Singlet oxygen-induced attenuation of growth factor signaling: possible role of ceramides

Singlet oxygen, an electronically excited form of molecular oxygen, is a primary mediator of the activation of stress-activated protein kinases elicited by ultraviolet A (UVA; 320-400 nm). Here, the effects of singlet oxygen (₁O₂) on the extracellular signal-regulated kinase (ERK) 1/2 and Akt/protein kinase B pathways were analyzed in human dermal fibroblasts. While basal ERK 1/2 phosphorylation was lowered in cells exposed to either ₁O₂, UVA or photodynamic treatment, Akt was moderately activated by photochemically generated ₁O₂ in a phosphoinositide 3-kinase (PI3K)-dependent fashion, resulting in the phosphorylation of glycogen synthase kinase-3 (GSK3). The activation of ERK 1/2 and Akt as induced by stimulation with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) was inhibited by ₁O₂ generated intracellularly upon photoexcitation of rose Bengal (RB). Photodynamic therapy (PDT)-induced apoptosis is known to be associated with increased formation of ceramides. Likewise, both ₁O₂ and UVA induced ceramide generation in human skin fibroblasts. The attenuation of EGF- and PDGF-induced activation of ERK 1/2 and Akt by ₁O₂ was mimicked by stimulation of fibroblasts with the cell-permeable C₂-ceramide. Interestingly, EGF-induced tyrosine phosphorylation of the EGF receptor was strongly attenuated by ₁O₂ but unimpaired by C₂-ceramide, implying that, although ceramide formation may mediate the above attenuation of ERK and Akt phosphorylation induced by ₁O₂, mechanisms beyond ceramide formation exist that mediate impairment of growth factor signaling by singlet oxygen. In summary, these data point to a novel mechanism of ₁O₂ toxicity: the known ₁O₂-induced activation of proapoptotic kinases such as JNK and p38 is paralleled by the prevention of activation of growth factor receptor-dependent signaling and of anti-apoptotic kinases, thus shifting the balance towards apoptosis.     

Singlet oxygen production in herbicide-treated photosystem II

Photo-generated reactive oxygen species in herbicide-treated photosystem II were investigated by spin-trapping. While the production of .OH and O2-* was herbicide-independent, 1O2 with a phenolic was twice that with a urea herbicide. This correlates with the reported influence of these herbicides on the redox properties of the semiquinone QA-* and fits with the hypothesis that 1O2 is produced by charge recombination reactions that are stimulated by herbicide binding and modulated by the nature of the herbicide. When phenolic herbicides are bound, charge recombination at the level of P+*Pheo-* is thermodynamically favoured forming a chlorophyll triplet and hence 1O2. With urea herbicides this pathway is less favourable.     

A Pathophysiologic Role for Epidermal Growth Factor Receptor in Pemphigus Acantholysis

The pemphigus family of autoimmune bullous disorders is characterized by autoantibody binding to desmoglein 1 and/or 3 (dsg1/dsg3). In this study we show that EGF receptor (EGFR) is activated following pemphigus vulgaris (PV) IgG treatment of primary human keratinocytes and that EGFR activation is downstream of p38 mitogen-activated protein kinase (p38). Inhibition of EGFR blocked PV IgG-triggered dsg3 endocytosis, keratin intermediate filament retraction, and loss of cell-cell adhesion in vitro. Significantly, inhibiting EGFR prevented PV IgG-induced blister formation in the passive transfer mouse model of pemphigus. These data demonstrate cross-talk between dsg3 and EGFR, that this cross-talk is regulated by p38, and that EGFR is a potential therapeutic target for pemphigus. Small-molecule inhibitors and monoclonal antibodies directed against EGFR are currently used to treat several types of solid tumors. This study provides the experimental rationale for investigating the use of EGFR inhibitors in pemphigus.     

Retinal damage by light: Possible implication of singlet oxygen

A new hypothesis is proposed in an attempt to explain the mechanism of the irreversible damage which can be induced in the retina by visible light. Upon illumination, retinal generates singlet oxygen and this reactive species can produce lipid peroxidation which in turn may induce membrane instability.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Sugar-induced tolerance to the herbicide atrazine in Arabidopsis seedlings involves activation of oxidative and xenobiotic stress responses

Exogenous sucrose confers to Arabidopsis seedlings a very high level of tolerance to the herbicide atrazine that cannot be ascribed to photoheterotrophic growth. Important differences of atrazine tolerance between sucrose and glucose treatments showed that activation of chloroplast biogenesis per se could not account for induced tolerance. Sucrose-induced acquisition of defence mechanisms was shown by the gene expression pattern of a chloroplastic iron superoxide dismutase and by enhancement of whole-cell glucose-6-phosphate dehydrogenase activity. Activation of these defence mechanisms depended on both soluble sugar and atrazine. Moreover, acquisition of sucrose protection was shown to unmask atrazine-induced gene expression, such as that of a cytosolic glutathione-S-transferase, which remained otherwise cryptic because of the lethal effects of atrazine in the absence of soluble sugars.     

Nonmuscle Myosin II Is Required for Internalization of the Epidermal Growth Factor Receptor and Modulation of Downstream Signaling

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) is an important process for regulating signal transduction, cellular dynamics, and cell-cell communication. Here, we demonstrate that nonmuscle myosin II (NM II) is required for the internalization of the EGFR and to trigger the EGFR-dependent activation of ERK and AKT. The EGFR was identified as a protein that interacts with NM II by co-immunoprecipitation and mass spectrometry analysis. This interaction requires both the regulatory light chain 20 (RLC20) of NM II and the kinase domain of the EGFR. Two paralogs of NM II, NM II-A, and NM II-B can act to internalize the EGFR, depending on the cell type and paralog content of the cell line. Loss (siRNA) or inhibition (25 μm blebbistatin) of NM II attenuates the internalization of the EGFR and impairs EGFR-dependent activation of ERK and AKT. Both internalization of the EGFR and downstream signaling to ERK and AKT can be partially restored in siRNA-treated cells by introduction of wild type (WT) GFP-NM II, but cannot be restored by motor mutant NM II. Taken together, these results suggest that NM II plays a role in the internalization of the EGFR and EGFR-mediated signaling pathways.     

Involvement of Singlet Oxygen in the Breakdown of Photosynthetic Pigments in the Leaves of Rice Seedling Exposed to Osmotic Stress and Light

The combined effects of osmotic stress and light on the generation of singlet oxygen (102) and its relation to the breakdown of photosynthetic pigments in leaves of hybrid rice (Oryza sativa L. subsp, indica cv. Shanyou 63) seedlings were studied under the condition of incubating the leaves with –0.8 MPa polyethylene glycol (PEG) solution. Under osmotic stress and increasing light intensity, the production of ¹O2 monitored as p-nitrosodimethylaniline (NDA) bleaching were increased in chloroplasts, degradation of chloro- phyll (Chl) and carotenoid (Car) were accelerated and Car loss preceded Chl causing a significant increase of Chl/Car ratio. A close correlation was observed between ¹O2 production and the contents of Chl, Car and malondialdehyde (MDA). Pretreatment with scavengers for ¹O2. such as β-carotene (β-Car) and histidine (His) reduced MDA content and retarded the degradation of photosynthetic pigments in rice leaves exposed to osmotic stress of -0.8 MPa and light intensity of 250 μmol · m- 2 · s-1, in contrast to that with photosensitizer riboflavin (RF). These results indicate that ¹O2 which generated in chloroplast from photosensitized reactions involving triplet Chl may play a significant role in the breakdown of photo- synthetic pigments and the preferential destruction of Car in the leaves under combined osmotic stress with light.     

A novel redox-based switch: LMW-PTP oxidation enhances Grb2 binding and leads to ERK activation

Low molecular weight-PTP has been reported as a redox-sensitive protein during both platelet-derived growth factor and integrin signalling. In response to oxidation the phosphatase undergoes a reversible inactivation, which in turn leads to the increase in tyrosine phosphorylation of its substrates and the properly executed anchorage-dependent proliferation program. Here, we report that an exogenous oxidative stress enhances LMW-PTP tyrosine phosphorylation, through oxidation/inactivation of the enzyme, thus preventing its auto-dephosphorylation activity. In particular, we observed a selective hyper-phosphorylation of Tyr132, that acts as a docking site for the adaptor protein Grb2. The redox-dependent enhancement of Grb2 recruitment to LMW-PTP ultimately leads to an improvement of ERK activation, likely triggering a prosurvival signal against the oxidant environment.     

Fibroblast Growth Factor Signaling Is Essential for Self-renewal of Dental Epithelial Stem Cells

A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.     

Singlet Oxygen-Trapping Reaction as a Method of 1O2 Detection: Role of Some Reducing Agents

The production of singlet oxygen by H₂O₂ disproportionation and via the oxidation of H₂O₂ by NaOCl in a neutral medium was monitored by spin trapping with 2,2,6,6 tetramethyl-4-piperidone (TMPone). The singlet oxygen formed in both reactions oxidized 2,2,6,6 tetramethyl-4-piperidone to give nitroxide radicals. However the production of nitroxide radicals was relatively small considering the concentrations of H₂O₂ and NaOCl used in the reaction systems. Addition of electron donating agents: ascorbate, Fe²⁺ and desferrioxamine leads to an increase in the production of nitroxide radicals. We assumed that a very slow step of the reaction sequence, the homolytic breaking of the O-O bond of N-hydroperoxide (formed as an intermediate product during the reaction of ¹O₂ with TMPone) could be responsible for the relatively small production of nitroxide radicals. Electron donating agents added to the reaction system probably raise the rate of the hydroperoxide decomposition by allowing a more rapid heterolytic cleavage of the O-O bond leading to a greater production of nitroxide radicals. The largest effect was observed in the presence of desferrioxamine. Its participation in this process is proved by the concomitant appearance of desferrioxamine nitroxide radicals. The results obtained demonstrate that the method proposed by several authors and tested in this study to detect singlet oxygen is not convenient for precise quantitative studies. The reactivity of TMPone towards O₂^-7HO₂' and 'OH has been also investigated. It has been found that both O₂^-7HO₂' and 'OH radicals formed in a phosphate buffer solution (pH 7.4, 37°C), respectively by a xanthine-oxidase/hypoxanthine system and via H₂O₂ UV irradiation, do not oxidize 2,2,6,6 tetramethyl-4-piperidone to nitroxide radicals.     

Protein Phosphotyrosine in Mouse Brain: Developmental Changes and Regulation by Epidermal Growth Factor, Type I Insulin-Like Growth Factor, and Insulin

Using antiphosphotyrosine antibodies, we have investigated protein phosphorylation in mouse brain during development in intact animals and in reaggregated cerebral cultures. Under basal conditions, in vivo and in vitro, the levels of two main phosphoproteins, of Mr 120,000 and 180,000 (pp180), increased with development, reaching a maximum in the early postnatal period and decreasing thereafter. In adult forebrain, pp180 was still highly phosphorylated, but it was not detected in cerebellum or in peripheral tissues. In reaggregated cortical cultures, epidermal growth factor (EGF), type I insulin-like growth factor (IGF-I), and insulin enhanced protein tyrosine phosphorylation of several proteins, which were specific for EGF or IGF-I/insulin. In highly enriched neuronal or astrocytic monolayer cultures, some proteins phosphorylated in basal conditions, or in response to EGF and IGF-I, were found in both types of culture, whereas others appeared cell type specific. In addition, in each cell type, some proteins were phosphorylated under the action of both growth factors. These results indicate that tyrosine protein phosphorylation is maximal in mouse brain during development and is regulated by growth factors in neurons as well as in astrocytes.     

表皮生长因子受体信号通路在维吾尔族妇女宫颈癌中的表达研究

目的:随着表皮生长因子受体(epidermal growth factor receptor,EGFR)靶点治疗药物在其它肿瘤治疗中的应用,能否在妇科肿瘤中应用并取得疗效,已经得到学者们的重视。但在临床应用之前,必须明确EGFR与妇科肿瘤发生发展的关系及其机制。因此,我们来探讨表皮生长因子受体EGFR信号通路在维吾尔族不同级别宫颈病变组织中的差异表达及其临床意义。方法:应用免疫组织化学方法检测EGFR、P-EGFR、ERK 1/2、P-ERK1/2在108例子宫颈癌,47例CIN,79例正常宫颈组织中的表达,并分析其与临床病理参数的关系。结果:EGFR在子宫颈癌、CIN及正常宫颈组织中的阳性表达率分别是74.10%、66%、27.80%(P0.01);P-EGFR在三组间的阳性表达率分别是55.60%、74.50%、20.30%(P0.01);ERK1/2在三组间的阳性表达率分别是60.10%、76.60%、25.30%(P0.01);P-ERK1/2在三组间的阳性表达率分别是44.40%、66%、24.10%(P0.01),其在宫颈癌中EGFR与与浸润深度、淋巴结转移有关;ERK与肿瘤的临床分期、远处转移有关,而且在宫颈癌中EGFR与P-EGFR、ERK 1/2、P-ERK1/2的表达成正相关(P0.05)。结论:EGFR、P-EGFR、ERK1/2、P-ERK1/2的表达与宫颈癌的发生、发展相关,促进宫颈癌的浸润和转移,抑制EGFR信号通路可能为多靶点联合治疗宫颈癌提供新的方向。     

Singlet Oxygen Production from the Reactions of Superoxide Ion in Aprotic Solvents: Implications for Hydrophobic Biochemistry

A number of reactions of superoxide ion in aprotic solvents have been reported to produce singlet oxygen. There is strong evidence for singlet oxygen generation from the reactions of superoxide ion with chlorine-containing halocarbons, bromine-containing halocarbons, hexafluorobenzene, diacylperoxides, lead tetraacetate. iodobenzene diacetate, cerium (IV) cation and tetranitromethane. Earlier studies reporting singlet oxygen generation from the reactions of superoxide ion with iodine. ferricenium ion, thianthrene cation radical, tris (N,N'-dioxobipyridyl) manganese (III) cation and di-μ-oxo-bis(phenanthrolinato manganese (IV)] cation need to be repeated because of the methods used to detect singlet oxygen had low specificity.

The results of studies of superoxide ion chemistry in aprotic solvents should not be uncritically extrapolated to hydrophobic biological microenvironments. such as the cell membrane.     

Opposite Regulation of Basic Fibroblast Growth Factor and Nerve Growth Factor Gene Expression in Rat Cortical Astrocytes Following Dexamethasone Treatment

Abstract: Growth factors are peptides that exert different activities in the CNS, supporting the survival of different cell populations and playing an important role in the maintenance of cell homeostasis. Much evidence has suggested that these molecules can protect neurons from degeneration induced by mechanical injury or excitotoxic stimuli. Different factors can contribute to the regulation of neurotrophic factor expression in the brain. Such mechanisms may therefore be important in the manipulation of the levels of these peptides in specific brain areas as a therapeutic intervention in acute and chronic neurodegenerative diseases. We have used a primary culture of rat cortical astrocytes to investigate the regulation of basic fibroblast growth factor (bFGF) gene expression in comparison with other neurotrophic molecules. Our results indicate that the glucocorticoid analogue dexamethasone markedly elevates bFGF mRNA levels but reduces the expression of nerve growth factor. The induction of bFGF was transient, as it peaked after 6 h and returned to basal levels within 24 h and was not blocked by coincubation of cycloheximide, thus indicating that it did not require de novo protein synthesis. This effect was also observed in vivo, as systemic injection of dexamethasone (1 or 10 mg/kg) produced a significant increase in the amount of bFGF mRNA in cerebral cortex and hippocampus. The effect we describe can contribute to the regulation of bFGF expression in the brain and may be important in relation to the protective effect exerted by this growth factor in different models of neuronal injury.     

Synergistic Signaling of Tumor Cell Invasiveness by Hepatocyte Growth Factor and Hypoxia

Hepatocyte growth factor (HGF) signaling promotes tumor invasiveness in renal cell carcinoma (RCC) and other cancers. In clear cell RCC, VHL loss generates pseudohypoxia that exacerbates HGF-driven invasion through β-catenin deregulation. Hypoxia also enhances HGF-driven invasiveness by papillary RCC cells, but in the absence of VHL, loss signaling integration involves three parallel routes: 1) hypoxia-induced reactive oxygen species production and decreased DUSP2 expression, leading to enhanced mitogen-activated protein kinase (MAPK) cascade activation; 2) reactive oxygen species-induced diacylglycerol production by phospholipase Cγ, leading to protein kinase C activation and increased protein phosphatase-2A activity, thereby suppressing HGF-induced Akt activation; and 3) a profound shift from HGF-enhanced, proliferation-oriented metabolism to autophagy-dependent invasion and suppression of proliferation. This tripartite signaling integration was not unique to RCC or HGF; in RCC cells, invasive synergy induced by the combination of hypoxia and epidermal growth factor occurred through the same mechanism, and in estrogen receptor-positive breast cancer cells, this mechanism was suppressed in the absence of estrogen. These results define the molecular basis of growth factor and hypoxia invasive synergy in VHL-competent papillary RCC cells, illustrate the plasticity of invasive and proliferative tumor cell states, and provide signaling profiles by which they may be predicted.     

Induction of Abiotic Stress Tolerance by Salicylic Acid Signaling

The role of salicylic acid (SA) as a key molecule in the signal transduction pathway of biotic stress responses has already been well described. Recent studies indicate that it also participates in the signaling of abiotic stresses. The application of exogenous SA could provide protection against several types of stresses such as high or low temperature, heavy metals, and so on. Although SA may also cause oxidative stress to plants, partially through the accumulation of hydrogen peroxide, the results published so far show that the preliminary treatment of plants with low concentrations of SA might have an acclimation-like effect, causing enhanced tolerance toward most kinds of abiotic stresses due primarily to enhanced antioxidative capacity. The effect of exogenous SA depends on numerous factors such as the species and developmental stage of the plant, the mode of application, and the concentration of SA and its endogenous level in the given plant. Recent results show that not only does exogenous SA application moderate stress effects, but abiotic stress factors may also alter the endogenous SA levels in the plant cells. This review compares the roles of SA during different abiotic stresses.     

Regulation by Interleukin-1 of Nerve Growth Factor Secretion and Nerve Growth Factor mRNA Expression in Rat Primary Astroglial Cultures

Primary cultures of neonatal rat cortical astrocytes contain low cellular levels (about 2 pg/mg of protein) of nerve growth factor (NGF), but secrete significant amounts of NGF into the culture medium (about 540 pg of NGF/mg of cell protein/38-h incubation). Incubation of astrocytes with interleukin-1 (IL-1) increased the cellular content of NGF and the amount secreted by about threefold. In comparison, cerebellar astrocytes secreted significant amounts of NGF, and the secretion was also stimulated by IL-1. The stimulatory action of IL-1 on astrocytes prepared from cortex was dose- and time-dependent. Concentrations of IL-1 causing half-maximal and maximal stimulation of NGF secretion were 1 and 10 U/ml, respectively). Maximal NGF secretion induced by IL-1 (10 U/ml) was seen following 38 h of incubation. The basal secretion of NGF was reduced by about 50% under Ca2(+)-free conditions; however, the percent stimulation of NGF secretion by IL-1 was the same in the absence or presence of Ca2+. The stimulatory action of IL-1 was specific, because other glial growth factors and cytokines were almost ineffective in stimulating NGF secretion from cortical astroglial cells. IL-1 treatment also increased cellular NGF mRNA content twofold. The results indicate that IL-1 specifically triggers a cascade of events, independent of cell growth, which regulate NGF mRNA content and NGF secretion by astrocytes.