibitors of plasma membrane ROS sources, such as diphenylene iodonium and apocynin, and by small interference RNA for Nox2. Moreover, IL-3 incubation stimulated the synthesis of Nox2 cytosolic sub-unit p47phox and glucose transporter Glut1. Thus, the inhibition of ROS generation by Nox inhibitors stimulated apoptosis showing that ROS production, induced by IL-3 via Nox2, protects leukaemic cells from cell death. Also incubation with receptor tyrosine kinase inhibitors, such as anti-leukaemic drugs blocking the stem cell factor receptor (c-kit), showed similar effects, hinting that IL-3 transmodulates c-kit phosphorylation. These mechanisms may play an important role in acute myeloid leukaemia treatment, representing a novel therapeutic target.     

Blue Light Exposure Targets NADPH Oxidase to Plasma Membrane and Nucleus in Wheat Coleoptiles

Wheat coleoptile tips generate superoxide radical as a part of the phototropic response to blue light, but the source of this free radical generation is not known. We evaluated the presence and involvement of homologs of neutrophil NADPH oxidase (NOX), including gp91phox, p22phox, p67phox, p47phox, and p40phox, in wheat coleoptiles using Western blot analysis and immunofluorescence microscopy. Blue light augmented the expression levels of all these subunits and targeted NOX subunits onto the plasma membrane and to the nucleus. gp91phox, p22phox, p67phox, and p40phox showed entry into the nucleus and exhibited physical closeness with DNA. CuZnSOD was also present in the coleoptile tip, which also showed a blue-light-dependent elevation in expression. Superoxide production and phototropic response were both abrogated by DPIC and staurosporine, indicating their cause-and-effect relationship. We conclude that blue light mediates a phototropic response in wheat coleoptiles through modulation of expression of NOX and SOD as well as the translocation of NOX subunits onto the plasma membrane and nuclear membrane. Thus, this study provides a mechanistic explanation for superoxide production during the photoresponse in wheat coleoptiles.     

Advanced Glycation End Products Induce Human Corneal Epithelial Cells Apoptosis through Generation of Reactive Oxygen Species and Activation of JNK and p38 MAPK Pathways

Advanced Glycation End Products (AGEs) has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS) and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA) induced Human telomerase-immortalized corneal epithelial cells (HUCLs) apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE). AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC) or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.