Silencing of the gene for homeobox protein HOXB13 by siRNA confers resistance to methylmercury on HEK293 cells

Abstract

-Methylmercury is an environmental pollutant that causes severe central nervous system disorders. We searched for transcription factors involved in the development of methylmercury toxicity and found that the decreased expression of a homeobox protein, HOXB13, in HEK293 cells conferred strong resistance to methylmercury. Key words: Methylmercury, Homeobox protein, HOXB13, Transcription factorCorrespondence: Akira Naganuma (E-mail: naganuma@mail.pharm.tohoku.ac.jp) Toxicogenomics/proteomics ReportThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.35, No.6, 941-944, 2010 Vol. 35 No. 6 941 cells/well in 6-well plates) were rinsed with 1 × PBS. Then, the total RNA was isolated using a FastPure ® RNA kit (Takara, Shiga, Japan), and first-strand cDNA synthesis was performed using the PrimeScrip ® RT reagent kit (Takara) in accordance with the manufacturer's protocol (Ikawa et al., 2008;Chen et al., 2009;Kawakami et al., 2009). We performed real-time PCR reactions (Thermal Cycler Dice ® , Takara) with the following primers: HOXB13-F, 5'-CTGGGTAGGTGGACAATTG-TA-3', and HOXB13-R, 5'-TACGCTGATGCCTGCT-GTCAA-3', for the HOXB13 gene; and GAPDH-F, 5'-GCACCGTCAAGGCTGAGAAC-3', and GAPDH-R, 5'-TGGTGAAGACGCCAGTGGA-3', for the GAP-DH gene. The fold decreases in HOXB13 mRNA levels were determined from standard curves after calibration of the assay. RESULTS AND DISCUSSIONWe selected about 1,000 proteins that are expected to function as transcription factors from DBD, a transcription factor prediction database (http://www.transcriptionfactor.org). Two double-stranded siRNAs targeting protein-coding genes were designed and introduced into HEK293 cells. The cells with introduced siRNAs were cultured for 48 hr in the presence of methylmercuric chloride (4 μM), which suppresses the proliferation of normal cells by about 60%. HOXB13 was identified as a factor whose reduction conferred strong methylmercury resistance to the cells. We introduced double-stranded siRNAs (siRNA Nos. 1 and 2) targeting HOXB13 into HEK293 cells. Cells harboring HOXB13 siRNA No. 1 exhibited slightly stronger methylmercury resistance than control cells. Cells harboring HOXB13 siRNA No. 2 exhibited marked methylmercury resistance (Fig. 1A). Cells harboring both siRNAs exhibited stronger methylmercury resistance than those harboring HOXB13 siRNA No. 2 alone (Fig. 1A) and the lowest HOXB13 mRNA level (about 16% of the level in control cells; Fig. 1B). HOXB13 mRNA levels in the cells harboring HOXB13 siRNA No. 1 or 2 were about 64% and 25% of the levels in the control cells, respectively (Fig. 1B). These results suggest that HOXB13 increases methylmercury toxicity. Decreased HOXB13 expression had little effect on the susceptibility of the cells to three metallic compounds other than methylmercury (Fig. 2). This suggests that HOXB13 may specifically increase methylmercury toxicity. We introduced HOXB13 with a V5 tag fused at the C terminus (HOXB13-V5) into HEK293 cells to investigate the intracellular distribution of HOXB13 and found HOXB13 predomi...

The Nuclear Protein HOXB13 Enhances Methylmercury Toxicity by Inducing Oncostatin M and Promoting Its Binding to TNFR3 in Cultured Cells

“…Sequences of siRNAs, used in this study, are listed in Figure S1. Knockdown efficiency of HOXB13 siRNA was confirmed in previous report [5,6]. Specificity of all siRNAs was predicted by manufactures.…”

“…Previously, we identified the homeobox protein B13 (HOXB13), a transcription factor, as an intracellular factor involved in the enhancement of methylmercury toxicity [5] and established its relationship to cytotoxicity caused by an oxidative stress inducer and a thiol group modifier [6]. HOXB13 is highly expressed in the developmental stages of mice, especially in the chorda and posterior mesoderm, and has been suggested to be involved in the formation of normal nerve tubes by the induction of apoptosis [7].…”

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

“…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. For example, they found that L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT), which is an essential enzyme for catalyzing the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate, is a factor that confers MeHg-resistance and that GFAT is the intracellular target molecule for MeHg toxicity Naganuma et al, 2000).…”

Methylmercury induces a brain-specific increase in chemokine CCL4 expression in mice

“…First-strand cDNA synthesis was carried out using the PrimeScript TM RT reagent kit (Takara, Shiga, Japan). Quantitative real-time PCR was performed using SYBR Premix EX Taq (Takara) and a Thermal Cycler Dice ® (Takara) (Hwang et al, 2010a;Tokumoto et al, 2011). PCR primers used were: CCL2, 5'-TTGTCACCAAGCTCAAGAGAGA-3' (sense) and 5'-GAGGTGGTTGTGGAAAAGGTAG-3' (antisense); CCL4, 5'-CAAACCTAACCCCGAGCAACAC-3' (sense) and 5'-GGTCTCATAGTAATCCATCACAAAGC-3' (antisense); CCL7, 5'-AATGCATCCACATGCTGCTA-3' (sense) and 5'-CTTTGGAGTTGGGGTTTTCA-3' (antisense); CCL9 5'-TGTTTCACATGGGCTTTCAA-3' (sense) and 5'-TTGTAGGTCCGTGGTTGTGA-3' (antisense); CCL12, 5'-GTCCTCAGGTATTGGCTGGA-3' (sense) and 5'-GGGTCAGCACAGATCTCCTT-3', (antisense); and GAPDH, 5'-ATCACCATCTTCCAGGAGCGA-3' (sense) and 5'-AGGGGCCATCCACAGTCTT-3' (antisense).…”