Opposite effects of tumor necrosis factor alpha on the sphingomyelin- ceramide pathway in two myeloid leukemia cell lines: role of transverse sphingomyelin distribution in the plasma membrane

Abstract

Tumor necrosis factor alpha (TNF alpha) mediates proliferation, functional activation, and apoptotic cell death depending on the target cell type. Although sphingomyelin (SPM) hydrolysis and ceramide generation may function as an important mediator in TNF alpha signaling, the molecular mechanisms of the signaling pathway(s) are still not well understood. The aim of the present study is to compare the effect of TNF alpha on SPM metabolism and cell growth in two myeloid leukemic cell lines (U937 and KG1a) that differ in their sensitivity to TNF alpha. Our results show that TNF alpha induced apoptosis in U937 but not in KG1a cells. TNF alpha triggered in KG1a cells neither SPM hydrolysis nor ceramide generation, but induced SPM synthesis and ceramide breakdown as well as dose-dependent cell proliferation. In contrast, TNF alpha induced in U937 SPM hydrolysis and ceramide generation as well as dose-dependent cell death. Synthetic cell permeant ceramide, as well as natural ceramide, generated by treatment with bacterial sphingomyelinase (SPMase), all induced apoptosis in both U937 and KG1a cells. These findings indicate that the SPM-ceramide pathway is altered in KG1a cells upstream of the ceramide generation. Analysis of the transverse distribution of SPM in the plasma membrane showed that the SPM pool involved in cell signaling (inner leaflet) was markedly reduced in KG1a cells; it is 7-fold lower than that found in the inner leaflet of U937 cells. Therefore, our study strongly suggests that the different responses induced by TNF alpha in myeloid cells are dependent on the SPM plasma membrane transverse asymmetry.

Restoration of TNF-α-induced ceramide generation and apoptosis in resistant human leukemia KG1a cells by the P-glycoprotein blocker PSC833

“…Analysis of transverse SM distribution in the plasma membrane was performed as previously described (16,(30)(31)(32). This method has been applied to various cell models such as Krebs-II ascitic cells, HL-60 cells, A431 cells, and neutrophils (31)(32)(33)(34)(35).…”

“…In both cases, plasma membrane inner leaflet SM content was deduced by subtracting the SM content of SMase-treated plasma membranes from the total plasma membrane SM content (16).…”

“…Since we and others (8,16) have previously described that in TNF-a sensitive myeloid leukemic cells such as U937, apoptosis was triggered via CER generation, we investigated whether CER was generated in KG1a cells treated with both PSC833 and TNF-a. To ascertain whether the combination of PSC833 and TNF-a could activate the SM cycle in KG1a cells, cells were prelabeled with [ 3 H]palmitic acid to equilibrium for 48 h. KG1a cells were then pretreated for 24 h with 1 mM PSC833 and then challenged every 24 h with 10 ng/ml TNF-a.…”

“…To ascertain whether the combination of PSC833 and TNF-a could activate the SM cycle in KG1a cells, cells were prelabeled with [ 3 H]palmitic acid to equilibrium for 48 h. KG1a cells were then pretreated for 24 h with 1 mM PSC833 and then challenged every 24 h with 10 ng/ml TNF-a. CER production was monitored at 48 h, 10 min after the addition of TNF-a (corresponding to the peak CER generation in TNF-a sensitive U937 cells) (16). As shown in Fig.…”

“…KG1a cells were then pretreated for 24 h with 1 mM PSC833 and challenged every 24 h with 10 ng/ml TNF-a. SMase activities were monitored 10 min after the addition of TNF-a (corresponding to the peak neutral SMase activation in TNF-a-sensitive U937 cells) (16). As shown in Fig.…”