We studied the regulation of the hamster CYP11B2 gene in the NCI-H295 cell line, which is known to produce aldosterone in response to stimulation by angiotensin II (AII) and KCl. Ten deletion plasmids harboring the 5'-untranslated region of the CYP11B2 gene were used for chloramphenicol acetyltransferase (CAT) assays. Transient transfections showed progressively increasing basal promoter activity by constructs beyond the TATA box, with a peak occurring with the -167 bp construct which contains putative Adl, Ad2, Ad5 and the newly reported -143/-161 cis-element sequences. The promoter activity was lower with the construct containing the putative Ad3 cis-element and increased with longer constructs. This indicates the presence of both inhibitory and stimulatory cis-elements in this area of the gene. Expression of the reporter gene of all constructs was stimulated by AII and KCl, with the exception of the construct containing only the TATA box, which showed 6-fold and 10-fold increases occurring with the -167 bp deletion plasmid. The patterns of increase in CAT activity with AII and KCI treatment were similar, showing that these two regulators can stimulate hamster CYP11B2 promoter activity through common cis-elements. The calcium channel antagonist nifedipine blocked the stimulatory effects of KCl on CAT activity, showing the involvement of calcium channels in the regulation of CYP11B2 gene transcription by KCl. 12-O-Tetradecanoylphorbol 13-acetate, a known stimulator of the protein kinase C (PKC) signaling pathway, was without significant effect on CAT activity. Bisindolylmaleimide, a specific inhibitor of PKC, had a significant enhancing effect (3.4- to 6-fold), indicating that PKC may negatively regulate the expression of the hamster CYP11B2 gene in NCI-H295 cells. A mutation was induced in the sequence -143/-161 of the - 350 bp construct in order to determine its importance in the regulation of hamster CYP11B2 promoter activity. The stimulatory effects of AII, KCl, forskolin and bisindolylmaleimide on CAT activity were significantly less in the mutant than in the wild type. These results confirm that this cis-element is necessary in maintaining a high level of transcriptional activity in stimulated NCI-295H cells. In conclusion, using NCI-295H transfected cells, we have found that the 5'-untranslated region of the hamster CYP11B2 gene possesses transcriptional activity with stimulatory and also inhibitory cis-elements; CYP11B2 promoter activity can be stimulated by AII, KCl, forskolin, dibutyryl cAMP and bisindolylmaleimide. Our results suggest that this gene is positively regulated through the protein kinase A signaling pathway and through calcium channels, whereas PKC may have a negative regulatory effect upon the transcription of the CYP11B2 gene. Furthermore, we have shown that the cis-element -143/-161 in the 5'-untranslated region of the hamster CYP11B2 gene is important in maintaining a high level of promoter activity in stimulated NCI-295H cells.
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J.-G. Lehoux and A. Lefebvre
ABSTRACT
Low-density lipoprotein (LDL) receptor mRNA was found in both rat and hamster adrenals. Within 30 min after ACTH administration a significant increase in the levels of both LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) mRNAs was observed in rat adrenals; these levels remained increased for up to 240 min. The increase in the levels of LDL receptor and HMG-CoA reductase mRNAs produced by ACTH was reduced by co-administration of aminoglutethimide while, at the same time, the adrenal cholesterol content of rats treated with both aminoglutethimide and ACTH was significantly increased compared with that in groups treated with ACTH alone. Cycloheximide also induced increased levels of rat adrenal mRNAs for LDL receptor and HMG-CoA reductase, but this effect was not additive with that of ACTH. These results suggest that, in the rat, the short-term effect of ACTH on the levels of mRNAs for the LDL receptor and HMG-CoA reductase is similarly controlled and might be mediated through changes in the adrenal cholesterol content. In the hamster adrenal, however, no significant fluctuations were found in the level of LDL receptor mRNA, although a marked increase was found in the level of HMG-CoA reductase mRNA, 2 h after ACTH administration. This indicates that an important effect of ACTH on cholesterol metabolism in the hamster adrenal is at the level of HMG-CoA reductase. In the hamster, therefore, where the main source of cholesterol for the adrenal gland is de-novo synthesis, it seems that a complex mechanism is involved in the control of LDL receptor gene expression.
A Fleury, L Ducharme, and JG LeHoux
In this study, we report the cDNA cloning of hamster adrenal steroidogenic acute regulatory (StAR) protein and the effect of adrenocorticotrophin (ACTH) on its expression in vivo. A hamster adrenal cDNA library was screened using an 852 bp fragment obtained by polymerase chain reaction; this fragment corresponds to the entire coding sequence (CDS) of the hamster adrenal StAR cDNA. Ten clones of different lengths were isolated and sequenced. The longest clone was 1564 bp and contained 34 bp in the 5'-untranslated region, 852 bp in the CDS, and 678 bp in the 3'-untranslated region (3'-UTR). Two polyadenylation signal sequences were found in the 3'-UTR. The CDS of the ten isolated clones was identical, but six of these lacked the last 132 nucleotides in the 3'-UTR, thus indicating that they had used the first polyadenylation signal. The hamster StAR protein contains 284 amino acid residues, and is 91.9% homologous to mouse, 90.5% to rat, 86.4% to human, 85% to porcine, and 82.5% to bovine StAR protein. Southern blot analysis indicated the presence of only one StAR gene in the hamster genome. Northern blotting analysis revealed the presence of the StAR mRNA in male and female steroidogenic tissues, namely adrenals and gonads, but not in the liver or in the kidneys of either sex. Three mRNA species of 1.7, 3.1 and 5.3 kb were found in whole hamster adrenals. Administration of ACTH to hamsters provoked increases (two- to threefold) in the adrenal content of the StAR mRNA within 1 h in vivo. Western blotting analysis on adrenal mitochondria showed that the level of StAR protein was also significantly elevated (1.5-fold) 1 h after ACTH treatment.
FM Rogerson, JG LeHoux, A Lefebre, and JI Mason
Complementary DNAs encoding the hamster type 2 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase were isolated from liver and kidney cDNA libraries. Nine clones were isolated containing identical coding and 3' untranslated sequences. However, six of the clones contained a 68-nucleotide stretch in the 5' untranslated region that was missing in the other three clones. Primers were designed to flank this region and the polymerase chain reaction (PCR) was performed on hamster liver and adrenal RNA. Two PCR products were amplified of the predicted molecular sizes and with the expected sequence. Primers were then designed to amplify sequences encompassing this region from hamster genomic DNA. Sequencing of the resultant PCR products demonstrated that the 68-nucleotide stretch missing in some transcripts corresponded exactly to the second of three exons identified. We conclude that the 5' untranslated region of this mRNA is transcribed from at least three exons, and that the sequence of the second of these exons is spliced out of some of the RNA transcripts.
AP Mathieu, A Fleury, L Ducharme, P Lavigne, and JG LeHoux
The steroidogenic acute regulatory protein (StAR) is the major entrance for cholesterol in mitochondria under acute stimulation. Under such circumstances, dysfunctional StAR activity can ultimately lead to lipoid congenital adrenal hyperplasia (LCAH). A complete understanding of the StAR's molecular structure and mechanism is essential to comprehend LCAH. Thus far, there is no mechanistic model that can explain experimental results at the molecular level. This is partly due to the lack of the molecular structure of StAR. The closest approximation to the StAR molecular structure is the human MLN64 which has a similar activity to StAR, has a highly homologous primary structure and for which an X-ray structure is known. In this context, we have modeled the structure of StAR through standard homology modeling procedures based on the MLN64 structure. Our StAR model shows the presence of a hydrophobic cavity of 783.9 A(2) in surface area, large enough to fit one molecule of cholesterol. In addition, we have identified a unique charged pair, as in MLN64, lining the surface of the cavity and which could play a key role in the binding of cholesterol through the formation of an H-bond with its OH moiety. This suggests that the cholesterol-binding site of StAR is located inside this cavity. Taking into account that internal cavities are destabilizing to native protein structures and that the lining of the cavity has to become accessible in order to allow cholesterol binding, we have explored the possibility that StAR could exist in equilibrium with partially unfolded states. Using a structure-based thermodynamics approach, we show that partially folded states (with an unfolded C-terminal alpha-helix, and an open cavity) can be significantly populated at equilibrium and therefore allow cholesterol binding. These results are supported by recent experiments that show a loss of StAR helical character upon binding of an analog of cholesterol. Moreover, we show that the replacement of the residues involved in the charged-pair located in the binding site results in the loss of StAR activity, supporting a key role for these residues. Taken together, our results are applicable to StAR functioning both in the mitochondrial intermembrane space as well as outside the mitochondria.
FM Rogerson, J Courtemanche, A Fleury, Head JR, JG LeHoux, and JI Mason
Western blot analyses of various hamster tissues reveal high levels of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 beta-HSD were isolated from hamster cDNA libraries. The type 1 isoform is a high-affinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 beta-HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 beta-HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.
