Multiple enhancer units mediate drug induction of CYP2H1 by xenobiotic-sensing orphan nuclear receptor chicken xenobiotic receptor.

Binding of nuclear receptors to drug-responsive enhancer units mediates transcriptional activation of cytochromes P-450 (P-450) by drugs and xenobiotics. In previous studies, a 264-base-pair (bp) phenobarbital-responsive enhancer unit (PBRU) located at -1671 to -1408 upstream of the chicken CYP2H1 transcriptional start-site increased gene expression when activated by the chicken xenobiotic-sensing orphan nuclear receptor CXR. In extension of these studies, we now have functionally analyzed a second distal drug-responsive element and delimited a 643- and a 240-bp PBRU located between 5 and 6 kilobases upstream of the transcriptional start site of CYP2H1. Both PBRUs were activated by CXR after treatment with different drugs. A nuclear receptor binding site, a direct repeat-4 (DR-4) hexamer repeat, was identified on the 240-bp PBRU. Site-directed mutagenesis of this DR-4 abolished activity in reporter gene assays in the chicken hepatoma cells leghorn male hepatoma as well as transactivation of the 240-bp PBRU by CXR in CV-1 cells. CXR bound to this PBRU in electromobility shift assays and the complex remained unaffected by unlabeled 240-bp PBRU with a mutated DR-4. In cross-species experiments, both the human xenobiotic-sensing nuclear receptors pregnane X receptor and constitutive androstane receptor bound to this element, suggesting sequence conservation between chicken and mammalian PBRUs and between the DNA binding domains of these receptors. Of two orphan nuclear receptors involved in cholesterol and bile acid homeostasis, only chicken liver X receptor (LXR) but not chicken farnesoid X receptor bound to the 240-bp PBRU. These results suggest that CYP2H1 induction is explained by the combined effect of multiple distal enhancer elements interacting with multiple transcription factors, including CXR and LXR.

Parallel and independent regulation of interleukin-3 mRNA turnover by phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase.

AU-rich elements (ARE) present in the 3' untranslated regions of many cytokines and immediate-early genes are responsible for targeting the transcripts for rapid decay. We present evidence from cotransfection experiments in NIH 3T3 cells that two signaling pathways, one involving phosphatidylinositol 3-kinase (PI3-K), and one involving the p38 mitogen-activated protein kinase (MAPK), lead to stabilization of interleukin-3 mRNA in parallel. Stabilization mediated by either of the two pathways was antagonized by tristetraprolin (TTP), an AU-binding protein known to promote constitutive decay of ARE-containing transcripts. Remarkably, the stabilizing AU-binding protein HuR, in collaboration with p38 MAPK but not with PI3-K, could overcome the destabilizing effect of TTP. These data argue that the stabilizing kinases PI3-K and p38 MAPK do not act through direct inactivation of TTP but via activating pathway-specific stabilizing AU-binding proteins. Our data suggest an integrated model of mRNA turnover control, where stabilizing (HuR) and destabilizing (TTP) AU-binding proteins compete and where the former are under the positive control of independent phosphokinase signaling pathways.

Somatic mRNA turnover mutants implicate tristetraprolin in the interleukin-3 mRNA degradation pathway.

Control of mRNA stability is critical for expression of short-lived transcripts from cytokines and proto-oncogenes. Regulation involves an AU-rich element (ARE) in the 3' untranslated region (3'UTR) and cognate trans-acting factors thought to promote either degradation or stabilization of the mRNA. In this study we present a novel approach using somatic cell genetics designed to identify regulators of interleukin-3 (IL-3) mRNA turnover. Mutant cell lines were generated from diploid HT1080 cells transfected with a reporter construct containing green fluorescent protein (GFP) linked to the IL-3 3'UTR. GFP was expressed at low levels due to rapid decay of the mRNA. Following chemical mutagenesis and selection of GFP-overexpressing cells, we could isolate three mutant clones (slowA, slowB, and slowC) with a specific, trans-acting defect in IL-3 mRNA degradation, while the stability of IL-2 and tumor necrosis factor alpha reporter transcripts was not affected. Somatic cell fusion experiments revealed that the mutants are genetically recessive and form two complementation groups. Expression of the tristetraprolin gene in both groups led to reversion of the mutant phenotype, thereby linking this gene to the IL-3 mRNA degradation pathway. The genetic approach described here should allow identification of the defective functions by gene transfer and is also applicable to the study of other mRNA turnover pathways.

The deep-sea as a final global sink of semivolatile persistent organic pollutants? Part II: Organochlorine pesticides in surface and deep-sea dwelling fish of the north and south Atlantic and the Monterey Bay Canyon (California).

The understanding of the global environmental multiphase distribution of persistent organic pollutants (POPs) as a result of the physico-chemical properties of the respective compounds is well established. We have analysed the results of a vertical transport of POPs from surface water to deepwater in terms of the contamination of the biota living in the respective environmental compartments. Samples were taken from the North and the South Atlantic and from the uprising water region of the continental shelf of California (Marine Sanctuary Monterey Bay and its Canyon). The contents of persistent organochlorine pesticides (DDTs, chlordanes, toxaphenes, HCHs, and HCB) in surface-living fish are compared to those in deepwater fish of the same geographic area. The deepwater biota show significantly higher burdens as compared to surface-living species of the same region. There are also indications for recycling processes of POPs of the class of organochlorine pesticides in the biophase of the abyss as well. It can be concluded that the bio- and geophase of the deep-sea may act as an ultimate global sink for persistent semivolatile contaminants in the marine environment like the soil on the continents.

Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60.

Chaperonins are essential for the folding of proteins in bacteria, mitochondria, and chloroplasts. We have functionally characterized the yeast mitochondrial chaperonins hsp60 and hsp10. In the presence of ADP, one molecule of hsp10 binds to hsp60 with an apparent Kd of 0.9 nM and a second molecule of hsp10 binds with a Kd of 24 nM. In the presence of ATP, the purified yeast chaperonins mediate the refolding of mitochondrial malate dehydrogenase. Hsp10 inhibits the ATPase activity of hsp60 by about 40%. Hsp10(P36H) is a point mutant of hsp10 that confers temperature-sensitive growth to yeast. Consistent with the in vivo phenotype, refolding of mitochondrial malate dehydrogenase in the presence of purified hsp10(P36H) and hsp60 is reduced at 25 degrees C and abolished at 30 degrees C. The affinity of hsp10(P36H) to hsp60 as well as to Escherichia coli GroEL is reduced. However, this decrease in affinity does not correlate with the functional defect, because hsp10(P36H) fully assists the GroEL-mediated refolding of malate dehydrogenase at 30 degrees C. Refolding activity, rather, correlates with the ability of hsp10(P36H) to inhibit the ATPase of GroEL but not that of hsp60. Based on our findings, we propose that the inhibition of ATP hydrolysis is mechanistically coupled to chaperonin-mediated protein folding.

Hsp60-independent protein folding in the matrix of yeast mitochondria.

Proteins that are imported from the cytosol into mitochondria cross the mitochondrial membranes in an unfolded conformation and then fold in the matrix. Some of these proteins require the chaperonin hsp60 for folding. To test whether hsp60 is required for the folding of all imported matrix proteins, we monitored the folding of four monomeric proteins after import into mitochondria from wild-type yeast or from a mutant strain in which hsp60 had been inactivated. The four precursors included two authentic matrix proteins (rhodanese and the mitochondrial cyclophilin Cpr3p) and two artificial precursors (matrix-targeted variants of dihydrofolate reductase and barnase). Only rhodanese formed a tight complex with hsp60 and required hsp60 for folding. The three other proteins folded efficiently without, and showed no detectable binding to, hsp60. Thus, the mitochondrial chaperonin system is not essential for the folding of all matrix proteins. These data agree well with earlier in vitro studies, which had demonstrated that only a subset of proteins require chaperones for efficient folding.