Selenium-dependent peroxidases suppress 5-lipoxygenase activity in B-lymphocytes and immature myeloid cells. The presence of peroxidase-insensitive 5-lipoxygenase activity in differentiated myeloid cells.
Differentiation of HL-60 cells by dimethylsulfoxide induces 5-lipoxygenase protein expression, but only low cellular 5-lipoxygenase activity. Similarly, B-lymphocytes express 5-lipoxygenase protein and show activity in cell homogenates but not in intact cells. Here, we demonstrate that suppression of cellular 5-lipoxygenase activity in these cell lines is serum dependent and that the serum effect can be mimicked by selenium. Selenium-dependent inhibition of 5-lipoxygenase activity was also observed in the corresponding cell homogenates or 100,000 x g supernatants when dithiothreitol or glutathione (GSH) was added. The properties of the endogenous selenium-dependent inhibitor, i.e., molecular mass, utilization of GSH and dithiothreitol as substrates, sensitivity to iodacetate, inhibition of 5-lipoxygenase activity in the presence of the GPx-1 inhibitor mercaptosuccinate, suggest that a selenoenzyme with properties of the phospholipid hydroperoxide glutathione peroxidase (GPx-4) is responsible for the 5-lipoxygenase inhibition in BL41-E95-A and immature HL-60 cells. Differentiation of HL-60 cells in the presence of 1,25-dihydroxyvitamin D3 and transforming growth factor-beta (TGF beta) upregulated cellular 5-lipoxygenase activity regardless of whether the cell were grown with or without serum or selenium. Also, 5-lipoxygenase activity in homogenates or 100,000 x g supernatants of 1,25-dihydroxyvitamin D3/TGF beta differentiated HL-60 cells and of human granulocytes was not inhibited by dithiothreitol or GSH. Thus, after 1,25-dihydroxyvitamin D3/TGF beta differentiation, HL-60 cells resemble normal granulocytes with respect to the high 5-lipoxygenase activity in intact cells and to the dithiothreitol effects in broken cell preparations. Combination experiments with 100000 x g supernatants of BL41-E95-A cells and neutrophils revealed that the high 5-lipoxygenase activity of granulocytes is due to stability of the 5-lipoxygenase catalytic activity against selenium-dependent peroxidases, but not to low peroxidase activity. Our data suggest that the capability of mature myeloid cells to release large amounts of leukotrienes after stimulation is due to a peroxidase-insensitive 5-lipoxygenase catalytic activity.
Werz
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Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe-University Frankfurt, Germany.
Steinhilber
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D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001402:B-Lymphocytes / Q000201:enzymology*; D002117:Calcitriol / Q000378:metabolism; D002454:Cell Differentiation; D004229:Dithiothreitol / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000378:metabolism*; D006098:Granulocytes / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D012643:Selenium / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism
D016212:Transforming Growth Factor beta; D005979:Glutathione Peroxidase; D001094:Arachidonate 5-Lipoxygenase; D002117:Calcitriol; D012643:Selenium; D004229:Dithiothreitol