FIGURE 6.

Up-regulation of TPP1 by fibrate drugs involves both PPARα and RXRα. A–C, mouse primary astrocytes were treated with different concentrations of all-trans-retinoic acid (RA) and gemfibrozil (Gem) and the combination of the two in serum-free DMEM/F-12 medium for 24 h followed by monitoring of mRNA expression of Cln2 by quantitative RT-PCR (A) and protein expression of TPP1 by Western blot (B). C, densitometric analysis of TPP1 (relative to β-actin) with RA and gemfibrozil treatment. ^a, p <0.05 versus WT control; ^b, p <0.05 versus 0.5 μm RA-only treatment; ^c, p <0.05 versus 10 μm gemfibrozil-only treatment. D and E, mouse primary astrocytes were untransfected and transfected with scrambled siRNA (1.0 μg) or RXRα siRNA (1.0 μg) for 36 h followed by treatment with RA (0.5 μm) and gemfibrozil (10 μm) alone and in combination for 24 h of serum-free DMEM/F-12 medium followed by RT-PCR for RXRα, RXRβ, and RXRγ (D) and quantitative real time PCR for Cln2 (E). *, p < 0.05 versus untransfected control; **, p < 0.05 versus scrambled siRNA transfected control; ^d, p < 0.05 versus untransfected and gemfibrozil-treated sample; ^e, p < 0.05 versus untransfected and RA-treated sample; ^f, p <0.05 versus scrambled siRNA-transfected and gemfibrozil-treated sample; ^g, p < 0.05 versus scrambled siRNA-transfected and RA-treated sample; ns, not significant with respect to RXR-α siRNA transfected control. F, mouse primary astrocytes were transfected with scrambled siRNA (1.0 μg) or RXRα siRNA (1.0 μg) and treated with gemfibrozil (10 μm) and RA (0.5 μm) alone and in combination under similar culture conditions, and the protein expressions of TPP1 were estimated by Western blot. G, densitometric analysis of TPP1 (relative to β-actin) with RA and gemfibrozil treatment. **, p < 0.05 versus scrambled siRNA-transfected control; ^h, p <0.05 versus only gemfibrozil treatment; ⁱ, p <0.05 versus only RA treatment; ns, not significant w.r.t. RXR-α siRNA-transfected control. H, mouse primary astrocytes isolated from wild type, PPARα^−/−, and PPARβ^−/− mice were treated with RA (0.5 μm) alone and in combination with gemfibrozil (10 μm) under similar culture conditions and cells, were subjected to Western blot for TPP1. I, densitometric analysis of TPP1 levels (relative to β-actin) in PPARα^−/− and PPARβ^−/− and wild type astrocytes after RA and gemfibrozil + RA treatment. †, p <0.05 versus WT control; ††, p <0.05 versus PPARβ^−/− control; ^j, p <0.05 versus only RA treatment in WT cells; ^k, p <0.05 versus only RA treatment in PPARβ^−/− cells; ns, not significant w.r.t. PPARα^−/− control. All results are means ± S.E. of at least three independent experiments.