Yasukazu Takanezawa

Vacuolar sequestration via tonoplast-targeted MerC enhances Cd tolerance; expression in roots plays a primary role, while expression in mesophyll is also crucial for protecting the chloroplasts. Targeting metal transporters to the vacuole via genetic engineering offers a strategy to enhance plant tolerance to toxic metals like cadmium (Cd) by promoting vacuolar sequestration. Understanding the influence of different expression patterns of metal transporters is crucial for elucidating Cd-tolerance mechanisms. This study investigated how ubiquitous (p35S promoter) versus mesophyll-specific (pRBCS1A promoter) expression of a tonoplast-targeted bacterial metal transporter, MerC-AtVAM3 (CV), impacts Cd tolerance, nutrient homeostasis, and subcellular Cd distribution in Arabidopsis. While the short-term plate assays revealed only slight tolerance improvements, the long-term hydroponic Cd treatments (0.5 µM and 1 µM) resulted in significant enhancement in the CV-expressing lines. Notably, the p35S-CV line, which ubiquitously expresses the transgene, exhibited stronger tolerance (improved growth, mitigated chlorosis confirmed by higher SPAD values) compared to the mesophyll-specific pRBCS1A-TCV lines, particularly under 1 µM Cd. Nutritional profiling indicated that CV expression alleviated some Cd-induced nutrient imbalances. Although root Cd accumulation was similar across lines, p35S-CV shoots displayed approximately 30% lower Cd concentration compared to the wild-type (Col-0) and pRBCS1A lines under 1 µM Cd conditions. In Col-0 mesophyll cells, subcellular analysis using the Leadmium Green dye showed Cd was preferentially localized in the peripheral cytoplasm. The Leadmium Green signal also exhibited strong co-localization with chloroplasts. Conversely, CV expression effectively redirected Cd to the central vacuole, confirming efficient sequestration by the tonoplast-targeted MerC. The superior tolerance of the p35S-CV line strongly suggests that vacuolar Cd sequestration in roots plays a primary role in conferring robust Cd tolerance in Arabidopsis, while vacuolar sequestration in shoots provides supportive protection.

p62/SQSTM1 (p62) and neighbor of BRCA1 gene 1 (NBR1) are two important cargo receptors involved in selective autophagy. While p62 is known to safeguard cells against the toxic effects of the environmental toxicant methylmercury (MeHg), the specific functions of p62 and NBR1 in MeHg-exposed cells remain unclear. In this study, we aimed to elucidate the distinct roles of p62 and NBR1 in conferring protection against cytotoxicity induced by MeHg. We found that MeHg increased both the mRNA and protein levels of p62 while decreasing those of NBR1. Upon exposure to MeHg, p62-knockout (KO) cells exhibited an approximately 30 % reduction in cell viability compared to wild-type (WT) cells; however, no such reduction was observed in NBR1KO cells. Additionally, p62KO cells exhibited a 1.5-fold increase in intracellular mercury (Hg) concentration compared to the WT following MeHg exposure, whereas NBR1KO cells had Hg levels comparable to those of WT cells. Upon exposure to MeHg, Nrf2 signaling activation was significantly reduced in p62KO cells compared to that in WT cells, whereas NBR1KO cells displayed Nrf2 activation levels similar to those of WT cells. Overall, these results suggest that p62, rather than NBR1, plays a crucial role in mitigating MeHg-induced cytotoxicity by reducing intracellular Hg levels through the activation of the Nrf2 signaling pathway.

We previously reported that in Arabidopsis, the phytochelatin-mediated metal-detoxification machinery is also essential for organomercurial phenylmercury (PheHg) tolerance. PheHg treatment causes severe root growth inhibition in cad1-3, an Arabidopsis phytochelatin-deficient mutant, frequently accompanied by abnormal root tip swelling. Here, we examine morphological and physiological characteristics of PheHg-induced abnormal root tip swelling in comparison to Hg(II) stress and demonstrate that auxin homeostasis disorder in the root is associated with the PheHg-induced root tip swelling. Both Hg(II) and PheHg treatments severely inhibited root growth in cad1-3 and simultaneously induced the disappearance of starch-containing plastid amyloplasts in columella cells. However, further confocal imaging of the root tip revealed distinct effects of Hg(II) and PheHg toxicity on root cell morphology. PheHg treatment suppressed most major genes involved in auxin homeostasis, whereas these expression levels were up-regulated after 24 h of Hg(II) treatment. PheHg-triggered suppression of auxin transporters PIN1, PIN2, and PIN3 as GFP-fusion proteins was observed in the root tip, accompanied by an auxin reporter DR5rev::GFP signal reduction. Supplementation of indole-3-acetic acid (IAA) drastically canceled the PheHg-induced root swelling, however, Hg(II) toxicity was not mitigated by IAA. The presented results show that the collapse of auxin homeostasis especially in root tips is a cause for the abnormal root tip swelling under PheHg stress conditions.

Methylmercury (MeHg) is widely distributed in nature and is known to cause neurotoxic effects. This study aimed to examine the anti-MeHg activity of oleanolic acid-3-glucoside (OA3Glu), a synthetic oleanane-type saponin derivative, by evaluating its effects on motor function, pathology, and electrophysiological properties in a mouse model of MeHg poisoning. Mice were orally administered 2 or 4 mg·kg-1·d-1 MeHg with or without 100 µg·kg-1·d-1 OA3Glu 5x/week for four weeks. Motor function was evaluated using beam-walking and dynamic weight-bearing (DWB) tests. High-dose MeHg exposure significantly increased the frequency of stepping off the hind leg while crossing the beam in the beam-walking test, and increased weight on forelegs when moving freely in the DWB test. OA3Glu treatment alleviated motor abnormality caused by high-dose MeHg exposure in both motor function tests. Additionally, OA3Glu treatment reduced the number of contracted Purkinje cells frequently observed in the cerebellum of MeHg-treated groups, although cerebrum histology was similar in all experimental groups. The synaptic potential amplitude in the cerebellum decreased as MeHg exposure increased, which was restored by OA3Glu treatment. Even in the cerebrum, where the effects of MeHg were not observed, the amplitude of the field potential was suppressed with increasing MeHg exposure but was restored with OA3Glu treatment. Taken together, the study findings suggest that OA3Glu improves neurotransmission and movement disorders associated with MeHg exposure via protection of Purkinje cells in the cerebellum while ameliorating pre/post-synaptic deficits in the cerebral cortex in which no changes were observed at the tissue level, potentially providing a treatment to mitigate MeHg toxicity.

Gadolinium-based contrast agents (GBCAs) are widely used in magnetic resonance imaging (MRI) to improve the sensitivity and enhance diagnostic performance. GBCAs are mostly eliminated from the body through the kidney after administration; however small amounts of gadolinium are retained in the brain and other tissues. Although there is increasing concern about the adverse health effects of gadolinium, the cellular effects of GBCAs remains poorly understood. Here, we elucidated the potential cytotoxicity of the GBCAs Omniscan and Gadovist in 12 different cell lines, especially 3T3-L1 adipocyte cell line. Omniscan and Gadovist treatments significantly increased intracellular gadolinium levels in 3T3-L1 cells in a time- and dose-dependent manner. Additionally, Omniscan and Gadovist treatments downregulated the expression of adipocyte differentiation markers, including peroxisome proliferator-activated receptor γ (PPARG), adiponectin (ADIPOQ), and fatty acid-binding protein (FABP4), in 3T3-L1 cells, especially during early differentiation (day 0-2). Moreover, histological analysis using Oil red O staining showed that gadolinium chloride (GdCl3) treatment suppressed lipid droplet accumulation and the expression of adipocyte differentiation markers. Overall, the results showed that Omniscan and Gadovist treatment suppressed adipocyte differentiation in 3T3-L1 cells, contributing to the understanding of the potential toxic effects of GBCA exposure.

Chronic exposure to methylmercury (MeHg) is positively associated with obesity and metabolic syndromes. However, the effect of MeHg on adipogenesis has not been thoroughly investigated. This study investigated the effects of continuous exposure to 0.5 µM MeHg on adipocyte differentiation in 3T3-L1 cells. Oil Red O staining and triglycerides (TG) assays demonstrated that MeHg enhanced the TG content in 3T3-L1 cells. MeHg enhanced the mRNA and protein expression of adipocyte differentiation markers including peroxisome proliferator-activated receptor γ, adiponectin, and fatty acid-binding protein, and their expression levels were prominent during the late stages (days 6-8) after the induction of differentiation. In addition, 0.5 µM MeHg promoted the expression of autophagy-related genes, including light chain 3 B-II and p62, after induction of differentiation. Treatment of 3T3-L1 cells with chloroquine (CQ), an autophagy inhibitor, during the early stages (days 0-2) after induction of differentiation inhibited cellular lipid accumulation in the presence of 0.5 µM MeHg. However, treatment with CQ during the late stages (days 6-8) had little effect on the MeHg-induced increase in TG content and the expression of adipocyte differentiation markers. Although the underlying mechanisms in the late stages remain to be completely elucidated, but the present data suggest that autophagy and other mechanisms play critical roles in adipogenesis during MeHg-induced differentiation. Collectively, our results suggest that continuous exposure to MeHg induces TG accumulation and expression of genes related to adipogenesis, especially during the late stages of 3T3-L1 differentiation, which may contribute to an improved understanding of MeHg-induced adipogenesis.

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.

Methylmercury (MeHg), an environmental pollutant, disrupts and impairs cellular function. MeHg binds to various cellular proteins, causing dysfunction and misfolding, which are considered underlying causes of MeHg toxicity. The p62 protein, also termed SQSTM1, is a ubiquitin-binding protein that targets ubiquitinated substrates to undergo autophagy and plays a key role in ameliorating MeHg toxicity. p62 also delivers ubiquitinated substrates to proteasomes. However, the role of these degradation systems in mitigating MeHg toxicity remains unknown. Herein, we explored the impact of the proteasome inhibitor MG132 on MeHg toxicity and examined the toxicity of co-treatment with MG132 and MeHg in p62KO mouse embryonic fibroblasts (MEFs) by analyzing cell viability, immunoblotting, mRNA levels, immunofluorescence, and the mercury content. The proteasome inhibitor MG132 enhanced MeHg-induced cytotoxicity while reducing intracellular mercury levels in MEFs. Co-treatment with MG132 and MeHg markedly increased levels of p62 and ubiquitinated proteins. Furthermore, co-treatment with MG132 and MeHg reduced p62KO MEF viability compared to that of wild-type MEFs. Our findings suggest that the proteasome participates in mitigating MeHg cytotoxicity, while p62 may play an important role in transporting MeHg-induced ubiquitinated proteins to the proteasome, as well as in autophagy. Collectively, these results imply that p62, and proteasome, and autophagy are vital for cytoprotection against MeHg toxicity.

SQSTM1/p62, hereinafter referred to as p62, is a stress-induced cellular protein that interacts with various signaling proteins as well as ubiquitinated proteins to regulate a variety of cellular functions and cell survival. Methylmercury (MeHg) exposure increases the levels of p62, the latter playing a protective role in MeHg-induced toxicity. However, the underlying mechanism by which p62 alleviates MeHg toxicity remains poorly understood. Herein, we report the interaction of p62 with neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), a HECT E3 ubiquitin ligase. The region of p62 where NEDD4 binds is located at the proline- and arginine (PR)-rich region (amino acids: 102-119), C-terminal extension of the Phox and Bem1 (PB1) domain. To evaluate the importance of the p62-NEDD4 complex, we examined the compensation of deletion mutant (GFP-Δ102-119 p62) for the lack of endogenous p62 in MEFs. GFP-p62/p62KO cells exhibited significantly higher cell viability than GFP-Δ102-119 p62/p62KO cells after treatment with MeHg. Our findings suggest novel mechanisms to alleviate MeHg toxicity through p62-NEDD4 complex formation.

Gadolinium-based contrast agents (GBCAs) are widely used to improve tissue contrast during magnetic resonance imaging. Exposure to GBCAs can result in gadolinium deposition within human tissues and has become a clinical concern because of the potential toxic effects of free gadolinium (Gd3+). Here, we report the impact of a single administration of GBCAs (Omniscan and Gadovist), and Gd3+ on mouse tissues. Five-week-old male BALB/c mice were injected intravenously with GBCAs or Gd3+. Seven days after injection, relatively high levels of gadolinium were detected in the spleen (118.87 nmol/g tissue), liver (83.00 nmol/g tissue), skin (48.56 nmol/g tissue), and kidneys (25.59 nmol/g tissue) of the Gd(NO3)3 (high dose: 0.165 mmol/kg) group; in the bones (11.12 nmol/g tissue), kidneys (7.49 nmol/g tissue), teeth (teeth: 6.18 nmol/g tissue), and skin (2.43 nmol/g tissue) of the Omniscan (high dose: 1.654 mmol/kg) group and in the kidneys (16.36 nmol/g tissue) and skin (4.88 nmol/g tissue) of the Gadovist (high dose: 3.308 mmol/kg) group. Enlargement of the spleen was observed in the Gd3+ group (p < 0.05), but not in the Omniscan or Gadovist groups. Gd3+ caused iron accumulation around the white pulp of the spleen, suggesting that enlargement of the spleen is, at least in part, associated with Gd3+ and/or iron accumulation. Our results may help elucidate the relative risks of different types of gadolinium agents, the mechanisms involved, and even recognition of potential toxic effects of GBCAs.

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.

Methylmercury (MeHg) is a hazardous environmental pollutant that causes serious toxicity in humans and animals, as well as proteotoxic stress. In our previous study, we found that MeHg induces the expression of p62/sequestosome 1 (p62) that selectively targets ubiquitinated proteins for degradation via autophagy, and that p62 might protect cells against MeHg toxicity. To further investigate the role of p62 in MeHg-induced stress responses, we evaluated the role of p62 in MeHg-induced endoplasmic reticulum (ER) stress in p62 knockout (p62KO) mouse embryonic fibroblasts (MEFs). Treatment of wild-type (WT) MEFs were treated with MeHg (1 μM) increased mRNA levels of Chop encoding C/EBP homologous protein, Trib3 encoding Tribbles homolog 3, and Dnajb9 encoding DnaJ heat-shock protein family (Hsp40) member B9 increased, suggesting that ER stress is elicited by MeHg stress. Additionally, p62KO MEFs treated with MeHg showed a higher mRNA expression of Chop and Trib3 relative to that in WT MEFs. Furthermore, knock-in of GFP-p62 to p62KO cells diminished the Chop and Trib3 induction responses to MeHg stress and resulted in a higher cell viability than that of p62KO MEFs. These results suggest that the protective role of p62 against MeHg toxicity is partly mediated by suppressing the ER stress response.

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.

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.

Gadolinium-based contrast agents (GBCAs) are widely used in clinical magnetic resonance imaging (MRI). Free gadolinium ions (Gd3+) released from GBCAs potentially increase the risk of GBCA-related toxicity. However, the cellular responses to Gd3+ and the underlying mechanisms responsible for protection against Gd3+ remain poorly understood. Recently, autophagy has been considered a cell survival mechanism against various toxic metals. Here, we investigated the relationship between Gd3+ and autophagy, as well as the effect of autophagy inhibition on the survival of cells exposed to Gd3+. We found that the increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II, a marker protein of autophagy, in Gd3+-exposed human embryonic kidney 293 (HEK293) cells. Moreover, we found a greater accumulation of LC3-II after exposure to an autophagy inhibitor, chloroquine (CQ), combined with Gd3+ than that after exposure to CQ alone, suggesting that Gd3+ activated autophagy in HEK293 cells. Furthermore, we found that Gd3+ reduced cell viability, which was more pronounced after CQ treatment. Our findings indicated that autophagy exerted a cytoprotective effect against Gd3+ toxicity, suggesting a potential link between autophagy and GBCA-associated adverse events.

For researchers in the plant metal field, the agar reagent used for the solid plate medium is a problematic factor because application of different agar types and even a different lot of the same agar type can mask the plant metal-related phenotypes and impair the reproducibility. In this study, we systematically assessed effects of different agar reagents on metal(loid) sensitivity and element accumulation of the Arabidopsis metal sensitive mutants. Three established mutants (cad1-3, cad1-6, and abcc1/2), and three different types of purified agar reagents (Type A, Type E, and Nacalai) with two independent batches for each reagent were subjected to the analyses. First, we found that element concentrations in the agar reagents largely varied among the agar types. Then the effects of agar reagents on the mutant metal(loid)-sensitivity were examined under As(III), Hg(II), Cd(II), and excess Zn(II) conditions. A significant variation of the mutant metal(loid)-sensitivity was observed among the different agar plates but the variation depended on the combination of metal(loid) stress and agar reagents. Briefly, the type-dependent variation was more evident under As(III) and Hg(II) than Cd(II) or excess Zn(II) conditions. A lot-dependent variation was also observed for Type A and Type E but not for Nacalai: hypersensitive phenotypes of cad1-3, cad1-6, and abcc1/2 under As(III) or Hg(II) treatments were diminished when different batches of the Type A or Type E agar types were used. We also found a significant variation of As and Hg accumulation in the wild-type and cad1-3. Plant As and Hg concentrations were remarkably higher and the difference between the genotypes was more evident when grown with Type A agar plates. We finally analyzed ionomic profiles in the plants exposed to As(III) stress. Agar-type specific ionomic changes in cad1-3 were more observed with the Type A plates than with the Nacalai plates. The presented results overall suggest that suitability of agar reagents for metal(loid)-related phenotyping depends on the experimental design, and an inappropriate selection of agar reagents can mask even very clear phenotypes of the established mutant like cad1-3. We also discuss perspectives on the agar problem in the plant metal study.

Some methylmercury (MeHg) is converted to inorganic mercury (Hg2+) after incorporation into human and animal tissues, where it can remain for a long time. To determine the overall toxicity of MeHg in tissues, studies should evaluate low concentrations of Hg2+. Although demethylation is involved, the participating enzymes or underlying mechanisms are unknown; in addition, the low cell membrane permeability of Hg2+ makes these analyses challenging. We established model cell lines to assess toxicities of low concentrations of Hg2+ using bacterial organomercury lyase (MerB). We engineered MerB-expressing HEK293 and HeLa cell lines that catalyze MeHg demethylation. These cells were significantly more sensitive to MeHg exposure compared to the parental cells. MeHg treatment remarkably induced metallothioneins (MTs) and hemeoxygenase-1 (HMOX-1) mRNAs and modest expression of superoxide dismutase 1, whereas catalase and glutathione peroxidase 1 mRNAs were not up-regulated. merB knockdown using small interfering RNA supported the induction of MT and HMOX-1 mRNA by MerB enzymatic activity. Pretreatment with Trolox, a water-soluble vitamin E analog, did not inhibit MeHg-induced elevation of MT-Ix and HMOX-1 mRNAs in MerB-expressing cells, suggesting that Hg2+ works independently of reactive oxygen species generation. Similar results were obtained in cells expressing MerB, suggesting that high MTs and HMOX-1 induction and cytotoxicity are common cellular responses to low intracellular Hg2+ concentrations. This is the first study to establish cell lines that demethylate intracellular MeHg to Hg2+ using bacterial MerB for overcoming the low membrane permeability of Hg2+ and exploring the intracellular responses and toxicities of low Hg2+ concentrations.

Methylmercury (MeHg) is a highly toxic pollutant, and is considered hazardous to human health. In our previous study, we found that MeHg induces autophagy and that Atg5-dependent autophagy plays a protective role against MeHg toxicity. To further characterize the role of autophagy in MeHg-induced toxicity, we examined the impact of autophagy on microtubules and nuclei under MeHg exposure using Atg5KO mouse embryonic fibroblasts (MEFs). Low concentrations of MeHg induced a decrease in α-tubulin and acetylated-tubulin in both wild-type and Atg5KO cells. While α-tubulin acetylation was promoted by treatment with tubacin, a selective inhibitor of histone deacetylase 6, MeHg treatment inhibits the increase of tubacin-induced acetylated-tubulin. However, similar effects were observed for treatment with either tubacin or tubacin + MeHg in wild-type and Atg5KO cells. We also found a significant increase in the number of multinuclear cells upon MeHg exposure in Atg5KO MEFs compared to wild-type MEFs. In addition, DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), markedly increased in Atg5KO MEFs compared to wild-type MEFs. Our results therefore suggest that autophagy is not a simple elimination pathway of MeHg-induced damaged proteins, but that it also plays a protective role in the context of MeHg-associated DSBs.

Methylmercury (MeHg) is one of the most toxic environmental pollutants, presenting a serious health hazard worldwide. In this study, we examined the potential of derivatives of oleanolic acid (OA), such as OA 3-glucoside, OA 28-glucoside, and OA 3,28-diglucoside, to mitigate MeHg toxicity in vitro and in vivo. We found that OA 3-glucoside suppressed the cellular MeHg uptake by 63.4% compared with that of the control and improved the cell viability from 75.4% to 107.9% upon exposure to cytotoxic MeHg in Caco-2 cells. To verify the anti-MeHg activity of OA 3-glucoside, mice were orally administered MeHg (0, 1.0, or 5.0 mg kg-1·d-1), with or without OA 3-glucoside, and then mercury accumulation was measured in various organs of the mice. The mice co-treated with MeHg and OA 3-glucoside showed significantly lower mercury content in organs such as the cerebrum, cerebellum, liver, kidney, and spleen, with 83.1%, 68.7%, 71.7%, 82.1%, and 18.2% of those in the OA 3-glucoside-untreated group, respectively. This suggested OA 3-glucoside had the potential as an anti-MeHg compound, owing to its ability to suppress the distribution of MeHg into organs. Supporting this hypothesis, the mice treated with MeHg and OA 3-glucoside showed a tendency to survive one day longer than the control mice. Our findings suggest OA 3-glucoside administration alleviates the toxicity of MeHg by suppressing MeHg accumulation in organs.

Fish consumption has both the risk of methylmercury (MeHg) poisoning and the benefit of obtaining n-3 polyunsaturated fatty acids (n-3 PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). However, the cellular interaction between MeHg and PUFAs remains unknown. Therefore, the aim of this study was to investigate the effects of MeHg and n-3 PUFA exposure on mouse embryonic fibroblasts (MEFs). The results showed that EPA had a negligible effect on MeHg-induced cell death, whereas DHA promoted it. Thiobarbituric acid reactive substance (TBARS) concentrations in cells exposed to DHA and MeHg were higher than in those exposed to EPA and MeHg. Treatment with DHA and MeHg markedly induced the expression of endoplasmic reticulum (ER) stress (CHOP and DNAJB9) and Nrf2 target gene (p62 and HMOX-1) mRNA levels. Unexpectedly, EPA supplementation in addition to DHA and MeHg attenuated DHA- and MeHg-induced cell death and suppressed ER stress and expression of Nrf2 target genes. Our results revealed a differential impact of DHA and EPA on MeHg-induced cell death, and combined treatment with DHA and EPA along with MeHg attenuated MeHg-induced toxicity.

For mercury phytoextraction, we previously demonstrated in Arabidopsis thaliana that a constitutive and ubiquitous promoter-driven expression of a bacterial mercury transporter MerC fused with SYP121, a plant SNARE for plasma membrane protein trafficking increases plant mercury accumulation. To advance regulation of ectopic expression of the bacterial transporter in the plant system, the present study examined whether merC-SYP121 expression driven by a root epidermis specific promoter (pEpi) is sufficient to enhance mercury accumulation in plant tissues. We generated five independent transgenic Arabidopsis plant lines (hereafter pEpi lines) expressing a transgene encoding MerC-SYP121 N-terminally tagged with a fluorescent protein mTRQ2 under the control of pEpi, a root epidermal promoter. Confocal microscopy analysis of the pEpi lines showed that mTRQ2-MerC-SYP121 was preferentially expressed in lateral root cap in the root meristematic zone and epidermal cells in the elongation zone of the roots. Mercury accumulation in shoots of the pEpi lines exposed to inorganic mercury was overall higher than the wild-type and comparable to the over-expressing line. The results suggest that cell-type specific expression of the bacterial transporter MerC in plant roots sufficiently enhances mercury accumulation in shoots, which could be a useful phenotype for improving efficiency of mercury phytoremediation.

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.

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.

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.

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.

Methylmercury (MeHg) is a widely distributed environmental pollutant that causes a series of cytotoxic effects. However, molecular mechanisms underlying MeHg toxicity are not fully understood. Here, we report that sequestosome1/p62 protects mouse embryonic fibroblasts (MEFs) against low-dose MeHg cytotoxicity via clearance of MeHg-induced ubiquitinated proteins. p62 mRNA and protein expression in MEFs were temporally induced by MeHg exposure p62-deficient MEFs exhibited higher sensitivity to MeHg exposure compared to their wild-type (WT) counterparts. An earlier and higher level of accumulation of ubiquitinated proteins was detected in p62-deficient cells compared with WT MEFs. Confocal microscopy revealed that p62 and ubiquitinated proteins co-localized in the perinuclear region of MEFs following MeHg treatment. Further analysis of MEFs revealed that ubiquitinated proteins co-localized with LC3-positive puncta upon co-treatment with MeHg and chloroquine, an autophagy inhibitor. In contrast, there was minimal co-localization in p62-deficient MEFs. The present study, for the first time, examined the expression and distribution of p62 and ubiquitinated proteins in cells exposed to low-dose MeHg. Our findings suggest that p62 is crucial for cytoprotection against MeHg-induced toxicity and is required for MeHg-induced ubiquitinated protein clearance.

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.

MerC, encoded by merC in the transposon Tn21 mer operon, is a heavy metal transporter with potential applications for phytoremediation of heavy metals such as mercuric ion and cadmium. In this study, we demonstrate that MerC also acts as a transporter for methylmercury. When MerC was expressed in Escherichia coli XL1-Blue, cells became hypersensitive to CH3Hg(I) and the uptake of CH3Hg(I) by these cells was higher than that by cells of the isogenic strain. Moreover, transgenic Arabidopsis plants expressing bacterial MerC or MerC fused to plant soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) accumulated CH3Hg(I) effectively and their growth was comparable to the wild-type plants. These results demonstrate that when the bacterium-derived merC gene is ectopically introduced in genetically modified plants, MerC expression in the transgenic plants promotes the transport and sequestration of methylmercury. Thus, our results show that the expression of merC in Arabidopsis results in transgenic plants that could be used for the phytoremediation and elimination of toxic methylmercury from the environment.

Methylmercury (MeHg) is a widespread environmental pollutant and causes a serious hazard to health worldwide. However, molecular mechanisms underlying MeHg toxicity remain elusive. We show that MeHg reduced mouse embryonic fibroblast (MEF) viability in a dose-dependent manner. Furthermore, MeHg treatment increased levels of autophagy markers LC3-II and p62, possibly by acting on the MAPKs signaling pathway in several cell types. MeHg exposure elevated the number of LC3 puncta in stable GFP-LC3 MEFs and the number of autophagic vacuoles. The accumulation of LC3-II and p62 increased further when complementing MeHg with autophagy inhibitor, chloroquine. Moreover, we found that autophagy-related gene 5-deficient (Atg5-/-) MEFs exhibited higher sensitivity and higher levels of p62 compared to their wild-type counterparts following MeHg exposure. This suggested that p62 was upregulated at the transcription level by MeHg and degraded by Atg5-dependent autophagy. Our data demonstrate that MeHg exposure promotes autophagy, and Atg5-dependent autophagy serves to protect cells from MeHg cytotoxicity.

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.

Plasma membranes are essential components of living cells, and phospholipids are major components of cellular membranes. Here, we used liquid chromatography/mass spectrometry to investigate changes in the membrane phospholipid content that occur in association with aging. Our results indicate that the levels of a particular species of phosphatidylcholine comprised of stearic acid and arachidonic acid increased with age. To determine the reason for the increased levels of this particular phosphatidylcholine, we examined the effect of highly unsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, on cellular aging. Applied arachidonic acid was incorporated into phosphatidylcholine molecules, but neither arachidonic acid nor other related unsaturated fatty acids had any effect. We conclude that increased levels of this distinctive phosphatidylcholine are a result of in vitro senescence.

Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanisms associated with MeHg-dependent neurotoxicity remains are unclear, it is known that MeHg leads to neurotoxicity in cerebellar granule cells (CGCs). In vitro exposure of murine CGC primary cultures to MeHg resulted in time- and concentration-dependent cell death. The present study was designed to assess the effect of fat-soluble antioxidant tocopherols and tocotrienols (unsaturated vitamin E) on MeHg-induced neurotoxicity using cultured CGCs. Significant protection from MeHg-induced neuronal cell death was observed with both tocopherols and tocotrienols. Moreover, we observed that tocotrienols are multi-fold more potent than tocopherols in protecting CGCs against MeHg neurotoxicity. At micromolar concentration, tocotrienols, but not tocopherols, showed complete protection by an antioxidant mechanism. Similarly, tocopherols and tocotrienols showed a protective effect on CGCs migration against MeHg-toxicity. These results suggested that oxidative events may contribute to MeHg toxicity in isolated cerebellar granule neurons, and that tocotrienols are potent supplements for pharmacological protection of the developing brain exposed to MeHg.

LIS1, a causative gene product for type I lissencephaly, binds to and regulates the dynein motor and the centrosome. LIS1 also forms a complex with the catalytic subunits alpha1 and alpha2 of type I platelet-activating factor acetylhydrolase [PAF-AH (I)]. However, the cellular function of the catalytic subunits remains unknown. In this study, we showed that over-expression of the catalytic subunits, especially alpha2, in cultured cells induced dramatic phenotypical changes including nuclear shape change, centrosomal amplification and microtubule disorganization. We examined if these effects were due to the catalytic activity and/or binding of alpha2 to LIS1. Substitution of a single amino acid Glu39 of murine alpha1 and alpha2 by Asp (alpha2-E39D) did not affect catalytic activity but completely abolished LIS1 binding. Over-expression of either alpha2-E39D or the catalytically inactive alpha2-S48C revealed that alpha2-E39D, but not alpha2-S48C, lost its ability to induce above-mentioned phenotypic changes. Biochemical analyses showed that LIS1 present in the precipitate fraction of murine brain homogenates could be translocated to the soluble fraction by alpha2, but not by alpha2-E39D. These results suggest that over-expression of the PAF-AH (I) catalytic subunits induces centrosomal amplification and microtubule disorganization by disturbing intracellular localization of LIS1.

ABCA3 protein is expressed predominantly at the limiting membrane of the lamellar bodies in alveolar type II cells, and mutations in the ABCA3 gene cause lethal respiratory distress in newborn infants. To investigate the function of ABCA3 protein, we generated Abca3-deficient mice by targeting Abca3. Full-term Abca3(-/-) newborn pups died within an hour after birth because of acute respiratory failure. Ultrastructural analysis revealed abnormally dense lamellar body-like organelles and no normal lamellar bodies in Abca3(-/-) alveolar type II cells. TLC and electrospray ionization mass spectrometry analyses of lipids in the pulmonary interstitium showed that phosphatidylcholine and phosphatidylglycerol, which contain palmitic acid and are abundant in normal surfactant lipids, were dramatically decreased in Abca3(-/-) lung. These findings indicate that ABCA3 plays an essential role in pulmonary surfactant lipid metabolism and lamellar body biogenesis, probably by transporting these lipids as substrates.

UNLABELLED: Human ABCB4 (multidrug resistance [MDR]3 P-glycoprotein) is expressed in the canalicular membrane of the hepatocyte. ABCB4 has been shown to be required for phosphatidylcholine (PC) secretion into the bile and to translocate PC across the plasma membrane. To further investigate the function of ABCB4, we established a cell line stably expressing ABCB4 (human embryonic kidney [HEK]/ABCB4). The efflux of phospholipids from HEK/ABCB4 cells was remarkably increased by the addition of taurocholate. In addition, the cholesterol efflux from HEK/ABCB4 cells was also enhanced in the presence of taurocholate. Light scattering measurements suggested that the taurocholate monomer plays an important role in ABCB4-mediated lipid secretion. On the other hand, the efflux of phospholipids and cholesterol was not mediated by ABCB1 (MDR1) even in the presence of taurocholate. Taurocholate promoted the efflux of phospholipids and cholesterol from HEK/ABCB4 cells more efficiently than glycocholate and cholate. ABCB4-K435M and ABCB4-K1075M, Walker A lysine mutants, did not mediate the phospholipid and cholesterol efflux in the presence of taurocholate, suggesting that ATP hydrolysis is essential for the efflux. Verapamil completely inhibited the taurocholate-dependent efflux of phospholipids and cholesterol from HEK/ABCB4 cells. Mass spectrometry revealed that, in the presence of taurocholate, HEK/ABCB4 cells preferentially secreted PC compared to sphingomyelin. PC vesicles induced cholesterol diffusion from cell membrane, but did not accept cholesterol from ABCB4. CONCLUSION: ABCB4 mediates the efflux of phospholipids into the canalicular lumen in the presence of bile salts, and plays a crucial role in bile formation and lipid homeostasis.

In an effort to elucidate the functions of secreted phospholipase A2 (sPLA2) enzymes in vivo, we generated transgenic (Tg) mice for group V sPLA2 (sPLA2-V) and group X sPLA2 (sPLA2-X), which act potently on phosphatidylcholine in vitro. We found that sPLA2-V Tg mice died in the neonatal period because of respiratory failure. The lungs of sPLA2-V Tg mice exhibited atelectasis with thickened alveolar walls and narrow air spaces, accompanied by infiltration of macrophages and only modest changes in eicosanoid levels. This severe pulmonary defect in sPLA2-V Tg mice was attributable to marked reduction of the lung surfactant phospholipids, phosphatidylcholine and phosphatidylglycerol. Given that the expression of sPLA2-V is greatly elevated in human lungs with severe inflammation, our present results raise the intriguing possibility that this isozyme may contribute to ongoing surfactant hydrolysis often observed in the lungs of patients with respiratory distress syndrome. In contrast, sPLA2-X Tg neonates displayed minimal abnormality of the respiratory tract with normal alveolar architecture and surfactant composition. This unexpected result was likely because sPLA2-X protein existed as an inactive zymogen in most tissues. The active form of sPLA2-X was detected in tissues with inflammatory granulation in sPLA2-X Tg mice. These results suggest that sPLA2-X mostly remains inactive under physiological conditions and that its proteolytic activation occurs during inflammation or other as yet unidentified circumstances in vivo.

Disrupted-In-Schizophrenia-1 (DISC1) is a unique susceptibility gene for major mental conditions, because of the segregation of its genetic variant with hereditary psychosis in a Scottish pedigree. Genetic association studies reproducibly suggest involvement of DISC1 in both schizophrenia and bipolar disorder in several ethnic groups. The DISC1 protein is multifunctional, and a pool of DISC1 in the dynein motor complex is required for neurite outgrowth in PC12 cells as well as proper neuronal migration and dendritic arborization in the developing cerebral cortex in vivo. Here, we show that a specific interaction between DISC1 and nuclear distribution element-like (NDEL1/NUDEL) is required for neurite outgrowth in differentiating PC12 cells. Among several components of the dynein motor complex, DISC1 and NDEL1 are selectively upregulated during neurite outgrowth upon differentiation in PC12 cells. The NDEL1 binding site of DISC1 was narrowed down to a small portion of exon 13, corresponding to amino acids 802-835 of DISC1. We demonstrate that genetic variants of DISC1, proximal to the NDEL1 binding site, affect the interaction between DISC1 and NDEL1.

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.

Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose tissue, are not fully understood. A database search for enzymes with alpha/beta hydrolase folds, the GXSXG motif for serine esterase and the His-Gly dipeptide motif, has provided a previously unannotated gene that is induced during 3T3-L1 adipocytic differentiation. Because of its remarkable structural resemblance to triacylglycerol hydrolase (TGH) with 70.4% identity, we have tentatively designated this enzyme as TGH-2 and the original TGH as TGH-1. TGH-2 is also similar to TGH-1 in terms of tissue distribution, subcellular localization, substrate specificity, and regulation. Both enzymes are predominantly expressed in liver, adipose tissue, and kidney. In adipocytes, they are localized in microsome and fatcake. Both enzymes hydrolyzed p-nitophenyl butyrate, triolein, and monoolein but not diolein, cholesteryl oleate, or phospholipids; hydrolysis of short-chain fatty acid ester was 30,000-fold more efficient than that of long-chain fatty acid triacylglycerol. Fasting increased the expression of both genes in white adipose tissue, whereas refeeding suppressed their expression. RNA silencing of TGH-2 reduced isoproterenol-stimulated glycerol release by 10% in 3T3-L1 adipocytes, while its overexpression increased the glycerol release by 20%. Thus, TGH-2 may make a contribution to adipocyte lipolysis during period of increased energy demand.

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.

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.

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.

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.