Biomedical subjects

Marion B Sewer

Publications and source records attributed to Marion B Sewer.

9 recordsLinked to original sources

PubMed2006

Steroidogenic factor-1 is a sphingolipid binding protein.

Steroidogenic factor (SF1, NR5A1, Ad4BP) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Studies have found that the ability of this receptor to increase target gene expression can be regulated by post-translational modification, subnuclear localization, and protein-protein interactions. Recent crystallographic studies and our mass spectrometric analyses of the endogenous receptor have demonstrated an integral role for ligand-binding in the control of SF1 transactivation activity. Herein, we discuss our findings that sphingosine is an endogenous ligand for SF1. These studies and the structural findings of others have demonstrated that the receptor can bind both sphingolipids and phospholipids. Thus, it is likely that multiple bioactive lipids are ligands for SF1 and that these lipids will differentially act to control SF1 activity in a context-dependent manner. Finally, these findings highlight a central role for bioactive lipids as mediators of trophic hormone-stimulated steroid hormone biosynthesis.

Animals↗

PubMed2006

Coregulator exchange and sphingosine-sensitive cooperativity of steroidogenic factor-1, general control nonderepressed 5, p54, and p160 coactivators regulate cyclic adenosine 3',5'-monophosphate-dependent cytochrome P450c17 transcription rate.

Transcription of the cytochrome P450 17 (CYP17) gene is regulated by cAMP-dependent binding of steroidogenic factor-1 (SF-1) to its promoter in the adrenal cortex. Using temporal chromatin immunoprecipitation and mammalian two-hybrid experiments, we establish the reciprocal presence of coactivators [general control nonderepressed (GCN5), cAMP response element-binding protein-binding protein, p300, p300/cAMP response element-binding protein-binding protein CBP associated factor, p160s, polypyrimidine tract associated splicing factor, and p54(nrb)], corepressors (class I histone deacetylases, receptor interacting protein, nuclear receptor corepressor, and Sin3A), and SWI/SNF (human homolog of yeast mating type switching/sucrose nonfermenting) and imitation SWI chromatin remodeling ATPases on the CYP17 promoter during transcription cycles in the H295R adrenocortical cell line. A ternary GCN5/SRC-1/SF-1 complex forms on the CYP17 promoter with cAMP-dependence within 30 min of cAMP stimulation, and corresponds with SWI/SNF chromatin remodeling. This complex is sensitive to the SF-1 antagonist sphingosine and results in decreased transcription of CYP17. GCN5 acetyltransferase activity and carboxy terminus binding proteins alternatively mediate disassembly of the complex. This work establishes the temporal order of cAMP-induced events on the promoter of a key steroidogenic gene during SF-1-mediated transcription.

Acetylation↗

PubMed2006

Sphingosine regulates the transcription of CYP17 by binding to steroidogenic factor-1.

Steroidogenic factor (SF1, Ad4BP, NR5A1) is a nuclear receptor that is essential for steroid hormone biosynthesis and endocrine development. Recent crystallographic studies have found that phospholipids are ligands for SF1. In the present study, our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using tandem mass spectrometry, we show that in H295R adrenocortical cells, SF1 is bound to sphingosine (SPH) and lyso-sphingomyelin (lysoSM) under basal conditions and that cAMP stimulation decreases the amount of SPH and lysoSM bound to the receptor. Silencing both acid and neutral ceramidases using small interfering RNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. SPH antagonized the ability of cAMP and the coactivator steroid receptor coactivator-1 to increase CYP17 reporter gene activity. These studies demonstrate that SPH is a bonafide endogenous ligand for SF1 and a negative regulator of CYP17 gene expression.

Binding Sites↗

PubMed2005

Cyclic adenosine 5'-monophosphate-dependent sphingosine-1-phosphate biosynthesis induces human CYP17 gene transcription by activating cleavage of sterol regulatory element binding protein 1.

In the human adrenal cortex, ACTH activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondrion and chronically increasing the transcription of steroidogenic genes (including CYP17) via a cAMP-dependent pathway. In the present study, we characterized the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) and the content of several sphingolipid species quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of several sphingolipids, including sphingomyelin, ceramides, and sphingosine and stimulating the activity of sphingosine kinase and increasing the release of sphingosine-1-phosphate (S1P) into the media. S1P increased CYP17 mRNA expression by promoting the cleavage and nuclear localization of sterol regulatory element binding protein (SREBP) 1. Chromatin immunoprecipitation assays revealed that Bt2cAMP and S1P increased acetylation of histone H3 and promoted binding of SREBP1 to the -520/-331 region of the CYP17 promoter. In summary, our studies demonstrate a role for sphingolipid metabolism and SREBP1 in ACTH-dependent CYP17 regulation and steroidogenesis.

Blotting, Northern↗

PubMed2004

ACTH regulates steroidogenic gene expression and cortisol biosynthesis in the human adrenal cortex via sphingolipid metabolism.

Sphingolipids are a diverse family of phospholipids and glycolipids that mediate cell-cell interactions, participate in signal transduction pathways and modulate the activity of various cellular proteins and receptors. The objective of the present studies was to characterize the role of the sphingolipid biosynthetic pathway in adrenocorticotropin (ACTH)-dependent steroidogenic gene expression and cortisol production. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) for various time periods and the content of sphingolipids was quantified by mass spectrometry. Treatment of H295R cells with ACTH and Bt2cAMP activated sphingolipid metabolism within five minutes. Decreases were found in the cellular levels of several sphingolipids, including sphingomyelin (SM) and glucosylceramide. ACTH/cAMP rapidly decreased levels of the signaling molecules ceramide, sphingosine and sphingosine-1-phosphate (S1P). The effect of these bioactive sphingolipids on steroidogenic gene expression was also examined. Both sphingosine and S1P were found to increase endogenous CYP17 mRNA and activate the transcriptional activity of CYP17-luciferase reporter constructs. Further, sphingosine and S1P rapidly increase cortisol biosynthesis in H295R cells. In summary, our studies establish a link between ACTH/cAMP-dependent steroidogenesis and sphingolipid metabolism in the human adrenal cortex. Finally, these findings suggest that sphingolipids may serve as signaling mediators in ACTH-stimulated cortisol biosynthesis.

Adrenal Cortex↗

PubMed2003

ACTH modulation of transcription factors responsible for steroid hydroxylase gene expression in the adrenal cortex.

Steroid hormone biosynthesis in the adrenal cortex and gonads involves the coordinated transcription of the genes encoding the steroid hydroxylases, 3beta-hydroxysteroid dehydrogenase (3betaHSD), the steroidogenic acute regulatory protein (StAR), and adrenodoxin (Adx). Transcriptional regulation of steroidogenic genes is multifactorial, entailing developmental, tissue-specific, constitutive, and cAMP-dependent mechanisms. Optimal steroidogenic capacity is achieved by the actions of ACTH which exerts transcriptional pressure on all steroidogenic genes. The actions of ACTH in the adrenal cortex have been studied in great detail and is mediated by cAMP and protein kinase A (PKA) via two temporally distinct pathways. The acute response leads to mobilization of cholesterol, the initial substrate for all steroidogenic pathways, from cellular stores to the inner mitochondrial membrane where cholesterol sidechain cleavage cytochrome P450 (P45011A1) resides. The slower, chronic response of ACTH in the adrenal cortex directs transcription of the genes encoding the steroidogenic enzymes. Although steroidogenic gene transcription in response to ACTH is cAMP-dependent, the consensus cAMP response pathway (CRE/CREB) is not involved. Instead, each steroidogenic gene utilizes unique cAMP-responsive sequences (CRS) found in the promoters of each gene, which bind a diverse array of transcription factors. Moreover, once specific transcription factors are bound to the promoters of the steroidogenic genes, increased gene expression requires posttranslational modification (phosphorylation/dephosphorylation) of the transcription factors and binding of coactivator proteins. This review provides a general view (with emphasis on the human) of the important factors involved in regulating steroidogenic gene expression and ultimately steroid hormone biosynthesis.

Adrenal Cortex↗

PubMed2002

CAMP-dependent protein kinase enhances CYP17 transcription via MKP-1 activation in H295R human adrenocortical cells.

Steroid hormone biosynthesis in the adrenal cortex is controlled by adrenocorticotropin (ACTH), which increases intracellular cAMP, resulting in the activation of cAMP-dependent protein kinase(PKA) and subsequent increase in steroidogenic gene transcription. We have found that a dual-specificity phosphatase is essential for conveying ACTH/cAMP-stimulated transcription of several steroidogenic genes in the human adrenal cortex. In the present study, the role of mitogen-activated protein kinase phosphatase-1 (MKP-1), a nuclear dual-specificity phosphatase, in the transcriptional activation of human CYP17 (hCYP17) in H295R human adrenocortical cells is established. Stimulation of H295R cells with dibutyryl-cAMP (Bt(2)cAMP) induces MKP-1 mRNA and protein expression within 30 min of exposure. In transient-transfection studies, transcriptional activity of an hCYP17 promoter-reporter construct was increased by Bt(2)cAMP and by overexpression of PKA or MKP-1. Furthermore, PKA phosphorylated an MKP-1-glutathione S-transferase fusion protein in in vitro assays and Bt(2)cAMP increased (32)P associated with MKP-1 that was immunoprecipitated from H295R cells. Finally, silencing MKP-1 expression using antisense oligonucleotides attenuated cAMP-stimulated hCYP17 expression, whereas silencing of ERK1/2 increased hCYP17 expression. These findings demonstrate integral roles for MKP-1 and ERK1/2 via regulation of the phosphorylation state of steroidogenic factor-1 (SF-1) in mediating ACTH/cAMP-dependent transcription of hCYP17, thereby maintaining the balance between transcriptional activation and repression.

Adrenal Cortex↗

PubMed2002

Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription.

The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.

Adrenal Cortex↗

PubMed2002

Adrenocorticotropin/cyclic adenosine 3',5'-monophosphate-mediated transcription of the human CYP17 gene in the adrenal cortex is dependent on phosphatase activity.

cAMP-dependent transcription of steroid hydroxylase genes involves activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream target proteins. Although the requirement for the activation of PKA is well established, none of the transcription factors required for steroid hydroxylase gene transcription have been found to be PKA phosphoproteins. In this study we examined the role of changes in phosphorylation state on the expression and transcriptional activity of the human CYP17 gene (hCYP17). Using inhibitors of serine/threonine phosphatase activity (okadaic acid) and phosphotyrosine phosphatase activity (peroxyvanadate), we can inhibit the cAMP-inducible binding of the steroidogenic factor-1 (SF-1), p54(nrb)/NonO, and polypyrimidine tract-binding protein-associated splicing factor (PSF) complex required for regulation of transcription to the promoter of hCYP17. Further, both okadaic acid and peroxyvanadate attenuate cAMP-stimulated increases in endogenous hCYP17 mRNA expression and in hCYP17 promoter-reporter construct luciferase activity. In vivo phosphorylation and immunoprecipitation of SF-1 show a cAMP-stimulated decrease in (32)P-labeled SF-1. Our findings demonstrate that activation of protein phosphatase(s) is essential for cAMP-dependent transcription of hCYP17 in H295R cells and suggest a role for PKA in phosphatase activation, which leads to dephosphorylation of SF-1 and increased gene transcription.

Adrenal Cortex↗