In vivo and mechanistic evidence of nuclear receptor CAR induction by artemisinin.

Artemisinin (a sesquiterpene lactone endoperoxide) has become important in multi-drug treatment of malaria. There is evidence that artemisinin induces drug metabolism which could result in drug-drug interactions. The objective of this study was to characterize the inductive properties of artemisinin on drug-metabolizing cytochrome P450 (CYP450) enzymes. The possibility of artemisinin to induce CYP450 was studied in artemisinin-treated (orally for four days) and vehicle-treated rats using reverse transcriptase polymerase chain reaction (RT-PCR). The effect on enzymatic activities in mouse microsomes from multiple artemisinin administration (intraperitonally) to mice were also studied as well as the effect on the expression in mouse primary hepatocytes and HEK293 cells. Increased CYP2B1 mRNA levels in rats could be seen after artemisinin treatment as well as a weak but reproducible increase in the intensity of CYP1A2. Administration of artemisinin to mice up-regulated hepatic CYP2B10-dependent, and to a lesser extent, CYP2A5-dependent enzyme activities. In primary hepatocyte culture, artemisinin significantly increased the CYP2B10 mRNA levels whereas the CYP2A5 mRNA levels were increased to a lesser extent. No significant changes were seen in the levels of other CYP enzymes. Artemisinin was an activator of constitutive androstane receptor (CAR) but not pregnane X receptor (PXR) in HEK293 cells. The results demonstrate that the drug exerts its effects on drug metabolism via the CAR receptor that results in up-regulation of genes such as the Cyp2b. The weaker up-regulation of CYP2A5 might also be CAR-dependent or alternatively, a consequence of artemisinin toxicity. The results of this study are of importance when predicting potential drug-drug interactions in multi-drug therapies with artemisinin.

Analysis of RNA-protein interactions of mouse liver cytochrome P4502A5 mRNA.

In our previous studies we have identified a 37/39 kDa, pyrazole-inducible, cytochrome P4502A5 (CYP2A5) mRNA binding protein and provided evidence that it may play a role in the stabilization and processing of the RNA [Geneste, Rafalli and Lang (1996) Biochem. J. 313, 1029-1037; Thulke-Gross, Hergenhahn, Tilloy-Ellul, Lang and Bartsch (1998) Biochem. J. 331, 473-481]. Details of the RNA-protein interactions are, however, not known. In this report we have performed an analysis of the interaction between the CYP2A5 mRNA and the 37/39 kDa protein. With UV-cross linking experiments, using RNA probes corresponding to various parts of the CYP2A5 mRNA, and with antisense oligonucleotides complementary to certain areas of the 3'-untranslated region (3'UTR), we could map the primary binding site to the tip of a 71 nt hair-pin loop at the 3'-UTR. This analysis also showed that the protein may have more than one site of interaction with the RNA and/or that, within the binding region, there could be more than one protein molecule binding to the RNA. Analysis of the probable conformations of the various probes used in the UV cross-linking experiments, in combination with the estimated binding affinities of the protein to the different probes, suggests that important factors in the high-affinity binding are the UAG triplet flanked by GA-rich sequences at the tip of the hair-pin loop, in addition to the conformation of the loop itself. Within the binding region, similarities with known binding sites of heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in other RNA molecules were revealed by sequence alignment analysis. Moreover, competition experiments with an oligoribonucleotide corresponding to a known high-affinity binding site of hnRNP A1, and immunoprecipitation of the UV cross-linked 37/39 kDa complex showed that the protein binding to the CYP2A5 mRNA could be hnRNP A1 or its close analogue. It was also shown that the 37/39 kDa protein binds with less affinity to CYP2A4 mRNA than to CYP2A5 mRNA. This is in accordance with experiments characterizing the binding site, since these two otherwise highly homologous genes are kown to have a three nucleotide difference within the region important for the high binding affinity. Since the response of CYP2A4 to pyrazole is known to be weak, as compared with CYP2A5, this observation provides further evidence for a regulatory role of the 37/39 kDa protein in CYP2A5 mRNA metabolism.

Characterization of two nuclear proteins that interact with cytochrome P-450 1A2 mRNA. Regulation of RNA binding and possible role in the expression of the Cyp1a2 gene.

Regulation of the expression of the cytochrome P-450 la2 gene (cyp1a2) occurs mainly at the transcriptional level, but the molecular events involved in the induction process are partly unknown. Some reports have proposed involvement of post-transcriptional mechanisms [Adesnik, M. & Atchison, M. (1986) Crit. Rev. Biochem. 19, 247-305; Silver, G. & Krauter, K. S. (1990) Mol. Cell. Biol. 10, 6765-6768]. Here we report the identification of two proteins in the nuclear fraction of mouse liver, with specific binding characteristics towards CYP1A2 mRNA. The proteins have apparent molecular masses of 37 kDa and 46 kDa and exhibit a high affinity for a poly(U) motif in the 3' untranslated region of CYP1A2 mRNA. This motif seems to be important for their specific and apparently competitive binding to CYP1A2 mRNA. Treatment of mice with an inducer of CYP1A2, 3-methylcholanthrene, increases the binding of the 46-kDa protein and decreases the binding of the 37-kDa protein to the mRNA, suggesting that changes in the binding of the proteins to the mRNA could play a role in the upregulation of CYP1A2 mRNA by 3-methylcholanthrene. Phosphorylation of the 46-kDa protein, or of an intermediary factor, may play a role in its binding activity. Furthermore, the 46-kDa but not the 37-kDa protein is recognized by a monoclonal antibody against the heterogeneous nuclear ribonucleoprotein C, a nuclear protein probably involved in pre-mRNA processing. While more work is needed to understand the function of the proteins that bind to the 3' untranslated region of CYP1A2, it is possible that the 37-kDa protein has a role in the maintenance of uninduced levels of CYP1A2 mRNA, while the 46-kDa protein could be important in the maturation of elevated levels of CYP1A2 pre-mRNA, during induction.