Mercuric ion attenuates nuclear factor-kappa B 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, media tes the toxicity associated with elemental, inorganic, and organic mercuria l compounds. Studies of cellular events associated with Hg2+ toxicity have focused largely on disruption of cell membranes and impairment of mitochond rial functions. In contrast, few studies have sought to define the specific molecular mechanisms through which Hg2+ might affect toxicity via alterati on of thiol-dependent signal transduction pathways that regulate cell proli feration 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 step s 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)-inducibl e form of NF-kappaB was readily detected in kidney cells and has been chara cterized 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)phosp hine hydrochloride (TCEP). Similarly, Hg2+ at the same concentrations preve nted DNA binding of a human recombinant wild-type p50p50 homodimer in bindi ng 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 compet itive thiols DTT, GSH, and L-cysteine in binding reactions. In contrast, co mpetitive 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 kid ney cells substantially diminished, in a the nucleus following LPS administ ration. Additionally, Hg2+ pretreatment impaired both the phosphorylation a nd degradation of I kappaB alpha, suggesting a specific effect on NF-kappaB activation at the level of I kappaB alpha proteolysis. Finally, Hg2+ at co ncentrations as low as 5 muM significantly diminished NF-kappaB-mediated tr anscriptional activity when administered to kidney cells transiently transf ected with an NF-kappaB-driven luciferase reporter gene (pLuc-4xNF-kappaB) prior to LPS treatment. These findings demonstrate that Hg2+, at low cellul ar concentrations, attenuates NF-kappaB activation at sites associated with I kappaB alpha phosphorylation and degradation, nuclear translocation of t he p50p65 heterodimer, and association of p50-cys(62), with the DNA kappaB binding site. Attenuation of NF-kappaB activation by Hg2+ through these mec hanisms 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.