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Biomedical subjects

P McQuiddy

Publications and source records attributed to P McQuiddy.

9 recordsLinked to original sources
PubMed1997

Thyroid regulation of NADPH:cytochrome P450 oxidoreductase: identification of a thyroid-responsive element in the 5'-flank of the oxidoreductase gene.

The current study demonstrates that T3-activated transcription of the NADPH:cytochrome P450 oxidoreductase (P450R) gene is dependent on the thyroid hormonal status of the animal, with both transcriptional and post-transcriptional pathways being important in regulating the cellular P450R mRNA level. The region required for transcriptional activation of the P450R gene by T3 has been identified. Nuclear run-on experiments demonstrated that the effects of T3 on P450R transcription are dependent on thyroid status, with a transcriptional enhancement obtained in T3-treated hypothyroid rat liver (1.8-fold increase) but not in T3-treated euthyroid animals. Transient cotransfection of P450R promoter/chloramphenicol acetyl transferase (CAT) constructs and the thyroid hormone receptor beta1 (TR beta1) expression plasmid into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter activity that was both T3 and TR beta1 dependent. Analysis of promoter deletion constructs identified a P450R-thyroid response region (P450R-TRE; bases, -564 to -536) containing three imperfect direct repeats of the thyroid response motif, AGGTCA. Mutational analysis further established that T3 induction was dependent only on the upstream direct repeat, having the sequence AGGTGAgctgAGGCCA. Footprint analysis showed that all three motifs were protected by proteins present in rat liver nuclear extracts, and a direct interaction between P450R-TRE and T3 receptors TR alpha1 and TR beta1 was demonstrated by gel-shift analysis. In vitro binding studies with P450R-TRE revealed the formation of heterodimeric complexes when TR alpha1 was coincubated with either the retinoic X receptor alpha or nuclear extract from rat liver, COS, or H4IIE cells. In addition, placement of the P450R-TRE upstream of the T3-nonresponsive heterologous thymidine kinase promoter resulted in a 2.7-fold transcriptional enhancement that was both T3 and TR beta1 dependent. Previous studies have demonstrated that T3 augments P450R mRNA levels approximately 20-30-fold and approximately 12-fold, respectively, in hypothyroid and euthyroid rats. Hence, for the hypothyroid state, transcriptional and post-transcriptional events contribute to the T3-induced mRNA increases; however, the marked increase in message level in T3-treated euthyroid animals depends primarily on post-transcriptional pathways.

Animals↗
PubMed1996

Transcriptional regulation of the TATA-less NADPH cytochrome P-450 oxidoreductase gene.

Multiple cis-acting DNA sequences regulating expression of the rat liver NADPH-cytochrome P-450 oxidoreductase gene have been identified in transient transfection assays using promoter deletion constructs linked to the chloramphenicol acetyl transferase gene. The TATA-less promoter possesses nine GC-boxes which contain the consensus sequence for the transcription factor Sp1. While loss of the seven distal GC-boxes had minimal effect on transcriptional activity, deletion of the next 35 bp, from -206 to -172, resulted in approximately 90% loss of promoter activity. Contained within this region is an Sp1 binding site indicating that either (1) this particular consensus sequence was essential for transcription, (2) the two proximal GC boxes act in concert, or (3) a yet unidentified regulatory element resides within this 35-bp stretch. In addition, transfection experiments demonstrated that two separate distal regions (-622 to -1167 and -1500 to -2300) contain negative regulatory elements which down-regulate gene transcription in a position-independent manner. Mobility-shift analyses and DNase footprinting identified sequences in the proximal region of the promoter that bound proteins present in nuclear extracts. Four protected segments were observed within the first 100 bp upstream of the transcription start site; these include (1) the region encompassing the transcription start site (-7 to +4), (2) the region normally occupied by a TATA-box (-38 to -18), (3) the bases from -78 to -60 which contain the regulatory element CACC, and (4) bases -105 to -92 which include an Sp1 binding site. Hence, regulation of the NADPH-cytochrome P-450 oxidoreductase gene is controlled by both positive and negative regulatory elements, and, of the nine Sp1 consensus sites, the two proximal sites are sufficient to support basal transcription.

Animals↗
PubMed1996

Dexamethasone responsiveness of a major glucocorticoid-inducible CYP3A gene is mediated by elements unrelated to a glucocorticoid receptor binding motif.

Elements responsible for dexamethasone responsiveness of CYP3A23, a major glucocorticoid-inducible member of the CYP3A gene family, have been identified. DNase I footprint analysis of the proximal promoter region revealed three protected sites (sites A, B, and C) within the sequence defined by -167 to -60. Mutational analysis demonstrated that both sites B and C were necessary for maximum glucocorticoid responsiveness and functioned in a cooperative manner. Interestingly, neither site contained a glucocorticoid responsive element. Embedded in site C was an imperfect direct repeat (5'-AACTCAAAGGAGGTCA-3'), showing homology to an AGGTCA steroid receptor motif, typically recognized by the estrogen receptor family, while site B contained an ATGAACT direct repeat; these core sequences were designated dexamethasone response elements 1 and 2 (DexRE-1 and -2), respectively. Neither element has previously been associated with a glucocorticoid-activated transcriptional response. Conversion of the DexRE-1 to either a perfect thyroid hormone or vitamin D3 responsive element further enhanced induction by dexamethasone. Gel-shift analysis demonstrated that glucocorticoid receptor did not associate with either DexRE-1 or -2; hence, glucocorticoid receptor does not directly mediate glucocorticoid induction of CYP3A23. These unusual features suggest an alternate pathway through which glucocorticoids exert their effects.

Amino Acid Sequence↗
PubMed1990

Glucocorticoid repression and basal regulation of the epoxide hydrolase promoter.

Through a series of promoter deletions and gene transfer experiments we have examined the basal regulation and glucocorticoid-mediated repression of the rat epoxide hydrolase gene. Three regions of the 5' flanking sequence were found to influence the basal level of promoter function in H4IIE hepatoma cells. Region A (-891 to -355 bp) contains an apparent repressor of epoxide hydrolase expression, while regions B (-271 to -171 bp) and C (-141 to -85) were found to contain important sequences required for optimal promoter activity. Previous work has demonstrated that dexamethasone represses epoxide hydrolase transcription by approximately 50% in isolated rat liver nuclei, and, in this study, we have demonstrated that the ability of the epoxide hydrolase promoter to drive CAT expression is similarly repressed in H4IIE cells treated with 1 microM dexamethasone. Furthermore, the level of endogenous epoxide hydrolase mRNA is decreased by 70-88% in nontransfected H4IIE cells treated with dexamethasone. Interestingly, promoter activity was not decreased by dexamethasone in COS cells, which lack glucocorticoid receptors. The current data show that sequences from -42 to +110 bp are sufficient to support the dexamethasone response, and, furthermore, they suggest that repression may not require direct interaction of the ligand-receptor complex with the promoter region.

Animals↗
PubMed1987

Structure and organization of the microsomal xenobiotic epoxide hydrolase gene.

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.

Amino Acid Sequence↗
PubMed1987

Induction of the hepatic mixed-function oxidase system by synthetic glucocorticoids. Transcriptional and post-transcriptional regulation.

Quantitative evaluation of transcriptional rates in isolated nuclei and intranuclear and cytoplasmic mRNA levels demonstrates that the catatoxic steroids pregnenolone-16 alpha-carbonitrile (PCN) and dexamethasone modulate microsomal drug metabolizing enzymes both positively and negatively at the transcriptional level. Additionally, a strong post-transcriptional influence, believed to be message stabilization, was found to be exerted by dexamethasone. Both NADPH-cytochrome P-450b-homologous mRNA are induced 7- and 12-fold, respectively, presumably through message stabilization. Under these conditions, however, the oxidoreductase content of the cell is only marginally increased while the cytochrome P-450b level is actually decreased by 40% within three days after a single injection of dexamethasone. In contrast, genes coding for cytochromes P-450PCN show a definite increase in transcription following administration of either pre gnenolone-16 alpha-carbonitrile or dexamethasone, whereas transcription of the epoxide hydrolase gene is markedly reduced by administration of these compounds.

Animals↗