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Papers by Xun Shen

The International Journal of Biochemistry & Cell Biology, 2006

It has been reported that over-expression of human heat shock protein 27 (hsp27) in murine cells ... more It has been reported that over-expression of human heat shock protein 27 (hsp27) in murine cells decreased the intracellular iron level [

Journal of Leukocyte Biology, 2003

To define the role of phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI-3K), signaling ... more To define the role of phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI-3K), signaling pathways in arachidonic acid (AA)-stimulated respiratory burst in human neutrophils, the AA-stimulated respiratory burst, Ins(1,4,5)P3 production, PI-3K activation, and cytoplasmic Ca2+ mobilization were investigated. It was found that Ins(1,4,5)P3 production and PI-3K activity in AA-stimulated cells were increased in a dose-dependent manner. U73122, the PLC inhibitor, effectively inhibited the AA-stimulated respiratory burst and Ca2+ release from th intracellular calcium store but not the activity of PI-3K, indicating the independence of PI-3K signaling on PLC activation. Wortmannin, the PI-3K inhibitor, at the concentration sufficient to inhibit PI-3K activity, can only partially inhibit Ca2+ release from the internal store, indicating a partial regulation of PLC signaling by PI-3K and the existence of two pathways initiated by different PLC subfamilies. One is regulated by PI-3K activ...

Journal of Biological Chemistry, 2001

The effects of phenylarsine oxide and a monoclonal antibody directed against type II phosphatidyl... more The effects of phenylarsine oxide and a monoclonal antibody directed against type II phosphatidylinositol 4-kinase (PI4K) on the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated respiratory burst and the PI4K activity in neutrophils were investigated. Fluorescence microscopic imaging showed that the antibody labeled with IANBD amide (N,N-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine) could enter into the cytosol possibly by endocytosis. It was found that the antibody inhibited the fMLP-stimulated respiratory burst but had little effect on the phorbol myristate acetate-activated respiratory burst in neutrophils, whereas phenylarsine oxide inhibited both. It was found that even at higher concentration, the antibody could not completely inhibit the cell response. Using cells preincubated with human immunoglobulin G of the same concentration as the control, the maximal inhibition of the fMLP-stimulated respiratory burst by the antibody against type II PI4K was found to be about 70%, whereas the PI4K activity was inhibited by only about 40%. The discrepancy in depressing the cell response and the enzyme activity may be the result of depletion of the phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate pools during the incubation of cells with the antibody. Both the 40% inhibition of PI4K activity and 70% depression of the respiratory burst by the type II PI4K antibody may imply that at least 40% of the phosphatidylinositol 4,5-biphosphate was synthesized promptly by all forms of PI4K and phosphatidylinositol-4-phosphate 5-kinase in the fMLP-activated cells. The results suggest that PI4K plays a central role in either phospholipase C or PI3K signaling and that PI3K, PI4K, and phosphatidylinositol 4-phosphate 5-kinase must be considered as an integrated family for the phosphatidylinositol 3,4,5-trisphosphate initiated signaling. Chemoattractant-mediated recruitment of leukocytes is a key step in the process of inflammation. Chemokines and chemotactic peptides, such as N-formyl-methionyl-leucyl-phenyl

Journal of Biological Chemistry, 2006

The p38 MAPK and heat shock protein 27 (hsp27) form a signaling complex with serine/threonine kin... more The p38 MAPK and heat shock protein 27 (hsp27) form a signaling complex with serine/threonine kinase Akt and MAPK-activated protein kinase-2 (MK2), which plays an important role in controlling stress-induced apoptosis and reorganizing actin cytoskeleton. However, regulation of the complex is poorly understood. In this study, the interaction between p38 and hsp27 was visualized in single living L929 cells using fluorescence resonance energy transfer technology, while their association with Akt was examined by immunoprecipitation analysis. Under normal growth conditions, p38 kinase constitutively interacts with hsp27. When cells were exposed to H 2 O 2 or stimulated by arachidonic acid, this interaction was disrupted. However, inhibition of the activation of p38 and Akt by selective inhibitors or overexpression of the kinase-dead mutant of p38 diminished such effects. Furthermore, mutation of phosphorylation sites of hsp27 renders the interaction resistant to H 2 O 2 and arachidonic acid. It was interesting to find that the interaction disappeared in the cells from MK2-knockout mice or the cells treated with lemptomycin B that blocks export of MK2 from nucleus to cytosol. However, MK2 is not required for the association of hsp27 with Akt. This study suggests that MK2 mediates the incorporation of p38 into the pre-existing complex of hsp27 with Akt. Phosphorylation of hsp27 finally breaks the signaling complex. The stress-activated p38 mitogen-activated protein kinase (MAPK) 2 and the stress-responsive heat shock protein 27 (hsp27) are two important proteins involved in cellular processes responding to extracellular stimuli and various stress

Clinical Cancer Research, 2007

Purpose: Heat shock protein 27 (Hsp27) is up-regulated in multiple malignancies and implicated in... more Purpose: Heat shock protein 27 (Hsp27) is up-regulated in multiple malignancies and implicated in cisplatin resistance. It is attempted to know how Hsp27 endues cell with cisplatin resistance by interfering with upstream of both apoptosis signal–regulating kinase 1 (ASK1)/p38 mitogen-activated protein kinase–activated apoptotic signaling and serine/threonine kinase Akt-dependent survival signaling. Experimental Design: The mouse L929 cells stably transfected with human Hsp27 or its dominant-negative mutant and the human cervical cancer HeLa cells transfected with Hsp27 siRNA were used. The cisplatin-induced apoptosis and activation of ASK1, p38, and Akt were compared in control cells, cells overexpressing Hsp27, and cells with their endogenous Hsp27 knocked down. Results: Hsp27 effectively protected the cells from cisplatin-induced DNA fragmentation. The p38 inhibitors obviously decreased whereas Akt inhibitors markedly increased the apoptotic fraction in cisplatin-treated cells. Ov...

Antioxidants & Redox Signaling, 2010

Actin is a highly conserved protein in eukaryotic cells, and has been identified as one of the ma... more Actin is a highly conserved protein in eukaryotic cells, and has been identified as one of the main redox targets by redox proteomics under oxidative stress. However, little is known about the mechanisms of regulation of the redox state of actin. In this study, we investigated how thioredoxin-1 (Trx1) affected the redox state of actin and its polymerization under oxidative stress in SH-SY5Y cells. Trx1 decreased the levels of reactive oxygen species (ROS) in the cells, and cysteine residues at positions 32, 35, and 69 of the Trx1 protein were active sites for redox regulation. Actin could be kept in a reduced state by Trx1 under H 2 O 2 stimulation. A physical interaction was found to exist between actin and Trx1. Cysteine 62 in Trx1 was the key site that interacted with actin, and it was required to maintain cellular viability and anti-apoptotic function. Taken together, these results suggested that Trx1 could protect cells from apoptosis under oxidative stress not only by increasing the total antioxidant capability and decreasing the ROS levels, but also by stabilizing the actin cytoskeletal system, which cooperatively contributed to the enhancement of cell viability and worked against apoptosis. Antioxid. Redox Signal. 13, 565-573.

Trends in Molecular Medicine, 2001

Despite years of investigation, it is still not known why iron levels are abnormally high in some... more Despite years of investigation, it is still not known why iron levels are abnormally high in some regions of the brain in neurodegenerative disorders. Also, it is not clear whether iron accumulation in the brain is an initial event that causes neuronal death or is a consequence of the disease process. Here, we propose that iron and iron-induced oxidative stress constitute a common mechanism that is involved in the development of neurodegeneration. Also, we suggest that, at least in some neurodegenerative disorders, brain iron misregulation is an initial cause of neuronal death and that this misregulation might be the result of either genetic or non-genetic factors.

Progress in Natural Science, 2009

Journal of Cellular and Molecular Medicine, 2010

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to... more The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10-fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA-stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA-stimulated respiratory burst. Using 1-butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA-stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA-stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA-stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA-stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2-dioctanoyl-sn-glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA-stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA-stimulated respiratory burst.

FEMS Microbiology Letters, 1991

A remarkable spontaneous photon emission was observed in isolated bacteroids of three strains of ... more A remarkable spontaneous photon emission was observed in isolated bacteroids of three strains of soybean rhizobia from different genera, but not for the same rhizobia when cultured in liquid medium. The photon emission is oxygendependent and can be inhibited by desferal or dipyridyl (both good iron-chelating agents), superoxide dismutase or /3-carotene. It is enhanced by catalase. The emission spectrum indicates that singlet oxygen is partly responsible for the luminescence.

Experientia, 1993

The hypothesis that biophotons display a high degree of coherence was tested by measuring photoco... more The hypothesis that biophotons display a high degree of coherence was tested by measuring photocount statistics (PCS) of the ultraweak photon emission from three living organisms (cucumber seedling, mungbean seedling and soybean rhizobium bacteroids) with a high-sensitivity single-photon counter. For comparison, the same experiments were performed for laser beam, randomized laser beam, chemiluminescence from autoxidation of luminol and the dark counts of the equipment. Photocount distributions, close to Poissonian, were observed for the three tested biological systems but not for the pure chemiluminescence of luminol.

Biochemical Journal, 2000

The mechanism of Fe 2 +-initiated lipid peroxidation in liposomes : the dual function of ferrous ... more The mechanism of Fe 2 +-initiated lipid peroxidation in liposomes : the dual function of ferrous ions, the roles of the pre-existing lipid peroxides and the lipid peroxyl radical

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2007

Our previous study showed that the adhesion molecule CD146 as a biomarker is over-expressed on ac... more Our previous study showed that the adhesion molecule CD146 as a biomarker is over-expressed on activated endothelium during angiogenesis, which was induced by tumor conditional medium and inhibited by anti-CD146 monoclonal antibody (mAb AA98). However, the CD146 molecular organization on the cells is unknown. Here, using immunoprecipitation, we found that the dimerization of CD146 occurs in both normal and tumor cells. However, the dimer/monomer ratio was higher in tumor cells than in normal cells. Moreover, we found that CD146 dimerization was up-regulated by tumor conditional medium through the NF-kappa B pathway and down-regulated by mAb AA98. To further confirm that CD146 dimerization occurs in living cells, we used fluorescence resonance energy transfer (FRET) with melanoma Mel888 cells co-expressing CFP/YFP-tagged CD146 fusion proteins. By acceptor photobleaching, we observed a strong FRET signal produced by these two fluorescencetagged proteins. The FRET efficiency reached 20.1%. Our data provide the first evidence that CD146 dimerization occurs in living cells and is regulated within the tumor microenvironment, implying that dimerization of CD146 may be associated with malignancy.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1998

The role of extracellular calcium in the activation of respiratory burst in human neutrophils was... more The role of extracellular calcium in the activation of respiratory burst in human neutrophils was studied by using the receptor agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the activator of protein kinase C phorbol myristate acetate (PMA). The level of intracellular free calcium was measured by using both cell suspensions and single cells in the presence and absence of extracellular calcium. The Ca 2-ATPase inhibitor, thapsigargin, was used to activate higher Ca 2 influx, while a novel calcium channel blocker, panax notoginseng saponins (PNGS) was used to block the Ca 2 entry from extracellular space during the responding period of cells. It was found that about two-thirds of the activation of respiratory burst initiated by the receptor agonist were attributed to the Ca 2 influx under normal physiological conditions. The higher Ca 2 influx resulted in tremendous enhancement of the intensity of respiratory burst initiated by fMLP and marked acceleration of the onset of the respiratory burst stimulated by PMA. It is evident that both intra-and extracellular Ca 2 are required for full activation of the respiratory burst of human neutrophils, and the Ca 2 influx from extracellular space plays an important role either in generation of reactive oxygen metabolites or in activation of protein kinase C.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2008

Constitutively activated NF-κB occurs in many inflammatory and tumor tissues. Does it interfere w... more Constitutively activated NF-κB occurs in many inflammatory and tumor tissues. Does it interfere with anti-inflammatory or anti-tumor signaling pathway? Here, we report that NF-κB p65 subunit repressed the Nrf2-antioxidant response element (ARE) pathway at transcriptional level. In the cells where NF-κB and Nrf2 were simultaneously activated, p65 unidirectionally antagonized the transcriptional activity of Nrf2. In the p65overexpressing cells, the ARE-dependent expression of heme oxygenase-1 was strongly suppressed. However, p65 inhibited the ARE-driven gene transcription in a way that was independent of its own transcriptional activity. Two mechanisms were found to coordinate the p65-mediated repression of ARE: (1) p65 selectively deprives CREB binding protein (CBP) from Nrf2 by competitive interaction with the CH1-KIX domain of CBP, which results in inactivation of Nrf2. The inactivation depends on PKA catalytic subunit-mediated phosphorylation of p65 at S276. (2) p65 promotes recruitment of histone deacetylase 3 (HDAC3), the corepressor, to ARE by facilitating the interaction of HDAC3 with either CBP or MafK, leading to local histone hypoacetylation. This investigation revealed the participation of NF-κB p65 in the negative regulation of Nrf2-ARE signaling, and might provide a new insight into a possible role of NF-κB in suppressing the expression of anti-inflammatory or anti-tumor genes.

American Journal of Physiology-Cell Physiology, 2009

To know whether thioredoxin 1 (Trx1) works for an antioxidant defense mechanism in atherosclerosi... more To know whether thioredoxin 1 (Trx1) works for an antioxidant defense mechanism in atherosclerosis, the effect of Trx1 on the release of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for recruitment and accumulation of monocytes/macrophages in the intima of artery vessel, was investigated in human endothelial-like EA.hy 926 cells. It was found that overexpression of Trx1 suppressed, whereas knockdown of endogenous Trx1 enhanced, oxidized low-density lipoprotein (oxLDL)-stimulated MCP-1 release and expression in the cells. It was also observed that overexpression of Trx1 suppressed, whereas depletion of endogenous Trx1 greatly promoted, nuclear translocation of c-Jun and the redox factor-1 (Ref-1). Electrophoretic mobility shift assay showed significantly reduced DNA-binding activity of activator protein-1 (AP-1) in Trx1-overexpressing cells but apparently enhanced DNA binding activity of AP-1 in Trx1-knockdown cells, indicating that nuclear Ref-1 rather than T...

American Journal of Physiology-Cell Physiology, 2007

Short-term hypoxic pretreatment is an effective approach to protect the lung from subsequent prol... more Short-term hypoxic pretreatment is an effective approach to protect the lung from subsequent prolonged hypoxic injury under conditions such as lung transplantation, shock, and trauma. However, the signaling pathways are not well understood. By use of high-throughput, two-dimensional electrophoresis combined with mass spectrometry, we found that short-term hypoxic treatment upregulated calreticulin (CRT), an endoplasmic-reticulum stress protein, in A549 human type II alveolar epithelial cells. Genetic manipulation of CRT expression in A549 cells through small interferring RNA inhibition or overexpression demonstrated a positive correlation between CRT expression level and cell viability in subsequent prolonged hypoxia, which indicates that CRT is a key mediator of short-term hypoxia-induced cell protection. Importantly, CRT overexpression prevented reactive oxygen species (ROS) accumulation during prolonged hypoxia by inducing the expression of thioredoxin (TRX), an antioxidant, in A...

FEBS Letters, 2008

We have previously shown that homocysteine (Hcy) can induce monocyte chemoattractant protein-1 (M... more We have previously shown that homocysteine (Hcy) can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes. Here, we show that Hcy upregulates expression of an important antioxidative protein, thioredoxin (Trx), via NADPH oxidase in human monocytes in vitro. The increase of Trx expression and activity inhibited Hcy-induced ROS production and MCP-1 secretion. Of note, 2-week hyperhomocysteinemia (HHcy) ApoE À/À mice showed accelerated lesion formation and parallel lower Trx expression in macrophages than ApoE À/À mice, suggesting that HHcy-induced sustained oxidative stress in vivo might account for impaired Trx and hence increased ROS production and MCP-1 secretion from macrophages, and subsequently accelerated atherogenesis.

Journal of neurochemistry, 2006

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell mode... more 6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of...

Molecular Biology of the Cell, 2006

PPARα, a member of the nuclear receptor superfamily, and thioredoxin, a critical redox-regulator ... more PPARα, a member of the nuclear receptor superfamily, and thioredoxin, a critical redox-regulator in cells, were found to form a negative feedback loop, which autoregulates transcriptional activity of PPARα. Thioredoxin was identified as a target gene of PPARα. Activation of PPARα leads to increase of thioredoxin expression as well as its translocation from cytoplasm to nucleus, whereas ectopic overexpression of thioredoxin in the nucleus dramatically inhibited both constitutive and ligand-dependent PPARα activation. As PPARα-target genes, the expression of muscle carnitine palmitoyltransferase I, medium chain acyl CoA dehydrogenase, and apolipoprotein A-I were significantly down-regulated by nucleus-targeted thioredoxin at transcriptional or protein level. The suppression of PPARα transcriptional activity by Trx could be enhanced by overexpression of thioredoxin reductase or knockdown of thioredoxin-interacting protein, but abrogated by mutating the redox-active sites of thioredoxin...