StearoylCoA Desaturase-5: A Novel Regulator of Neuronal Cell Proliferation and Differentiation

Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1), a Δ9-desaturase that converts saturated fatty acids (SFA) into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5), a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls). De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFR)Akt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key lipogenic pathways and transduction signaling cascades, SCD5 participates in the regulation of neuronal cell growth and differentiation

Overexpression of human SCD5 increases the ratio n-7 MUFA to SFA.

<p>Neuro2a cells were stably transfected with human SCD5 cDNA in a pCDNA4 expression plasmid (SCD5 cells) or with empty plasmid alone (pCDNA4 cells). Expression of SCD5 was determined by immunoblot analysis of cell homogenates (<i>A</i>). Fatty acids from total cell lipids were assessed by HPLC and ratios MUFA to SFA were calculated. <i>B</i>, palmitoleic acid (16:1n-7) to palmitic acid (16∶0); <i>C</i>, palmitoleic acid plus cis-vaccenic(18:1n-7) to palmitic acid (16∶0); D, oleic acid (18:1n-9) to stearic acid (18∶0). *, p<0.05 or less.</p

Effect of retinoic acid on the expression of SCD5 in human cells.

<p>WS-1 human normal skin fibroblasts were treated with increasing concentration of retinoic acid (RA) up to 10 µM or vehicle DMSO (0.1% by vol) for 24 h. SCD5 and β-actin in cell homogenates were detected by immunoblot analysis and were quantified by densitometry (<i>A</i>). SH-SY5Y human neuroblastoma cells were incubated with 20 µM RA or DMSO for 4 days and content of SCD5 protein was assessed by Western blot (<i>B</i>). Levels of SCD5 were normalized using β-actin values. Data represent the mean ± SD of triplicate samples. *, p<0.05 or less vs control, by Student's t test.</p

Expression of human SCD5 in Neuro2a cells increases cell proliferation. Effect of retinoic acid.

<p>Neuro2a cells stably transfected with human SCD5 cDNA (SCD5 cells) or with empty plasmid (pCDNA4 cells) were seeded in 12-well plates and grown for different time points up to 120 h (<i>A</i>). At each time point, cell proliferation was determined by Crystal violet assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039787#s2" target="_blank">Materials and Methods</a>. <i>B</i>, SCD5-expressing Neuro2a cells and their controls were incubated with 10 µM retinoic acid or DMSO vehicle for 48 h. Cell growth was estimated by Crystal violet staining method. Values represent the mean ± S.D. of triplicate determinations. *, p<0.05 or less vs control, by Student's t test. <i>C</i>, Western blot determination of cyclin D1 and β-actin levels in control (pCDNA4) and SCD5-expressing Neuro-2a cells incubated in serum-free DMEM with 0.1% BSA, in presence or absence of 10µM retinoic acid (RA), for 1 h or 48 h.</p

SCD5 activity blocks retinoic acid-induced differentiation of Neuro2a cells.

<p>SCD5-expressing Neuro2a cells and empty plasmid-carrying controls (pCDNA4) were seeded in 60-mm dishes and incubated in 10%FBS DMEM. Twenty-four hours later, media was removed and cells were incubated with differentiation media (DMEM supplemented with 2%FBS and10 µM retinoic acid) for 48 h. Cells were fixed, stained with coomassie brilliant blue proliferation and photographed in a phase-contrast microscope (<i>A</i>). <i>B</i>, percentage (%) of cells bearing one or more neurites equal to or longer than cell body diameter.</p

SCD5 expression impairs the activation of EGFR signaling cascade.

<p>Neuro2a cells, stably transfected with SCD5 cDNA (SCD5) or empty plasmid, were incubated in serum-free DMEM with 0.1BSA, in presence or absence of 10 µM retinoic acid (RA), for 1 h or 48 h. A group of cells incubated with RA for 48 h was stimulated with 100 ng/mL EGF for 5 minutes. Cells were harvested and homogenized and phosphorylation of EGFR, ERK, and Akt was determined by Western blot.</p