Transcription factors Sp1 and AP-2 mediate induction of acid sphingomyelinase during monocytic differentiation

Cells from the human monocytic leukemia cell line THP-I differentiate towar ds a macrophage-like phenotype when stimulated with phorbol 12-myristate-13 -acetate (PMA), 1,25-dihydroxy-vitamin D-3, and various other agents, We de monstrate here that the expression of the lysosomal enzyme acid sphingomyel inase (ASM; E.C. 3.1.4.12) is induced during this process and is strongly e levated in differentiated THP-I cells, as well as in differentiated human m ononuclear phagocytes. Using Northern blotting, RNase protection assay, and nuclear run-on analyses, we show that the up-regulation of ASM expression is regulated mainly at the level of transcription and that new protein synt hesis is required for enhanced ASM activity, This cell-type specific induct ion by PMA treatment was further investigated with respect to transcription al control. A series of 5' deletion derivatives of the upstream regulatory region were used in transient transfection assays to identify promoter elem ents required for basal and inducible gene expression. A PMA responsive ele ment was localized to a region between -319 and -219 bp upstream of the ini tiation codon and co-transfections with transcription factor expression pla smids for AP-2 and Spl resulted in augmented ASM promoter activity, which w as abolished when the binding sites for these two factors were deleted, Usi ng electrophoretic mobility shift assays and supershift assays we demonstra te that this region is specifically bound by Spl and AP-2, These factors ar e present in nuclear extracts prepared from both induced and uninduced THP- I cells. However, the intensity of the complex formed appeared to increase when nuclear extracts from PMA-treated cells were used. From these studies, we conclude that a concerted action of the transcription factors AP-2 and Spl is essential for the up-regulation of ASM expression during the process of macrophage differentiation.