Resolution of multiple AP-1 complexes in HL-60 cells induced to differentiate by 1,25-dihydroxyvitamin D3

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Abstract
Activator protein‐1 (AP‐1) complex plays a central role in the regulation of both growth and differentiation in many cell types. Monocytic differentiation of HL‐60 cells by TPA (12‐0‐tetradecanoyl phorbol‐13‐acetate) has been reported to be paralleled by increased AP‐1 binding to DNA and by elevated c‐jun expression, suggesting transcriptional level of control. We show that two forms of AP‐1 complex, designated AP‐1/1 and AP‐1/2, can be demonstrated in logarithmically growing HL‐60 cells, that the exposure of these cells to 10−8 M 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) results in increased binding of these complexes to the AP‐1 DNA element, and that the AP‐1 complex can be resolved into at least three forms in differentiated cells. Binding to, or competition with, a mutated form of the AP‐1 binding site shows that the most slowly migrating complex (AP‐1/3) binds to DNA with greater specificity than do complexes AP‐1/1 and AP‐1/2, while antibody inhibition and binding studies performed at 37°C indicate that jun proteins predominate in AP‐1/2 complexes. Exposure of extracts from differentiated, but not untreated, HL‐60 cells to 2 mM ATP increases the prominence of AP‐1/3 complexes, and reduces the DNA binding of AP‐1/1 complexes. Treatment of the extracts with phosphatases abolishes the binding of AP‐1/2 and AP‐1/3 to DNA, and increases the binding intensity of AP‐1/1. When extracts from differentiated cells are mixed with extracts from undifferentiated cells the AP‐1/3 complexes become less prominent, suggesting than an inhibitory activity in undifferentiated cells prevents the formation of AP‐1/3 complexes. These studies show the association of multiple forms of AP‐1 complex with the mature monocytic phenotype, and suggest several levels of control of monocytic differentiation.