Mercuric ion attenuates nuclear factor-κB activation and DNA binding in normal rat kidney epithelial cells:: Implications for mercury-induced nephrotoxicity

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-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 Hg2+ on the expression of NF-kappaB in normal rat kidney epithelial (NRK52E) cells, a principal target of Hg2+ 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 Hg2+ (0-55 muM) 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 cys(62)-->ser(62) 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-kappaB-DNA binding affinity when cells were pretreated in vivo with Hg2+ at concentrations as low as 2 muM prior to LPS administration. Immunoblot analyses indicted that Hg2+ pretreatment of kidney cells substantially diminished, in a the nucleus following LPS administration. Additionally, Hg2+ pretreatment impaired both the phosphorylation and degradation of I kappaB alpha, suggesting a specific effect on NF-kappaB activation at the level of I kappaB alpha proteolysis. Finally, Hg2+ at concentrations as low as 5 muM 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 Hg2+, at low cellular concentrations, attenuates NF-kappaB activation at sites associated with I kappaB alpha 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 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. (C) 2001 Academic Press.