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Reick, Martin
Mechanisms of AH receptor down-regulation: Involvement of a labile protein, a calcium dependent protease, and a protein kinase

Order No. 9633803

In this dissertation we present evidence that CYP1A1 transcription and in vivo DNA-protein interactions at XREs are down-regulated in parallel with the DNA-binding activity of the ligand activated AH receptor complex (AHRC). This indicates that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription, and that the AHRC is required continuously to maintain transcription. We show also that the down-regulation process depends on protein synthesis, and that it involves degradation of the AHR subunit but not of ARNT. AHRC down-regulation is a Ca$\sp{2+}$ dependent process, since both depletion of intracellular Ca$\sp{2+}$ stores, and interference with Ca$\sp{2+}$ currents can inhibit down-regulation in Hepa-1 cells. We also demonstrate that a specific inhibitor of the Ca$\sp{2+}$ dependent protease calpain, as well as the protein kinase inhibitors H-7, calphostin C, and bisindolylmaleimide can block AHRC down-regulation. These findings are functionally relevant, since treatments that block down-regulation increase in AHRC dependent CAT gene expression substantially. Ca$\sp{2+}$ measurements reveal a very rapid and transient change in intracellular free (Ca$\sp{2+}$) in response to 2,3,7,8- tetrachlorodibenzo- (p) -dioxin (TCDD).

Thus, TCDD orchestrates a second response, that starts with a rapid rise in (Ca$\rm\sp{2+}\rbrack\sb{i}$ and results in the activation of a Ca$\sp{2+}$ dependent protease, which in turn is instrumental in AHRC down-regulation. Down-regulation involves components in addition to calpain since: (1) In vitro calpain digestion of the AHRC results in partial digestion products not observed in vivo, which implicates secondary proteases. (2) Calpains are too stable to mediate cycloheximide action. (3) Down-regulation can be blocked by cycloheximide after TCDD-induced $\rm\lbrack Ca\sp{2+}\rbrack\sb{i}$ transients have passed.

Finally, we present data suggesting that ionomycin might induce AHRC/ARNT complex formation in a ligand independent manner. Also, experiments with caffeine show that AHRC dependent CAT gene expression can be elevated without changing the levels of liganded AHRC, the kinetics of AHRC activation, or down- regulation. This increase in AHRC mediated transactivation is probably due to elevated $\rm\lbrack Ca\sp{2+}\rbrack\sb{i}.$ Source: DAI, 57, no. 06B (1996): p. 3580

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