Andrée Lefebvre

De novo synthesis of corticosteroids in hamster adrenal glands.

Abstract The role of endogenous cholesterol in corticosteroid biosynthesis has been examined in the hamster adrenal. (A) In hamsters sacrificed at different times of the day the following have been determined: plasma corticosteroid and cholesterol levels, endogenous cholesterol content of the adrenal, the corticosteroid secretion pattern in the adrenal incubated for 90 min. and the adrenal cholesterol content after 90 min of incubation. (B) The capacity of the hamster adrenal to incorporate 3H2O into digitonin precipitable sterols has been compared with that of the rat. The concentration of adrenal free cholesterol is significantly lower at 19:00 h than at 11:00 h. The in vitro secretion pattern of corticosteroids parallels that of plasma corticosteroids. both having a maximum around 18:00 h. Hamster adrenals can incorporate about nine-fold more 3H2O into sterols than rat adrenals and five- to seven-fold more than rat liver. Hamster adrenals seem to be largely capable of synthesizing cholesterol from small molecules of substrate. Endogenous adrenal free cholesterol content may well be involved in regulating its own synthesis.

Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms

We reported previously that the protein kinase C (PKC) inhibitor GF109203X stimulated the hamsterCYP11B2 promoter activity in transfected NCI-H295 cells. PKCα, -ε, and -ζ were detected in hamster adrenal zona glomerulosa and NCI-H295 cells, and PKCθ in NCI-H295 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibited basal and stimulated cytochrome P450 aldosterone synthase mRNA expression by angiotensin (AII), dibutyryl cyclic adenosine 3′:5′-monophosphate (Bt2cAMP), or KCl in NCI-H295 cells. BasalCYP11B2 promoter activity was inhibited in cells cotransfected with constitutively active (CA) PKCα, -ε, and -θ mutants, whereas it was increased with CA-PKCζ. Dominant negative (DN) PKCα, -θ, -ε, and -ζ mutants stimulated the promoter activity. AII-, KCl-, and Bt2cAMP-stimulatory effects were abolished in cells cotransfected with CA-PKCα, -ε, or -θ. The effect of Bt2cAMP was abolished by CA-PKCζ but AII and KCl were still able to enhance the promoter activity. DN-PKCα, -ε, -θ, or -ζ did not inhibit these effects. Gö6976 enhanced promoter activity, providing further evidence that PKCα was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKCα, -ε, or -θ abolished the effects of AII, KCl, and Bt2cAMP on the activity of −102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (α) and atypical (ζ) PKC.

The hamster adrenal cytochrome P450C11 has equipotent 11β-hydroxylase and 19-hydroxylase activities, but no aldosterone synthase activity

We have isolated a hamster adrenal P45OC11 cDNA which shared 90 and 84% homology, respectively, with the nucleotide sequence and the amino acid sequence of the hamster adrenal P450aldo. Both P450C11 and P450aldo cDNA coding sequences were inserted in the plasmid pBluescript SK, transcribed and then translated using a rabbit reticulocyte system in the presence of [35S]methionine. The reaction products were immunoprecipitated with an anti-bovine P450C11 antibody for P450C11 and with an anti-hamster P450aldo for P450aldo. Immunoprecipitated proteins were analyzed by polyacrylamide gel electrophoresis. A single 35S-labeled protein band was detected for P450C11 and for P450aldo, respectively. P450C11 and P450aldo cDNAs were then both inserted into the expression vector pCMV5 containing a viral sequence specific for the attachment of ribosomes to mRNA. These constructions were transfected in COS-1 cells. 24 h after transfection, the presence of P450C11 and P450aldo mRNAs was determined by Northern blot analysis. In a time study experiment we found that P450C11 transformed the labeled-steroid into [14C]corticosterone, [14C]19-OH-deoxycorticosterone and [14C]18-OH-deoxycorticosterone in ratios of 1:1.11:0.07, after 2 h of incubation; no [14C]aldosterone could be detected. Cells transfected with plasmids harboring the P450aldo cDNA transformed [14C]deoxycorticosterone to [14C]corticosterone, [14C]aldosterone, [14C]18-OH-corticosterone, [14C]18-OH-deoxycorticosterone, [14C]19-OH-deoxycorticosterone and [14C]11-dehydrocorticosterone in ratios of 1:0.25:0.45:0.04:0.04:0.04 after 12 h of incubation. These results indicate that one P450 catalyzes the ultimate step of glucocorticoid formation and a separate P450 is involved in the final steps of aldosterone formation in hamster adrenals. The capacity of the hamster adrenal P450C11 to hydroxylate at positions 11beta and 19 in nearly equal ratio makes this animal an excellent model to study the mechanism of synthesis and inhibition of 19-OH-deoxycorticosterone, the precursor of 19-nor-deoxycorticosterone, a very potent mineralocorticoid involved in the development of essential hypertension.

StAR-related lipid transfer domain protein 5 binds primary bile acids.

Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4–6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored. The SAR by NMR [1H-15N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the 1H-15N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10−4 M−1 and 6.3·104 M−1 were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol.

Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5

STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA>LCA>CDCA>GDCA>TDCA>CA>UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.

ADRENOCORTICOTROPIN REGULATES THE LEVEL OF THE STEROIDOGENIC ACUTE REGULATORY (STAR) PROTEIN MRNA IN HAMSTER ADRENALS

In this study, we report the cloning of a StAR cDNA from a hamster adrenal cDNA library. The library was screened using a PCR fragment specific for the hamster adrenal StAR cDNA. Several clones of different lengths were obtained and one of these was sequenced. Northern blotting analysis revealed the presence of the StAR mRNA in male and female adrenals, in tests and ovaries, but not in the liver or kidneys of either sex. Whole hamster adrenals revealed the presence of four mRNAs of 0.65, 1.7, 3.1 and 5.25 kb, respectively. In addition, ACTH regulates the expression of StAR mRNA in hamster adrenals. Indeed, when groups of hamsters were injected with ACTH and sacrificed at different times after treatment, only the 0.65 kb form of the StAR mRNA did not increase, whereas the other forms increased at varying levels. These results might suggest that the expression of the StAR protein in hamster adrenals depends upon different genes, different promoters, or different polyadenylation signal sites. In conclusion, these results indicate that in vivo, StAR is regulated by ACTH, suggesting the participation of this protein in controlling the transformation of cholesterol to pregnenolone, a key regulatory step in corticosteroidogenesis.

The binding site specificity of STARD4 subfamily: Breaking the cholesterol paradigm.

Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain proteins display diverse expression patterns and cellular localisations. They bind a large variety of lipids and sterols and are involved in lipid metabolism, lipid transfer and cell signalling. The START domain tertiary structure is an α-helix/β-grip fold module of approximately 210 amino acids delimiting an internal cavity forming the binding site. However, the determinants that dictate ligand specificity and the mechanism of ligand entry and exit are ill-defined. Herein, we review and discuss the current knowledge on ligand specificity and binding mechanism of START domains. More specifically, we highlight that the conserved residues of STARD1, STARD3, STARD4, STARD5 and STARD6 START domains binding sterol play an important structural role for the global protein fold, whereas the residues forming the cavity that fits the shape of their respective ligand are divergent, suggesting their participation in ligand specificity. We also explore the potential binding of steroids to STARD6 in the context of ligand selectivity.

Effect of ACTH on cholesterol and steroid synthesis in adrenocortical tissues

Previous studies have established that under normal conditions, adrenal HMG-CoA reductase activity is higher in hamsters than in rats and humans. The hamster reductase activity follows a diurnal rhythm corresponding to that of plasma ACTH and glucocorticoids [Endocrinology 107 (1980) 215] but not to that of aldosterone. ACTH treatments to hamsters increased reductase activity after a latency of 60 min; this enhancement was prevented by cycloheximide [J. steroid Biochem. 24 (1986) 325]. Immunotitration and immunoblotting studies confirmed that ACTH caused an increase in reductase protein synthesis. In rats, long-term (1-9 days) and short-term (3 h) treatments with ACTH also induced increase in adrenal HMG-CoA reductase activity and reductase protein. In the presence of iodoacetamide and inhibitors of proteolytic enzyme, a main specific band of enzyme was evinced in the area of 102 +/- 6 kDaMr, by Western blotting, for both hamster homogenate and microsomal preparations (Endocrinology, 120 (1987]. Similarly Mr values were found with rat adrenal preparations. The concentration of mRNA, analyzed using the c-DNA pRed-10 coding for the Chinese hamster ovary reductase, was increased in adrenals of hamsters treated with ACTH. The reductase mRNA levels also fluctuated during the day in parallel with those of reductase activity and reductase protein. In conclusion, these results indicate that ACTH and other conditions inducing a change in hamster adrenal HMG-CoA reductase activity provoke parallel changes in reductase mRNA and reductase protein content. ACTH acts on the adrenal reductase of species synthesizing large as well as small quantities of cholesterol, thus indicating the general importance of this hormonal control.

The effect of ACTH on HMG-CoA reductase activity in hamster adrenals

Abstract Injection to hamsters of various low doses of ACTH resulted in gradual increases in adrenal HMG-CoA reductase activity, in plasma and adrenal corticosteroid concentrations but produced no change in adrenal cholesterol content. These data indicate that under physiological conditions, ACTH could regulate HMG-CoA reductase activity through a mechanism which does no apparently involve a change in the cholesterol content of the gland.

Hormonal regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase mRNA in the rat adrenal gland

We studied the effect of ACTH on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase enzyme. Reductase activity and reductase mass were enhanced by 22- and 6.2-fold respectively in one series of experiments, whereas in another the levels of reductase activity, reductase mass, and reductase mRNA were increased 6.6-, 3.6- and 2.2-fold respectively, following daily administration of exogenous ACTH for 3 days. Daily injection of 4-aminopyrazolopyrimidine (4-APP) to rats for 3 days increased circulating ACTH level 5.4-fold, whereas adrenal HMG-CoA reductase activity, reductase mass and reductase mRNA levels were greatly increased 36-, 10- and 16-fold, respectively. To counteract the effect of elevated plasma ACTH, dexamethasone acetate (Dex) was administered to 4-APP treated rats. At 3 h post Dex administration, plasma ACTH and corticosteroids levels were effectively decreased by 58 and 59%, respectively. The levels of adrenal HMG-CoA reductase mRNA, reductase activity and reductase mass were also diminished by 38, 31 and 40%, respectively. Our results show that rat adrenal HMG-CoA reductase can respond rapidly to hormonal changes, presumably through variations in circulating ACTH levels.