Copper effects on metal regulatory factors of cultured human fibroblasts

The effects of copper on metal regulatory factors were studied in cultured human fibroblasts using mobility shift assays and protein blotting. A mobility shift was apparent on polyacrylamide gels when fibroblast whole cell extracts were reacted with a 32P-labeled 14-base-pair MREa sequence of the hMT IIA gene. Binding was specific, since the shifted band was not competed away by MREa analogs containing two base changes. Known transcription factors SP1, AP1, AP2, and OCT1 did not compete with the fibroblast metal regulatory element binding protein (MREBP) for binding to the MREa probe. When fibroblasts were grown in 200 or 800 microM copper, binding of the MREBP was enhanced. Protein blotting experiments were performed by electrophoresing cell extracts, blotting onto nitrocellulose, and probing with the 32P-labeled MREa 14-mer. A 112-kDa band, apparent in HeLa cell nuclear extract as well as fibroblast whole cell extract, was not influenced by the presence of copper in the growing fibroblasts. A 34-kDa protein, also present in both HeLa cells and fibroblasts, appeared to correspond with the protein causing the mobility shift. Copper added to the fibroblast growth media enhanced binding of the 34-kDa protein to MREa using the blotting procedure. We conclude that the fibroblast 34-kDa MREBP is a positive regulatory factor influenced by copper. Human fibroblasts can be employed to further study metal regulatory factors whose binding to metal regulatory elements is influenced by copper and other heavy metals.