Age-dependent decrease in 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity in hypertensive patients

BACKGROUND: The prevalence of arterial hypertension lacking a defined underlying cause increases with age. Age-related arterial hypertension is insufficiently understood, yet known characteristics suggest an aldosterone-independent activation of the mineralocorticoid receptor. Therefore, we hypothesized that 11beta-HSD2 activity is age-dependently impaired, resulting in a compromised intracellular inactivation of cortisol (F) with F-mediated mineralocorticoid hypertension. METHODS: Steroid hormone metabolites in 24-h urine samples of 165 consecutive hypertensive patients were analyzed for F and cortisone (E), and their TH-metabolites tetrahydro-F (THF), 5alphaTHF, TH-deoxycortisol (THS), and THE by gas chromatography-mass spectroscopy. Apparent 11beta-HSD2 and 11beta-hydroxylase activity and excretion of F metabolites were assessed. RESULTS: In 72 female and 93 male patients aged 18-84 years, age correlated positively with the ratios of (THF + 5alphaTHF)/THE (P = 0.065) and F/E (P < 0.002) suggesting an age-dependent reduction in the apparent 11beta-HSD2 activity, which persisted (F/E; P = 0.020) after excluding impaired renal function. Excretion of F metabolites remained age-independent most likely as a consequence of an age-dependent diminished apparent 11beta-hydroxylase activity (P = 0.038). CONCLUSION: Reduced 11beta-HSD2 activity emerges as a previously unrecognized risk factor contributing to the rising prevalence of arterial hypertension in elderly. This opens new perspectives for targeted treatment of age-related hypertension.

The role of adrenal steroidogenesis in arterial hypertension

Adrenal aldosterone production, the major regulator of salt and water retention, is discussed with respect to hypertensive diseases. Physiological aldosterone production is tightly regulated, either stimulated or inhibited, in the adrenal zona glomerulosa by both circulating factors and/or by locally derived endothelial factors. Arterial hypertension caused by volume overload is the leading clinical symptom indicating increased mineralocorticoid hormones. Excessive aldosterone production is seen in adenomatous disease of the adrenals. The balance between stimulatory/proliferative and antagonistic signaling is disturbed by expression of altered receptor subtypes in the adenomas. Increased aldosterone production without a detectable adenoma is the most frequent form of primary aldosteronism. Both increased sensitivity to agonistic signals and activating polymorphisms within the aldosterone synthase gene (CYP11B2) have been associated with excessive aldosterone production. 17alpha-Hydroxylase deficiency and glucocorticoidremediable aldosteronism can also cause excessive mineralocorticoid synthesis. In contrast, the severe form of pregnancy-induced hypertension, preeclampsia, is characterized by a compromised volume expansion in the presence of inappropriately low aldosterone levels. Initial evidence suggests that compromised CYP11B2 is causative, and that administration of NaCl lowered blood pressure in pregnant patients with low aldosterone availability due to a loss of function.

ROLE OF MICRORNA LET-7 IN PANCREATIC BETA CELLS

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression at transcriptional or post-transcriptional level. Let-7 family is among the first identified human miRNAs and regulates multiple cellular processes including glucose metabolism in multiple organs. It has been reported that overexpression of let-7 resulted in insulin resistance and impaired glucose tolerance through repressing insulin signaling pathway in both muscle and liver. However, the role and mechanism underlying let-7 function in pancreatic beta-cells have yet to be elucidated. Let-7 family contains nine members, which poses a significant challenge in complete deletion of this miRNA family. To study the function of let-7 and to overcome the functional redundancies of various let-7 members in pancreatic beta-cells, the highly expressed let-7a and let-7b were blocked simultaneously using short tandem target mimic (STTM) approach developed in our laboratory. Introducing STTM-let7 into beta-cells markedly increased the expression of Caspase 3, a direct target of let-7, confirming a sufficient functional knockdown of let-7a/b by STTM-let7. STTM-let7 enhanced apoptotic cell death induced by cytokine, indicating that let-7a/b is able to protect from apoptosis through attenuating Caspase 3 expression in pancreatic beta-cells. In contrast to the previous observation that let-7 silencing increases insulin signaling in muscle and liver, inhibition of let-7 with STTM-let7 significantly repressed glucose-stimulated insulin signaling in pancreatic beta-cells, leading to impaired insulin secretion and reduced beta-cell proliferation. Taken together, an appropriate level of let-7 is essential in maintaining beta-cell function and viability. Dysregulation of let-7 may contribute to the pathogenesis of type 2 diabetes.

Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents

The CYP17A1 gene is the qualitative regulator of steroidogenesis. Depending on the presence or absence of CYP17 activities mineralocorticoids, glucocorticoids or adrenal androgens are produced. The expression of the CYP17A1 gene is tissue as well as species-specific. In contrast to humans, adrenals of rodents do not express the CYP17A1 gene and have therefore no P450c17 enzyme for cortisol production, but produce corticosterone. DNA methylation is involved in the tissue-specific silencing of the CYP17A1 gene in human placental JEG-3 cells. We investigated the role of DNA methylation for the tissue-specific expression of the CYP17A1 gene in rodents. Rats treated with the methyltransferase inhibitor 5-aza-deoxycytidine excreted the cortisol metabolite tetrahydrocortisol in their urine suggesting that treatment induced CYP17 expression and 17alpha-hydroxylase activity through demethylation. Accordingly, bisulfite modification experiments identified a methylated CpG island in the CYP17 promoter in DNA extracted from rat adrenals but not from testes. Both methyltransferase and histone deacetylase inhibitors induced the expression of the CYP17A1 gene in mouse adrenocortical Y1 cells which normally do not express CYP17, indicating that the expression of the mouse CYP17A1 gene is epigenetically controlled. The role of DNA methylation for CYP17 expression was further underlined by the finding that a reporter construct driven by the mouse -1041 bp CYP17 promoter was active in Y1 cells, thus excluding the lack of essential transcription factors for CYP17 expression in these adrenal cells.

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.

Fetal ventral mesencephalon of human and rat origin maintained in vitro and transplanted to 6-hydroxydopamine-lesioned rats gives rise to grafts rich in dopaminergic neurons

Free-floating roller tube cultures of human fetal (embryonic age 6-10 weeks post-conception) and rat fetal (embryonic day 13) ventral mesencephalon were prepared. After 7-15 days in vitro, the mesencephalic tissue cultures were transplanted into the striatum of adult rats that had received unilateral injections of 6-hydroxydopamine into the nigrostriatal bundle 3-5 weeks prior to transplantation. Graft survival was assessed in tyrosine hydroxylase (TH)-immunostained serial sections of the grafted brains up to post-transplantation week 4 for the human fetal xenografts and post-transplantation week 11 for the rat fetal allografts. D-amphetamine-induced rotation was monitored up to 10 weeks after transplantation in the allografted animals and compared with that of lesioned-only control animals. All transplanted animals showed large, viable grafts containing TH-immunoreactive (ir) neurons. The density of TH-ir neurons in the human fetal xenografts and in rat fetal allografts was similar. A significant amelioration of the amphetamine-induced rotation was observed in the animals that received cultured tissue allografts. These results promote the feasibility of in vitro maintenance of fetal human and rat nigral tissue prior to transplantation using the free-floating roller tube technique.

Effects of GDNF pretreatment on function and survival of transplanted fetal ventral mesencephalic cells in the 6-OHDA rat model of Parkinson's disease

Transplantation of fetal dopaminergic (DA) neurons offers an experimental therapy for Parkinson's disease (PD). The low availability and the poor survival and integration of transplanted cells in the host brain are major obstacles in this approach. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with growth- and survival-promoting capabilities for developing DA neurons. In the present study, we examined whether pretreatment of ventral mesencephalic (VM) free-floating roller tube (FFRT) cultures with GDNF would improve graft survival and function. For that purpose organotypic cultures of E14 rat VM were grown for 2, 4 or 8 days in the absence (control) or presence of GDNF [10 ng/ml] and transplanted into the striatum of 6-hydroxydopamine-lesioned rats. While all groups of rats showed a significant reduction in d-amphetamine-induced rotations at 6 weeks posttransplantation a significantly improved graft function was observed only in the days in vitro (DIV) 4 GDNF pretreated group compared to the control group. In addition, no statistical significant differences between groups were found in the number of surviving tyrosine hydroxylase-immunoreactive (TH-ir) neurons assessed at 9 weeks posttransplantation. However, a tendency for higher TH-ir fiber outgrowth from the transplants in the GDNF pretreated groups as compared to corresponding controls was observed. Furthermore, GDNF pretreatment showed a tendency for a higher number of GIRK2 positive neurons in the grafts. In sum, our findings demonstrate that GDNF pretreatment was not disadvantageous for transplants of embryonic rat VM with the FFRT culture technique but only marginally improved graft survival and function.

Role of CYP27A1 in progesterone metabolism in vitro and in vivo

In the kidney, progesterone is inactivated to 20alpha-dihydro-progesterone (20alpha-DH-progesterone) to protect the mineralocorticoid receptor from progesterone excess. In an attempt to clone the enzyme with 20alpha-hydroxysteroid activity using expression cloning in CHOP cells and a human kidney expression library, serendipitously cDNA encoding CYP27A1 was isolated. Overexpression of CYP27A1 in CHOP cells decreased progesterone conversion to 20alpha-DH-progesterone in a dose-dependent manner, an effect enhanced by cotransfection with adrenodoxin and adrenodoxin reductase. Incubation of CHOP cells with 27-hydroxycholesterol, a product of CYP27A1, increased the ratio of progesterone/20alpha-DH-progesterone in a concentration-dependent manner, indicating that the effect of CYP27A1 overexpression was mediated by 27-hydroxycholesterol. In order to analyze whether these observations are relevant in vivo, progesterone and 20alpha-DH-progesterone were measured by GC-MS in 24-h urine of CYP27A1 gene knock out (ko) mice and their control wild type (wt) and heterozygote (hz) littermates. In CYP27A1 ko mice, urinary progesterone concentrations were decreased, 20alpha-DH-progesterone increased and the progesterone/20alpha-DH-progesterone ratio decreased threefold (p<0.001). Thus, CYP27A1 modulates progesterone concentrations. The underlying mechanism is inhibition of 20alpha-hydroxysteroid dehydrogenase by 27-hydroxycholesterol. Key words: Progesterone, sterol 27-hydroxylase, 27-hydroxycholesterol, 20a-steroid dehydrogenase, 20a-DH-progesterone.

Synthesis of arylated highly congested indans using a domino sequence

A novel and efficient regioselective synthesis of various arylated highly congested 7-aryl-5-methylsulfanylindan-4-carbonitriles (3a-(), methyl 7-aryl-5-methylsulfanylindan-4-carboxylates (lOa-e) and 7-aryl-5-methylsulfanylindan-4-carboxylic acids (lla-e) through base-catalyzed reaction of 6-aryl-4-methylsulfanyl-2-oxo-2H-pyran-3-carbonitriles (la-() and methyl 6-aryl-4-methylsulfanyl-2-oxo-2Hpyran-3-carboxylates (9a-e) by cyclopentanone (2) has been delineated. The synthetic potential of 2-pyranone was explored further to generate mo'iecular diversity using 6-aryl-4-secamino- 2-oxo-2H-pyran-3-carbonitriles (7a-h), 5,6-diaryl-4-methylsulfanyl-2-oxo2H-pyran-3-carbonitriles (Sa,b) and methyl 5,6-diaryl-4- methylsulfanyl-2-oxo-2H-pyran-3-carboxylates (12a,b) as precursors for the ring transformation by cyclopentanone to assess the effects of substituents on the course of the reaction to obtain highly congested indans, 6,7diaryl-5-methylsulfanylindan-4-carbonitriles (6a,b), 7-aryl-5-(piperidin-I-yl)indancarbonitriles (8a-h) and methyl 6,7-4- diaryl-5-methylsulfanylindan-4-carboxylate 13a,b).

Mechanistic insights from the crystal structures of a feast/famine regulatory protein from Mycobacterium tuberculosis H37Rv

Rv3291c gene from Mycobacterium tuberculosis codes for a transcriptional regulator belonging to the (leucine responsive regulatory protein/regulator of asparigine synthase C gene product) Lrp/AsnC-family. We have identified a novel effectorbinding site from crystal structures of the apo protein, complexes with a variety of amino acid effectors, X-ray based ligand screening and qualitative fluorescence spectroscopy experiments. The new effector site is in addition to the structural characterization of another distinct site in the protein conserved in the related AsnC-family of regulators. The structures reveal that the ligandbinding loops of two crystallographically ndependent subunits adopt different conformations to generate two distinct effector-binding sites. A change in the conformation of the binding site loop 100–106 in the B subunit is apparently necessary for octameric association and also allows the loop to interact with a bound ligand in the newly identified effector-binding site. There are four sites of each kind in the octamer and the protein preferentially binds to aromatic amino acids. While amino acids like Phe, Tyr and Trp exhibit binding to only one site, His exhibits binding to both sites. Binding of Phe is accompanied by a conformational change of 3.7A ° in the 75–83 loop, which is advantageously positioned to control formation of higher oligomers. Taken together, the present studies suggest an elegant control mechanism for global transcription regulation involving binding of ligands to the two sites, individually or collectively.

P450 oxidoreductase deficiency: a new disorder of steroidogenesis

Microsomal P450 enzymes, which metabolize drugs and catalyze steroid biosynthesis require electron donation from NADPH via P450 oxidoreductase (POR). POR knockout mice are embryonically lethal, but we found recessive human POR missense mutations causing disordered steroidogenesis and Antley-Bixler syndrome (ABS), a skeletal malformation syndrome featuring craniosynostosis. Dominant mutations in exons 8 and 10 of fibroblast growth factor receptor 2 (FGFR2) cause phenotypically related craniosynostosis syndromes and were reported in patients with ABS and normal steroidogenesis. Sequencing POR and FGFR2 exons in 32 patients with ABS and/or hormonal findings suggesting POR deficiency showed complete genetic segregation of POR and FGFR2 mutations. Fifteen patients carried POR mutations on both alleles, four carried POR mutations on 1 allele, nine carried FGFR2/3 mutations on one allele and no mutation was found in three patients. The 34 affected POR alleles included 10 with A287P, 7 with R457H, 9 other missense mutations and 7 frameshifts. These 11 missense mutations and 10 others identified by database mining were expressed in E. coli, purified to apparent homogeneity, and their catalytic capacities were measured in four assays: reduction of cytochrome c, oxidation of NADPH, and support of the 17alpha-hydroxylase and 17,20 lyase activities of human P450c17. As assessed by Vmax/Km, 17,20 lyase activity provided the best correlation with clinical findings. Modeling human POR on the X-ray crystal structure of rat POR shows that these mutant activities correlate well with their locations in the structure. POR deficiency is a new disease, distinct from the craniosynostosis syndromes caused by FGFR mutations.

Diversity and function of mutations in p450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis

P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 "private" mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17alpha-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR's support of catalysis by P450c17--the enzyme most closely associated with the hormonal phenotype--provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.

P450 oxidoreductase deficiency: a new disorder of steroidogenesis affecting all microsomal P450 enzymes

Combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase activities was first described in 1985; however the genes for P450c17 and P450c21 in these patients lack mutations. In 1986 we postulated that this disorder might be due to mutations in P450 oxidoreductase (POR), the flavoprotein that donates electron to these and all other microsomal P450 enzymes, but this hypothesis was not tested until the POR gene sequence became available through the genome database. We found five POR missense mutations in our first four patients. In vitro assays of the activities of these mutations showed that the standard assay of POR activity, reduction of cytochrome c, correlated poorly with the patients' phenotypes, but that assays of POR-supported 17alpha-hydroxylase and 17,20 lyase activities correlated well. POR deficiency is a new disorder of adrenal and gonadal steroidogenesis that affects all microsomal cytochrome P450 enzymes, hence may have important implications for genetic differences in drug metabolism.