Primate Monocytes - CD14, CD16 - Ziegler-Heitbrock

The 5-lipoxygenase promoter is regulated by DNA methylation

Abstract

5-Lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is expressed in a tissue- and cell differentiation-specific manner. The 5-LO core promoter required for basal promoter activity has a unique G+C-rich sequence which contains five tandem Sp1 consensus sequences. The mechanisms involved in the regulation of cell-type specific 5-LO expression are unknown. Here we show, that 5-LO expression is regulated by DNA methylation. Treatment of the 5-LO negative cell lines U937 and HL-60TB with the demethylating agent 5-aza-2'-deoxycytidine (AdC) upregulated expression of 5-LO primary transcripts and mature mRNA in a similar fashion indicating that AdC stimulates 5-LO gene transcription. Analysis of the methylation status of the 5-LO promoter revealed that the core promoter region was methylated in U937 and HL-60TB cells, whereas it was unmethylated in the 5-LO positive parent HL-60 cell line. Reporter gene assays with 5-LO promoter constructs gave an up to 68- and 655-fold repression of 5-LO promoter activity in HeLa and Mono Mac 6 cells by methylation. 1,25-dihydroxyvitamin D3 and TGFbeta that are potent inducers of the 5-LO pathway in myeloid cell lines, increased 5-LO RNA expression in HL-60TB and U937 cells but co-treatment with AdC was required to achieve 5-LO expression levels in HL-60TB cells that were comparable to wild-type HL-60 cells. In reporter gene assays, 1,25-dihydroxyvitamin D3 and TGFbeta were unable to induce promoter activity when the 5-LO promoter constructs were methylated, which suggests that 5-LO promoter demethylation is a prerequisite for the high level induction of 5-LO gene expression by 1,25?dihydroxyvitamin D3 and TGFbeta and that the effects of both agents on 5-LO mRNA expression are not related to DNA methylation.