Nickel-induced proteins in human HaCaT keratinocytes: annexin II and phosphoglycerate kinase

It has been established in previous in vitro experiments with human HaCaT k eratinocytes that nickel becomes cytotoxic at concentrations higher than 10 0 muM and that it is accumulated mainly in the cytosolic fraction (Ermolli et al., 2000). The aim of this work was to search possible biomarkers of me tal insult, i.e. nickel-binding proteins or proteins differentially express ed in the cytosolic fraction of nickel-exposed cells (up to 1 mM nickel) as compared to controls. Cytosolic proteins were studied by isoelectric focus ing (IEF) and two-dimensional gel electrophoresis (2-DE). Separation by IEF revealed nickel-induced changes in the abundance of cytosolic proteins as visualised with nickel-nitrilo-triacetic-alkaline phosphatase (Ni-NTA-AP) i n blots. The cytosolic fraction of cells incubated with nickel, at concentr ations over 100 muM, showed nickel binding components which were absent or present in significantly lower amounts in control cells. These proteins had isoelectric points (pls) 6.9, 7.7 and 8.5. After 2-DE silver- and protein staining significantly increased abundance of four proteins was observed. T heir pr values corresponded to those of the nickel binding ones seen after IEF. PI protein with pi 6.9 had a molecular weight estimated to 38 kDa, two proteins with pi around 7.7 showed molecular weights of 57 and 22 kDa, res pectively and another protein with pI of 8.5 had a molecular weight of 33 k Da. The increased abundance of these components, both in IEF experiments an d in 2-DE, correlated with the nickel concentration in the culture media. N -terminal amino acid sequencing and database search allowed identification of one protein as phosphoglycerate kinase and another one as annexin II. Th e involvement of these proteins in cellular functions and their possible im plications in the mechanism of nickel toxicity in keratinocytes are discuss ed. Some of these proteins may be biomarker candidates for effects of nicke l exposure in human keratinocytes. (C) 2001 Elsevier Science Ireland Ltd. A ll rights reserved.