Sphingomyelin synthesis is involved in adherence during macrophage differentiation of HL-60 cells

Prior studies demonstrated that sphingomyelin degradation via a sphingomyelinase antagonized phorbol ester-mediated differentiation of HL-60 cells into macrophages (Kolesnick, R.N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies show that phorbol esters induce early sphingomyelin synthesis in HL-60 cells and that this event may play a direct role in development of an adherent macrophage population. A maximally effective concentration of the potent phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 1 x 10(-7) M) stimulated an elevation in the sphingomyelin level at 24 h from 560 to 700 pmol/10(6) cells; a peak level of 1400 pmol/10(6) cells was achieved at 48 h. Phosphatidylcholine levels did not change significantly, indicating sphingomyelin synthesis was selective. The phosphatidylcholine:sphingomyelin ratio decreased from 10.3 to 7.9 at 24 h and to 5.3 at 48 h. Phorbol ester-induced sphingomyelin synthesis was biphasic. A burst of synthesis, detectable within 1 h and linear for 4 h, was followed by a prolonged phase at a slower rate. Ceramide synthesis was also biphasic. Ceramide levels decreased initially consistent with activation of the enzyme, phosphatidylcholine:ceramide cholinephosphotransferase and increased during the prolonged phase of sphingomyelin synthesis. During phorbol ester-induced differentiation, an adherent macrophage population was demonstrable by 14 h. This population contained the entire elevation of sphingomyelin levels. This demonstrates that early sphingomyelin synthesis defines a population of cells destined to become adherent macrophages. Studies were performed to directly manipulate sphingomyelin levels. Small unilamellar vesicles containing sphingomyelin did not directly induce macrophage differentiation but rather potentiated the effect of submaximal concentrations of phorbol ester. Sphingomyelin vesicles (2 x 10-6 M) enhanced TPA (5 x 10-10 M)-induced adherence 2-fold from 12 to 24% of the total population. Sphingosylphosphorylcholine (5 x 10-6 M), which may be acylated to sphingomyelin, was similarly effective. Further, exogenous sphingomyelinase, but not various phospholipases A2 and C, induced detachment of adherent macrophages. In sum, these studies demonstrate that phorbol esters induce early, selective synthesis of sphingomyelin in HL-60 cells. This event defines a population of cells destined to become adherent macrophages and may play a direct role in the adherence process.