Cell selective cAMP induction of rat CYP1B1 in adrenal and testis cells. Identification of a novel cAMP-responsive far upstream enhancer and a second Ah receptor-dependent mechanism

CYP1B1 is unique among P450 cytochromes in exhibiting inductive responses mediated by both the Ah receptor (AhR) and cAMP. cAMP induction was mediated either by a 189bp far upstream enhancer region (FUER, -5110 to -5298) or by a 230bp AhR-responsive enhancer region (AhER) (-797 to -1026). CYP1B1 luciferase reporters respond selectively to cAMP and TCDD in adrenal Y-1 cells (only cAMP), testis MA10 cells (cAMP>TCDD), and C3H10T1/2 mouse embryo fibroblasts (only TCDD). In Y-1 cells, which lack AhR, cAMP induction is totally dependent on the FUER, including absolute requirements for upstream and downstream halves of this region, and for CREB activity at a CRE sequence located at the 3(')-end. cAMP stimulation of the FUER was remarkably high (27-fold) and equally effective when linked to an HSV-TK promoter, indicating direct cAMP activation of the FUER. Binding of CREB to the essential CRE was demonstrated along with dominant negative effects of functionally impaired mutants. cAMP induction in MA10 cells was partially mediated by the FUER mechanism but was regulated additionally by AhER through AhR activity. MA10 cells also exhibit cAMP-dependent AhR down-regulation and AhR/Arnt complex formation. Mutations in AhER including XRE5 were similarly inhibitory to cAMP stimulation in MA10 cells and to TCDD stimulation in C3H10T1/2 cells. Transfection of AhR into the AhR-deficient Y-1 cells did not introduce this second mechanism, which indicated a need for additional components that are present in MA10 cells.     

Lack of an absolute requirement for the native aryl hydrocarbon receptor (AhR) and AhR nuclear translocator transactivation domains in protein kinase C-mediated modulation of the AhR pathway.

Protein kinase C (PKC)-mediated modulation of the aryl hydrocarbon receptor (AhR) pathway was examined in CHOK1-derived L10.I cells stably transfected with the pGUDLUC6.1 reporter; pGUDLUC6.1 is solely controlled by four dioxin-responsive enhancer elements. Co treatment of L10.I cells with 10 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and 81 nM phorbol 12-myristate 13-acetate (PMA), an activator of sn-1,2-diacylglyerol binding PKCs, enhanced transactivation of the reporter construct several-fold relative to cells treated with a saturating 10 nM TCDD dose alone; this effect was dubbed the "PMA effect." A domain swapping and deletional analysis of the native AhR and AhR nuclear translocator (ARNT) protein transactivation domains (TADs) was performed to determine if these domains are absolutely required for the AhR x ARNT dimer-mediated PMA effect in the L10.I model system; controls demonstrate the suitability of the L10.I model for these analyses and that endogenous AhR and ARNT levels are extremely low in this model. Transient coexpression of the AhR and ARNT-474-FLAG, an ARNT protein lacking the native ARNT TAD, in L10.I cells reveals the native ARNT TAD is not absolutely required for the AhR x ARNT-474-FLAG dimer to mediate the PMA effect. Transient coexpression of AhRDeltaCVP, a chimeric AhR protein in which the native AhR TAD has been replaced with the VP16 (herpes simplex virus protein 16) TAD (which control experiments demonstrate is unaffected by PMA), and ARNT in L10.I cells indicates that the native AhR TAD is not absolutely required for this AhRDeltaCVP x ARNT dimer to mediate the PMA effect. These observations strongly suggest that PKC-mediated modulation of the AhR pathway is not absolutely dependent on coactivators recruited to the AhR. ARNT dimer by the native TADs of the AhR and its heterodimerization partner ARNT.     

Aryl hydrocarbon receptor (AhR)-mediated reporter gene expression systems in transgenic tobacco plants

In mammals, the aryl hydrocarbon receptor (AhR) mediates expression of certain genes, including CYP1A1, in response to exposure to dioxins and related compounds. We have constructed a mouse AhR-mediated gene expression systems for a β-glucuronidase (GUS) reporter gene consisting of an AhR, an AhR nuclear translocator (Arnt), and a xenobiotic response element (XRE)-driven promoter in transgenic tobacco plants. On treatment with the AhR ligands 3-methylcholanthrene (MC), β-naphthoflavone (βNF), and indigo, the transgenic tobacco plants exhibited enhanced GUS activity, presumably by inducible expression of the reporter gene. The recombinant AhR (AhRV), with the activation domain replaced by that of the Herpes simplex virus protein VP16, induced GUS activity much more than the wild-type AhR in the transgenic tobacco plants. Plants carrying AhRV expressed the GUS reporter gene in a dose- and time-dependent manner when treated with MC; GUS activity was detected at 5 nM MC on solid medium and at 12 h after soaking in 25 μM MC. Histochemical GUS staining showed that this system was active mainly in leaf and stem. These results suggest that the AhR-mediated reporter gene expression system has potential for the bioassay of dioxins in the environment and as a novel gene expression system in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.