I , %-Dihydroxyvitamin D ,-Regulated Binding of Nuclear Proteins to a c-) ) 2 yc Intron Element *

The steroid hormone 1,25-dihydroxyvitamin D:, I1,25-(OH),D,, / induces maturation of many cells, including HL-60 promyelocytic leukemia cells that are differentiated to monocytesilnacrophages. This process involves changes in transcription of genes such as c-r,x,yc. We investigated the effects of 1,25(OH),D,j on nuclear protein binding to the c-vzyc intron element (MIE), a region of DNA within the c-myc gene. A mutation in this MIE sequence has been shown to be associated with uncontrolled expression of the c-myc gene in various cell lines. In this report, we demonstrate for the first time that 1,25-(OH),D, induces increased binding of nuclear proteins to this MIE in HL-60 cells. The majot MIE-binding proteins were approximately 32 kDa in size. Interestingly, phorbol 12.myristate 13-acetate Induced a similar increase in binding of the 32-kDa doublet protein to the MIE. In addition, we showed that the level of 13%kDa MIE-binding protein was increased by I ,25-(OH),D, and phorbol 12.myristate 13-acetate. However, the extent of MIE binding by the 13%kDa protein is significantly less than that of the 32-kDa doublet binding species. MIE binding by the 32-kDa doublet protein was significantly increased within 12 h of 1,25(OH),D, treatment. The time course of this increase was similar to that of 1,25-(OH),D,-induced inhibition of c-myc gene transcription. We also demonstrated that dephosphorylation of the 32.kDa doublet protein inhibited its blnding to the MIE. Thus, this study shows that the mechanism employed by 1,25-(OH),D, for regulation of c-myc expression may involve an increase in protein binding to the MIE, which has been shown to be the site for control of wnyc gene expression. (Endocrinology 137: 4154-4160, 1996) 1,25-DIHYDROXYVITAMIN D, [1,25-(OH&D,] mediates a variety of cell processes. In addition to its ability to regulate serum calcium and phosphate levels in the intestine, bone, and kidneys (l), 1,25-(OH),D, affects the growth and differentiation processes of numerous cell types, including keratinocytes (2), bone cells (3), leukemia cells (4, 5), and cardiac myocytes (6). HL-60 promyelocytic leukemia cells are induced to differentiate into monocytes (4) and osteoclast-like cells (3) upon exposure to 1,25-(OH),D,. Classically, the mechanism of action of 1,25-(OH)2D, involves binding to a nuclear receptor, which itself binds to a specific response element, ultimately mediating gene transcription (1, 7). Interestingly, processes localized at the plasma membrane may also be important in conveying the signal for 1,25-(OH),D,induced differentiation. Studies have shown that in osteoblast-like cells (8), keratinocytes (9), intestinal cells (10, ll), and HL-60 cells (12, 13), 1,25-(OH),D, increases lipid turnover and intracellular calcium levels. These events, which subsequently activate protein kinase C (PKC) and other enzymes, may also be employed by 1,25-(OH),D, to regulate gene transcription and cellular differentiation. Such studies suggest a role for PKC in the regulation of gene transcription during the process of HL-60 cell differentiation. It has been shown that the expression of several genes, including c-nzyc, c-fos, and PKC, is regulated by 1,25-(OH),D, before the appearance of the mature monocytic phenotype (14-17). SevReceiv& April 19, 1996. Address all correspondence and requests for reprints to: Dr. Robert U. Simpson, Department of Pharmacology, University of Michigan, 1301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0632. * This work was supported by NIH Grants CA-43859 and DE-10337. t Predoctoral Fellow: Medical Student Fellowship in Pharmacology from the Pharmaceutical Manufactures Association Foundation. $ Predoctoral Fellow: Interdepartmental Training Grant in the Pharmacological Sciences, NIH / NIGMS Grant GM-07767. era1 studies have reported that c-myc expression is decreased whereas c-fos expression is transiently increased during the process of HL-60 cell differentiation (15,18,19). Additionally, PKQ expression is increased as early as 6 h and maximally at 48 h after exposure of these cells to 1,25-(OH),D, (16, 17, 20,21). Considering the nature of these early events and the accepted importance of these gene products in cell signaling and growth, it is likely that the regulation of these genes is a critical event in 1,25-(OH),D,-induced HL-60 cell differentiation (22, 23). Several studies demonstrated that c-myc plays an important role in the process of cellular differentiation. Differentiation of Friend erythroleukemia cells and 3T3-Ll preadipocytes can be inhibited by constitutive expression of the c-myc gene (24-26). Similarly, HL-60 cells can be induced to differentiate by inhibiting c-nzyc messenger RNA translation with specific anti-c-lnyc DNA probes (27-29). In contrast, others have suggested that rapid down-regulation of c-myc gene expression is not sufficient to induce HL-60 differentiation, whereas prolonged down-regulation may be more important (30, 31). In the presence of 1,25-(OH),D,, HL-60 cell transcription of c-myc was abolished by 48 h (15). We previously showed that this reduction in transcription is a result of a transcriptional elongation block at a site within the first intron (22). Using HeLa cells, Zajac-Kaye et al. (32) established the importance of binding of a 138-kDa protein to a sequence within the first intron of the c-myc gene called the MIE for control of c+zyc gene transcription (32). Our study was designed to investigate the effect of 1,25-(OH&D, on the interaction of nuclear proteins from HL-60 cells to this MIE. We demonstrate that a 32-kDa doublet nuclear protein is the major factor in HL-60 cells that binds to the MIE. Interestingly, phorbol 12-myristate 13acetate (PMA), an activator of PKC, induces a similar increase in the 32-kDa doublet protein-MIE interaction. We also dem-