Sphingomyelin turnover induced by vitamin D3 in HL-60 cells

TLDR

Sphingolipid cycles are thought to participate in signal transduction and cellular differentiation. The study examined whether sphingomyelin hydrolysis drives differentiation of HL‑60 cells by adding exogenous sphingomyelinase. Sphingolipid turnover was assessed in HL‑60 cells treated with 1α,25‑(OH)₂‑vitamin D₃, measuring choline‑labelled species, ceramide, phosphorylcholine, and neutral sphingomyelinase activity, and by adding bacterial sphingomyelinase to mimic hydrolysis. Vitamin D₃ induced a transient sphingomyelin cycle in HL‑60 cells, with peak ceramide and phosphorylcholine production at 2 h, a 90 % rise in neutral sphingomyelinase activity, and exogenous sphingomyelinase potentiating subthreshold vitamin D₃‑induced differentiation, confirming a sphingomyelin cycle in human cells.

Abstract

Sphingolipid metabolism was examined in human promyelocytic leukemia HL-60 cells. Differentiation of HL-60 cells with 1 alpha, 25-dihydroxyvitamin D3 (vitamin D3; 100 nM) was accompanied by turnover. Maximum turnover of [3H]choline-labeled occurred 2 h following vitamin D3 treatment, with levels decreasing to 77 +/- 6% of control and returning to base-line levels by 4 h. Ceramide and phosphorylcholine were concomitantly generated. Ceramide mass levels increased by 55% at 2 h following vitamin D3 treatment and returned to base-line levels by 4 h. The amount of phosphorylcholine produced equaled the amount of hydrolyzed, suggesting the involvement of a sphingomyelinase. Vitamin D3 treatment resulted in a 90% increase in the activity of a neutral sphingomyelinase from HL-60 cells. The inferred role of hydrolysis in the induction of cell differentiation was investigated using an exogenous sphingomyelinase. When a bacterial sphingomyelinase was added at concentrations that caused a similar degree of hydrolysis as 100 nM vitamin D3, it enhanced the ability of subthreshold levels of vitamin D3 to induce HL-60 cell differentiation. This study demonstrates the existence of a sphingomyelin cycle in human cells. Such sphingolipid cycles (Hannun, Y., and Bell, R. (1989) Science 243, 500-507) may function in a signal transduction pathway and in cellular differentiation.