Both Sphingomyelin Synthases SMS1 and SMS2 Are Required for Sphingomyelin Homeostasis and Growth in Human HeLa Cells

Fikadu Geta Tafesse,Klazien Huitema,Martin Hermansson,Seleéne Van Der Poel,Joep Van Den Dikkenberg,Andreas Uphoff,Pentti Somerharju,Joost C.M Holthuis

doi:10.1074/jbc.m702423200

Fikadu Geta Tafesse, Klazien Huitema + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m702423200

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Abstract

Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM.

Highlights

Several lines of evidence indicate that SM synthesis plays a critical role in cell growth and survival
Reverse transcription-PCR revealed the presence of both SMS1 and SMS2 transcripts in all five cell lines analyzed. These results indicate that SMS1 and SMS2 are encoded by ubiquitously expressed genes
In this study we showed that SMS1 and SMS2 function as the principal Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively

Summary

EXPERIMENTAL PROCEDURES

Chemicals—NBD-hexanoylceramide (C6-NBD-ceramide) was from Molecular Probes (Eugene, OR). Equal amounts per fraction were subjected to immunoblotting and analyzed for GlcCer and SM synthase activity To this end, gradient fractions (100 ␮l) were incubated for 1 h at 37 °C in Hanks’ buffered saline solution (HBSS) containing 10 ␮M C6-NBD-Cer, 1 mM UDP-glucose, 1 mM MgCl2, and 1 mM MnCl2 in a total volume of 0.5 ml. Cells were washed in phosphate-buffered saline and subjected to lipid extraction using the method of Bligh and Dyer [22]. After 6 h of treatment, the medium was changed for Opti-MEM containing 3% normal or 3% delipidated fetal calf serum supplemented with 80 ␮M exogenous SM or PC as described previously [3]. At the indicated time points, cells were washed with phosphate-buffered saline, trypsinized, and resuspended in HBSS containing 1% fetal calf serum. After 15 min of incubation, 100 ng/ml propidium iodine was added, and the cells were analyzed by flow cytometry as above

RESULTS

Lipid class

DISCUSSION