Inhibition of p38 Mitogen-Activated Protein Kinases Attenuates Methylmercury Toxicity in SH-SY5Y Neuroblastoma Cells.

Methylmercury (MeHg) is a toxic metal that causes irreversible damage to the nervous system, making it a risk factor for neuronal degeneration and diseases. MeHg activates various cell signaling pathways, particularly the mitogen-activated protein kinase (MAPK) cascades, which are believed to be important determinants of stress-induced cell fate. However, little is known about the signaling pathways that mitigate the neurotoxic effects of MeHg. Herein, we showed that pretreatment with a p38 MAPK-specific inhibitor, SB203580, attenuates MeHg toxicity in human neuroblastoma SH-SY5Y cells, whereas pretreatment with the extracellular signaling-regulated kinase inhibitor U0126 and the c-Jun N-terminal kinase inhibitor SP600125 does not. Specifically, we quantified the levels of intracellular mercury (Hg) and found that pretreatment with SB203580 reduced Hg levels compared to MeHg treatment alone. Further analysis showed that pretreatment with SB203580 increased multidrug resistance-associated protein 2 (MRP2) mRNA levels after MeHg treatment. These results indicate that detoxification of MeHg by p38 MAPK inhibitors may involve an efflux function of MeHg by inducing MRP2 expression.

Phytochelatin-mediated metal detoxification pathway is crucial for an organomercurial phenylmercury tolerance in Arabidopsis.

KEY MESSAGE: An organomercurial phenylmercury activates AtPCS1, an enzyme known for detoxification of inorganic metal(loid) ions in Arabidopsis and the induced metal-chelating peptides phytochelatins are essential for detoxification of phenylmercury. Small thiol-rich peptides phytochelatins (PCs) and their synthases (PCSs) are crucial for plants to mitigate the stress derived from various metal(loid) ions in their inorganic form including inorganic mercury [Hg(II)]. However, the possible roles of the PC/PCS system in organic mercury detoxification in plants remain elusive. We found that an organomercury phenylmercury (PheHg) induced PC synthesis in Arabidopsis thaliana plants as Hg(II), whereas methylmercury did not. The analyses of AtPCS1 mutant plants and in vitro assays using the AtPCS1-recombinant protein demonstrated that AtPCS1, the major PCS in A. thaliana, was responsible for the PheHg-responsive PC synthesis. AtPCS1 mutants cad1-3 and cad1-6, and the double mutant of PC-metal(loid) complex transporters AtABCC1 and AtABCC2 showed enhanced sensitivity to PheHg as well as to Hg(II). The hypersensitivity of cad1-3 to PheHg stress was complemented by the own-promoter-driven expression of AtPCS1-GFP. The confocal microscopy of the complementation lines showed that the AtPCS1-GFP was preferentially expressed in epidermal cells of the mature and elongation zones, and the outer-most layer of the lateral root cap cells in the meristematic zone. Moreover, in vitro PC-metal binding assay demonstrated that binding affinity between PC and PheHg was comparable to Hg(II). However, plant ionomic profiles, as well as root morphology under PheHg and Hg(II) stress, were divergent. These results suggest that PheHg phytotoxicity is different from Hg(II), but AtPCS1-mediated PC synthesis, complex formation, and vacuolar sequestration by AtABCC1 and AtABCC2 are similarly functional for both PheHg and Hg(II) detoxification in root surficial cell types.

Development of affinity bead-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione.

For a better understanding of metal-ligand interaction and its function in cells, we developed an easy, sensitive, and high-throughput method to quantify ligand-metal(loid) binding affinity under physiological conditions by combining ligand-attached affinity beads and inductively coupled plasma-optical emission spectrometry (ICP-OES). Glutathione (GSH) and two phytochelatins (PC2 and PC3, small peptides with different numbers of free thiols) were employed as model ligands and attached to hydrophilic beads. The principle of the assay resembles that of affinity purification of proteins in biochemistry: metals binding to the ligand on the beads and the rest in the buffer are separated by a spin column and quantified by ICP-OES. The binding assay using the GSH-attached beads and various metal(loid)s suggested the different affinity of the metal-GSH interactions, in accordance with the order of the Irving-Williams series and the reported stability constants. The binding assay using PC2 or PC3-attached beads suggested positive binding between PCs and Ni(II), Cu(II), Zn(II), Cd(II), and As(III) in accordance with the number of thiols in PC2 and PC3. We then conducted the competition assay using Cd(II), Mn(II), Fe(II), Cu(II), and Zn(II), and the results suggested a better binding affinity of PC2 with Cd(II) than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and Cd(II) compared to GSH and Cd(II). These results suggested the dominance of PC-Cd complex formation in vitro, supporting the physiological importance of PCs for the detoxification of cadmium in vivo. We also discuss the potential application of the assay.

Cadmium transport activity of four mercury transporters (MerC, MerE, MerF and MerT) and effects of the periplasmic mercury-binding protein MerP on Mer-dependent cadmium uptake.

Mercury superfamily proteins, i.e. inner membrane-spanning proteins (MerC, MerE, MerF and MerT) and a periplasmic mercury-binding protein (MerP), transport mercury into the cytoplasm. A previous study demonstrated that a Mer transporter homolog exhibits cadmium transport activity; based on this, the present study aimed to evaluate the cadmium transport activity of MerC, MerE, MerF and MerT and the effects of MerP co-expression in Escherichia coli. Bacteria expressing MerC, MerE, MerF or MerT without MerP were more sensitive to cadmium and significantly absorbed more cadmium than did the control strain. Expression of MerP in combination with MerC, MerE, MerF or MerT increased the bacterial sensitivity to cadmium and cadmium accumulation compared to a single expression of MerC, MerE, MerF or MerT. Cadmium uptake mediated by MerC, MerE, MerF or MerT was inhibited under cold or acidic conditions. These findings suggest that MerC, MerE, MerF and MerT are broad-spectrum heavy metal transporters that mediate both mercury and cadmium transport into cells and that MerP accelerates the cadmium transport ability of MerC, MerE, MerF and MerT.

Cytochalasin E increased the sensitivity of human lung cancer A549 cells to bortezomib via inhibition of autophagy.

Cancer cells enhance autophagic activity as a survival measure against metabolic and therapeutic stresses. The inhibition of autophagy may represent a valuable sensitizing target for cancer treatment. Recently, we examined the ability of various cytochalasins to inhibit autophagy and demonstrated the potent inhibitory effect of cytochalasin E (CE) on autophagic flux. The present study was conducted to investigate whether CE inhibited autophagosome-lysosome fusion, and to determine whether CE enhanced chemotherapy-induced cell death. Cell exposure to CE led to the accumulation of microtubule-associated protein light chain 3-II (LC3-II) and sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62) in a dose- and time-dependent manner. Cells treated with CE exhibited distinct formation of p62-positive structures on lysosome-associated membrane protein 2 (LAMP2)-positive lysosomal vesicles. CE treatment following serum starvation robustly reduced cell viability and increased expression levels of LC3-II and p62, in comparison to those of cells treated with CE alone. Furthermore, combination treatment with CE and bortezomib, an inhibitor of the 26S proteasome, showed a synergistic effect in targeting human lung cancer A549 cells. Altogether, our results demonstrated that CE treatment inhibited autophagosome-lysosome fusion, and this activity, in part, augmented bortezomib-induced cell death. Therefore, we concluded that CE may be a potentially effective therapeutic agent against lung cancer, especially in a combination therapy with proteasome inhibitors.

Identification of C-terminal Regions in Arabidopsis thaliana Phytochelatin Synthase 1 Specifically Involved in Activation by Arsenite.

Phytochelatins (PCs) are major chelators of toxic elements including inorganic arsenic (As) in plant cells. Their synthesis confers tolerance and influences within-plant mobility. Previous studies had shown that various metal/metalloid ions differentially activate PC synthesis. Here we identified C-terminal parts involved in arsenite- [As(III)] dependent activation of AtPCS1, the primary Arabidopsis PC synthase. The T-DNA insertion in the AtPCS1 mutant cad1-6 causes a truncation in the C-terminal regulatory domain that differentially affects activation by cadmium (Cd) and zinc (Zn). Comparisons of cad1-6 with the AtPCS1 null mutant cad1-3 and the double mutant of tonoplast PC transporters abcc1/2 revealed As(III) hypersensitivity of cad1-6 equal to that of cad1-3. Both cad1-6 and cad1-3 showed increased As distribution to shoots compared with Col-0, whereas Zn accumulation in shoots was equally lower in cad1-6 and cad1-3. Supporting these phenotypes of cad1-6, PC accumulation in the As(III)-exposed plants were at trace level in both cad1-6 and cad1-3, suggesting that the truncated AtPCS1 of cad1-6 is defective in PCS activity in response to As(III). Analysis of a C-terminal deletion series of AtPCS1 using the PCS-deficient mutant of fission yeast suggested important regions within the C-terminal domain for As(III)-dependent PC synthesis, which were different from the regions previously suggested for Cd- or Zn-dependent activation. Interestingly, we identified a truncated variant more strongly activated than the wild-type protein. This variant could potentially be used as a tool to better restrict As mobility in plants.

Cysteine and histidine residues are involved in Escherichia coli Tn21 MerE methylmercury transport.

Bacterial resistance to mercury compounds (mercurials) is mediated by proteins encoded by mercury resistance (mer) operons. Six merE variants with site-directed mutations were constructed to investigate the roles of the cysteine and histidine residues in MerE protein during mercurial transport. By comparison of mercurial uptake by the cell with intact and/or variant MerE, we showed that the cysteine pair in the first transmembrane domain was critical for the transport of both Hg(II) and CH 3Hg(I). Also, the histidine residue located near to the cysteine pair was critical for Hg(II) transport, whereas the histidine residue located on the periplasmic side was critical for CH 3Hg(I) transport. Thus, enhanced mercurial uptake mediated by MerE may be a promising strategy for the design of new biomass for use in the bioremediation of mercurials in the environment.

Variation in the activity of distinct cytochalasins as autophagy inhibitiors in human lung A549 cells.

Autophagy is a cell survival process that represents a therapeutic target in cancer treatment. Many types of cytochalasins have been identified and some of them have been reported to interfere with the formation of the autophagosome, although only limited data are available to assess their potential effects. Therefore, in this study, we examined the effects of cytochalasins and structurally related compounds on cell survival and the regulation of autophagy in human lung A549 adenocarcinoma cells. Cytochalasin D (CD) and cytochalasin E (CE) prominently inhibited the growth of A549 cells in a dose-dependent manner. Following treatment with CE, F-actin filaments were disrupted, and the proportion of binucleated cells increased, whereas no such effects were observed with the seven other cytochalasins tested. We found that cytochalasin H (CH), CD, and especially CE could induce the up-regulation of autophagy-related protein (LC3-II) and SQSTM1/p62. Using bafilomycin A1, we demonstrated that CD, CE, and CH inhibited autophagosome turnover, resulting in a dysfunctional autophagic process. The results of this study reveal that CE is the most potent cytochalasin in terms of its ability to induce cell death and inhibit autophagy. CE may therefore be an effective therapeutic agent against lung cancer.

Phytochelatin Synthase has Contrasting Effects on Cadmium and Arsenic Accumulation in Rice Grains.

Phytochelatin (PC) synthesis has been well demonstrated as a major metal tolerance mechanism in Arabidopsis thaliana, whereas its contribution to long-distance element transport especially in monocots remains elusive. Using rice as a cereal model, we examined physiological roles of Oryza sativa phytochelatin synthase 1 (OsPCS1) in the distribution and detoxification of arsenic (As) and cadmium (Cd), two toxic elements associated with major food safety concerns. First, we isolated four different transcript variants of OsPCS1 as well as one from OsPCS2. Quantitative real-time reverse transcription-PCR (RT-PCR) of each OsPCS transcript in rice seedlings suggested that expression of OsPCS1full, the longest OsPCS1 variant, was most abundant, followed by OsPCS2. Heterologous expression of OsPCS variants in PCS-deficient mutants of Schizosaccharomyces pombe and A. thaliana suggested that OsPCS1full possessed PCS activity in response to As(III) and Cd while the activity of other PCS variants was very low. To address physiological functions in toxic element tolerance and accumulation, two independent OsPCS1 mutant rice lines (a T-DNA and a Tos17 insertion line) were identified. The OsPCS1 mutants exhibited increased sensitivity to As(III) and Cd in hydroponic experiments, showing the importance of OsPCS1-dependent PC synthesis for rice As(III) and Cd tolerance. Elemental analyses of rice plants grown in soil with environmentally relevant As and Cd concentrations showed increased As accumulation and decreased Cd accumulation in grains of the T-DNA line. The Tos17 mutant also exhibited the reduced Cd accumulation phenotype. These contrasting effects on As and Cd distribution to grains suggest the existence of at least partially distinct PC-dependent pathways for As and Cd.

ATP-binding cassette transporter A1 is involved in hepatic alpha-tocopherol secretion.

Vitamin E (alpha-tocopherol) is an essential fat-soluble nutrient with antioxidant properties. alpha-Tocopherol transfer protein (alpha-TTP), the product of the gene responsible for familial isolated vitamin E deficiency, plays an important role in maintaining the plasma alpha-tocopherol level by mediating the secretion of alpha-tocopherol by the liver. However, the mechanisms underlying hepatic alpha-tocopherol secretion are not fully understood. This study was undertaken to elucidate the mechanism of alpha-tocopherol re-efflux from hepatocytes, the cells that have the most important role in regulating plasma-alpha-tocopherol concentrations. From in vitro experiments using [(3)H]alpha-tocopheryl acetate and McARH7777 cells that stably express alpha-tocopherol transfer protein (alpha-TTP), the following results were obtained. First, addition of apolipoprotein A-I (apoA-I), a direct acceptor of the ATP-binding cassette transporter A1 (ABCA1)-secreted lipids, increased alpha-tocopherol secretion in a dose-dependent manner. Second, probucol, an antiatherogenic compound reported to be an inactivator of ABCA1 reduced hepatic alpha-tocopherol secretion. Third, ABCA1-RNAi suppressed hepatic alpha-tocopherol secretion. In a mouse in vivo experiment, addition of 1% probucol to the diet decreased plasma alpha-tocopherol concentrations. These results strongly suggest that ABCA1 is substantially involved in hepatic alpha-tocopherol secretion.

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1.

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.

Structure-activity relationships of fluorinated lysophosphatidic acid analogues.

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions with seven-transmembrane G-protein-coupled receptors (GPCRs) and the nuclear hormone receptor PPARgamma. To identify receptor-selective LPA analogues, we describe a series of fluorinated LPA analogues in which either the sn-1 or sn-2 hydroxyl group was replaced by a fluoro or fluoromethyl substituent. We also describe stabilized phosphonate analogues in which the bridging oxygen of the monophosphate was replaced by an alpha-monofluoromethylene (-CHF-) or alpha-difluoromethylene (-CF(2)-) moiety. The sn-2- and sn-1-fluoro-LPA analogues were unable to undergo acyl migration, effectively "freezing" them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively. We first tested these LPA analogues on insect Sf9 cells induced to express human LPA(1), LPA(2), and LPA(3) receptors. While none of the analogues were found to be more potent than 1-oleoyl-LPA at LPA(1) and LPA(2), several LPA analogues were potent LPA(3)-selective agonists. In contrast, 1-oleoyl-LPA had similar activity at all three receptors. The alpha-fluoromethylene phosphonate analogue 15 activated calcium release in LPA(3)-transfected insect Sf9 cells at a concentration 100-fold lower than that of 1-oleoyl-LPA. This activation was enantioselective, with the (2S)-enantiomer showing 1000-fold more activity than the (2R)-enantiomer. Similar results were found for calcium release in HT-29 and OVCAR8 cells. Analogue 15 was also more effective than 1-oleoyl-LPA in activating MAPK and AKT in cells expressing high levels of LPA(3). The alpha-fluoromethylene phosphonate moiety greatly increased the half-life of 15 in cell culture. Thus, alpha-fluoromethylene LPA analogues are unique new phosphatase-resistant ligands that provide enantiospecific and receptor-specific biological readouts.

Neuronal expression and neuritogenic action of group X secreted phospholipase A2.

Although individual mammalian secreted phospholipase A(2) (sPLA(2)) enzymes exhibit unique tissue and cellular distributions, the cell type-specific functions of each enzyme remain largely unknown. In this study, we found by immunohistochemistry that group X sPLA(2) (sPLA(2)-X) is uniquely located in the peripheral neuronal fibers, an observation that was supported by detection of its transcript and protein in the neuronal cell line PC12 and in primary dorsal root ganglia neurons. Adenoviral expression of sPLA(2)-X in PC12 cells facilitated neurite outgrowth, particularly when combined with a suboptimal concentration of nerve growth factor. In neuronally differentiated PC12 cells, sPLA(2)-X was preferentially localized in the Golgi apparatus and growth cones, and proteolytic conversion of the proenzyme to mature enzyme mainly occurred after the secretion process. The neurite-extending ability of sPLA(2)-X depended on the production of its catalytic product, lysophosphatidylcholine. Moreover, nerve growth factor-induced neurite extension of PC12 cells was modestly but significantly attenuated by an anti-sPLA(2)-X antibody or by a small interfering RNA for sPLA(2)-X. These observations suggest the potential contribution of sPLA(2)-X to neuronal differentiation, and possibly repair, under certain conditions.

Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2.

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.

Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma.

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti-apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non-specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA-synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 degrees C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA-degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA-phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer.

Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide.

Scavenger receptor expressed by endothelial cells I (SREC-I) is a novel endocytic receptor for acetylated low density lipoprotein (LDL). Here we show that SREC-I is expressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly stimulated the expression of SREC-I in macrophages. In an initial attempt to clarify the role of SREC-I in the uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with a targeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming effect of the type A scavenger receptor (SR-A) on the uptake of Ac-LDL, we further generated mice lacking both SR-A and SREC-I (SR-A(-/-);SREC-I(-/-)) by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 90 and 6%, respectively. LPS increased the absolute contribution of SR-A and SREC-I by 1.9- and 2.3-fold, respectively. On the other hand, LPS decreased the absolute contribution of other pathways by 31%. Consistently, LPS did not increase the expression of other members of the scavenger receptor family such as CD36. In conclusion, SREC-I serves as a major endocytic receptor for Ac-LDL in LPS-stimulated macrophages lacking SR-A, suggesting that it has a key role in the development of atherosclerosis in concert with SR-A.