Stimulation of myelin proteolipid protein gene expression by eicosapentaenoic acid in C6 glioma cells

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Abstract

In this study, the role of exogenous fatty acids in the regulation of proteolipid protein (PLP) gene expression was investigated using the following model culture system: C6 glioma cells expressing the green-fluorescent protein (eGFP) driven by different segments of PLP promoter. Eicosapentanoic acid (EPA; 20:5 n-3), but not arachidonic acid (AA; 20:4 n-6), induced a significant increase in medium fluorescence intensity (MFI) determined by fluorescence-activated cell sorting (FACS). The induction of PLP promoter was time-dependent showing maximal activity between 24 and 48 h after EPA exposure. PLP promoter activation was dependent on fatty acid concentration, with maximum activation at 200 μM. Northern blot analysis confirmed the fluorescence data in C6 cells incubated with EPA. Furthermore, this treatment increased the adenylyl cyclase-cyclic AMP (cAMP) levels and the mitogen-activated protein kinase (MAPK) activation in C6 cells. PLP promoter activity was inhibited by pre-treatment with H89 (protein kinase A (PKA) inhibitor), but not with PD98059 (MAPK inhibitor), suggesting that EPA stimulates the expression of PLP via cAMP-mediated pathways.

Introduction

Proteolipid protein (PLP) is the major protein component (50%) of CNS myelin (Lees and Brostoff, 1984). PLP is essential for the compaction of the myelin sheath which, in turn, is crucial for normal brain function as shown by the severe consequences of PLP mutation/deficiency in rodents (Hogan and Greenfield, 1984, Gardinier et al., 1986, Boison and Stoffel, 1989, Shiota et al., 1991) and humans (Hudson et al., 1989, Koeppen and Robitaille, 2002). The expression of myelin genes including PLP has been reported to be affected by dietary lipids. DeWille and Farmer (1992) have shown that postnatal essential fatty acids (EFAs) deficiency reduces PLP and myelin basic protein (MBP) mRNAs levels in the neonatal brain.

Our studies (Salvati et al., 1984, Salvati et al., 1996a) have shown that odd-chain fatty acids, included within EFAs by Berthuis et al. (1968), accelerate the developmental expression of PLP and myelin oligodendrocyte protein (MOG) genes in newborns whose mothers were fed diets supplemented with these fatty acids during pregnancy and throughout lactation.

Taken together, these results indicate that dietary fatty acids can modulate the expression levels of PLP and other myelin genes during brain development. However, the molecular and cellular mechanisms induced by dietary fatty acids and involved in myelin gene expression are still not well understood. Furthermore, since diets contain a mixture of EFAs, in vivo studies are not suitable to detect which specific lipid component produces the effects.

To identify the fatty acids involved in myelin gene regulation and to clarify their mechanisms, we developed a model system to study PLP gene expression: rat C6 glioma cells were stably transfected with the gene for green-fluorescent protein (GFP) driven by the promoter region of PLP gene. C6 cells were chosen because they retain certain characteristics of glial precursors, express some astrocyte and oligodendrocyte genes including PLP, and are capable of differentiation in vitro under certain experimental conditions (Milner et al., 1985, Konat et al., 1991, Ye et al., 1992).

This approach has permitted the screening of fatty acids involved in PLP gene regulation on the basis of their GFP expression levels evaluated by cytofluorimetric analysis. Our attention was focused on polyunsaturated fatty acids (PUFAs), for there is growing evidence that PUFAs may regulate gene expression in some tissues (Price et al., 2000, Clarke, 2001) and in the brain (Kitajka et al., 2002). We analyzed arachidonic acid (AA; 20:4 n-6) and eicosapentanoic acid (EPA; 20:5 n-3), metabolites of linoleic (18:2 n-6) and linolenic acids (18:3 n-3), respectively, and precursors for eicosanoids biosynthesis which can exert a wide range of biological actions.

Section snippets

PLP constructs

The eGFP-1476 PLP and eGFP-276 PLP constructs were obtained with a two-step strategy to simplify the cloning in the correct orientation of sequences from the mouse PLP promoter in the pEGFP-1 expression vector (Clontech Laboratories).

The B6-CAT plasmid (a kind gift of Nave) containing the sequence of 1476 bp of mouse PLP promoter (between −1385 and +90 bp) was digested with restriction enzymes Pst I and Xho I to excise the entire sequence or with Xba I and Xho I to excise a fragment of 276 bp

Activation of PLP promoter by fatty acids

To search for physiological regulators of PLP gene expression, rat C6 glioma cells, stably transfected with a vector in which GFP expression is driven by a fragment of 276 bp of the PLP promoter (eGFP-276 PLP), were treated with AA and EPA. The time course and dose dependence of fatty acids on PLP promoter modulation are shown in Fig. 1. The treatment with EPA resulted in a sharp increase in medium fluorescence intensity (MFI) as from 12 h, reaching its maximum effect at 48 h (Fig. 1A). FK, an

Discussion

Our study showed that C6 glioma cells transfected with eGFP driven by the PLP promoter can be used as a simple model to identify molecules and to research into the mechanisms involved in gene regulation. We chose C6 cell line as a model system to study PLP expression because these immortalized cells express the PLP gene. Moreover, Milner et al. (1985) showed that the transcription of the PLP gene in C6 cells is active even though it is considerably lower than in oligodendrocytes. This

Acknowledgements

This work was supported by a grant from the Italian Ministry of Health—finalized project: genotype–phenotype ratio in the neuronal and myelin pathologies: diagnostic, prognostic and therapeutic implications. We thank Prof. E.V. Avvedimento for the critical reading of the manuscript.

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