Tracking the Induction of Five Cytochrome P450 (CYP450) Enzymes using a Platform of Parallel Reporter Assays

Transcription

1 APPLICATION NOTE Tracking the Induction of Five Cytochrome P (CYP) Enzymes using a Platform of Parallel Reporter Assays Bill Ho, Ph.D., Jeff Johnson, Ph.D., and Maria I. Morano, Ph.D. Cayman Chemical Company Key Features Induction of five CYP enzymes was monitored using Cayman s reverse transfection reporter assay (RTA) technology. RTA was applied to express different nuclear receptors individually, or in combination, for a rapid investigation into the molecular mechanisms of CYP induction. An additive and potentially synergistic effect on CYPA induction was observed after co-activation of PXR and CAR. Cayman Chemical () -997 E. Ellsworth Road Ann Arbor, MI

2 Introduction Xenobiotics may interact with specific nuclear receptors to modulate the expression levels of the CYP drug metabolizing enzymes. The interplay of multiple CYPs may lead to adverse effects on the enzyme s ability to properly metabolize or clear drugs/compounds., The constitutive androstane receptor (CAR) and pregnane x receptor (PXR) are two key nuclear receptors that can induce several CYPs, including CYPB, CYPA, and CYPC9., CAR is the main CAR isoform, of more than identified variants, found in hepatocytes. CAR shows strong constitutive activity when heterologously expressed in cell lines while two less abundant splice variants, CAR and CAR, display no constitutive activity and can trans-activate CYPB and CYPA upon ligand binding. To investigate the molecular mechanism of CAR-mediated CYP induction in hepatocytes, we assembled secreted alkaline phosphatase (SEAP) reporter gene constructs using the native promoter sequences for CYPs. These reporters were co-transfected by reverse transfection into HepG cells in various combinations of CARs, PXR, and HNFα. Reverse transfection complexes with optimized stoichiometries of DNAs were coated on microplates. This approach allowed the direct comparison of reporter activity with different combinations of nuclear receptors without the cell lineage differences associated with stable cell lines. The reporter constructs were functionally verified by expression of PXR or CAR in HepG cells that were stimulated with their corresponding ligands. As expected, in the presence of CAR, both CYPB and CYPA reporters exhibited strong constitutive activity that masked the induction effect of the CAR ligand CITCO (-(-chlorophenyl)imidazo[,-b][,]thiazole--carbaldehyde-o-(,-dichlorobenzyl)oxime). The co-expression of PXR and CAR drastically suppressed the basal constitutive activities of CAR on both reporters, resulting in a greater than -fold increase in CITCO induced activation of CAR on the CYPB reporter. Although CITCO binding to CAR or CAR triggered approximately a -fold induction of the CYPA reporter, the co-activation of CAR and PXR led to a more robust (>-fold) induction of CYPA than by activation of either nuclear receptor alone, suggesting a synergistic effect. This study demonstrated the interaction of nuclear receptors in the regulation of gene expression by using the original genomic promoter sequences with multiple native regulatory elements in reporter gene assays. Methods Reverse Transfection, CYP Induction, and SEAP Reporter Assay CYP SEAP Reporter Constructs Genomic DNA fragments containing all known critical regulatory elements for CYPA, CYPB, CYPC9, CYPC9, and CYPA gene regulation were amplified by PCR and assembled into SEAP reporter constructs (Figure ). Each reporter construct contained - kb of genomic fragments that have been mapped to be necessary for induction. The use of the SEAP reporter gene allows sample analysis at multiple time points.

3 CYPA X X X X X X X 9 PRB X PRC CYPA CYPA Promoter Ex Ex SEAP CYPB XREM PBREM 9 7 CA E-BOX DR CYPB 79 CYPB Proximal Promoter SEAP CYPC9 97 X 9 X H GR H 9 CE H 99 7 H H H CYPC9 CYPC9 Promoter SEAP CYPC9 X? 9 X GR H H 9 H 7 7 H H E CYPC9 CYPC9 Promoter SEAP CYPA CYPA Promoter SEAP HNF PXR ERE HNF PXR(ER) CYPA Figure. CYP SEAP reporter constructs. Xenobiotic response elements are labeled as XREM, X, X, X, and PXR. HNF/H and HNF/H are denoted for corresponding hepatic nuclear factor binding sites. Glucocorticoid receptor binding sites are marked with GR in CYPC9 and CYPC9. Phenobarbital response element is highlighted as PBREM in CYPB.

4 Reverse transfection plates were coated with transfection complexes containing a SEAP reporter gene supplemented with the necessary transcription factor(s) and nuclear receptor(s). HepG cells were added to the plates the day before applying testing compounds. For a more detailed explanation of the reserve transfection assay methodology please see the technical brief titled: User Friendly, No-Maintenance Reporter Assays - The Technology and Utility of Reverse Transfection. Results CYP Induction Assays Induction of reporters for five CYP genes, CYPA, CYPB, CYPC9, CYPC9, and CYPA, were examined using reverse transfection on HepG cells by activating individual nuclear receptors with prototypical small molecule inducers (Figure ). Induction of the CYPA reporter was monitored up to 9 hours after stimulation of the endogenous aryl hydrocarbon receptor (AHR) by -methylcholanthrene (-MC). A -fold increase in reporter activity was already observed at hours with an EC of nm and a Z value of >.7 while the up-regulation persisted throughout the 9 hours of stimulation. The reporter for CYPB was tested in a CAR-mediated induction assay. Although a plateau response by CITCO was not achieved due to limited solubility, a strong induction was observed after hours incubation and it reached over -fold at 7 hours. With µm CITCO, the Z value at 7 hours was >.. A highly robust induction of CYPC9 reporter was observed after the activation of glucocorticoid receptor (GR) with dexamethasone for hours with an EC of. nm, and a Z value of >.7. The fold of stimulation decreased with prolonged incubation. The CYPC9 reporter was also examined using a similar approach where hydrocortisone treatment resulted in a smaller but noticeable increase in reporter activity with an EC of nm and a Z value well above.7 at 7 hours. The CYPA reporter was tested with PXR supplementation and rifampicin stimulation. At µm, a near-saturated response was obtained. Higher doses resulted in severe cytotoxicity and quenching of the luminescent signal (data not shown). The calculated Z values were >. at 7 hours and near.7 at 9 hours at both µm and µm rifampicin. The inducibility of all five CYP reporter constructs was confirmed.

5 CYPA-SEAP Induction (Fold of control) CYPA hr hr 7hr 9hr CAR/CYPB CAR / CYPB-SEAP Induction (Fold of control) hr hr 7hr hgr / CYPC9-SEAP Activity (Fold of control) GR/CYPC9 hr hr 7hr 9hr Log[-MC] (M) Log[CITCO] (M) Log[Dexamethasone] (M) GR/CYPC9 hgr / CYPC9-SEAP Induction (Fold of control) hr 7hr 9hr Log[Hydrocortisone] (M) PXR/CYPA PXR / CYPA-SEAP Induction (Fold of control) hr hr 7hr 9hr Log[] (M) Figure. Induction assays for CYPA, CAR/CYPB, GR/CYPC9, hgr/cypc9, and PXR/CYPA reporter genes. CAR Effect on CYP Reporter Gene Expression CAR has been reported to be the major modulator for CYPB and, to a lesser extent, for CYPA. It has also been implicated to mediate small inductions in CYPC9, CYPC9, and CYPA/. The effects of CAR expression on the activations of these reporters were evaluated after 7 hour incubation with µm CITCO or control medium. The constitutive activity of CAR was observed when it was co-transfected with each of the five CYP reporter constructs resulting in significant elevation of reporter activities (Figure ). The smallest upregulation by CAR was on the CYPA-SEAP where the basal reporter activity was increased by only.-fold. It was also noted that an endogenous mechanism mediated a stimulatory effect of CITCO in the absence of CAR supplementation leading to a.-fold higher reporter activity. Such CITCO effect was detected on top of the constitutive activity by CAR. The strongest effect of CAR was observed with the CYPB-SEAP reporter where the basal reporter activity was augmented by more than -fold. The constitutive activity of CAR on the CYPA-SEAP reporter was not as

6 prominent as on CYPB-SEAP, however, both CYPB-SEAP and CYPA-SEAP reporters exhibited a -fold CAR-dependent CITCO effects. In addition, CAR also exhibited a -fold increase in basal reporter gene activities of CYPC9-SEAP and CYPC9-SEAP but with no further induction by the addition of CITCO. The CITCO effect on CYPC9 reporter in the absence of CAR was not examined here but it was shown to have no effect in a separate experiment (data not shown). CYPA CYPB CYPC9,, 7, CYPA-SEAP Activity (RLU),,,, CITCO CYPB-SEAP Activity (RLU),, 7,,, CITCO CYPC9-SEAP Activity (RLU),,,,,, CITCO No Receptor + CAR No Receptor + CAR No Receptor + CAR 9, CYPC9, CYPA CYPC9-SEAP Activity (RLU), 7,,,,,,, CITCO CYPA-SEAP Activity (RLU),,,,, CITCO No Receptor + CAR No Receptor + CAR Figure. Constitutive activity of CAR when co-transfected with CYPA, CYPB, CYPC9, CYPC9, and CYPA reporter constructs. PXR-Mediated Modulation of CYP Reporter Gene Expression In contrast to the constitutive activity of CAR, the expression of PXR resulted in a % to % suppression of basal activity of all the CYP reporters, presumably due to competition between the promoters for the transcription factors (Figure ). Without supplementation of PXR, µm rifampicin did not show any effect on reporter gene activities driven by CYPB, CYPC9, CYPC9, and CYPA promoters, but a small decrease in CYPA-SEAP. With the expression of PXR, rifampicin produced strong inductions of CYPB-SEAP and CYPA-SEAP reporters and moderate inductions of CYPC9-SEAP and CYPC9-SEAP reporters, whereas such an effect on the CYPA-SEAP reporter was not statistically significant.

7 CYPA CYPB CYPC9 CYPA-SEAP Activity (RLU) 9,, 7,,,,,,, No Receptor + PXR CYPB-SEAP Activity (RLU),,,, 9 7 No Receptor + PXR CYPC9-SEAP Activity (RLU),,,, 9,, 7,,,,,,, No Receptor + PXR CYPC9-SEAP Activity (RLU),,,,,,,, CYPC9 No Receptor + PXR CYPA-SEAP Activity (RLU),7,,,,,,, 9 7 CYPA No Receptor + PXR Figure. Induction of CYPA, CYPB, CYPC9, CYPC9, and CYPA reporter activity when co-transfected with PXR in the presence of µm rifampicin. PXR Uncovers CAR-Mediated CYP Induction The effect of PXR co-expression on CAR constitutive activity was examined in several CYP reporter assays (Figure ). Without PXR supplementation, the basal activities of CYPA-SEAP, CYPB-SEAP, CYPC9-SEAP, and CYPA-SEAP elevated by CAR were slightly suppressed by rifampicin. Compared to the % to % reduction in the basal reporter gene activities by PXR, as shown in Figure, the suppressive effect of PXR on the CAR-elevated reporter activity was much stronger. In contrast, the co-expression of CAR did not noticeably impact PXR-mediated CYPB and CYPA reporter gene inductions by rifampicin. 7

8 CYPA-SEAP Activity (RLU),,,, CYPA CYPB CYPC9 CYPA, CAR CAR + PXR CYPB-SEAP Activity (RLU) 7, 7,,,,,,,,,,,,, CAR CAR + PXR CYPC9-SEAP Activity (RLU) 7,,,,,,, CAR CAR + PXR Figure. PXR co-expression suppresses CAR constitutive activity in CYPA, CYPB, CYPC9, and CYPA reporter assays. CYPA-SEAP Activity (RLU),,,9,,7,,,,,,, 9 7 CAR CAR + PXR To further investigate the PXR effect on the down-regulation of CAR constitutive activity, various levels of PXR cdna were titrated into the transfection complex containing a constant amount of CAR cdna in the same expression vector (Figure ). This experiment was conducted with the CYPB-SEAP reporter, which exhibited the strongest CAR constitutive activity. With a CAR to PXR ratio of as little as :, the constitutive activity was suppressed nearly 7% (Figure A). The suppression of PXR cdna in the transfection complex with a : ratio of CAR to PXR abolished the constitutive activity due to CAR expression. The suppressive effect of PXR was specific to the basal constitutive activity of CAR which amplified the CITCO-stimulated induction mediated by CAR (Figure B). In other words, in the presence of PXR, the CYPB-SEAP reporter assay with CAR becomes a very robust assay to test for potential inducers. A CYPB-SEAP Basal Activity (RLU),,,,, B CYPB-SEAP Induction (Fold of ) 7 : CAR:PXR : CAR:PXR : CAR:PXR : CAR:PXR : CAR:PXR : CAR:PXR : CAR:PXR : CAR:PXR Log[CITCO] (M) Figure. A) PXR suppression of CAR constitutive activity at variable concentrations of PXR cdna. B) CITCO-induced activity of CAR at variable concentrations of PXR cdna.

9 Interactions between Activated PXR and CAR Receptors The interaction between PXR and CAR activation was further examined on both CYPB-SEAP (Figure 7) and CYPA-SEAP (Figure ) reporter gene assays where stronger inductions by the activation of each nuclear receptor were revealed. The stoichiometry of DNA mixtures in reverse transfection complexes was modified to detect the activation of both nuclear receptors. When supplemented with only PXR, µm of rifampicin only produced a -fold induction of CYPB-SEAP. A smaller increase in CYPB-SEAP reporter activity by µm CITCO was also observed in the absence of exogenous CAR. When the cells were incubated with both µm rifampicin and µm CITCO, a nearly -fold induction was exhibited. In the presence of CAR, there was no significant induction of reporter by rifampicin or CITCO due to the high level of constitutive activity (. ±.-fold at hours). In the presence of PXR + CAR, the response to rifampicin was much lower than with PXR alone, while the CITCO effect on CYPB induction became much more prominent (>-fold) than with either nuclear receptor supplement separately. Co-incubation of rifampicin and CITCO did not produce further induction. PXR CYPB CAR PXR + CAR CYPB Reporter Activity (Fold of ) CYPB Reporter Activity (Fold of ) μm rifampicin μm rifampicin μm CITCO. μm CITCO μm rifampicin + μm CITCO μm rifampicin +. μm CITCO μm rifampicin μm rifampicin μm CITCO. μm CITCO μm rifampicin + μm CITCO μm rifampicin +. μm CITCO hr hr 7hr 9hr CYPB Reporter Activity (Fold of ) μm rifampicin μm rifampicin μm CITCO. μm CITCO μm rifampicin + μm CITCO μm rifampicin +. μm CITCO Figure 7. PXR and CAR interactions in a CYPB-SEAP reporter gene assay. Similar to the CYPB-SEAP reporter experiment above, the effect of PXR-CAR interaction on CYPA induction was also examined. With supplementation of PXR alone, µm of rifampicin produced a nearly -fold induction of the reporter activity while CITCO resulted in a very small induction without CAR supplementation. In the presence of CAR, the constitutive activity led to a. ±.-fold increase in basal activity; however, a - to -fold further induction by CITCO was also observed. With the co-expression of PXR and CAR, the basal activity dropped below the no receptor control level (data not shown). 9

10 Nevertheless, the response to either rifampicin or CITCO alone was not affected by the co-expression of the other nuclear receptor (PXR or CAR). In contrast, the co-stimulation of both nuclear receptors with µm CITCO and µm rifampicin produced an. ±.-fold induction at 9 hours revealing a potentially synergistic effect. CYPA CYPA Reporter Activity (Fold of ) 9 7 PXR CYPA Reporter Activity (Fold of ) 9 7 CAR hr hr 7hr 9hr CYPA Reporter Activity (Fold of ) 9 7 PXR + CAR μm rifampicin μm rifampicin μm CITCO. μm CITCO μm rifampicin + μm CITCO μm rifampicin +. μm CITCO μm rifampicin μm rifampicin μm CITCO. μm CITCO μm rifampicin + μm CITCO μm rifampicin +. μm CITCO μm rifampicin μm rifampicin μm CITCO. μm CITCO μm rifampicin + μm CITCO μm rifampicin +. μm CITCO Figure. PXR and CAR interactions in a CYPA-SEAP reporter gene assay. Conclusions In conclusion, Cayman s reverse transfection technology resulted in superior co-transfection efficiency and reproducibility in the CYP induction reporter assay. This platform is flexible and allows rapid assessment of compounds in CYP gene induction by evaluating activation of different transacting factors, expressed individually or in combination, at multiple time points. In HepG cells, CAR expression showed a strong constitutive activity in CYPA, CYPB, CYPC9, CYPC9, and CYPA reporter gene assays. The constitutive activity of CAR can be suppressed by co-expression of PXR. As a result, the co-expression of PXR in the CAR/CYPB-SEAP assay enhanced the ligand response by CAR and allowed the screening of inducers with the major CAR isoform. Moreover, the co-activation of PXR and CAR produced an additive, and potentially synergistic, effect on CYPA induction.

11 References. Sinz, M., Wallace, G., and Sahi, J. AAPS J. (), 9- ().. Polasek, T.M., Lin, F.P.Y., Miners, J.O., et al. Br. J. Clin. Pharmacol. 7(), 77-7 ().. Pelkonen, O., Turpeinen, M., Hakkola, J., et al. Arch. Toxicol. (), 7-7 ().. Tompkins, L.M. and Wallace, A.D. J. Biochem. Mol. Toxicol. (), 7- (7).. Anderson, L.E., Dring, A.M., Hamel, L.D., et al. Toxicol. Lett. (), - ().. Molnár, F., Küblbeck, J., Jyrkkärinne, J., et al. Drug Metabol. Drug Interact. (), 79-9 (). Reverse Transfection Assays Available from Cayman Item No Product Name CYPA/ Induction Reporter Assay Kit CYPB Induction Reporter Assay Kit CYPC9 Induction Reporter Assay Kit EP Receptor (human) Reporter Assay Kit* EP Receptor (human) Reporter Assay Kit* EP Receptor (rat) Activation Assay Kit (camp) FFAR (GPR) Reporter Assay Kit FFAR (GPR) Reporter Assay Kit Melanocortin- Receptor Reporter Assay Kit Melanocortin- Receptor Reporter Assay Kit Orexin Receptor Reporter Assay Kit Orexin Receptor Reporter Assay Kit TGR (GP-BAR) Reporter Assay Kit *Also available for rat homologs (Item Nos. and ) View a complete list of our reverse transfection assays at Cayman Contract Research Services Our team of experts can design and run a customized screening project utilizing reverse transcription assay technology. Don t see your target of interest? Let us build a customized reverse transfection assay catered to your particular experimental plan. Contact us at contractresearch@caymanchem.com or visit

12 Cayman Chemical () -997 E. Ellsworth Road Ann Arbor, MI