TCDD-inducible poly(ADP-ribose) polymerase: a novel response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes pleotropic effects in mammalian species through modulating gene expression. Here we analyzed TCDD-induced mRNA expression by using mRNA differential display and report the cloning of a novel TCDD-inducible poly(ADP-ribose) polymerase (TiPARP). TiPARP cDNA contains an open reading frame of 657 amino acid residues; the carboxyl half shares sequence similarity to the catalytic domain of PARP, a family of enzymes that catalyze poly ADP-ribosylation of proteins. Expression of the cDNA by in vitro transcription/translation reveals a protein of approximately 75 kDa. The expressed TiPARP exhibits PARP activity toward histone. TiPARP is highly homologous to RM1 which is induced during long-term potentiation, a memory formation process, and to TIL which is induced in T cells infiltrating progressing tumors. TiPARP mRNA is expressed in a broad range of mouse tissues. Together, these data demonstrate that TiPARP is a novel target of TCDD that may contribute to multiple responses to TCDD by modulating protein function through poly ADP-ribosylation.

Superinduction of CYP1A1 gene expression. Regulation of 2,3,7, 8-tetrachlorodibenzo-p-dioxin-induced degradation of Ah receptor by cycloheximide.

Cycloheximide superinduces the transcription of CYP1A1 in the presence of an agonist for the Ah receptor (AhR). To investigate the molecular target for "superinduction," we analyzed the agonist-induced degradation of AhR. Whereas 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a potent agonist of AhR, induces a rapid reduction of the AhR protein, cycloheximide blocks the down-regulation of steady state AhR. Analyses of the turnover of AhR reveal that cycloheximide blocks the shortening of the half-life of AhR by TCDD. Blocking of the TCDD-induced AhR degradation requires inhibition of protein synthesis, because (a) cycloheximide inhibits protein synthesis at the concentration at which it causes superinduction and inhibition of AhR degradation; and (b) puromycin, an inhibitor of protein synthesis by mimicking aminoacyl-tRNA, also blocks the TCDD-induced AhR degradation. The blocking of the TCDD-induced AhR degradation correlates with the superinduction of CYP1A1 gene expression in a time- and dose-dependent manner. Furthermore, cycloheximide is shown to increase the accumulation of the TCDD-activated AhR and the functional AhR x Arnt complex in nucleus. Collectively, our results reveal a mechanism of superinduction by cycloheximide by enhancing the stability of agonist-activated AhR. The finding that inhibition of protein synthesis blocks the TCDD-induced AhR turnover implicates a cycloheximide-sensitive, labile factor (designated as AhR degradation promoting factor, or ADPF) in controlling the removal of agonist-activated AhR in nucleus.

2,3,7,8-tetrachlorodibenzo-p-dioxin-induced degradation of aryl hydrocarbon receptor (AhR) by the ubiquitin-proteasome pathway. Role of the transcription activaton and DNA binding of AhR.

Activation of the aryl hydrocarbon receptor (AhR) by 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a potent agonist of AhR, induces a marked reduction in steady state AhR. To analyze the mechanism of regulation of ligand-activated AhR, we examined the biochemical pathway and function of the down-regulation of the receptor by TCDD. Pulse-chase experiments reveal that TCDD shortens the half-life (t1/2) of AhR from 28 to 3 h in mouse hepatoma cells. Inhibitors of the 26 S proteasome, lactacystin and MG132, block the TCDD-induced turnover of AhR. The TCDD-induced degradation of AhR involves ubiquitination of the AhR protein, because (a) TCDD induces formation of high molecular weight, ubiquitinated AhR and (b) degradation of AhR is inhibited in ts20 cells, which bear a temperature-sensitive mutation in the ubiquitin-activating enzyme E1, at a nonpermissive temperature. Inhibition of proteasomal degradation of AhR increases the amount of the nuclear AhR.Arnt complex and "superinduces" the expression of endogenous CYP1A1 gene by TCDD, indicating that the proteasomal degradation of AhR serves as a mechanism for controlling the activity of the activated receptor. We also show that deletion of the transcription activation domain of AhR abolishes the degradation, whereas a mutation in the DNA-binding region of AhR or Arnt reduces the degradation; these data implicate the transcription activation domain and DNA binding in AhR degradation. Our findings provide new insights into the regulation of TCDD-activated AhR through ubiquitin-mediated protein degradation.