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C‐Jun‐N‐terminal kinase (JNK): Novel regulator of PXR‐mediated CYP3A4 induction
Author(s) -
Taneja Guncha,
Chu Chun,
Moorthy Bhagavatula,
Ghose Romi
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.713.6
Subject(s) - pregnane x receptor , cyp3a4 , kinase , activator (genetics) , mapk/erk pathway , pharmacology , p38 mitogen activated protein kinases , chemistry , transfection , gene knockdown , reporter gene , microbiology and biotechnology , biology , nuclear receptor , cytochrome p450 , receptor , gene expression , transcription factor , biochemistry , enzyme , gene
Cytochrome P450 3A4 (CYP3A4) is the most abundant human drug metabolizing enzyme (DME) that is responsible for metabolism of up to 50% of clinically available drugs. Alteration in CYP3A4 enzyme levels impacts the disposition of its substrates and could ultimately lead to harmful clinical consequences such as failure of therapy or adverse drug reactions. Clinical studies report that CYP3A4 was up‐regulated by xenobiotics such as rifampicin (RIF) or drugs of abuse (e.g. marijuana) and this led to failure of oral contraceptive and anti‐retroviral therapy respectively. In order to prevent such undesirable consequences because of drug‐drug interactions, we need to better understand the molecular mechanisms of regulation of CYP3A4. So far, CYP3A4 gene expression is known to be upregulated by activation of nuclear receptors, mainly pregnane‐X‐receptor (PXR). However, post‐translational modifications such as phosphorylation modulate the activity of PXR. Recent studies have shown that mitogen activated protein kinases (MAPKs) are involved in regulation of DMEs. MAPKs comprise of JNK, ERK and p38 . In this study, HepG2 cells (human hepatocellular carcinoma cells) were transfected with CYP3A4‐Luciferase and hPXR plasmid followed by sequential treatment with MAPK inhibitors and rifampicin (specific PXR activator). Interestingly, we found that treatment with JNK inhibitor (30 μM SP600125) for 30 minutes significantly attenuated rifampicin‐mediated CYP3A4 reporter activity as well as gene expression and enzyme activity in HepG2 cells. Genetic knockdown using custom siRNA for JNK 1/2 corroborated that JNK is required for optimum CYP3A4 reporter activity. Pretreatment with ERK inhibitor also attenuated CYP3A4 luciferase signal but no change was observed with p38 kinase inhibitor. Immunoblot analysis revealed that rifampicin activated both JNK 1 and 2 starting at 60 minutes up to 8 hours and SP600125 treatment inhibited JNK activation at the same time points. Moreover, we found that PXR protein expression decreased in HepG2 nuclear extracts treated with SP600125 , which indicates that JNK may be required for nuclear translocation of PXR. Taken together, our study shows, that JNK is activated by rifampicin, which activates PXR and ultimately up‐regulates CYP3A4. In conclusion, this research is the first study to show that JNK plays an important role in regulation of CYP3A4 via PXR . This novel mechanism of induction of CYP3A4 will further enhance our understanding about drug‐drug interactions and altered drug disposition due to changes in CYP3A4 levels. Support or Funding Information This work was supported by NIH Grant: 1R21DA035751‐01A1 to RG and BM.PXR‐mediated CYP3A4 reporter gene expression is suppressed by specific JNK inhibitor‐ SP600125 and JNK pathway inhibitor –Curcumin. * indicates p<0.05.PXR‐mediated CYP3A4 gene expression is suppressed by specific JNK inhibitor‐ SP600125 at 2h, 4h, 6h, 8h and 12h. * indicates p<0.05 as compared to DMSO/RIF 0h group.PXR mediated CYP3A4 activity is suppressed by JNK inhibitor as assessed using P450 Glo assay kit having specific CYP3A4 substrate‐Luciferin IPA. * indicates p<0.05.PXR mediated CYP3A4 reporter activity is suppressed when JNK is knocked down. * indicates p<0.05.