Student Research: Francisco Dieguez

Inhibition of Nuclear Factor Kappa B DNA Binding by Mercury and Effects of Competitive Thiol Reagents in Normal Rat Renal Epithelial Cells
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Nuclear Factor kappa B (NFkB) is a mammalian transcriptional activator known to be involved in the inducible expression of a variety of genes, particularly those involved in immune, inflammatory and acute phase responses. In most cell types, NFkB exists as a heterodimer composed of two subunits with molecular mass values of 50 and 65 kDa, referred to as p50 (NFkB1) and p65 (Rel A), respectively. In cells which have inducible NFkB activity, NFkB exists as an inactive cytoplasmic form consisting of a p50-p65 heterodimer bound to an inhibitory subunit, IkB. The transcriptional activity of NFkB-dependent genes involves disassociation of IkB and from NFkB heterodimer in the cytoplasm and subsequent translocation of active NFkB into the nucleus, where it binds with the k enhancer in the promoter region of various genes to effect transcription. Depending on the cell type, NFkB is activated by a wide range of stimuli including viruses, bacterial lipopolysaccharides (LPS), cytokines and reactive oxygen species (ROS). NFkB-mediated gene transcription requires the interaction of a critical reduced cysteine moiety on the p50 subunit (p50-cys62) with the DNA kB binding site. We evaluated the effects of mercuric ion (Hg2+), a potent thiol binding agent, on the expression of NFkB in normal rat kidney epithelial (NRK) cells. The DNA binding of NFkB in nuclear extracts was determined by electrophoretic mobility shift (gel shift) assays using a 32P-labeled oligonucleotide containing the kB consensus sequence. Both constitutive and LPS-inducible foRMS of NFkB were readily detected in NRK cells. The expression of the inducible form of NFkB was prevented in a dose-related manner by Hg2+ (100mM to 250 mM) added to DNA binding reactions. Given the presence of ~250mM DTT in the binding reaction, the concentration of free Hg2+ which elicited full inhibition of NFkB binding was estimated to be as low as 25mM. The Hg2+ block of NFkB-DNA binding was completely prevented by competitive thiols, DTT, and bME at 0.588 and 3.0 mM, respectively. In contrast, the non-thiol antioxidants, BHA and vitamin E, were unable to prevent the effects of Hg2+ even at concentrations as high as 3.1 mM in the binding reaction. Using commercially supplied p50WT human recombinant protein and the non-thiol reducing agent (Tris(2-carboxyethyl) phosphine hydrochloride (TCEP·HC1) in binding reactions, NFkB DNA binding was prevented in a dose-related manner by Hg2+ in the range of 10 to 150 mM. The Hg2+ block of NFkB-DNA binding was reversed by the competitive thiol DTT at 104 mM, and in a dose-related manner by GSH and L-cysteine at 0.1 to 3.5 mM. In contrast, Hg2+ did not inhibit DNA binding of a mutant form of the p50 protein containing a cys62--->ser62 mutation. These results indicated that Hg2+, at very low levels, prevents NFkB activation, at least in part by blocking the interaction of the NFkB binding domain (p50-cys62) with the DNA kB binding site. Prevention of the NFkB activation may underlie apoptotic or other cytotoxic responses associated with low level Hg2+ exposure in kidney epithelial cells.