Methylmercury induces CCL2 expression through activation of NF-κB in human 1321N1 astrocytes

Abstract

-Methylmercury is an environmental pollutant that is toxic to the central nervous system; however, the molecular mechanisms underlying its toxicity remain unclear. Methylmercury increases expression of several chemokines in the cerebellum of mice treated with methylmercury. The present study analyzes the mechanism underlying methylmercury-induced chemokine expression using human 1321N1 astrocytes, and shows that methylmercury increases CCL2 expression in these cells. The transcription factor NF-κB is involved in the induction of chemokine expression. Methylmercury increased the level of the NF-κB p65 subunit in the nuclei of 1321N1 cells. The methylmercury-induced increase in CCL2 expression was significantly decreased by suppression of p65 expression by RNA interference. These results suggest that methylmercury induces chemokine expression through activation of NF-κB in human astrocytes.Key words: Methylmercury, Chemokine, CCL2, NF-κB Correspondence: Akira Naganuma (E-mail: naganuma@m.tohoku.ac.jp) LetterThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.37, No.6, 1275-1278 Vol. 37 No. 6 1275 facturer's protocol. Lentivirus was produced in HEK293T cells by co-transfection of the lentiviral vector pGIPZ encoding p65-shRNA and the Trans-Lentiviral Packaging Plasmid Mix (Thermo Fisher Scientific). 1321N1 cells were infected with lentivirus in the presence of polybrene (5 μg/ml) and selected in medium containing puromycin (1 μg/ml). The target sequence of p65 shRNA used was 5'-ACCATCAAGATCAATGGCT-3'. Quantitative real-time PCR (qPCR)Total RNA was isolated from cells using the Isogen II kit (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. First-strand cDNA synthesis was carried out using the PrimeScript TM RT reagent kit (Takara, Shiga, Japan). qPCR was performed using SYBR Premix EX Taq (Takara) (Hwang et al., 2011a;Takahashi et al., 2011). PCR primers used were GAPDH, 5'-GGGGAAGCTTGTCAATGG-3' (sense) and 5'-GGCAGTGATGGCATGGACTC-3' (antisense); p65, 5'-CTGCAGTTTGATGATGAAGA-3' (sense) and 5'-TAGGCGAGTTATAGCCTCAG-3' (antisense); and CCL2, 5'-CATTGTGGCCAAGGAGATCTG-3' (sense) and 5'-CTTCGGAGTTTGGGTTTGCTT-3' (antisense). Levels of P65 and CCL2 mRNA were normalized to that of GAPDH. Enzyme-linked immunosorbent assay (ELISA)CCL2 secreted into culture medium was quantified using the Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Preparation of nuclear and post-nuclear fractions1321N1 cells were lysed in ice-cold hypotonic buffer (10 mM HEPES-KOH [pH 7.9], 1.5 mM MgCl 2 , 10 mM KCl, 1 mM Dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) containing protease inhibitors (Roche Applied Science, Indianapolis, IN, USA) and 10% Nonidet P-40, and centrifuged at 15,000 × g at 4°C. The supernatant (post-nuclear fraction) was removed, and the pellet was resuspended in nuclear lysis buffer (10 mM HEPES-KOH [pH 7.9], 400 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl 2 , 1 mM DTT, 5% glycerol) containing protease inhibitors (Roche App...

Suppression of methylmercury-induced IL-6 and MCP-1 expressions by N-acetylcysteine in U-87MG human astrocytoma cells

“…The present study showed that exposure to a non-cytotoxic dose (4 μM) of MeHg activated IL-6 and MCP-1 mRNA expressions at 6 h, and subsequently, protein synthesis at 12 h. This MeHg level was within the range reported in previous studies, from 2.5 to 10 μM, in which these cytokines were induced in various glial cell lines [7,14,16,26]. Recently IL-6 production due to low dose of MeHg-treatment in astrocytes was reported to have a protective role against neurotoxicity [29,33].…”

“…Monocyte chemoattractant protein (MCP)-1 also seems to be important in various neuroinflammatory diseases [10]. Recently, an elevation of MCP-1 expression in response to MeHg exposure was observed in mice experiment (10 mg/kg/day for 7 days) [22] and human 1321N1 astrocytoma cells (10 μM for 6 h) [26]. It was also reported to work as an alert system for MeHg exposure, using MCP-1-knockout mice [18].…”

<i>S</i>-Mercuration of cellular proteins by methylmercury and its toxicological implications

“…The biological factors that are altered by MeHg have been explored with the aim of shedding light on the molecular mechanisms that underlie MeHg toxicity (Hwang et al, 2011a(Hwang et al, , 2013aHwang and Naganuma, 2006;Kim et al, 2012Kim et al, , 2013Lee et al, 2012;Takahashi et al, 2013;Toyama et al, 2011). Furthermore, the research group led by Dr. Naganuma has found a large number of proteins that are resistant or hypersensitive to MeHg (Hwang et al, 2007(Hwang et al, , 2010a(Hwang et al, , 2010b(Hwang et al, , 2010c(Hwang et al, , 2011b(Hwang et al, , 2012a(Hwang et al, , 2012b(Hwang et al, , 2013b.…”

Specific tumor-derived CCL2 mediated by pyruvate kinase M2 in colorectal cancer cells contributes to macrophage recruitment in tumor microenvironment

“…Multiple studies have reported that NF-κB signaling has a central role in the transactivation of CCL2, [39][40][41][42][43] which attracted us to examine whether PKM2-dependent expression of CCL2 could be attributed to the upregulation of NF-κB signaling. Luciferase activities were highly significantly upregulated in all p65 expression groups compared to control group, while the group treated with siPKM2 and p65 plasmid were decreased compared to paired control.…”