Descripción: 11α,12α-aziridinosteroids (2a, b, c) were prepared from 5β-H-11-pregnene-3, 20-dione (1) using different iminophenyliodinanes and cloramine aziridination reagents.

Descripción: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90-95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients. © 2011 Minutolo et al.

Descripción: Purification of the ethanolic extract of the starfish L. annulatus led to the isolation of two sulfated glycosides and a pentahydroxylated steroid. One of the saponins contains a novel pentasaccharide chain attached to C-6 of the steroidal aglycone.

Descripción: Glucocorticoid receptor (GR) is cytoplasmic in the absence of ligand and localizes to the nucleus after steroid binding. Previous evidence demonstrated that the hsp90-based heterocomplex bound to GR is required for the efficient retrotransport of the receptor to the nuclear compartment. We examined the putative association of GR and its associated chaperone heterocomplex with structures of the nuclear pore. We found that importin β and the integral nuclear pore glycoprotein Nup62 interact with hsp90, hsp70, p23, and the TPR domain proteins FKBP52 and PP5. Nup62 and GR were able to interact in a more efficient manner when chaperoned by the hsp90-based heterocomplex. Interestingly, the binding of hsp70 and p23 to Nup62 does not require the presence of hsp90, whereas the association of FKBP52 and PP5 is hsp90 dependent, as indicated by the results of experiments where the hsp90 function was disrupted with radicicol. The ability of both FKBP52 and PP5 to interact with Nup62 was abrogated in cells overexpressing the TPR peptide. Importantly, GR cross-linked to the hsp90 heterocomplex was able to translocate to the nucleus in digitonin-permeabilized cells treated with steroid, suggesting that GR could pass through the pore in its untransformed state. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Descripción: Rapid, ligand-dependent movement of glucocorticoid receptors (GR) from cytoplasm to the nucleus is hsp90-dependent, and much of the movement system has been defined. GR-hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus. The immunophilins link to dynein indirectly via the dynamitin component of the dynein-associated dynactin complex (Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) J. Biol. Chem. 279, 22483-22489). Although it is known that rapid, hsp90-dependent GR movement requires intact microtubules, it has not been shown that the movement is dynein-dependent. Here, we show that overexpression of dynamitin, which blocks movement by dissociating the dynein motor from its cargo, inhibits ligand-dependent movement of the GR to the nucleus. We show that native GR·hsp90·immnunophilin complexes contain dynamitin as well as dynein and that GR heterocomplexes isolated from cytosol containing paclitaxel and GTP to stabilize microtubules also contain tubulin. The complete movement system, including the dynein motor complex and tubulin, can be assembled under cell-free conditions by incubating GR immune pellets with paclitaxel/GTP-stabilized cytosol prepared from GR - L cells. This is the first evidence that the movement of a steroid receptor is dynein-dependent, and it is the first isolation of a steroid receptor bound to the entire system that determines its retrograde movement.

Descripción: 17(13→18)-Abeo and D-homo analogs of the natural neurosteroid 3α-hydroxy-5αH-pregnan-20-one were prepared by anionic or radical (mercury (II) hydride mediated) rearrangements of steroidal cyclopropylketones respectively.

Descripción: Diosgeninlactone (1), a natural product from Solanum vespertilio, was stereo-selectively synthesized in high yield from 3β-hydroxy-5-androstene.

Descripción: 3α-Hydroxy-2,19-oxido-5αH-pregnan-20-one was synthesized in 6 steps from the easily available 19-hydroxypregnenolone 3-acetate. The 2,19-oxido bridge was formed in a single step upon spontaneous cyclization and desilylation of a TBDMS protected 19-hydroxy-2α,3α-epoxypregnane. The 4,19-oxido analogue was obtained as a byproduct upon cyclization of the isomeric 3α,4α-epoxide.

Descripción: The three related 160-kDa proteins, SRC-1, TIF-2 and RAC-3, were initially identified as factors interacting with nuclear receptors. They have also been reported to potentiate the activity of other transcription factors such as AP-1 or NF-κB. The aim of this work was to identify whether SRC-1 interferes with the TGF-β/Smad signaling pathway, and if so, to identify its underlying mechanisms of action. Using transient cell transfection experiments performed in human dermal fibroblasts with the Smad3/4-specific (SBE) 4-lux reporter construct, as well as the human PAI-1 promoter, we determined that SRC-1 enhances TGF-β-induced, Smad-mediated, transcription. Likewise, SRC-1 overexpression potentiated TGF-β-induced upregulation of PAI-1 steady-state mRNA levels. Using a mammalian two-hybrid system, we demonstrated that SRC-1 interacts with the transcriptional co-activators p300/CBP, but not with Smad3. Overexpression of the adenovirus E1A oncoprotein, an inhibitor of CBP/p300 activity, prevented the enhancing effect of SRC-1 on Smad3/4-mediated transcription, indicating that p300/CBP may be required for SRC-1 effect. Such hypothesis was validated, as expression of a mutant form of SRC-1 lacking the CBP/p300-binding site failed to upregulate Smad3/4-dependent transcription, while full-length SRC-1 potentiated p300-Smad3 interactions. These results identify SRC-1 as a novel Smad3/4 transcriptional partner, facilitating the functional link between Smad3 and p300/CBP. © 2005 Nature Publishing Group All rights reserved.

Descripción: The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-XL (antiapoptotic)/bcl-XS (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-X L/bcl-XS favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: The alkylation of amino groups of the mineralocorticoid receptor (MR) with pyridoxal 5′-phosphate or 2,4,6-trinitrobenzenesulphonate (TNBS) under controlled conditions modifies only one lysyl residue, which accounts for a 70% inhibition of steroid binding capacity. The Kd of aldosterone for MR is not affected by the treatment, but the total number of binding sites is greatly decreased. The modified receptor is capable of dynamically conserving its association with the hsp90-based heterocomplex. Importantly, the binding of natural agonists protects the hormone binding capacity of the MR from the inactivating action of alkylating agents. In contrast, antagonistic steroids are totally incapable of providing such protection. Like the antagonistic ligands, and despite its potent mineralocorticoid biological effect, the sole MR specific synthetic agonist known to date, 11,19-oxidoprogesterone (11-OP), shows no protective effect upon treatment of the MR with pyridoxal 5′-phosphate or TNBS. Limited digestion of the MR with α-chymotrypsin generates a 34 kDa fragment, which becomes totally resistant to digestion upon binding of natural agonists, but not upon binding of antagonists. Interestingly, the synthetic 21-deoxypregnanesteroid 11-OP exhibits an intermediate pattern of proteolytic degradation, suggesting that the conformational change generated in the MR is not equivalent to that induced by antagonists or natural agonists. We conclude that in the first steps of activation, the MR changes its conformation upon binding of the ligand. However, the nature of this conformational change depends on the nature of the ligand. The experimental evidence shown in this work suggests that a single lysyl group can determine the hormone specificity of the MR. © 2002 Elsevier Science B.V. All rights reserved.

Descripción: Nur factors are critical for proopiomelanocortin (POMC) induction by CRH in corticotrophs, but the pathways linking CRH to Nur are unknown. In this study we show that in AtT-20 corticotrophs CRH and cAMP induce Nur77 and Nurr1 expression and transcription at the NurRE site by protein kinase A (PKA) and calcium-dependent and -independent mechanisms. Calcium pathways depend on calmodulin kinase II (CAMKII) activity, and calcium-independent pathways are accounted for in part by MAPK activation (Rap1/B-Raf/MAPK-ERK kinase/ERK1/2), demonstrated by the use of molecular and pharmacological tools. ATT-20 corticotrophs express B-Raf, as do other cells in which cAMP stimulates MAPK. CRH/cAMP stimulated ERK2 activity and increased transcriptional activity of a Gal4-Elk1 protein, which was blocked by overexpression of dominant negative mutants and kinase inhibitors and stimulated by expression of B-Raf. The MAPK kinase inhibitors did not affect Nur77 and Nurr1 mRNA induction but blocked CRH or cAMP-stimulated Nur transcriptional activity. Moreover, MAPK stimulated phosphorylation and transactivation of Nur77. The functional impact of these pathways was confirmed at the POMC promoter. In conclusion, in AtT-20 corticotrophs the CRH/cAMP signaling that leads to Nur77/Nurr1 mRNA induction and transcriptional activation, and thus POMC expression, is dependent on protein kinase A and involves calcium/calmodulin kinase II (Nur induction/activation) and MAPK calcium-dependent and -independent (Nur phosphorylation-activation) pathways.

Descripción: Background: The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. Methodology/Principal Findings: Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GRDNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. Conclusions/Significance: The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior. © 2010 Presman et al.

Descripción: A number of natural and synthetic substances are used in the treatment of immunological disorders. The immunosuppressive drugs are widely utilized in clinical treatments of autoimmune disorders, in the prevention of transplant rejection as well as in non-autoimmune diseases such as allergy. The design of immunosuppressive therapies is based on the control of the exacerbated immune response. The pathophysiologic mean of this concept is to modulate the action of mononuclear cells, being T cells the main targets. Immunosuppressive agents have different molecular targets, and an important drawback in their use is that they also inhibit the normal immune system response. Depending on their mode of action, immunosuppressive drugs can be classified in four different groups: antinflammatory drugs of the corticosteroid family, inhibitors of the calcineurin pathway, cytototoxic or antiproliferative drugs and specific antibodies. In this article, we focus on the molecular action of immunosuppressive drugs such as steroids, cyclosporine, tacrolimus, azathioprine, cyclophosphamide, sirolimus, mycophenolate mofetil, leflunomide and specific antibodies, providing data to characterize and improve the use of these agents.

Descripción: Testicular macrophages as well as endothelial cells, which are intimately associated with Leydig cells, constitute a potential source of paracrine nitric oxide (NO) in the testis. In the present study, we investigated the effect of NO donors on MA-10 murine Leydig tumor cell line and rat Leydig cell steroidogenesis. We show that NO donors inhibit human CG- induced steroidogenesis in both type of cells. We also studied NO mechanism of action. Contrary to what is observed in many other systems, NO inhibitory effect on Leydig cell steroidogenesis is not mediated by cyclic GMP (cGMP) because NO fails to increase cGMP production, and cGMP analogs do not reproduce NO effect. NO does not modify the production of cAMP, the main second messenger that mediates gonadotropin action. When we studied NO effect over the steroidogenic pathway in MA-10 cells, we found that NO was inhibiting the conversion of cholesterol to pregnenolone. Taken together these results show an inhibitory effect of NO donors on Leydig cell steroidogenesis, and suggest that NO can be directly inhibiting cholesterol side-chain cleavage enzyme (cytochrome P450(scc)) as it does with other heme proteins, including different cytochromes P459.

Descripción: Messenger RNA (mRNA) for enzymes involved in adrenal steroid biosynthesis are expressed in the brain, and the coded enzymes have been shown to be active. The expression of mRNA for the cytochrome P-450 enzyme aldosterone synthase, crucial for the final step in the synthesis of aldosterone and the synthesis of aldosterone was studied in several anatomic areas of the rat brain. Expression of the mRNA for the aldosterone synthase was demonstrated by RT-PCR/Southern blot in adrenal, aorta, hypothalamus, hippocampus, amygdala, cerebrum, and cerebellum. Incubation of brain minces from intact and adrenalectomized rats demonstrated the synthesis of corticosterone and aldosterone from endogenous precursors. Incubations of brain mince, with [1,23H]-deoxycorticosterone, followed by extraction and three different successive TLCs, demonstrated the presence of labeled aldosterone, corticosterone, and 18-hydroxy-deoxycorticosterone. Incubation, in the presence of 10 μM cortisol or metyrapone, inhibited the synthesis of aldosterone or both aldosterone and corticosterone, respectively. These studies indicate that the rat brain has the enzymatic machinery for the synthesis of adrenal corticosteroids and is capable of synthesizing aldosterone. Aldosterone synthesized in the brain might play a paracrine role in the regulation of blood pressure.

Descripción: Brassinosteroids (BRs) are involved in numerous physiological processes associated with plant development and especially with cell expansion. Here we report that two BRs, 28-homobrassinolide (HBL) and its direct precursor 28-homocastasterone (HCS), promote cell expansion of Arabidopsis thaliana suspension cells. We also show that cell expansions induced by HBL and HCS are correlated with the amplitude of the plasma membrane hyperpolarization they elicited. HBL, which promoted the larger cell expansion, also provoked the larger hyperpolarization. We observed that membrane hyperpolarization and cell expansion were partially inhibited by the proton pump inhibitor erythrosin B, suggesting that proton pumps were not the only ion transport system modulated by the two BRs. We used a voltage clamp approach in order to find the other ion transport systems involved in the PM hyperpolarization elicited by HBL and HCS. Interestingly, while anion currents were inhibited by both HBL and HCS, outward rectifying K+ currents were increased by HBL but inhibited by HCS. The different electrophysiological behavior shown by HBL and HCS indicates that small changes in the BR skeleton might be responsible for changes in bioactivity. © 2005 JSPP.

Descripción: Interactions between steroid hormone receptors and signal transducer and activator of transcription (Stat)-mediated signaling pathways have already been described. In the present study, we explored the capacity of progestins to modulate Stat3 transcriptional activation in an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in BALB/c mice and in the human breast cancer cell line T47D. We found that C4HD epithelial cells, from the MPA-induced mammary tumor model, expressed Stat3 and that MPA treatment of C4HD cells up-regulated Stat3 protein expression. In addition, MPA induced rapid, nongenomic Stat3, Jak1, and Jak2 tyrosine phosphorylation in C4HD and T47D cells. MPA treatment of C4HD cells also resulted in rapid c-Src tyrosine phosphorylation. These effects were completely abolished by the progestin antagonist RU486. Abrogation of Jak1 and Jak2 activity by transient transaction of C4HD cells with dominant negative (DN) Jak1 or DN Jak2 vectors, or inhibition of Src activity by preincubation of cells with the Src family kinase inhibitor PP2, blocked the capacity of MPA to induce Stat3 phosphorylation. Treatment of C4HD cells with MPA induced Stat3 binding to DNA. In addition, MPA promoted strong Stat3 transcriptional activation in C4HD and T47D cells that was inhibited by RU486 and by blockage of Jak1, Jak2, and Src activities. To investigate the correlation between MPA-induced Stat3 activation and cell growth, C4HD cells were transiently transfected with a DN Stat3 expression vector, Stat3Y705-F, or with a constitutively activated Stat3 mutant, Stat3-C. While expression of Stat3Y705-F mutant had an inhibitory effect on MPA-induced growth of C4HD cells, transfection with the constitutively activated Stat3-C vector resulted in MPA-independent proliferation. Finally, we addressed the effect of targeting Stat3 in in vivo growth of C4HD breast tumors. Blockage of Stat3 activation by transfection of C4HD cells with the DN Stat3Y705-F expression vector significantly inhibited these cells' ability to form tumors in syngeneic mice. Our results have for the first time demonstrated that progestins are able to induce Stat3 transcriptional activation, which is in turn an obligatory requirement for progestin stimulation of both in vitro and in vivo breast cancer growth. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Descripción: The antiapoptotic effect of melatonin (MEL) has been described in several systems. In particular, MEL inhibits glucocorticoid-mediated apoptosis. Our group previously demonstrated that in the thymus, MEL inhibits the release of Cytochrome C from mitochondria and the dexamethasone-dependent increase of bax mRNA levels. In this study we analyzed the ability of MEL to regulate the activation of the glucocorticoid receptor (GR) in mouse thymocytes. We found that even though the methoxyindole does not affect the ligand binding capacity of the receptor, it impairs the steroid-dependent nuclear translocation of the GR and also prevents transformation by blocking the dissociation of the 90-kDa heat shock protein. Coincubation of the methoxyindole with dexamethasone did not affect the expression of a reporter gene in GR-transfected Cos-7 cells or HC11 and L929 mouse cell lines that express Mel-1a and retinoid-related orphan receptor-α (RORα) receptors. Therefore, the antagonistic effect of MEL seems to be specific for thymocytes, in a Mel 1a- and RORα-independent manner. In summary, the present results suggest a novel mechanism for the antagonistic action of MEL on GR-mediated effects, which involves the inhibition of 90-kDa heat shock protein dissociation and the cytoplasmic retention of the GR. Copyright © 2006 by The Endocrine Society.