Mercuric Ion Attenuates Nuclear Factor-κB Activation and DNA Binding in Normal Rat Kidney Epithelial Cells: Implications for Mercury-Induced Nephrotoxicity

doi:10.1006/taap.2001.9195

Toxicology and Applied Pharmacology

Volume 173, Issue 3, 15 June 2001, Pages 176-187

https://doi.org/10.1006/taap.2001.9195 Get rights and content

Abstract

Mercuric ion (Hg²⁺), one of the strongest thiol-binding agents known, mediates the toxicity associated with elemental, inorganic, and organic mercurial compounds. Studies of cellular events associated with Hg²⁺ toxicity have focused largely on disruption of cell membranes and impairment of mitochondrial functions. In contrast, few studies have sought to define the specific molecular mechanisms through which Hg²⁺ might affect toxicity via alteration of thiol-dependent signal transduction pathways that regulate cell proliferation and survival. Of particular interest in this regard is the effect of Hg²⁺ on nuclear factor-κB (NF-κB), a pleiotropic transcriptional factor that is known to require reduced cysteine moieties at critical steps of activation and DNA binding. Here, we evaluated the effects of Hg²⁺ on the expression of NF-κB in normal rat kidney epithelial (NRK52E) cells, a principal target of Hg²⁺ toxicity. The lipopolysaccharide (LPS)-inducible form of NF-κB was readily detected in kidney cells and has been characterized as the p50p65 heterodimer. NF-κB–DNA binding was prevented in a dose-related manner by Hg²⁺ (0–55 μM) in vitro when added to DNA binding reactions containing the nonthiol reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Similarly, Hg²⁺ at the same concentrations prevented DNA binding of a human recombinant wild-type p50p50 homodimer in binding reactions, and this effect was attenuated using a mutant form of the p50 protein containing a cys₆₂→ser₆₂ mutation. The inhibition of p50–DNA binding by Hg²⁺ was reversible in a dose-related manner in vitro by competitive thiols DTT, GSH, and l-cysteine in binding reactions. In contrast, competitive thiols added to nuclear binding reactions were unable to reverse attenuation of LPS-mediated NF-κB–DNA binding affinity when cells were pretreated in vivo with Hg²⁺ at concentrations as low as 2 μM prior to LPS administration. Immunoblot analyses indicted that Hg²⁺ pretreatment of kidney cells substantially diminished, in a dose-related manner, the concentration of p65 translocated into the nucleus following LPS administration. Additionally, Hg²⁺ pretreatment impaired both the phosphorylation and degradation of IκBα, suggesting a specific effect on NF-κB activation at the level of IκBα proteolysis. Finally, Hg²⁺ at concentrations as low as 5 μM significantly diminished NF-κB-mediated transcriptional activity when administered to kidney cells transiently transfected with an NF-κB-driven luciferase reporter gene (pLuc-4×NF-κB) prior to LPS treatment. These findings demonstrate that Hg²⁺, at low cellular concentrations, attenuates NF-κB activation at sites associated with IκBα phosphorylation and degradation, nuclear translocation of the p50p65 heterodimer, and association of p50-cys₆₂ with the DNA κB binding site. Attenuation of NF-κB activation by Hg²⁺ through these mechanisms may underlie apoptotic or other cytotoxic responses that are known to be associated with low level Hg²⁺ exposure in kidney epithelial cells.

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It has also been demonstrated that Hg2+-induced inhibition of NF-κB-p50 binding to DNA may involve direct interaction between Hg and Cys62, which may significantly affect the susceptibility of renal cells to toxicity and apoptosis. It is also notable that binding Hg2+ to the other five Cys residues on the molecule may also possess an inhibitory effect, although being less significant [210]. In view of the antiapoptotic effect of p50 binding to Bcl3 and subsequent antiapoptotic effects, Hg-induced inhibition of p50 binding activity may predispose the cell to apoptosis.
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Hg2+ is potent thiol-reactive agent and chemically modified enzymes can be prepared by incorporating Hg2+-attached ligands into cysteine residues through Hg-S interactions (Bagger and Byberg, 1991). Thiol-directed modification of a critical cysteine residue by Hg2+ was reported to attenuates the DNA-binding activity of a human recombinant NF-kB based on EMSA (Dieguez-Acuña et al., 2001). NF-kB is a redox-sensitive transcription factor known to require reduced cysteine moieties for DNA binding and transcriptional activation.
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Quantitative proteomic analysis reveals the mode-of-action for chronic mercury hepatotoxicity to marine medaka (Oryzias melastigma)
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The potent toxicity of Hg compounds is often associated with the high affinity of Hg for sulfur, causing an efficient biding to cysteine residues in proteins and enzymes, thereby perturbing their functions (Sørmo et al., 2011). Although Hg could produce multi-toxicities in organisms, most attention has been paid to Hg neurotoxicity and nephrotoxicity (Berg et al., 2010; Castoldi et al., 2001; Dieguez-Acuña et al., 2001; Keyvanshokooh et al., 2009; Nøstbakken et al., 2012; Torres et al., 2011). Consequently, there have been several molecular mechanistic studies on neurological (Castoldi et al., 2001) and renal (Zalups, 2000) toxicities induced by this metal.
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In two human mast cell lines, mercury exposure was shown to increase IL-6 production (Kempuraj et al., 2010). Further, mercury interferes with cytokine-related signaling cascades including NF-κB and p38 MAPK (Dieguez-Acuna et al., 2001; Kim et al., 2002b). Altered cytokine repertoires in response to mercury exposure may contribute to the development of autoimmunity.
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¹: To whom correspondence should be addressed at Department of Environmental Health, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98105. Fax: (206) 528-3550; E-mail: [email protected].

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Regular ArticleMercuric Ion Attenuates Nuclear Factor-κB Activation and DNA Binding in Normal Rat Kidney Epithelial Cells: Implications for Mercury-Induced Nephrotoxicity

Abstract

Kidney Int.

J. Biol. Chem.

Toxicol. In Vitro

Toxicol. Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Immunolbiology

J. Biol. Chem.

Kidney Int.

Cell

Biochem. Pharmacol.

Cell

Environ. Res.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Toxicol. Appl. Pharmacol.

Kidney Int.

Stimulation-dependent IκBα phosphorylation marks the -κB inhibitor for degradation via the ubiquitin–proteasome pathway

Proc. Natl. Acad. Sci. USA

A 65-kDa subunit of active NF-κB is required for inhibition of NF-κB by IκB

Genes Dev.

Mercury

New insights into the role of nuclear factor-κB, a ubiquitous transcription factor in the initiation of diseases

Clin. Chem.

Signal-induced site-specific phosphorylation targets IκB to the ubiquitin–proteasome pathway

Genes Dev.

Mitochondrial calcium release as induced by Hg2+

J. Biol. Chem.

NF-κB and apoptosis in mammary epithelial cells

J. Mam. Gland Biol. Neoplasm

A cytokine-responsive IκB kinase that activates the transcription factor NF-κB

Nature

Inorganic mercury chloride-induced apoptosis in the cultured porcine renal cell line LLC-PK1

J. Pharmacol. Exp. Ther.

Regular Article
Mercuric Ion Attenuates Nuclear Factor-κB Activation and DNA Binding in Normal Rat Kidney Epithelial Cells: Implications for Mercury-Induced Nephrotoxicity

Mitochondrial calcium release as induced by Hg²⁺