-
[show abstract]
[hide abstract]
ABSTRACT: The respiratory burst is an important physiological function of the neutrophils in killing the bacteria invading in human
body. We used chemiluminescence method to measure the exogenous arachidonic acid-stimulated respiratory burst, and measured
the cytosolic free calcium concentration in neutrophils by the fluorescence method. It was found that, on one hand, the arachidonic
acid-stimulated respiratory burst was enhanced by elevating the cytosolic free calcium concentration in neutrophils with a
potent endomembrane Ca2+-ATPase inhibitor, Thapsgargin; on the other hand, chelating the intracellular or extracellular calcium by EGTA or BAPTA inhibited
the respiratory burst. Results showed that calcium plays an important regulatory role in the signaling pathway involved in
the exogenous arachidonic acid-stimulated respiratory burst of neutrophils.
Keywordsarachidonic acid-neutrophils-respiratory burst-chemiluminescence-intracellular calcium-extracellular calcium
Chinese Science Bulletin 04/2012; 46(4):314-317. · 1.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 Trx1 itself finally dominates the regulation of AP-1 activity, although Trx1 is considered to upregulate AP-1 activity. It was also observed that Trx1 depressed intracellular generation of reactive oxygen species (ROS). Diphenyleneiodonium (DPI), the inhibitor of NADPH oxidase, suppressed MCP-1 secretion, whereas transient expression of Nox1 enhanced transcription of MCP-1 in endothelial cells. Assays with AP-1 and MCP-1 luciferase reporters further demonstrated that transient expression of Trx1 significantly depressed the transcriptional activity of c-Jun/c-Fos and consequent MCP-1 transcription. This study suggests that Trx1 inherently suppresses MCP-1 expression in vascular endothelium and may prevent atherosclerosis by depressing MCP-1 release. Besides the suppression of intracellular ROS generation, the inhibition of nuclear translocation of AP-1 and Ref-1 are mainly responsible for the downregulation of MCP-1 by Trx1.
AJP Cell Physiology 05/2010; 298(5):C1170-9. · 3.54 Impact Factor
-
Xiaogang Wang,
Shukuan Ling,
Dingsheng Zhao,
Qiao Sun,
Qi Li,
Feng Wu,
Jielin Nie,
Lina Qu,
Bo Wang, Xun Shen,
Yanqiang Bai,
Yingxian Li,
Yinghui Li
[show abstract]
[hide abstract]
ABSTRACT: 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.
Antioxidants & Redox Signaling 03/2010; 13(5):565-73. · 8.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Cellular and Molecular Medicine 02/2010; 15(3):647-53. · 4.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To know if thioredoxin reductase 1 (TrxR1) plays a role in antioxidant defense mechanisms against atherosclerosis, effect of TrxR1 on expression/release of monocyte chemoattractant protein (MCP-1) was investigated in activated human endothelial-like EAhy926 cells. The MCP-1 release and expression, cellular generation of reactive oxygen species (ROS), nuclear translocation and DNA-binding activity of NF-kappaB subunit p65 were assayed in cells either overexpressing recombinant TrxR1 or having their endogenous TrxR1 knocked down. It was found that overexpression of TrxR1 enhanced, while knockdown of TrxR1 reduced MCP-1 release and expression. Upregulation of MCP-1 by TrxR1 was associated with increasing generation of intracellular ROS generation, enhanced nuclear translocation and DNA-binding activity of NF-kappaB. Assay using NF-kappaB reporter revealed that TrxR1 upregulated transcriptional activity of NF-kappaB. This study suggests that TrxR1 enhances ROS generation, NF-kappaB activity and subsequent MCP-1 expression in endothelial cells, and may promote rather than prevent vascular endothelium from forming atherosclerotic plaque.
Biochemical and Biophysical Research Communications 07/2009; 386(4):703-8. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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(-/-) mice showed accelerated lesion formation and parallel lower Trx expression in macrophages than ApoE(-/-) 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.
FEBS Letters 12/2008; 582(28):3893-8. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Constitutively activated NF-kappaB occurs in many inflammatory and tumor tissues. Does it interfere with anti-inflammatory or anti-tumor signaling pathway? Here, we report that NF-kappaB p65 subunit repressed the Nrf2-antioxidant response element (ARE) pathway at transcriptional level. In the cells where NF-kappaB and Nrf2 were simultaneously activated, p65 unidirectionally antagonized the transcriptional activity of Nrf2. In the p65-overexpressing 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-kappaB p65 in the negative regulation of Nrf2-ARE signaling, and might provide a new insight into a possible role of NF-kappaB in suppressing the expression of anti-inflammatory or anti-tumor genes.
Biochimica et Biophysica Acta 06/2008; 1783(5):713-27. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 A549 cells. Furthermore, CRT promoted the nuclear translocation of nuclear factor-E2-related factor 2, the transcription factor of TRX. Finally, overexpressing an inactive TRX mutant reversed the effects of CRT on ROS accumulation and cell protection. Our results demonstrate that CRT stimulates the anti-oxidant pathway and contributes to short-term hypoxia-induced protection in A549 type II alveolar epithelial cells, which may have potential therapeutic ramifications for hypoxic pulmonary diseases.
AJP Cell Physiology 02/2008; 294(1):C47-55. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
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.
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. Overexpression of Hsp27 doubly enhanced the drug-induced Akt activation while substantially depressing the drug-induced activation of ASK1 and p38. Knockdown of the endogenous Hsp27 in HeLa cells resulted in the effects opposite to that observed in the Hsp27-overexpressing cells. Enhancement of Akt activation is associated with complex formation between Akt and Hsp27, whereas depression of ASK1/p38 activation is attributed to a reversion of the drug-induced inhibition of thioredoxin reductase activity and subsequent oxidation of thioredoxin.
Hsp27 endues cells with cisplatin resistance via depression of the drug-induced ASK1/p38 activation and enhancement of the drug-induced Akt activation. This study revealed the intervention of Hsp27 in upstream of both ASK1/p38 apoptotic signaling and phosphatidylinositol 3-kinase/Akt survival signaling. Therefore, the inhibition of Hsp27 may be a novel strategy of cancer chemotherapy.
Clinical Cancer Research 06/2007; 13(10):2855-64. · 7.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 fluorescence-tagged 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 05/2007; 1773(4):513-20. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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-knock-out 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.
Journal of Biological Chemistry 01/2007; 281(48):37215-26. · 4.77 Impact Factor
-
12/2006: pages 39-52;
-
[show abstract]
[hide abstract]
ABSTRACT: 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 p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.
Journal of Neurochemistry 05/2006; 97(1):234-44. · 4.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: PPARalpha, 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 PPARalpha. Thioredoxin was identified as a target gene of PPARalpha. Activation of PPARalpha 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 PPARalpha activation. As PPARalpha-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 PPARalpha 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. Mammalian one-hybrid assays showed that thioredoxin inhibited PPARalpha activity by modulating its AF-1 transactivation domain. It was also demonstrated by electrophoretic mobility-shift assay that thioredoxin inhibited the binding of PPARalpha to the PPAR-response element. Together, it is speculated that the reported negative-feedback loop may be essential for maintaining the homeostasis of PPARalpha activity.
Molecular Biology of the Cell 05/2006; 17(4):1822-33. · 4.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It has been reported that over-expression of human heat shock protein 27 (hsp27) in murine cells decreased the intracellular iron level [Arrigo, A. P., Virot, S., Chaufour, S., Firdaus, W., Kretz-Remy, C., & Diaz-Latoud, C. (2005). Hsp27 consolidates intracellular redox homeostasis by upholding glutathione in its reduced form and by decreasing iron intracellular levels. Antioxidants & Redox Signalling, 7, 412-422]. However, the mechanism involved is unknown. In this study, the regulation of transferrin receptor 1 (TfR1)-mediated iron uptake by human hsp27 was investigated in CCL39 cells by overexpression of human hsp27 and its dominant-negative mutant (hsp27-3G). The results showed that overexpression of hsp27 diminished intracellular labile iron pool, increased the binding activity of iron regulatory protein (IRP) to iron responsive element (IRE) and the cell surface-expressed TfR1s. However, the increased surface-expressed TfR1s resulted in decrease rather than increase of iron uptake. Further study revealed that overexpression of hsp27 decelerated transferrin endocytosis and recycling, while overexpressed hsp27-3G had a reversal effect. Moreover, flowcytometric analysis showed an enhanced actin polymerization in the cells overexpressing hsp27. In particular, fluorescence imaging of cytoskeleton displayed highly stabilized microfilaments and preferential localization of hsp27 in cortical area of the actin cytoskeleton. In contrast, disruption of actin cytoskeleton by cytochalasin B resulted in acceleration of the endocytosis and recycling of Tf, as well as increase of iron uptake. Meanwhile, the possible involvement of ferroportin 1 in down-regulation of intracellular iron level by overexpression of hsp27 was checked. However, the outcome was negative. Our findings indicate that hsp27 down-regulates TfR1-mediated iron uptake via stabilization of the cortical actin cytoskeleton rather than the classical IRP/IRE mode. The study may also imply that hsp27 protects cells from oxidative stress by reducing cellular iron uptake.
The International Journal of Biochemistry & Cell Biology 02/2006; 38(8):1402-16. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Intracellular Ca2+ signaling controls many cellular functions. Understanding its regulation by selenoproteins is essential for understanding the role of selenoproteins in regulating cell functions. The activity of thioredoxin reductase (TrxR), thioredoxin (Trx) content, and the activity of glutathione peroxidase (GPx) in the human endothelial cells cultured in selenium-supplemented medium (refer as Se+ cells) was found 70%, 40%, and 20% higher, respectively than those in the cells cultured in normal medium (refer as Se0 cells). The intracellular Ca2+ signaling initiated by inositol 1,4,5-trisphosphate (IP3), histamine, thapsigargin (TG), carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone (FCCP), and cyclosporin A (CsA) was investigated in both Se+ and Se0 cells. It was interestingly found that the higher activity of selenoproteins reduced the sensitivity of IP3 receptor to the IP3-triggered Ca2+ release from intracellular stores, but enhanced activation of the receptor-coupled phospholipase C in histamine-stimulated Se+ cells by showing much more generation of IP3 and higher elevation of cytosolic Ca2+. The higher selenoprotein activity also reduced susceptibility of the uniporter to the mitochondrial uncoupler, susceptibility of the permeability transition pore (PTP) to its inhibitor, and the vulnerability of endoplasmic reticulum (ER) Ca2+-ATPase to its inhibitor in selenium-supplementing cells. The results suggest that cell calcium signaling is subjected to thiol-redox regulation by selenoproteins.
Journal of Cellular Physiology 11/2005; 205(1):97-106. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The mechanisms of H2O2-induced Ca2+ release from intracellular stores were investigated in human umbilical vein endothelial cells. It was found that U73122, the selective inhibitor of phospholipase C, could not inhibit the H2O2-induced cytosolic Ca2+ mobilization. No elevation of inositol 1,4,5-trisphosphate (IP3) was detected in cells exposed to H2O2. By loading mag-Fura-2, a Ca2+ indicator, into intracellular store, the H2O2-induced Ca2+ release from intracellular calcium store was directly observed in the permeabilized cells in a dose-dependent manner. This release can be completely blocked by heparin, a well-known antagonist of IP3 receptor, indicating a direct activation of IP3 receptor on endoplasmic reticulum (ER) membrane by H2O2. It was also found that H2O2 could still induce a relatively small Ca2+ release from internal stores after the Ca2+-ATPase on ER membrane and the Ca2+ uptake to mitochondria were simultaneously inhibited by thapsigargin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The later observation suggests that a thapsigargin-insensitive non-mitochondrial intracellular Ca2+ store might be also involved in H2O2-induced Ca2+ mobilization.
Redox report: communications in free radical research 02/2005; 10(1):29-36. · 1.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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)P(3) production, PI-3K activation, and cytoplasmic Ca(2+) mobilization were investigated. It was found that Ins(1,4,5)P(3) 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 Ca(2+) release from the 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 Ca(2+) 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 activation, and the other is independent of PI-3K signaling. It was observed that AA could still induce a noncapacitative Ca(2+) entry in the cells when Ca(2+) release from the intracellular store was blocked by a PLC inhibitor, or a capacitative Ca(2+) entry was induced by preincubation with thapsigargin. However, the AA-mediated, noncapacitative Ca(2+) entry seems to play a little, if any, role in the stimulated respiratory burst. The present study suggests that the PLC signaling pathway, which may be activated by PLC(beta) and PLC(gamma), respectively, and the PI-3K signaling pathway are involved in the AA-stimulated respiratory burst in human neutrophil.
Journal of Leukocyte Biology 10/2003; 74(3):428-37. · 4.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Fluorescence quenching was used to study the kinetics of the transferrin receptor (TfR)-mediated iron uptake in the calcein-loaded K562 cells. It was found that elevation of intracellular free Ca(2+) ([Ca(2+)](i)) by thapsigargin (TG) speeds up the initial rate of iron uptake and increases the overall capacity of the cells in taking up iron. Depletion of intracellular Ca(2+) or complete chelation of extracellular Ca(2+) results in complete inhibition of the iron uptake in cells. To gain insight into molecular mechanism, IANBD-labeled transferrin (Tf) and microscopic fluorescence imaging were used to observe the endocytosis and recycling of the Tf-TfR complex in single live cells. The study showed that the preincubation of cells with TG or phorbol myristate acetate (PMA), the direct activator of protein kinase C (PKC), accelerated the endocytosis and recycling of the complex in a dose-dependent manner. W-7, the calmodulin antagonist, and GF109203X, a selected cell-permeant inhibitor of PKC, can reverse the acceleration. Analysis of actin polymerization in controlled, [Ca(2+)](i)-elevated and W-7-treated cells revealed that the actin polymerization is enhanced as [Ca(2+)](i) is raised, but reduced by W-7. The results suggest that the regulation of actin polymerization by intracellular Ca(2+) may play a central role in Ca(2+)-dependent iron uptake.
Cell Calcium 05/2003; 33(4):257-66. · 3.77 Impact Factor
