Aryl hydrocarbon induction of rat cytochrome P-450d results from increased precursor RNA processing.

Abstract

We have previously demonstrated that cytochrome P45Od mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript.Exposure of most organisms to environmental pollutants results in the induction of sets of genes whose products act to metabolize these compounds. In rat liver exposed to aryl hydrocarbons such as 3-methylcholanthrene (MCA), a-napthaflovone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin, the hydrocarbon-metabolizing enzymes cytochromes P-450c and P-450d are the major induced proteins (12, 13). It has been determined that transcriptional regulation mediated by the cytosolic Ah receptor is primarily responsible for the hydrocarbon-induced accumulation of P-450c and its mouse homolog cytochrome P1-450 in intact liver (14, 26). However, it has been demonstrated that the Ah receptor-dependent 30-fold induction of P-450d mRNA and its mouse homolog P3-450 is mediated primarily at a posttranscriptional level (10,18,19). We and others (15,18,19) have also examined the MCA induction of the P-450c and P-450d genes in primary rat hepatocyte cultures grown in defined medium and have shown that both genes appear to be mainly posttranscriptionally regulated. Thus, the regulation of P-450d in cultured cells is identical with its regulation in liver cells, whereas P-450c loses much of its transcriptional inducibility and becomes regulated at a posttranscriptional step.To define the mechanism responsible for the hydrocarbon induction of P-450d mRNA, we have analyzed (i) the degradation rate, (ii) the relationship between protein synthesis and MCA induction, and (iii) the accumulation of nuclear mRNA processing intermediates in the presence and absence of MCA. These results show that the Ah receptormediated induction of P-450d mRNA is effected at an early step of processing of the nuclear precursor mRNA, perhaps at the process-versus-discard decision point, and not at the level of mature cytoplasmic mRNA turnover. Moreover, the inhibition of protein synthesis has no significant effect on the induction process, suggesting that the Ah receptor, which has been shown to enter the nucleus after hydrocarbon binding (22, 26), may act directly on an early event in the mRNA synthesis of P-450d.Degradati...

Ischaemia and reperfusion injury of rat liver increases expression of glutathione S-transferase A1/A2 in zone 3 of the hepatic lobule

“…Thus the values obtained from these methods are less than those from in situ hybridization, which allows for the exclusion of hepatocytes from zones 1 and 2 that do not respond to the I\R. Also, the finding that increased rates of transcription shown by nuclear run-offs assays are seen at 3 h after I\R and yet message levels peak at later times suggests that other processes, such as changes in RNA processing [44], may have affected transcript levels following I\R. The half-life of rGSTA1\A2 transcripts also may have been increased after I\R due to a reduction in RNA degradation.…”

Characterization of Two Nuclear Proteins that Interact with Cytochrome P‐450 1A2 mRNA

“…While further studies are required to examine what role the 46-kDahnRNP C protein could play in the expression of the Cypla2 gene, we can speculate that the increased binding of the 46-kDa protein to the 3'UTR of CYPlA2 mRNA after 3-methylcholanthrene treatment could result in modulation of the maturation of the pre-mRNA, possibly through altered splicing [16] of the pre-mRNA. We can also postulate that the 46-kDa protein modulates the polyadenylation efficiency or the stability of the CYPlA2 primary transcripts.…”

“…Moreover, although activation of transcription has been shown to be the major regulatory pathway for CYPlAl , the possibility that posttranscriptional mechanisms could be part of the regulation of the expression of CYPlA2 has been raised in several reports [14-161. Accordingly, alteration of CYPlA2 pre-mRNA processing was shown by Silver and Krauter [16].Several studies have shown that the 3' untranslated region (UTR) of mRNAs may play an important role in the overall regulation of gene expression [17], and some motifs have been characterized in the 3'UTR as highly important for the posttranscriptional regulation of the corresponding genes. These include the AU-rich elements present in many short-lived mRNAs [18, 191, polypyrimidine tracts [20], and stem-loops found in histone mRNAs (211 and transferrin-receptor mRNA 1221.…”

“…Moreover, although activation of transcription has been shown to be the major regulatory pathway for CYPlAl [lo-121 and CYPlA2 [13], the possibility that posttranscriptional mechanisms could be part of the regulation of the expression of CYPlA2 has been raised in several reports [14-161. Accordingly, alteration of CYPlA2 pre-mRNA processing was shown by Silver and Krauter [16].…”

Heterogeneous Nuclear Ribonucleoprotein A1 and Regulation of the Xenobiotic-Inducible GeneCyp2a5

“…The transcriptional activation of P450 genes has been widely studied; recently, a number of xenobiotic-activated receptors that act as specific transcription factors have been discovered (Waxman, 1999). Other than transcription, post-transcriptional control is important in the regulation of several P450s (Simmons et al, 1987;Silver and Krauter, 1990;Peng and Coon, 1998); however, the molecular mechanisms involved are poorly known.…”