Mercuric ion attenuates nuclear factor-kappaB activation and DNA binding in normal rat kidney epithelial cells: implications for mercury-induced nephrotoxicity.
Mercuric ion (Hg(2+)), 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 Hg(2+) 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 Hg(2+) 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 Hg(2+) on nuclear factor-kappaB (NF-kappaB), 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 Hg(2+) on the expression of NF-kappaB in normal rat kidney epithelial (NRK52E) cells, a principal target of Hg(2+) toxicity. The lipopolysaccharide (LPS)-inducible form of NF-kappaB was readily detected in kidney cells and has been characterized as the p50p65 heterodimer. NF-kappaB-DNA binding was prevented in a dose-related manner by Hg(2+) (0-55 microM) in vitro when added to DNA binding reactions containing the nonthiol reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Similarly, Hg(2+) 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 cys(62)-->ser(62) mutation. The inhibition of p50-DNA binding by Hg(2+) 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-kappaB-DNA binding affinity when cells were pretreated in vivo with Hg(2+) at concentrations as low as 2 microM prior to LPS administration. Immunoblot analyses indicted that Hg(2+) pretreatment of kidney cells substantially diminished, in a dose-related manner, the concentration of p65 translocated into the nucleus following LPS administration. Additionally, Hg(2+) pretreatment impaired both the phosphorylation and degradation of IkappaBalpha, suggesting a specific effect on NF-kappaB activation at the level of IkappaBalpha proteolysis. Finally, Hg(2+) at concentrations as low as 5 microM significantly diminished NF-kappaB-mediated transcriptional activity when administered to kidney cells transiently transfected with an NF-kappaB-driven luciferase reporter gene (pLuc-4xNF-kappaB) prior to LPS treatment. These findings demonstrate that Hg(2+), at low cellular concentrations, attenuates NF-kappaB activation at sites associated with IkappaBalpha phosphorylation and degradation, nuclear translocation of the p50p65 heterodimer, and association of p50-cys(62) with the DNA kappaB binding site. Attenuation of NF-kappaB activation by Hg(2+) through these mechanisms may underlie apoptotic or other cytotoxic responses that are known to be associated with low level Hg(2+) exposure in kidney epithelial cells.
Dieguez-Acuña
F J
FJ
Center for Ecogenetics and Environmental Health, University of Washington, Seattle, Washington, 98105, USA.
Ellis
M E
ME
Kushleika
J
J
Woods
J S
JS
D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli; D005978:Glutathione / Q000494:pharmacology; D020672:I-kappa B Proteins; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism*; D007674:Kidney Diseases / Q000139:chemically induced*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008628:Mercury / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA; D008156:Luciferases; D008628:Mercury; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol