Search

Stimulator of interferon genes (STING) is an ER-localized transmembrane protein and the receptor for 2',3'-cyclic guanosine monophosphateendash;adenosine monophosphate (cGAMP), which is a second messenger produced by cGAMP synthase (cGAS), a cytosolic double-stranded DNA sensor. The cGAS-STING pathway plays a critical role in the innate immune response to infection of a variety of DNA pathogens through the induction of the type I interferons. Pharmacological activation of STING is a promising therapeutic strategy for cancer, thus the development of potent and selective STING agonists has been pursued. Here we report that mouse STING can be activated by phenylarsine oxide (PAO), a membrane permeable trivalent arsenic compound that preferentially reacts with thiol group of cysteine residue (Cys). The activation of STING with PAO does not require cGAS or cGAMP. Mass spectrometric analysis of the peptides generated by trypsin and chymotrypsin digestion of STING identifies several PAO adducts, suggesting that PAO covalently binds to STING. Screening of STING variants with single Cys to serine residues (Ser) reveals that Cys88 and Cys291 are critical to the response to PAO. STING activation with PAO, as with cGAMP, requires the ER-to-Golgi traffic and palmitoylation of STING. Our results identify a non-nucleotide STING agonist that does not target the cGAMP-binding pocket, and demonstrate that Cys of STING can be a novel target for the development of STING agonist.Key words: STING agonist, cysteine modification, innate immunity, phenylarsine oxide.

Methylmercury is an environmental pollutant that induces potent neurotoxicity. We previously identified transcription factor 3 (TCF3) as a transcription factor that is activated in the brains of mice treated with methylmercury, and reported that methylmercury sensitivity was increased in cells in which TCF3 expression was suppressed. However, the mechanisms involved in the activation of TCF3 by methylmercury and in the reduction of methylmercury toxicity by TCF3 remained unclear. We found that treatment of mouse neuronal C17.2 cells with methylmercury increased TCF3 protein levels and promoted the binding of TCF3 to DNA consensus sequences. In cells treated with actinomycin D, a transcription inhibitor, an increase in TCF3 protein levels was also observed under methylmercury exposure. However, in the presence of cycloheximide, a translation inhibitor, methylmercury delayed the degradation of TCF3 protein. In addition, treatment with MG132, a proteasome inhibitor, increased TCF3 protein levels, and there was not significant increase in TCF3 protein levels by methylmercury under these conditions. These results suggest that methylmercury may activate TCF3 by increasing its levels through inhibition of TCF3 degradation by the proteasome. It has been previously reported that the induction of apoptosis in neurons is involved in methylmercury-induced neuronal damage in the brain. Although apoptosis was induced in C17.2 cells treated with methylmercury, this induction was largely suppressed by overexpression of TCF3. These results indicate that TCF3, which is increased in the brain upon exposure to methylmercury, may be a novel defense factor against methylmercury-induced neurotoxicity.

trans-Fatty acids (TFAs) are unsaturated fatty acids with at least one carbon-carbon double bond in trans configuration. TFA consumption has been epidemiologically associated with neurodegenerative diseases (NDs) including Alzheimer's disease. However, the underlying mechanisms of TFA-related NDs remain unknown. Here, we show a novel microglial signaling pathway that induces inflammation and cell death, which is dramatically enhanced by elaidic acid (EA), the most abundant TFA derived from food. We found that extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from injured cells, induced activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway, which is one of the major stress-responsive mitogen-activated protein (MAP) kinase signaling pathways, and subsequent caspase-3 cleavage and DNA ladder formation (hallmarks of apoptosis) in mouse microglial cell lines including BV2 and MG6 cells. Furthermore, we found that in these microglial cell lines, EA, but not its cis isomer oleic acid, facilitated extracellular ATP-induced ASK1/p38 activation and apoptosis, which was suppressed by pharmacological inhibition of either p38, reactive oxygen species (ROS) generation, P2X purinoceptor 7 (P2X7), or Ca2+/calmodulin-dependent kinase II (CaMKII). These results demonstrate that in microglial cells, extracellular ATP induces activation of the ASK1-p38 MAP kinase pathway and ultimately apoptosis downstream of P2X7 receptor and ROS generation, and that EA promotes ATP-induced apoptosis through CaMKII-dependent hyperactivation of the ASK1-p38 pathway, in the same manner as in macrophages. Our study may provide an insight into the pathogenesis of NDs associated with TFAs.

Selenoprotein P (SELENOP) is a major plasma selenoprotein that contains 10 Sec residues, which is encoded by the UGA stop codon. The mRNA for SELENOP has the unique property of containing two Sec insertion sequence (SECIS) elements, which is located in the 3′ untranslated region (3′UTR). Here, we coincidentally identified a novel gene, CCDC152, by sequence analysis. This gene was located in the antisense region of the SELENOP gene, including the 3′UTR region in the genome. We demonstrated that this novel gene functioned as a long non-coding RNA (lncRNA) that decreased SELENOP protein levels via translational rather than transcriptional, regulation. We found that the CCDC152 RNA interacted specifically and directly with the SELENOP mRNA and inhibited its binding to the SECIS-binding protein 2, resulting in the decrease of ribosome binding. We termed this novel gene product lncRNA inhibitor of SELENOP translation (L-IST). Finally, we found that epigallocatechin gallate upregulated L-IST in vitro and in vivo, to suppress SELENOP protein levels. Here, we provide a new regulatory mechanism of SELENOP translation by an endogenous long antisense ncRNA., Graphical Abstract Graphical AbstractL-IST directly interacts with SELENOP mRNA, inhibits the binding of SECIS-binding protein 2 (SBP2) and ribosome to SELENOP mRNA, and finally decreases SELENOP protein expression. Epigallocatechin gallate (EGCg) upregulated L-IST to suppress SELENOP protein levels.

We recently found that tumor necrosis factor-α (TNF-α) may be involved in neuronal cell death induced by methylmercury in the mouse brain. Here, we examined the cells involved in the induction of TNF-α expression by methylmercury in the mouse brain by in situ hybridization. TNF-α-expressing cells were found throughout the brain and were identified as microglia by immunostaining for ionized calcium binding adaptor molecule 1 (Iba1). Methylmercury induced TNF-α expression in mouse primary microglia and mouse microglial cell line BV2. Knockdown of apoptosis signal-regulating kinase 1 (ASK1), an inflammatory cytokine up-regulator that is responsible for reactive oxygen species (ROS), decreased methylmercury-induced TNF-α expression through decreased phosphorylation of p38 MAP kinase in BV2 cells. Suppression of methylmercury-induced reactive oxygen species (ROS) by antioxidant treatment largely abolished the induction of TNF-α expression and phosphorylation of p38 by methylmercury in BV2 cells. Finally, in mouse brain slices, the TNF-α antagonist (WP9QY) inhibited neuronal cell death induced by methylmercury, as did the p38 inhibitor SB203580 and liposomal clodronate (a microglia-depleting agent). These results indicate that methylmercury induces mitochondrial ROS that are involved in activation of the ASK1/p38 pathway in microglia and that this is associated with induction of TNF-α expression and neuronal cell death.

Anticancer drug gefitinib causes inflammation-based side effects, such as interstitial pneumonitis. However, its mechanisms remain unknown. Here, we provide evidence that gefitinib elicits pro-inflammatory responses by promoting mature-interleukin-1β (IL-1β) and high-mobility group box 1 (HMGB1) release. Mitochondrial reactive oxygen species (mtROS) driven by gefitinib stimulated the formation of the NLRP3 (NACHT, LRR and PYD-containing protein 3) inflammasome, leading to mature-IL-1β release. Notably, gefitinib also stimulated HMGB1 release, which is, however, not mediated by the NLRP3 inflammasome. On the other hand, gefitinib-driven mtROS promoted the accumulation of γH2AX, a hallmark of DNA damage, leading to the activation of poly (ADP-ribose) polymerase-1 (PARP-1) and subsequent active release of HMGB1. Together our results reveal the potential ability of gefitinib to initiate sterile inflammation via two distinct mechanisms, and identified IL-1β and HMGB1 as key determinants of gefitinib-induced inflammation that may provide insights into gefitinib-induced interstitial pneumonitis.

Redox regulation of proteins via cysteine residue oxidation is involved in the control of various cellular signal pathways. Pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, is critical for the metabolic shift from glycolysis to the pentose phosphate pathway under oxidative stress in cancer cell growth. The PKM2 tetramer is required for optimal pyruvate kinase (PK) activity, whereas the inhibition of inter-subunit interaction of PKM2 induced by Cys358 oxidation has reduced PK activity. In the present study, we identified three oxidation-sensitive cysteine residues (Cys358, Cys423 and Cys424) responsible for four oxidation forms via the thiol oxidant diamide and/or hydrogen peroxide (H2O2). Possibly due to obstruction of the dimer-dimer interface, H2O2-induced sulfenylation (-SOH) and diamide-induced modification at Cys424 inhibited tetramer formation and PK activity. Cys423 is responsible for intermolecular disulfide bonds with heterologous proteins via diamide. Additionally, intramolecular polysulphide linkage (–Sn–, n ≧ 3) between Cys358 and an unidentified PKM2 Cys could be induced by diamide. We observed that cells expressing the oxidation-resistant PKM2 (PKM2C358,424A) produced more intracellular reactive oxygen species (ROS) and exhibited greater sensitivity to ROS-generating reagents and ROS-inducible anti-cancer drugs compared with cells expressing wild-type PKM2. These results highlight the possibility that PKM2 inhibition via Cys358 and Cys424 oxidation contributes to eliminating excess ROS and oxidative stress.

Although homeobox protein B13 (HOXB13) is an oncogenic transcription factor, its role in stress response has rarely been examined. We previously reported that knockdown of HOXB13 reduces the cytotoxicity caused by various oxidative stress inducers. Here, we studied the role of HOXB13 in cytotoxicity caused by hydrogen peroxide in human lung epithelial A549 cells. The knockdown of HOXB13 reduced hydrogen peroxide-induced cytotoxicity, however, this phenomenon was largely absent in the presence of antioxidants (Trolox or N-acetyl cysteine (NAC)). This suggests that HOXB13 may be involved in the cytotoxicity caused by hydrogen peroxide via the production of reactive oxygen species (ROS). Hydrogen peroxide also increased both the mRNA and protein levels of HOXB13. However, these increases were rarely observed in the presence of a transcriptional inhibitor, which suggests that hydrogen peroxide increases protein levels via increased transcription of HOXB13. Furthermore, cell death occurred in A549 cells that highly expressed HOXB13. However, this cell death was mostly inhibited by treatment with antioxidants. Taken together, our findings indicate that HOXB13 may be a novel factor involved in the induction of oxidative stress, which causes cell death via intracellular ROS production.

Homeobox protein B13 (HOXB13), a transcription factor, is related to methylmercury toxicity, however, the downstream factors involved in enhancing methylmercury toxicity remain unknown. We performed microarray analysis to search for downstream factors whose expression is induced by methylmercury via HOXB13 in human embryonic kidney cells (HEK293), which are useful model cells for analyzing molecular mechanisms. Methylmercury induced the expression of oncostatin M (OSM), a cytokine of the interleukin-6 family, and this was markedly suppressed by HOXB13 knockdown. OSM knockdown also conferred resistance to methylmercury in HEK293 cells, and no added methylmercury resistance was observed when both HOXB13 and OSM were knocked down. Binding of HOXB13 to the OSM gene promoter was increased by methylmercury, indicating the involvement of HOXB13 in the enhancement of its toxicity. Because addition of recombinant OSM to the medium enhanced methylmercury toxicity in OSM-knockdown cells, extracellularly released OSM was believed to enhance methylmercury toxicity via membrane receptors. We discovered tumor necrosis factor receptor (TNF) receptor 3 (TNFR3) to be a potential candidate involved in the enhancement of methylmercury toxicity by OSM. This toxicity mechanism was also confirmed in mouse neuronal stem cells. We report, for the first time, that HOXB13 is involved in enhancement of methylmercury toxicity via OSM-expression induction and that the synthesized OSM causes cell death by binding to TNFR3 extracellularly.

Chronic exposure to methylmercury (MeHg), an environmental electrophilic pollutant, reportedly increases the risk of human cardiac events. We report that exposure to a low, non-neurotoxic dose of MeHg precipitated heart failure induced by pressure overload in mice. Exposure to MeHg at 10 ppm did not induce weight loss typical of higher doses but caused mitochondrial hyperfission in myocardium through the activation of Drp1 by its guanine nucleotide exchange factor filamin-A. Treatment of neonatal rat cardiomyocytes with cilnidipine, an inhibitor of the interaction between Drp1 and filamin-A, suppressed mitochondrial hyperfission caused by low-dose MeHg exposure. Modification of cysteine residues in proteins with polysulfides is important for redox signaling and mitochondrial homeostasis in mammalian cells. We found that MeHg targeted rat Drp1 at Cys624, a redox-sensitive residue whose SH side chain forms a bulky and nucleophilic polysulfide (Cys624-S(n)H). MeHg exposure induced the depolysulfidation of Cys624-S(n)H in Drp1, which led to filamin-dependent activation of Drp1 and mitochondrial hyperfission. Treatment with NaHS, which acts as a donor for reactive polysulfides, reversed MeHg-evoked Drp1 depolysulfidation and vulnerability to mechanical load in rodent and human cardiomyocytes and mouse hearts. These results suggest that depolysulfidation of Drp1 at Cys624-S(n)H by low-dose MeHg increases cardiac fragility to mechanical load through filamin-dependent mitochondrial hyperfission.

Methylmercury is an environmental pollutant that causes specific and serious damage to the central nervous system. We have previously shown that C-C motif chemokine ligand 4 (CCL4) protects cultured neural cells from methylmercury toxicity and expression of CCL4 is specifically induced in mouse brain by methylmercury. In this study, we examined the transcriptional regulatory mechanism that induces CCL4 expression by methylmercury using C17.2 mouse neural stem cells. The promoter region of the CCL4 gene was analyzed by a reporter assay, revealing that the region up to 50 bp upstream from the transcription start site was necessary for inducing expression of CCL4 by methylmercury. Nine transcription factors that might bind to this upstream region and be involved in the induction of CCL4 expression by methylmercury were selected, and the induction of CCL4 expression by methylmercury was suppressed by the knockdown of serum response factor (SRF). In addition, the nuclear level of SRF was elevated by methylmercury, and an increase in the amount bound to the CCL4 gene promoter was also observed. Furthermore, we examined the upstream signaling pathway involved in the induction of CCL4 expression by SRF, and confirmed that activation of p38 and ERK, which are part of the MAPK pathway, are involved. These results suggest that methylmercury induces the expression of CCL4 by activating SRF via the p38 and ERK signaling pathway. Our findings are important for elucidating the mechanism involved in the brain-specific induction of CCL4 expression by methylmercury.

Aims We had previously reported that addition of putrescine to the culture medium was reported to reduce methylmercury toxicity in C17.2 neural stem cells. Here, we have examined the inhibition of methylmercury-induced cytotoxicity by putrescine using ODC1-overexpressing C17.2 cells. Materials and methods We established stable ODC1-overexpressing C17.2 cells and evaluated methylmercury-induced apoptosis by examining the TUNEL assay and cleaved caspase-3 levels. Mitochondria-mediated apoptosis was also evaluated by reduction of mitochondrial membrane potential and recruitment of Bax and Bak to the mitochondria. Key findings ODC is encoded by ODC1 gene, and putrescine levels in ODC1-overexpressing cells were significantly higher than in control cells. Overexpression of ODC1 and addition of putrescine to the culture medium suppressed DNA fragmentation and caspase-3 activation, which are observed when apoptosis is induced by methylmercury. Moreover, mitochondrial dysfunction and reactive oxygen species (ROS) generation, caused by methylmercury, were also inhibited by the overexpression of ODC1 and putrescine; pretreatment with ODC inhibitor, however, promoted both ROS generation and apoptosis by methylmercury. Finally, we found that Bax and Bak, the apoptosis-promoting factors, to be increased in mitochondria, following methylmercury treatment, and the same was inhibited by overexpression of ODC1. These results suggest that overexpression of ODC1 may prevent mitochondria-mediated apoptosis by methylmercury via increase of putrescine levels. Significance Our findings provide important clues to clarify mechanisms involved in the defense against methylmercury toxicity and suggest novel biological functions of putrescine.

Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission–accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission–associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding–dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1–filamin A interaction by cilnidipine suppressed mitochondrial hyperfission–associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development.

Methylmercury is an environmental pollutant that shows selective toxicity to the central nervous system. We previously reported that brain-specific expression of chemokine CCL3 increases in mice administered methylmercury. However, the relationship between CCL3 and methylmercury toxicity has not been elucidated. Here, we confirmed that induction of CCL3 expression occurs before pathological change by methylmercury treatment was observed in the mouse brain. This induction was also observed in C17.2 mouse neural stem cells before methylmercury-induced cytotoxicity. In addition, cells in which CCL3 was knocked-down showed higher methylmercury sensitivity than did control cells. Moreover, activation of transcription factor NF-κB was observed following methylmercury treatment, and methylmercury-mediated induction of CCL3 expression was partially suppressed by knockdown of p65, an NF-κB subunit. Our results suggest that NF-κB plays a role in the induction of methylmercury-mediated CCL3 expression and that this action may be a cellular response to methylmercury toxicity.

Methylmercury (MeHg) is selectively toxic to the central nervous system, but mechanisms related to its toxicity are poorly understood. In the present study, we identified the chemokine, C-C motif Chemokine Ligand 4 (CCL4), to be selectively upregulated in the brain of MeHg-administered mice. We then investigated the relationship between CCL4 expression and MeHg toxicity using in vivo and in vitro approaches. We confirmed that in C17.2 cells (a mouse neural stem cell line) and the mouse brain, induction of CCL4 expression occurs prior to cytotoxicity caused by MeHg. We also show that the addition of recombinant CCL4 to the culture medium of mouse xprimary neurons attenuated MeHg toxicity, while knockdown of CCL4 in C17.2 cells resulted in higher MeHg sensitivity compared with control cells. These results suggest that CCL4 is a protective factor against MeHg toxicity and that induction of CCL4 expression is not a result of cytotoxicity by MeHg but is a protective response against MeHg exposure.

Electrophiles such as methylmercury (MeHg) affect cellular functions by covalent modification with endogenous thiols. Reactive persulfide species were recently reported to mediate antioxidant responses and redox signaling because of their strong nucleophilicity. In this study, we used MeHg as an environmental electrophile and found that exposure of cells to the exogenous electrophile elevated intracellular concentrations of the endogenous electrophilic molecule 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), accompanied by depletion of reactive persulfide species and 8-SH-cGMP which is a metabolite of 8-nitro-cGMP. Exposure to MeHg also induced S-guanylation and activation of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced activation of redox signaling and the consequent cell damage were attenuated by pretreatment with a reactive persulfide species donor. In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity impair the redox signaling regulatory mechanism, particularly of intracellular reactive persulfide species and therefore lead to cellular pathogenesis. Our results suggest that reactive persulfide species may be potential therapeutic targets for attenuating cell injury by electrophiles.

Methylmercury (MeHg) is an environmental electrophile that covalently modifies cellular proteins with reactive thiols, resulting in the formation of protein adducts. While such protein modifications, referred to asS-mercuration, are thought to be associated with the enzyme dysfunction and cellular damage caused by MeHg exposure, the current consensus is that (1) there is a cellular response to MeHg through the activation of NF-E2-related factor 2 (Nrf2) coupled toS-mercuration of its negative regulator, Kelch-like ECH-associated protein 1 (Keap1), and (2) the Keap1/Nrf2 pathway protects against MeHg toxicity. In this review, we introduce our findings and discuss the observations of other workers concerning theS-mercuration of cellular proteins by MeHg and the importance of the Keap1/Nrf2 pathway in protection against MeHg toxicity in cultured cells and mice.

Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.

The canonical Wnt signaling is frequently activated due to overexpression and/or mutations in components of this pathway in hepatocellular carcinoma (HCC). However, the biological role of noncanonical Wnt-mediated signaling in HCC with respect to the signaling pathways involved and their physiologic function is unknown. Here, we report the role of Wnt11, a member of the noncanonical cascade, in hepatic oncogenesis. The expression levels of Wnt11 mRNA and protein were significantly downregulated in human HCC tumors compared with the adjacent uninvolved liver as measured by quantitative real-time reverse transcription-PCR and Western blot analysis. In human HCC cell lines, overexpression of Wnt11 activated protein kinase C signaling. Protein kinase C antagonized the canonical signaling through phosphorylation of β-catenin and reduced T-cell factor–mediated transcriptional activity, resulting in a decrease of cell proliferation. Furthermore, ectopic expression of Wnt11 promotes RhoA/Rho kinase activation. We found that activated Rho kinase inhibited Rac1 to reduce cell motility and migration. These observations suggest a novel role for Wnt11 as a tumor suppressor during hepatocarcinogenesis because loss of expression promotes the malignant phenotype via both canonical and noncanonical Wnt signaling pathways. Mol Cancer Res; 8(2); 254–65

Human exposure to methylmercury (MeHg) from contaminated fish is a potential health risk. Because of its chemical properties as a soft electrophile, we investigated the participation of Nrf2 in the cellular response to and protection against MeHg with SH-SY5Y cells and with primary mouse hepatocytes from Nrf2- and Keap1-deficient mice. Exposure of SH-SY5Y cells to MeHg activated Nrf2 through the binding of MeHg and Keap1. Nrf2 overexpression attenuated MeHg-induced cytotoxicity in SH-SY5Y cells. In addition, primary mouse hepatocytes extracted from Nrf2-deficient mouse was susceptible, and hepatocyte-specific conditional Keap1-deficient mouse was resistant to MeHg-induced cytotoxicity. Consistent with this data, MeHg was accumulated by Nrf2 deficiency and reduced by Keap1 deficiency. Our findings indicate that MeHg activates Nrf2 and the activation of Nrf2 is essential for reduction of MeHg toxicity by facilitating its excretion into extracellular space.

Methylmercury (MeHg), an environmental pollutant, causes neuronal death via endoplasmic reticulum (ER) stress; however, the precise mechanism is not fully understood. The aim of this study was to elucidate the possible mechanism of MeHg-induced neurotoxicity. Treatment with MeHg resulted in a loss of cell viability in a concentration-dependent manner accompanying the expression of ER stress marker genes in human neuroblastoma SH-SY5Y cells. We next attempted to identify a target protein for MeHg in the ER. MeHg covalently modified protein disulfide isomerase (PDI), which is important for disulfide bond formation in nascent proteins in the ER lumen. S-Nitrosylation of the catalytic domains of PDI by nitric oxide was attenuated up to 50 % by a MeHg challenge in cells. The MeHg-modified C-terminal catalytic domain in PDI was detected by MALDI-TOF/MS. Furthermore, treatment with MeHg significantly attenuated the enzymatic activity of PDI. Taken together, these observations suggest that MeHg results in ER stress and following the unfolded protein response pathway via ER dysfunction due to S-mercuration of the C-terminus of PDI.

Growth factor receptor-bound protein-2 (GRB-2) is a protein linking receptor tyrosine kinase and Sos (Son of Sevenless gene; Ras GDP/GTP exchange protein), leading to activation of the Ras-mitogen-activated protein kinase (MAPK) cascade. So far, it remains unclear how GRB-2 plays a role in signal transduction pathways evoked by hepatotrophic factors. This study was attempted to evaluate the involvement of GRB-2 in signalling in rat hepatocyte growth. Using rat cultured hepatocytes stimulated by hepatotrophic factors and regenerating livers after partial hepatectomy (PH) we examined GRB-2-mediated linkage of hepatotrophic factor receptors to signal transducing molecules such as Sos or dynamin-II by immunoprecipitation and western blot analysis. In primary cultured hepatocytes stimulated with hepatocyte growth factor (HGF) or epidermal growth factor (EGF), GRB-2 linked HGF receptor or EGF receptor, respectively, to Sos which activated the mitogen-activated protein kinase (MAPK) cascade. In contrast, in primary cultured hepatocytes stimulated with insulin, GRB-2 linked insulin receptor substrate-1 (IRS-1) to dynamin-II as well as Sos. In the early phase after PH, GRB-2 activated the Ras-MAPK cascade by linking HGF receptor, IRS-1, or EGF receptor to Sos. In the late phase after PH, a complex of IRS-1-GRB-2 associated with dynamin-II, indicating that GRB-2 may transduce signals from IRS-1 to dynamin-II. We conclude that GRB-2 may play a role in transmitting signals from hepatotrophic factors to not only MAPK but also to other signalling pathways in hepatocyte growth.

Aquaporin (AQP) 9 is a member of the aquaglyceroporin subfamily of AQPs in the transfer of water and small solutes such as glycerol and arsenite. It is well recognized that arsenic toxicity is associated with intracellular accumulation of this metalloid. In the present study, we examined the contribution of AQP9 to the uptake of inorganic arsenite, thereby increasing arsenic-induced cytotoxicity in primary mouse hepatocytes. Pretreatment with sorbitol as a competitive inhibitor of AQP9 and siRNA-mediated knockdown of AQP9 resulted in a significant decrease of arsenite uptake in the cell and its cytotoxicity. Furthermore, overexpression of AQP9 in HEK293 cells led to the enhancement of intracellular arsenic concentration, resulting in enhanced cytotoxicity after arsenite exposure. These results suggest that AQP9 is a channel to define arsenite sensitivity in primary mouse hepatocytes.

Background/Aims Ninjurin1 is a novel adhesion molecule that has a role in promoting nerve regeneration. Although ninjurin1 is ubiquitously expressed in various human tissues, including the liver, the biologic functions of ninjurin1 in tissues other than the nervous system remain unknown. The aim of this study was to investigate the function of ninjurin1 in hepatocytes. Methods The effect of ninjurin1 overexpression was examined in Huh-7 hepatoma cells. Ninjurin1 expression was examined by Western blot in human hepatocellular carcinoma tissues as well as their adjacent liver tissues. Results Ninjurin1-overexpressing clones exhibited strong growth inhibition due to G1 cell cycle arrest, which is associated with a posttranscriptional increase in p21 WAF1/Cip1 , a decrease of cyclin-dependent kinase 2 activity and the hypophosphorylation of Rb. The ninjurin1-overexpressing clones had increased senescence-associated β-galactosidase activity and autofluorescent pigment, characteristic features of cellular senescence. The levels of ninjurin1 expression were higher in hepatocellular carcinoma tissues than those in adjacent liver tissues. Conclusions The present study provides the first evidence that ninjurin1 is able to induce the senescence program. Ninjurin1 may be involved in the regulation of cellular senescence in the liver during carcinogenesis.