Resolution of multiple AP-1 complexes in HL-60 cells induced to differentiate by 1,25-dihydroxyvitamin D3.
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 degrees 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.
Department of Laboratory Medicine and Pathology, UMD-New Jersey Medical School, Newark 07103.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D002762:Cholecalciferol / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D016755:Proto-Oncogene Proteins c-jun; D002762:Cholecalciferol; D000255:Adenosine Triphosphate