Abstract
Sphingomyelin or the products derived from its metabolism may constitute a signaling system involved in a variety of cellular processes. The activation of a plasma membrane neutral sphingomyelinase, which catalyzes the first step in sphingomyelin turnover, has been suggested to play an important role in cellular differentiation. We have studied the effect of exogenous staphylococcal sphingomyelinase on DNA synthesis and on the composition of membrane sphingolipids in quiescent Swiss 3T3 fibroblasts. Sphingomyelinase stimulated proliferation of Swiss 3T3 cells and potentiated the mitogenic action of other growth factors, such as insulin, epidermal growth factor, and bombesin. Treatment with sphingomyelinase produced a significant decrease in sphingomyelin accompanied by a corresponding increase in ceramide levels. No significant increases were detected in the levels of products derived from ceramide, i.e. ceramide 1-phosphate, sphingosine, or sphingosine 1-phosphate. To further investigate the role of ceramide in cellular proliferation, we studied the effect of cell-permeable analogs of ceramide on DNA synthesis in quiescent Swiss 3T3 cells. Both N-hexanoylsphingosine and N-acetylsphingosine at low concentrations stimulated [3H]thymidine incorporation and acted synergistically with a wide variety of growth factors known to induce proliferation of quiescent Swiss 3T3 fibroblasts. Similar effects were observed with bovine brain ceramides. These results suggest that ceramide may be involved in the regulation of cellular proliferation.
Highlights
(Merril and Stevens, 1989; Merrill and Jones, 1990; Kim et al, 1991; Koval and Pagano, 1991; Kolesnick, 1991)
We found that exogenous sphingomyelinase induced DNA synthesis and potentiated the action of other known growth factors in quiescent Swiss 3T3 fibroblasts
We investigated whether sphingomyelinase exhibited the same effects in Swiss 3T3 fibroblasts
Summary
Proliferation chloride (79.3 Ci/mmol), and [y-3'P]ATP (3000 Ci/mmol) were pur- located by autoradiography of the plates. The cells pended in cbloroform/methanol (k1, v/v), analyzed by TLC using were cultured in 6-well cluster plastic tissue culture dishes (6 X 34- the solvent system described above for ceramide 1-phosphate, and mm wells, Costar). Radioactive lipids with an &of 0.23 & 0.05, similar cells/cm' in DMEM supplemented with 2 mM glutamine, penicillin to that described for ceramide 1-phosphate Ceramide 1-phosphate and sphingosine 1-phosphatewere anamedium (1:l) supplemented with 20 pg/ml bovine serum albumin and lyzed by TLC as described above. Cells were treated with sphingomyematerial was measured as described Lipid Analyses-Cells were prelabeled in DMEM containing 32Pi described above for the preparation of ceramide 1-phosphate. Phosphatidic acid (RF= 0.45 k 0.05) from ceramide 1-phosphate (RF confluent cultures were labeled with [methyl-3H]choline(2 pCi/ml). Concentrated HCl(lO02001, v/v), and phases were separated exactly as described (Zhang et al, 1990a).The lipids in the chloroform phase
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