Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch.

Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last ß-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pKa value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pH-sensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals.

Nicotinamide phosphoribosyltransferase secreted from microglia via exosome during ischemic injury.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the salvage pathway of nicotinamide adenine dinucleotide synthesis. NAMPT can also be secreted and functions as a cytokine. We have previously shown that in the brain, NAMPT expression and secretion can be induced in microglia upon neuroinflammation and injury. Yet the mechanism for NAMPT secretion remains unclear. Here we show that NAMPT can be actively secreted from microglia upon the treatment of ischemia-like injury - oxygen-glucose deprivation and recovery (OGD/R). We confirmed that classical ER-Golgi pathway is not involved in NAMPT secretion. NAMPT secretion was further enhanced by ATP, and the secretion was mediated by P2X7 receptor and by intracellular Ca2+ . Importantly, we found that phospholipase D inhibitor, n-butanol, phospholipase D siRNA, and wortmannin significantly decreased OGD/R-induced and ATP-enhanced release of NAMPT in microglia. After excluding the mechanisms of involving secretory autophagy, endosomes, and secretory lysosome, we have concluded that microglial NAMPT is secreted mainly via exosome. Immune-electron microscopy identifies NAMPT in extracellular vesicles with the size and morphology characteristic of exosome. With the vesicles harvested by ultra-centrifugation, exosomal NAMPT is further confirmed by Western blotting analysis. Intriguingly, the amount of NAMPT relative to exosomal protein markers remains unchanged upon the treatment of OGD/R, suggesting a constant load of exosomal NAMPT in microglia. Taken together, we have identified NAMPT is actively secreted via exosome from microglia during neuroinflammation of ischemic injury.

Nicotinamide ribose ameliorates cognitive impairment of aged and Alzheimer's disease model mice.

Nicotinamide adenine dinucleotide (NAD) supplementation to repair the disabled mitochondria is a promising strategy for the treatment of Alzheimer's disease (AD) and other dementia. Nicotinamide ribose (NR) is a safe NAD precursor with high oral bioavailability, and has beneficial effects on aging. Here, we applied NR supplied food (2.5 g/kg food) to APP/PS1 transgenic AD model mice and aged mice for 3 months. Cognitive function, locomotor activity and anxiety level were assessed by standard behavioral tests. The change of body weight, the activation of microglia and astrocytes, the accumulation of Aß and the level of serum nicotinamide phosphoribosyltransferase (NAMPT) were determined for the evaluation of pathological processes. We found that NR supplementation improved the short-term spatial memory of aged mice, and the contextual fear memory of AD mice. Moreover, NR supplementation inhibited the activation of astrocytes and the elevation of serum NAMPT of aged mice. For AD model mice, NR supplementation inhibited the accumulation of Aß and the migration of astrocyte to Aß. In addition, NR supplementation inhibit the body weight gain of aged and APP/PS1 mice. Thus, NR has selective benefits for both AD and aged mice, and the oral uptake of NR can be used to prevent the progression of dementia.

Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques.

A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate. Using K63-linked diubiquitin, we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray scattering (SAXS), and smFRET techniques. CXMS allows the modeling of protein conformational states that are alternatives to the crystal structure. SAXS provides ensemble-averaged low-resolution shape information. Importantly, smFRET affords state-specific populations, and the FRET distances validate the ensemble structures obtained by refining against CXMS and SAXS restraints. Together, the integrative use of bulk and single-molecule techniques affords better insight into protein dynamics and shall be widely implemented in structural biology.

Nicotinamide phosphoribosyltransferase inhibitor APO866 induces C6 glioblastoma cell death via autophagy.

APO866 is a potent inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), and inhibits nicotinamide adenine dinucleotide (NAD) synthesis. Our previous study showed that APO866 inhibits the proliferation of C6 glioblastoma cells, but failed to induce apoptosis. Since APO866 inhibits cellular metabolism and such metabolic stress is closely related with autophagy, thus we determined whether APO866 can induce autophagy in C6 glioblastoma cells and whether the autophagy induced by APO866 is pro-death or pro-survival. Using LC3 immunofluorescence imaging and transmission electron microscopy detection, we found that APO866 at 1-100 nM induced autophagy in C6 glioblastoma cells. APO866 at 1 nM mainly induced initial autophagic vacuoles. Whereas APO866 at 100 nM induced degrading autophagic vacuoles, as well as induced nuclei malformation and mitochondria swelling. In addition, APO866 concentration-dependently decreased the cell viability of C6 glioblastoma cells, and this effect was attenuated by autophagy inhibitors, including 3-methyladenine and LY294002. APO866 concentration-dependently decreased intracellular NAD level. Interestingly, APO866 at 1 nM slightly decreased intracellular NAD level, but dramatically increased autophagy-positive cells. The dramatical cell viability decreasing required the decreasing of intracellular NAD level to a very low threshold. Thus, our results indicated that APO866 induced pro-death autophagy in C6 glioblastoma cells by decreasing intracellular NAD, and low concentration of APO866 can be used as an autophagy inducer in autophagic-death sensitive glioblastoma.

[Research progress on epithelial-mesenchymal transition in cancer recurrence and metastasis].

Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their morphology and function and gradually transformed into mesenchymal-like cells. It is considered that EMT is the main cause for tumor recurrence and metastasis. Many factors are involved in the regulation of EMT, such as E-cadherin, transforming growth factor-ß, Wnt signaling pathway, microRNA and EMT-related transcription factors. This article reviews the research progress on EMT and the involved mechanisms, and thus to provide a new perspective on cancer therapy in the future.

Cysteinyl leukotriene receptor 1 mediates LTD4-induced activation of mouse microglial cells in vitro.

AIM: To investigate the roles of cysteinyl leukotriene receptors CysLT1R and CysLT2R in leukotriene D4 (LTD4)-induced activation of microglial cells in vitro. METHODS: Mouse microglial cell line BV2 was transfected with pcDNA3.1(+)-hCysLT1R or pcDNA3.1(+)-hCysLT2R. The expression of relevant mRNAs and proteins in the cells was detected using RT-PCR and Western blotting, respectively. Phagocytosis was determined with flow cytometry analysis. The release of interleukin-1ß (IL-1ß) from the cells was measured using an ELISA assay. RESULTS: The expression of CysLT1R or CysLT2R was considerably increased in the transfected BV2 cells, and the receptors were mainly distributed in the plasma membrane and cytosol. Treatment of the cells expressing CysLT1R or CysLT2R with CysLT receptor agonist LTD4 (0.1-100 nmol/L) concentration-dependently enhanced the phagocytosis, and increased mRNA expression and release of IL-1ß. Moreover, the responses of hCysLT1R-BV2 cells to LTD4 were significantly larger than those of hCysLT2R-BV2 or WT-BV2 cells. Pretreatment of hCysLT1R-BV2 cells with the selective CysLT1R antagonist montelukast (1 µmol/L) significantly blocked LTD4-induced phagocytosis as well as the mRNA expression and release of IL-1ß, whereas the selective CysLT2R antagonist HAMI 3379 (1 µmol/L) had no such effects. CONCLUSION: CysLT1R mediates LTD4-induced activation of BV2 cells, suggesting that CysLT1R antagonists may exert anti-inflammatory activity in brain diseases.

Growth cones as soft and weak force generators.

Many biochemical processes in the growth cone finally target its biomechanical properties, such as stiffness and force generation, and thus permit and control growth cone movement. Despite the immense progress in our understanding of biochemical processes regulating neuronal growth, growth cone biomechanics remains poorly understood. Here, we combine different experimental approaches to measure the structural and mechanical properties of a growth cone and to simultaneously determine its actin dynamics and traction force generation. Using fundamental physical relations, we exploited these measurements to determine the internal forces generated by the actin cytoskeleton in the lamellipodium. We found that, at timescales longer than the viscoelastic relaxation time of τ = 8.5 ± 0.5 sec, growth cones show liquid-like characteristics, whereas at shorter time scales they behaved elastically with a surprisingly low elastic modulus of E = 106 ± 21 Pa. Considering the growth cone's mechanical properties and retrograde actin flow, we determined the internal stress to be on the order of 30 pN per µm(2). Traction force measurements confirmed these values. Hence, our results indicate that growth cones are particularly soft and weak structures that may be very sensitive to the mechanical properties of their environment.

[Research advances on the roles of nicotinamide phosphoribosyltransferase in inflammation].

Nicotinamide phosphoribosyltransferase (Nampt) is also called visfatin or pre-B-cell colony-enhancing factor. The functions of Nampt have been reported as a cytokine, an adipokine and the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. As a pleiotropic multifunctional protein, Nampt is involved in a variety of physiological and pathological conditions including innate immunity, metabolic disorders, and stress; and Nampt also participates in inflammatory disorders such as acute lung injury, atherosclerosis, myocardial infarct, obesity, type 2 diabetes, and rheumatoid arthritis. The studies indicate that Nampt might be a potential target for pharmacological intervention against inflammatory diseases. We review research advances on the roles of Nampt in inflammation.

[Effect of histone deacetylase inhibitor NL101 on rat neurons].

OBJECTIVE: To investigate the protective effect of histone deacetylase inhibitor NL101 on L-homocysteine (HCA)-induced toxicity in rat neurons, and the toxic effect on normal rat neurons. METHODS: In the presence of NL101 at various concentrations, HCA (5 mmol/L)-induced changes in cell density, necrosis, and viability were determined in the mixed cultures of rat cortical cells and the primary cultures of rat neurons. The direct effect of NL101 on primary neurons was also observed in the absence of HCA. Histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was used as the control. After the treatments, cell viability, the density, and morphology of neurons and glial cells, and cell necrosis were determined. RESULTS: In the mixed cultures of cortical cells, NL101 had no effect on HCA (5 mmol/L)-induced cell number reduction at 0.001-10µmol/L; however, it significantly attenuated necrosis at 1-10 µmol/L, and increased neuronal number at 1 µmol/L. NL101 had no effect on the mixed cortical cells in the absence of HCA. In the primary neurons, NL101 reduced neuronal viability and mildly increased necrosis at 1-10 µmol/L in the absence of HCA, while it significantly attenuated HCA-induced neuronal viability reduction at 0.01-10 µmol/L and reduced neuronal necrosis at 1-10 µmol/L. The effects of NL101 were apparently similar to those of SAHA. CONCLUSION: NL101 has protective effect on HCA-induced neuronal injury but it is neurotoxic at high concentrations, which is similar to the typical histone deacetylase inhibitor SAHA.

[Effects of leukotriene D4 on proliferation and migration of lung epithelial A549 cells in vitro].

OBJECTIVE: To investigate the effects of cysteinyl leukotriene (CysLT) receptor agonist leukotriene D4 (LTD4) on proliferation and migration in lung epithelial A549 cells. METHODS: The expression of CysLT1 receptor and CysLT2 receptor was determined by immunofluoresence staining in A549 cells. A549 cells were treated with LTD4 (0.01-100 nmol/L) for 24-72 h. Cell viability was detected by MTT reduction assay. Cell migration was determined by modified scratch and healing model. RESULTS: In A549 cells, CysLT1 receptor and CysLT2 receptor were mainly expressed in the cytoplasm, membrane and few in the nuclei. The treatment of LTD4 (0.01-100 nmol/L) for 24-72 h caused no effect on cell viability (Ps>0.05); when A549 cells were treated with 100 nmol/L LTD4 for 24, 48 and 72 h the cell viability was (103.00±4.46)%，(107.00±9.45)% and (105.00±9.02)% of control, respectively (Ps>0.05). The migration rate of A549 cells after scratching during the first 24 h was markedly greater than that during the second and third 24 h in the same concentration groups; however, no significant difference in migration rate was noticed when the cells were treated with different concentrations of LTD4 (0.01-100 nmol/L)(Ps>0.05). The migration of A549 cells was 1.15-fold, 1.21-fold and 1.06-fold of that of control when the cells were treated with 100 nmol/L LTD4 for 24, 48 and 72 h, respectively (Ps>0.05). CONCLUSION: The proliferation and migration of A549 cells are not changed when treated with 0.01-100 nmol LTD4 for up to 72h.

[Antioxidative effects of cysteinyl leukotriene receptor antagonists montelukast and HAMI 3379 on ischemic injury in rat cortical neurons in vitro].

OBJECTIVE: To investigate the antioxidative effects of two cysteinyl leukotriene receptors antagonists (CysLT1R and CysLT2R) montelukast and HAMI 3379 on ischemic injury of rat cortical neurons in vitro. METHODS: Cultured rat cortical neurons were pretreated with CysLT1R antagonist montelukast and CysLT2R antagonist HAMI 3379, and then exposed to oxygen-glucose deprivation/recovery (OGD/R）or H2O2. Reactive oxygen species (ROS) mitochondrial membrane potential (MMP) depolarization, neuronal viability and lactate dehydrogenase (LDH) release were determined. Meanwhile, RNA interference was used to inhibit the expression of CysLT1R and CysLT2R，and the effects were observed. RESULTS: ROS production in neurons was significantly increased after 1 h OGD, which reached the peak at 30 min and lasted for 1.5 h after recovery. Montelukast and HAMI 3379 at 0.01-1µmol/L moderately decreased OGD/R-induced ROS production (P<0.05). Montelukast mildly attenuated OGD/R-induced MMP depolarization (P<0.05)，but HAMI 3379 had no effect. H2O2 reduced neuronal viability and increased LDH release, namely inducing neuronal injury. Montelukast and HAMI 3379 at 0.1-1µmol/L moderately attenuated H2O2-induced neuronal injury (P<0.05). However, both CysLT1R siRNA and CysLT2R shRNA did not significantly affect the responses mentioned above. CONCLUSION: In ischemic neuronal injury, montelukast and HAMI 3379 exert a moderate antioxidative effect, and this effect may be receptor-independent.

[Effects of the water channel aquaporin 4 deficiency on bleomycin-induced lung fibrosis in mice].

OBJECTIVE: To evaluate the effect of water channel aquaporin 4 (AQP4) on bleomycin-induced lung fibrosis in mice. METHODS: In wild type and AQP4 gene knockout (AQP4-/-) mice, lung fibrosis was induced by injection of bleomycin (3 mg/kg) into the trachea and saline injection was used as a control. At d3, 7, 14, 28 after bleomycin-treatment, mice were randomly sacrificed in batch and the lung coefficient was determined. Serum levels of TGF-ß1 and TNF-α were measured by ELISA and hydroxyproline contents in lung tissue were determined by Alkaline hydrolysis method. H-E staining and Masson's staining were performed to examine the pathological changes of lung tissues after bleomycin-treatment. RESULTS: On d14 after bleomycin-treatment, the lung coefficients in wild type mice and AQP4-/- mice were 1.9-fold (12.69 ± 6.05 vs 6.80 ± 0.82, q=4.204, P<0.05) and 2.3-fold (14.05 ± 5.82 vs 6.05± 0.58, q=5.172, P<0.01) of that in control, respectively, but no significant difference was found between wild type and AQP4-/- mice in the lung coefficient value (P>0.05). The hydroxyproline contents in the lung increased after bleomycin-treatment; on d28, the lung hydroxyproline contents in wild type and in AQP4-/- mice were 1.55-fold (0.85 ± 0.22 g/mg vs 0.55 ± 0.14 µg/mg, q=4.313, P<0.05) and 1.4-fold (0.84 ± 0.13 µg/mg vs 0.60 ± 0.14µg/mg, q=4.595，P<0.05) of that in control, respectively, but no significant difference was noticed between wild type and AQP4-/- mice in lung hydroxyproline contents. There was a tendency that serum TGF-ß1 and TNF-α levels increased in bleomycin-treated mice, but no significant difference was found between wild type and AQP4-/- mice. AQP4-knockout showed no effects on pathological changes of lung tissues with H-E staining and Masson's staining in mice with bleomycin-induced lung fibrosis. CONCLUSION: AQP4 might not be involved in bleomycin-induced lung fibrosis in mice.

PARP-1 inhibitor alleviates cerebral ischemia/reperfusion injury by reducing PARylation of HK-1 and LDH in mice.

Poly (ADP-ribose) polymerase-1 (PARP-1) activity significantly increases during cerebral ischemia/reperfusion. PARP-1 is an NAD+-consumption enzyme. PARP-1 hyperactivity causes intracellular NAD+ deficiency and bioenergetic collapse, contributing to neuronal death. Besides, the powerful trigger of PARP-1 causes the catalyzation of poly (ADP-ribosyl)ation (PARylation), a posttranslational modification of proteins. Here, we found that PARP-1 was activated in the ischemic brain tissue during middle-cerebral-artery occlusion and reperfusion (MCAO/R) for 24 h, and PAR accumulated in the neurons in mice. Using immunoprecipitation, Western blotting, liquid chromatography-mass spectrometry, and 3D-modeling analysis, we revealed that the activation of PARP-1 caused PARylation of hexokinase-1 and lactate dehydrogenase-B, which, therefore, caused the inhibition of these enzyme activities and the resulting cell energy metabolism collapse. PARP-1 inhibition significantly reversed the activity of hexokinase and lactate dehydrogenase, decreased infarct volume, and improved neuronal deficiency. PARP-1 inhibitor combined with pyruvate further alleviated MCAO/R-induced ischemic brain injury in mice. As such, we conclude that PARP-1 inhibitor alleviates neuronal death partly by inhibiting the PARylation of metabolic-related enzymes and reversing metabolism reprogramming during cerebral ischemia/reperfusion injury in mice. PARP-1 inhibitor combined with pyruvate might be a promising therapeutic approach against brain ischemia/reperfusion injury.

HAMI 3379, a CysLT2 receptor antagonist, attenuates ischemia-like neuronal injury by inhibiting microglial activation.

The cysteinyl leukotrienes (CysLTs) are inflammatory mediators closely associated with neuronal injury after brain ischemia through the activation of their receptors, CysLT1R and CysLT2R. Here we investigated the involvement of both receptors in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemic neuronal injury and the effect of the novel CysLT2R antagonist HAMI 3379 [3-({[(1S,3S)-3- carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy)benzoic acid] in comparison with the CysLT1R antagonist montelukast. In primary neurons, neither the nonselective agonist leukotriene D4 (LTD4) nor the CysLT2R agonist N-methyl-leukotriene C4 (NMLTC4) induced neuronal injury, and HAMI 3379 did not affect OGD/R-induced neuronal injury. However, in addition to OGD/R, LTD4 and NMLTC4 induced cell injury and neuronal loss in mixed cultures of cortical cells, and neuronal loss and necrosis in neuron-microglial cocultures. Moreover, they induced phagocytosis and cytokine release (interleukin-1ß and tumor necrosis factor-α) from primary microglia, and conditioned medium from the treated microglia induced neuronal necrosis. HAMI 3379 inhibited all of these responses, and its effects were the same as those of CysLT2R interference by CysLT2R short hairpin RNA, indicating CysLT2R dependence. In comparison, montelukast moderately inhibited OGD/R-induced primary neuronal injury and most OGD/R- and LTD4-induced (but not NMLTC4-induced) responses in mixed cultures, cocultures, and microglia. The effects of montelukast were both dependent and independent of CysLT1Rs because interference by CysLT1R small interfering RNA had limited effects on neuronal injury in neuron-microglial cocultures and on cytokine release from microglia. Our findings indicated that HAMI 3379 effectively blocked CysLT2R-mediated microglial activation, thereby indirectly attenuating ischemic neuronal injury. Therefore, CysLT2R antagonists may represent a new type of therapeutic agent in the treatment of ischemic stroke.

Biomechanical properties of retinal glial cells: comparative and developmental data.

The biomechanical properties of Müller glial cells may have importance in understanding the retinal tissue alterations after retinal surgery with removal of the inner limiting membrane and during the ontogenetic development, respectively. Here, we compared the viscoelastic properties of Müller cells from man and monkey as well as from different postnatal developmental stages of the rat. We determined the complex Young's modulus E = E' + iE″ in a defined range of deforming frequencies (30, 100, and 200 Hz) using a scanning force microscope, where the real part E' reflects the elastic property (energy storage or elastic stiffness) and the imaginary part E″ reflects the viscous property (energy dissipation) of the cells. The viscoelastic properties were similar in Müller cells from man, monkey, and rat. In general, the elastic behavior dominated over the viscous behavior (E' > E″). The inner process of the Müller cell was the softest region, the soma the stiffest (Einnerprocess(')Eglia(')). These relations were also observed during the postnatal development of the rat. It is concluded that, generally, retinal cells display mechanics of elastic solids. In addition, the data indicate that the rodent retina is a reliable model to investigate retinal mechanics and tissue alterations after retinal surgery. During retinal development, neuronal branching and synaptogenesis might be particularly stimulated by the viscoelastic properties of Müller cell processes in the inner plexiform layer.

The Depletion of NAMPT Disturbs Mitochondrial Homeostasis and Causes Neuronal Degeneration in Mouse Hippocampus.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in the salvaging synthesis pathway of the nicotinamide adenine dinucleotide (NAD). Both NAMPT and NAD progressively decline upon aging and neurodegenerative diseases. The depletion of NAMPT induces mitochondrial dysfunction in motor neurons and causes bioenergetic stress in neurons. However, the roles of NAMPT in hippocampus neurons need to be further studied. Using floxed Nampt (Namptflox/flox) mice, we knocked out Nampt specifically in the hippocampus CA1 neurons by injecting rAAV-hSyn-Cre-APRE-pA. The depletion of NAMPT in hippocampus neurons induced cognitive deficiency in mice. Nevertheless, no morphological change of hippocampus neurons was observed with immunofluorescent imaging. Under the transmission electron microscope, we observed mitochondrial swollen and mitochondrial number decreasing in the cell body and the neurites of hippocampus neurons. In addition, we found the intracellular Aß (6E10) increased in the hippocampus CA1 region. The intensity of Aß42 remained unchanged, but it tended to aggregate. The GFAP level, an astrocyte marker, and the Iba1 level, a microglia marker, significantly increased in the mouse hippocampus. In the primary cultured rat neurons, NAMPT inhibition by FK866 decreased the NAD level of neurons at > 10-9 M. FK866 dropped the mitochondrial membrane potential in the cell body of neurons at > 10-9 M and in the dendrite of neurons at > 10-8 M. FK866 decreased the number and shortened the length of branches of neurons at > 10-7 M. Together, likely due to the injury of mitochondria, the decline of NAMPT level can be a critical risk factor for neurodegeneration.

Transforming growth factor ß1-induced astrocyte migration is mediated in part by activating 5-lipoxygenase and cysteinyl leukotriene receptor 1.

BACKGROUND: Transforming growth factor-ß 1 (TGF-ß 1) is an important regulator of cell migration and plays a role in the scarring response in injured brain. It is also reported that 5-lipoxygenase (5-LOX) and its products, cysteinyl leukotrienes (CysLTs, namely LTC4, LTD4 and LTE4), as well as cysteinyl leukotriene receptor 1 (CysLT1R) are closely associated with astrocyte proliferation and glial scar formation after brain injury. However, how these molecules act on astrocyte migration, an initial step of the scarring response, is unknown. To clarify this, we determined the roles of 5-LOX and CysLT1R in TGF-ß 1-induced astrocyte migration. METHODS: In primary cultures of rat astrocytes, the effects of TGF-ß 1 and CysLT receptor agonists on migration and proliferation were assayed, and the expression of 5-LOX, CysLT receptors and TGF-ß1 was detected. 5-LOX activation was analyzed by measuring its products (CysLTs) and applying its inhibitor. The role of CysLT1R was investigated by applying CysLT receptor antagonists and CysLT1R knockdown by small interfering RNA (siRNA). TGF-ß 1 release was assayed as well. RESULTS: TGF-ß 1-induced astrocyte migration was potentiated by LTD4, but attenuated by the 5-LOX inhibitor zileuton and the CysLT1R antagonist montelukast. The non-selective agonist LTD4 at 0.1 to 10 nM also induced a mild migration; however, the selective agonist N-methyl-LTC4 and the selective antagonist Bay cysLT2 for CysLT2R had no effects. Moreover, CysLT1R siRNA inhibited TGF-ß 1- and LTD4-induced astrocyte migration by down-regulating the expression of this receptor. However, TGF-ß 1 and LTD4 at various concentrations did not affect astrocyte proliferation 24 h after exposure. On the other hand, TGF-ß 1 increased 5-LOX expression and the production of CysLTs, and up-regulated CysLT1R (not CysLT2R), while LTD4 and N-methyl-LTC4 did not affect TGF-ß 1 expression and release. CONCLUSIONS: TGF-ß 1-induced astrocyte migration is, at least in part, mediated by enhanced endogenous CysLTs through activating CysLT1R. These findings indicate that the interaction between the cytokine TGF-ß 1 and the pro-inflammatory mediators CysLTs in the regulation of astrocyte function is relevant to glial scar formation.

Reactive glial cells: increased stiffness correlates with increased intermediate filament expression.

Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia-reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild-type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)) in which glial cells are consequently devoid of IFs, and normal Long-Evans rats. In response to ischemia-reperfusion, glial cells stiffened significantly in wild-type mice and rats but were unchanged in GFAP(-/-)Vim(-/-) mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up-regulation in reactive glial cells may facilitate neuroregeneration.

Phospholipase D participates in H(2)O(2)-induced A549 alveolar epithelial cell migration.

To investigate the effects of phospholipase D (PLD) on low-concentration hydrogen peroxide (H(2)O(2))-induced growth and migration in alveolar epithelial A549 cells, the cells were exposed to H(2)O(2) (3-100 µM) for 12-48 hours, cell proliferation was determined by MTT assay and cell migration was tested by a modified epithelial wound healing assay. We found that one bolus of H(2)O(2) (10-100 µM) did not affect proliferation, but significantly stimulated migration (143-161% of control) after a 12-hour exposure. Pretreatment with the antioxidants catalase (1000 U/ml), N-acetyl-cysteine (2 mM), or edaravone (10 µM) abolished the migration induced by 30 µM H(2)O(2); the PLD inhibitor 1-butanol (0.5%) also attenuated H(2)O(2)-induced migration to the control level; while exogenous phosphatidic acid (PA) (10(-7)-10(-4) M) mimicked the effects of PLD activation and induced migration in a dose-dependent manner. We suggest that the alveolar epithelial cell migration induced by exposure to low concentrations of H(2)O(2) benefits tissue repair during acute lung injury (ALI) and PLD is involved in the underlying mechanism.