Abstract
The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.
Highlights
Sphingolipids are considered predominant building blocks of biological membranes
Our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the Madin-Darby canine kidney (MDCK) cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway
Sphingolipid Metabolism in Nonstimulated Proliferating MDCK Cells—After a “lag” period (ϳ24 h, Fig. 1A), MDCK cells still proliferated at the rate of approximately one cycle of division every 24 h, they were submitted to a low concen
Summary
In contrast to the well documented importance of sphingolipid metabolism in determining cellular fate under stress conditions or under the effect of external stimuli, very little is known about its role in basal nonstimulated conditions. We showed differential branching of the sphingolipid metabolic pathways according to the developmental stage in rat renal papillae (18). We have shown that the developmental regulation of SK expression and activity leads sphingolipid metabolism to the formation of Sph-1-P in the neonatal period and of Cer in adult tissue. Because we used ex vivo tissue from neonatal and adult rats, we were unable to demonstrate whether such different branching of the sphingolipid metabolism was either a cause or a consequence of the developmental stage. The present study was undertaken to elucidate whether intracellular sphingolipid metabolism can determine the cellular fate in nonstimulated conditions. Our most important findings were that in nonstimulated proliferating MDCK cells: 1) the level of de novo synthesized Cer is a determining factor to decide cellular fate; and 2) the de novo synthesis of Cer is under the control of the Sph-1-P, which appears to be formed through a de novo synthesis-independent pathway
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