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Peroxisome proliferator-activated receptor alpha, PPAR α , directly regulates transcription of cytochrome P450 CYP2C8

View Article: PubMed Central

ABSTRACT

The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions –2762/–2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype.

No MeSH data available.


PPARα knockdown and activation in HepaRG cells. (A) mRNA levels were measured in HepaRG cultures cultured in three independent differentiation batches 72 h after PPARα activation using ligand WY14,643 (left part of the diagram) or siRNA-mediated PPARα knock-down (right part of the diagram) and compared with mRNA levels measured in cells treated with either DMSO (left) or non-targeting siRNA (right) set at 1.0. The graph shows the means from three (mRNA, activity) or two (protein) independent experiments, with error bars indicating standard deviations (mRNA, activity) and dots representing individual data points (protein). ∗, Statistically significant (P < 0.05, paired t-test). (B) Representative Western Blot analysis of the corresponding protein levels of CYP2C8 following either ligand-mediated induction (lanes DMSO and WY14,643) or siRNA-mediated knock-down (lanes siPPARa and siCTR) of PPARα.
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Figure 3: PPARα knockdown and activation in HepaRG cells. (A) mRNA levels were measured in HepaRG cultures cultured in three independent differentiation batches 72 h after PPARα activation using ligand WY14,643 (left part of the diagram) or siRNA-mediated PPARα knock-down (right part of the diagram) and compared with mRNA levels measured in cells treated with either DMSO (left) or non-targeting siRNA (right) set at 1.0. The graph shows the means from three (mRNA, activity) or two (protein) independent experiments, with error bars indicating standard deviations (mRNA, activity) and dots representing individual data points (protein). ∗, Statistically significant (P < 0.05, paired t-test). (B) Representative Western Blot analysis of the corresponding protein levels of CYP2C8 following either ligand-mediated induction (lanes DMSO and WY14,643) or siRNA-mediated knock-down (lanes siPPARa and siCTR) of PPARα.

Mentions: To directly investigate the functional impact of PPARα on CYP2C8 we applied two available strategies, namely stimulation with the canonical PPARα ligand, WY14,643, and depletion of PPARα using siRNA-mediate gene knock down. As shown in Figure 3A, treatment of HepaRG cells with 100 μM WY14,643 significantly induced the expression of CYP2C8 at mRNA (more than threefold), protein (Figure 3B, lane WY14,643) and activity (over twofold) levels, confirming earlier observations by Prueksaritanont et al. (2005). In the same experimental set-up, transfection of HepaRG cells with PPARα-targeting siRNA resulted in >50% reduction in the expression of mRNA and >40% decrease in protein levels (Figure 3B, lane siPPARa) of CYP2C8 as compared with cells treated with non-silencing siRNA. The measurement of corresponding CYP2C8 enzyme activity after PPARA gene silencing resulted in an average amodiaquine N-desethylation reduction over 45% as compared with non-targeting control. These findings demonstrated that PPARα mediates both basal and inducible regulation of CYP2C8 in human hepatocytes.


Peroxisome proliferator-activated receptor alpha, PPAR α , directly regulates transcription of cytochrome P450 CYP2C8
PPARα knockdown and activation in HepaRG cells. (A) mRNA levels were measured in HepaRG cultures cultured in three independent differentiation batches 72 h after PPARα activation using ligand WY14,643 (left part of the diagram) or siRNA-mediated PPARα knock-down (right part of the diagram) and compared with mRNA levels measured in cells treated with either DMSO (left) or non-targeting siRNA (right) set at 1.0. The graph shows the means from three (mRNA, activity) or two (protein) independent experiments, with error bars indicating standard deviations (mRNA, activity) and dots representing individual data points (protein). ∗, Statistically significant (P < 0.05, paired t-test). (B) Representative Western Blot analysis of the corresponding protein levels of CYP2C8 following either ligand-mediated induction (lanes DMSO and WY14,643) or siRNA-mediated knock-down (lanes siPPARa and siCTR) of PPARα.
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Figure 3: PPARα knockdown and activation in HepaRG cells. (A) mRNA levels were measured in HepaRG cultures cultured in three independent differentiation batches 72 h after PPARα activation using ligand WY14,643 (left part of the diagram) or siRNA-mediated PPARα knock-down (right part of the diagram) and compared with mRNA levels measured in cells treated with either DMSO (left) or non-targeting siRNA (right) set at 1.0. The graph shows the means from three (mRNA, activity) or two (protein) independent experiments, with error bars indicating standard deviations (mRNA, activity) and dots representing individual data points (protein). ∗, Statistically significant (P < 0.05, paired t-test). (B) Representative Western Blot analysis of the corresponding protein levels of CYP2C8 following either ligand-mediated induction (lanes DMSO and WY14,643) or siRNA-mediated knock-down (lanes siPPARa and siCTR) of PPARα.
Mentions: To directly investigate the functional impact of PPARα on CYP2C8 we applied two available strategies, namely stimulation with the canonical PPARα ligand, WY14,643, and depletion of PPARα using siRNA-mediate gene knock down. As shown in Figure 3A, treatment of HepaRG cells with 100 μM WY14,643 significantly induced the expression of CYP2C8 at mRNA (more than threefold), protein (Figure 3B, lane WY14,643) and activity (over twofold) levels, confirming earlier observations by Prueksaritanont et al. (2005). In the same experimental set-up, transfection of HepaRG cells with PPARα-targeting siRNA resulted in >50% reduction in the expression of mRNA and >40% decrease in protein levels (Figure 3B, lane siPPARa) of CYP2C8 as compared with cells treated with non-silencing siRNA. The measurement of corresponding CYP2C8 enzyme activity after PPARA gene silencing resulted in an average amodiaquine N-desethylation reduction over 45% as compared with non-targeting control. These findings demonstrated that PPARα mediates both basal and inducible regulation of CYP2C8 in human hepatocytes.

View Article: PubMed Central

ABSTRACT

The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPAR&alpha;), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPAR&alpha;. Two linked intronic SNPs in PPAR&alpha; (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPAR&alpha; in HepaRG human hepatocyte cells resulted in up to &sim;60 and &sim;50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPAR&alpha; agonist WY14,643 lead to an induction by &gt;150 and &gt;100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPAR&alpha;. Electromobility shift assay demonstrated direct binding of PPAR&alpha; to a DR-1 motif located at positions &ndash;2762/&ndash;2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/&beta;-catenin acts as a functional inhibitor of PPAR&alpha;-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPAR&alpha; in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/&beta;-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype.

No MeSH data available.