Caffeic acid phenethyl ester induces adrenoleukodystrophy (Abcd2) gene in human X-ALD fibroblasts and inhibits the proinflammatory response in Abcd1/2 silenced mouse primary astrocytes

Abstract

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene. Accumulation of very long chain fatty acids (VLCFA) that have been attributed to reduced peroxisomal VLCFA β-oxidation activity are the hallmark of the disease. Overexpression of ABCD2 gene, the closest homolog of ABCD1, has been shown to compensate for ABCD1, thus correcting the VLCFA derangement. The accumulation of VLCFA leads to a neuroinflammatory disease process associated with demyelination of the cerebral white matter. The present study underlines the importance of caffeic acid phenethyl ester (CAPE) in inducing the expression of ABCD2 (ALDRP), and normalizing the peroxisomal β-oxidation as well as the levels of saturated and monounsaturated VLCFAs in cultured human skin fibroblasts of X-ALD patients. The expression of ELOVL1, the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1), was also reduced by CAPE treatment. Importantly, CAPE upregulated Abcd2 expression and peroxisomal β-oxidation and lowered the VLCFA levels in Abcd1-deficient U87 astrocytes and B12 oligodendrocytes. In addition, using Abcd1/Abcd2-silenced mouse primary astrocytes we examined the effects of CAPE in VLCFA-induced inflammatory response. CAPE treatment decreased the inflammatory response as the expression of inducible nitric oxide synthase, inflammatory cytokine, and activation of NF-κB in Abcd1/Abcd2-silenced mouse primary astrocytes was reduced. The observations indicate that CAPE corrects both the metabolic disease of VLCFA as well as secondary inflammatory disease; therefore, it may be a potential drug candidate to be tested for X-ALD therapy in humans.

Figure 1

ABCD2 and ABCD3 mRNA is induced by CAPE in control normal human skin fibroblasts. ABCD1 (A), ABCD2 (B) and ABCD3 (C) expression levels in control normal human (n =3) treated with CAPE in a dose-dependent manner. ABCD2 and ABCD3 mRNA levels were determined by quantitative realtime RT–PCR and normalized to GAPDH. Data are represented as mean±SD. *P<0.05; **P<0.01; ***P<0.001; NS, non-significant.

Figure 2

CAPE stimulates the β-oxidation of lignoceric and palmitic acid in cultured control normal human skin fibroblasts in a dose-dependent manner. Cells were incubated in serum-containing DMEM with different concentrations of CAPE for 3 days. After every 24 h, medium was replaced with the addition of fresh reagents. Palmitic (A) and lignoceric acid (B) β-oxidation activity was measured as mentioned in Materials and Methods. Values are mean±S.D. of three different experiments. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

ABCD2 mRNA expression is induced by CAPE in human X-ALD skin fibroblasts. ABCD2 expression levels in human skin fibroblasts derived from X-ALD (GM04932, GM04934), and AMN (GM07530) treated with CAPE in a dose dependent manner. ABCD2 mRNA levels were determined by quantitative realtime RT–PCR and normalized to GAPDH. Data are represented as mean±SD of three different experiments. *P<0.05, **P<0.01; ***P<0.001 CAPE treated compared to respective untreated X-ALD fibroblasts; NS, non-significant

Figure 4

ABCD3 mRNA expression is induced by CAPE in human X-ALD skin fibroblasts. ABCD3 expression levels in human skin fibroblasts derived from X-ALD (GM04932, GM04934) and AMN (GM07530) treated with CAPE in a dose dependent manner. ABCD3 mRNA levels were determined by quantitative realtime RT–PCR and normalized to GAPDH. Data are represented as mean±SD of three different experiments. *P<0.05, **P<0.01, ***P<0.001 CAPE treated compared to respective untreated X-ALD fibroblasts; NS, non-significant.

Figure 5

CAPE upregulates the levels of ABCD2 and ABCD3 proteins in X-ALD human skin fibroblasts. Human fibroblasts derived from an X-ALD patient (GM04932) were cultured and used to analyze the levels of peroxisomal integral membrane protein transporters ABCD2 and ABCD3 by Western blot in membranes fractions obtained by carbonate treatment (membrane preparation containing integral membrane proteins), as indicated in Methods section. Na⁺/K⁺-ATPase (plasma membrane protein) was used as indicator of protein loading for plasma membrane fractions.

Figure 6

CAPE stimulates the β-oxidation activities and lowers the levels of saturated and monounsaturated VLCFA in human X-ALD skin fibroblasts. Human skin fibroblasts from ALD (GM04932, GMo04934) and AMN (GM07530) patients were incubated in serum-containing DMEM with different concentrations of CAPE and lignoceric (A) and palmitic (B) acid β-oxidation activities were measured after 3 days as described in Materials and Methods. After every 24 h, medium was replaced with the addition of fresh reagents. FAME was prepared directly from cells as described in Material and Methods. FAs were analyzed by GC after adding C_27:0 as an internal standard. C_26.0, C_26:1, and C_22:0 were measured as a percent of total FAs and expressed as ratio of C_26:0/C_22:0 (C) and C_26:1/C_22:0 (D). Results represent the means ± SD of duplicates from three different experiments.*P<0.05; **P<0.01; ***P<0.001; NS, non- significant.

Figure 7

CAPE treatment lowers the mRNA expression of ELOVL1 in X-ALD human skin fibroblasts. Control and X-ALD human skin fibroblasts were treated dose-dependently with CAPE for 3 days and mRNA expression of ELOVL1 was quantified by real time RT-PCR normalized to GAPDH. Data are represented as mean±SD. *P<0.05, **P<0.01 CAPE treatment compared with untreated human X-ALD fibroblasts; NS, non-significant.

Figure 8

CAPE treatment upregulates Abcd2 expression in mouse primary mixed glial cells and Abcd1-deficient U87 and B12 oligodendrocytes. Mouse mixed glial cultures were treated with CAPE everyday for three days and mRNA expression of Abcd2 (A) and MBP (B) was quatified with real time RT-PCR. Pool of three GFP-tagged lentiviral-shRNAs for Abcd1 was used for transduction of human U87 astrocytoma (C) and rat B12 oligodendrocytes (D). Cells were tranduced with either non-targeting (NT) or Abcd1-Lentiviral (Abcd1-Lenti) particles. Lentiviral-shRNAs induced gene silencing, indicated by a significant decrease in Abcd1 expression, analyzed by qRT-PCR (C and D). There was no change in Abcd1 mRNA expression (C and D) in Abcd1-deficient B12 oligodendrocytes treated with CAPE. Abcd2 mRNA expression in control, NT and Abcd1-deficient U87 astrocytes (E) and B12 oligodendrocytes (F) (n =3) treated with CAPE dose-dependently. Data are represented as mean±SD. **P<0.01; ***P<0.001; NS, Non-significant.

Figure 9

CAPE stimulates the β-oxidation activities of lignoceric and palmitic acid, and inhibits ELOVL1 expression in Abcd1-deficient U87 astrocytes and B12 oligodendrocytes. Palmitic and lignoceric β-oxidation activities were measured in Abcd1-deficient U87 astrocytes (A) and B12 oligodendrocytes (B) incubated in serum-containing DMEM with different concentrations (μM) of CAPE for 3 days as described in Materials and Methods. After every 24 h, medium was replaced with the addition of fresh reagents. Abcd1-deficient U87 astrocytes (C) B12 oligodendrocytes (D) were treated dose-dependently with CAPE for 3 days and mRNA expression of ELOVL1 was quantified in control, NT, and untreated/treated Abcd1-deficient cells by qRT-PCR normalized to GAPDH. FAME was prepared directly from cells as described in Material and Methods. FAs were analyzed in U87 astrocytes (E) and B12 oligodendrocytes (F) by GC after adding C_27:0 as an internal standard. C_26.0, C_26:1, and C_22:0 were measured as a percent of total FAs and expressed as ratio of C_26:0/C_22:0. *P<0.05; **P<0.01; ***P<0.001; NS, non-significant.

Figure 10

CAPE inhibits reactive oxygen species (ROS) and expression of proinflammatory cytokines (iNOS and TNF-α) in Abcd-1/2-silenced mouse primary astrocytes. ROS generation in primary astrocytes after silencing or CAPE treatment (A). Nitrite (B) and TNF-α (C) were measured by ELISA in the supernatant of primary astrocytes after silencing or CAPE treatment. For the detection of iNOS, 5LOX and p65 protein expression by immunoblot (D) in response to Abcd1/2 silencing or CAPE treatment, cell lysate from astrocytes was prepared as described in Material methods. ^*P<0.05; ^**P<0.01; ^***P<0.001; NS, non-significant.