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Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

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Abstract

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an in vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress.

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Acknowledgments

This work was financially supported by the Bulgarian National Fund for Scientific Research (Grant DOO2-212/2008).

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Authors and Affiliations

  1. Department of Lipid-Protein Interactions, Institute of Biophysics, Bulgarian Academy of Sciences, Sofia, 1113, Bulgaria

    Teodora Lupanova, Nadezhda Stefanova, Diana Petkova, Galya Staneva, Albena Jordanova, Kamen Koumanov & Albena Momchilova

  2. Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University, 8 Dragan Tzankov str, Sofia, 1164, Bulgaria

    Nadezhda Stefanova & Roumen Pankov

  3. Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA

    Roumen Pankov

Authors
  1. Teodora Lupanova
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  2. Nadezhda Stefanova
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  3. Diana Petkova
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  4. Galya Staneva
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  5. Albena Jordanova
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  6. Kamen Koumanov
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  7. Roumen Pankov
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  8. Albena Momchilova
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Correspondence to Albena Momchilova.

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Lupanova, T., Stefanova, N., Petkova, D. et al. Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix. Mol Cell Biochem 340, 215–222 (2010). https://doi.org/10.1007/s11010-010-0420-y

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  • DOI: https://doi.org/10.1007/s11010-010-0420-y

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