Fatty acid 2-hydroxylase regulates cAMP-induced cell cycle exit in D6P2T schwannoma cells

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.

Fig. 1.

Proposed biosynthetic pathway for hFA-sphingolipids. The de novo pathway is postulated based on our in vitro and in vivo evidence (20, 22). Salvage pathways (not shown) leading to formation of hFA-sphingolipids have not been studied. In addition to C₁₆ fatty acid, many other fatty acids (C₁₄–C₃₄) can be used. The N-acylation step is catalyzed by any of the six isoforms of ceramide synthase (38). GSL, glycosphingolipid; SM, sphingomyelin.

Fig. 2.

Effects of FA2H shRNA on DHC and ceramide. D6P2T cells were transfected with control shRNA (black bars) or FA2H shRNA (white bars) plasmid. Transfected cells were selected for puromycin resistance for 2 weeks. Lipids were quantified by LC/MS/MS and normalized against protein contents. The mean and SD of triplicate measurements are shown. (See supplementary Table I for the quantities of individual molecular species.) A: The sum of 13 nonhydroxy DHC and 10 hFA-DHC molecular species. B: The sum of 13 nonhydroxy ceramide and 12 hFA-ceramide molecular species.

Fig. 3.

FA2H silencing nullifies db-cAMP-induced upregulation of FA2H. D6P2T cells were transfected with control shRNA or FA2H shRNA plasmid as indicated. Transfected cells were selected for puromycin resistance for 2 weeks and then treated with vehicle only or 1 mM db-cAMP for 48 h. A: FA2H mRNA levels. FA2H mRNA levels were determined by quantitative PCR and normalized against 18s rRNA levels. The mean and SD of triplicate measurements are shown. B: FA2H activity. Crude cell lysates (50 μg protein) were used for FA2H assays. The mean and SD of triplicate measurements are shown.

Fig. 4.

FA2H silencing facilitates cell growth and inhibits db-cAMP-induced growth arrest in D6P2T cells. D6P2T cells were transfected with control shRNA or FA2H shRNA plasmid and selected for puromycin resistance for 2 weeks. A–D: Cells were treated with sham (A, C) or 0.1 mM db-cAMP (B, D) for 24 h before stained with calcein-AM. E: Cell counts in samples A–D. The counts were normalized against the cell number in A. The mean and SD of triplicate counts are shown. F: [³H]thymidine incorporation. Low confluency cells were exposed to [³H]thymidine for 5 h in a serum-free medium. After extensive washing, cells were lysed to quantify radioactivity by liquid scintillation counting. The mean and SD of sextuplicate measurements are shown.

Fig. 5.

FA2H silencing prevents upregulation of CDK inhibitors induced by db-cAMP. D6P2T cells were transfected with control shRNA or FA2H shRNA plasmid. Transfected cells were selected for puromycin resistance for 2 weeks. Cells were treated with sham, 0.1 mM, or 1 mM db-cAMP for 48 h and processed for SDS-PAGE (10 μg protein/lane).