Abstract
Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); thus, ceramidases control the levels of these bioactive sphingolipids in cells and tissues. Neutral ceramidase (nCDase) is highly expressed in colorectal tissues, and a recent report showed that nCDase activity is involved in Wnt/β-catenin signaling. In addition, the inhibition of nCDase decreases the development and progression of colorectal tumor growth. Here, to determine the action of nCDase in colorectal cancer cells, we focused on the subcellular localization and metabolic functions of this enzyme in HCT116 cells. nCDase was found to be located in both the plasma membrane and in the Golgi apparatus, but it had minimal effects on basal levels of ceramide, sphingosine, or S1P. Cells overexpressing nCDase were protected from the cell death and Golgi fragmentation induced by C6-ceramide, and they showed reduced levels of C6-ceramide and higher levels of S1P and sphingosine. Furthermore, compartment-specific metabolic functions of the enzyme were probed using C6-ceramide and Golgi-targeted bacterial SMase (bSMase) and bacterial ceramidase (bCDase). The results showed that Golgi-specific bCDase also demonstrated resistance against the cell death stimulated by C6-ceramide, and it catalyzed the metabolism of ceramides and produced sphingosine in the Golgi. Targeting bSMase to the Golgi resulted in increased levels of ceramide that were attenuated by the expression of nCDase, also supporting its ability to metabolize Golgi-generated ceramide. These results are critical in understanding the functions of nCDase actions in colorectal cancer cells as well as the compartmentalized pathways of sphingolipid metabolism.
Highlights
Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); ceramidases control the levels of these bioactive sphingolipids in cells and tissues
This study demonstrates that Neutral ceramidase (nCDase) is located in the Golgi in HCT116 cells in addition to its location in the plasma membrane
The results demonstrate the ability of nCDase in hydrolyzing the lipid in the Golgi complex and protecting cells from apoptosis induced by C6-ceramide
Summary
Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); ceramidases control the levels of these bioactive sphingolipids in cells and tissues. Compartment-specific metabolic functions of the enzyme were probed using C6-ceramide and Golgi-targeted bacterial SMase (bSMase) and bacterial ceramidase (bCDase). Targeting bSMase to the Golgi resulted in increased levels of ceramide that were attenuated by the expression of nCDase, supporting its ability to metabolize Golgi-generated ceramide. These results are critical in understanding the functions of nCDase actions in colorectal cancer cells as well as the compartmentalized pathways of sphingolipid metabolism.—Sakamoto, W., N. Alkaline ceramidases (ACERs) 1, 2, and 3 are primarily located in the endoplasmic reticulum (ER) and Golgi complexes
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