Abstract
Sphingolipids are ubiquitous components of eukaryotic cells that regulate various cellular functions. In many cell types, a fraction of sphingolipids contain 2-hydroxy fatty acids, produced by fatty acid 2-hydroxylase (FA2H), as the N-acyl chain of ceramide [hydroxyl fatty acid (hFA)-sphingolipids]. FA2H is highly expressed in myelin-forming cells of the nervous system and in epidermal keratinocytes. While hFA-sphingolipids are thought to enhance the physical stability of specialized membranes produced by these cells, physiological significance of hFA-sphingolipids in many other cell types is unknown. In this study, we report novel roles for FA2H and hFA-sphingolipids in the regulation of the cell cycle. Treatment of D6P2T Schwannoma cells with dibutyryl-cAMP (db-cAMP) induced exit from the cell cycle with concomitant upregulation of FA2H. Partial silencing of FA2H in D6P2T cells resulted in 60-70% reduction of hFA-dihydroceramide and hFA-ceramide, with no effect on nonhydroxy dihydroceramide and ceramide. Under these conditions, db-cAMP no longer induced cell cycle exit, and cells continued to grow and divide. Immunoblot analyses revealed that FA2H silencing prevented db-cAMP-induced upregulation of cyclin-dependent kinase inhibitors p21 and p27. These results provide evidence that FA2H is a negative regulator of the cell cycle and facilitates db-cAMP-induced cell cycle exit in D6P2T cells.
Highlights
Sphingolipids are ubiquitous components of eukaryotic cells that regulate various cellular functions
We previously showed that silencing fatty acid 2-hydroxylase (FA2H) by small interfering RNA or short hairpin RNA (shRNA) in rat Schwannoma D6P2T cells resulted in ?60% reduction of total hydroxyl fatty acid (hFA) content in the cell [23]
HFA-DHC/ ceramide and nonhydroxy DHC and ceramide were determined in D6P2T cells transfected with control shRNA or FA2H shRNA expression plasmid (Fig. 2)
Summary
Sphingolipids are ubiquitous components of eukaryotic cells that regulate various cellular functions. We provide evidence that FA2H regulates cAMP-induced cell cycle exit in D6P2T cells. Cells expressing control or FA2H shRNA were treated with vehicle only, 0.1, or 1 mM db-cAMP for 24 h. We previously showed that silencing FA2H by small interfering RNA or shRNA in rat Schwannoma D6P2T cells resulted in ?60% reduction of total hFA content in the cell [23].
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