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1.
Mol Cell Endocrinol ; 427: 73-85, 2016 May 15.
Article in English | MEDLINE | ID: mdl-26992564

ABSTRACT

Oxysterol binding protein-related protein 2 (ORP2) is a lipid binding protein that has been implicated in various cellular processes, including lipid sensing, cholesterol efflux, and endocytosis. We recently identified ORP2 as a member of a protein complex that regulates glucocorticoid biosynthesis. Herein, we examine the effect of silencing ORP2 on adrenocortical function and show that the ORP2 knockdown cells exhibit reduced amounts of multiple steroid metabolites, including progesterone, 11-deoxycortisol, and cortisol, but have increased concentrations of androgens, and estrogens. Moreover, silencing ORP2 suppresses the expression of most proteins required for cortisol production and reduces the expression of steroidogenic factor 1 (SF1). ORP2 silencing also increases cellular cholesterol, concomitant with decreased amounts of 22-hydroxycholesterol and 7-ketocholesterol, two molecules that have been shown to bind to ORP2. Further, we show that ORP2 binds to liver X receptor (LXR) and is required for nuclear LXR expression. LXR and ORP2 are recruited to the CYP11B1 promoter in response to cAMP signaling. Additionally, ORP2 is required for the expression of other LXR target genes, including ABCA1 and the LDL receptor (LDLR). In summary, we establish a novel role for ORP2 in regulating steroidogenic capacity and cholesterol homeostasis in the adrenal cortex.


Subject(s)
Cholesterol/metabolism , Hydrocortisone/biosynthesis , Receptors, Steroid/physiology , Adrenal Cortex/metabolism , Cell Line , Dehydroepiandrosterone/biosynthesis , Gene Knockdown Techniques , Gene Silencing , Homeostasis/drug effects , Humans , Receptors, Steroid/genetics , Steroids/metabolism
2.
Mol Cell Biol ; 35(7): 1223-37, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25605330

ABSTRACT

Glucocorticoid production in the adrenal cortex is activated in response to an increase in cyclic AMP (cAMP) signaling. The nuclear protein p54(nrb)/NONO belongs to the Drosophila behavior/human splicing (DBHS) family and has been implicated in several nuclear processes, including transcription, splicing, and RNA export. We previously identified p54(nrb)/NONO as a component of a protein complex that regulates the transcription of CYP17A1, a gene required for glucocorticoid production. Based on the multiple mechanisms by which p54(nrb)/NONO has been shown to control gene expression and the ability of the protein to be recruited to the CYP17A1 promoter, we sought to further define the molecular mechanism by which p54(nrb)/NONO confers optimal cortisol production. We show here that silencing p54(nrb)/NONO expression in H295R human adrenocortical cells decreases the ability of the cells to increase intracellular cAMP production and subsequent cortisol biosynthesis in response to adrenocorticotropin hormone (ACTH) stimulation. Interestingly, the expression of multiple phosphodiesterase (PDE) isoforms, including PDE2A, PDE3A, PDE3B, PDE4A, PDE4D, and PDE11A, was induced in p54(nrb)/NONO knockdown cells. Investigation of the mechanism by which silencing of p54(nrb)/NONO led to increased expression of select PDE isoforms revealed that p54(nrb)/NONO regulates the splicing of a subset of PDE isoforms. Importantly, we also identify a role for p54(nrb)/NONO in regulating the stability of PDE transcripts by facilitating the interaction between the exoribonuclease XRN2 and select PDE transcripts. In summary, we report that p54(nrb)/NONO modulates cAMP-dependent signaling, and ultimately cAMP-stimulated glucocorticoid biosynthesis by regulating the splicing and degradation of PDE transcripts.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/genetics , Cyclic AMP/metabolism , Glucocorticoids/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Octamer Transcription Factors/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Cell Line , DNA-Binding Proteins , Gene Expression Regulation , Gene Silencing , Glucocorticoids/genetics , Humans , Hydrocortisone/genetics , Hydrocortisone/metabolism , Nuclear Matrix-Associated Proteins/genetics , Octamer Transcription Factors/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proteolysis , RNA-Binding Proteins/genetics
3.
Biochim Biophys Acta ; 1841(4): 552-62, 2014 Apr 04.
Article in English | MEDLINE | ID: mdl-24369117

ABSTRACT

Diacylglycerol kinase theta (DGKθ) plays a pivotal role in regulating adrenocortical steroidogenesis by synthesizing the ligand for the nuclear receptor steroidogenic factor 1 (SF1). In response to activation of the cAMP signaling cascade nuclear DGK activity is rapidly increased, facilitating PA-mediated, SF1-dependent transcription of genes required for cortisol and dehydroepiandrosterone (DHEA) biosynthesis. Based on our previous work identifying DGKθ as the enzyme that produces the agonist for SF1, we generated a tetracycline-inducible H295R stable cell line to express a short hairpin RNA (shRNA) against DGKθ and characterized the effect of silencing DGKθ on adrenocortical gene expression. Genome-wide DNA microarray analysis revealed that silencing DGKθ expression alters the expression of multiple genes, including steroidogenic genes, nuclear receptors and genes involved in sphingolipid, phospholipid and cholesterol metabolism. Interestingly, the expression of sterol regulatory element binding proteins (SREBPs) was also suppressed. Consistent with the suppression of SREBPs, we observed a down-regulation of multiple SREBP target genes, including 3-hydroxy-3-methylglutary coenzyme A reductase (HMG-CoA red) and CYP51, concomitant with a decrease in cellular cholesterol. DGKθ knockdown cells exhibited a reduced capacity to metabolize PA, with a down-regulation of lipin and phospholipase D (PLD) isoforms. In contrast, suppression of DGKθ increased the expression of several genes in the sphingolipid metabolic pathway, including acid ceramidase (ASAH1) and sphingosine kinases (SPHK). In summary, these data demonstrate that DGKθ plays an important role in steroid hormone production in human adrenocortical cells.


Subject(s)
Adrenal Cortex/metabolism , Cholesterol/metabolism , Dehydroepiandrosterone/biosynthesis , Diacylglycerol Kinase/metabolism , Hydrocortisone/biosynthesis , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Diacylglycerol Kinase/antagonists & inhibitors , Diacylglycerol Kinase/genetics , Gene Expression Regulation , Humans , Lipid Metabolism/drug effects , Phosphorylation , Promoter Regions, Genetic , RNA Splicing Factors , Signal Transduction/drug effects , Sterol Regulatory Element Binding Proteins/biosynthesis , Transcription Factors/genetics , Transcription Factors/metabolism
4.
Biochem J ; 454(2): 267-74, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23767959

ABSTRACT

DGKs (diacylglycerol kinases) catalyse the conversion of diacylglycerol into PA (phosphatidic acid), a positive modulator of mTOR (mammalian target of rapamycin). We have found that chenodeoxycholic acid and the synthetic FXR (farnesoid X receptor) ligand GW4064 induce the mRNA and protein expression of DGKθ in the HepG2 cell line and in primary human hepatocytes. Reporter gene studies using 1.5 kB of the DGKθ promoter fused to the luciferase gene revealed that bile acids increase DGKθ transcriptional activity. Mutation of putative FXR-binding sites attenuated the ability of GW4046 to increase DGKθ luciferase activity. Consistent with this finding, ChIP (chromatin immunoprecipitation) assays demonstrated that bile acid signalling increased the recruitment of FXR to the DGKθ promoter. Furthermore, GW4064 evoked a time-dependent increase in the cellular concentration of PA. We also found that GW4064 and PA promote the phosphorylation of mTOR, Akt and FoxO1 (forkhead box O1), and that silencing DGKθ expression significantly abrogated the ability of GW4046 to promote the phosphorylation of these PA-regulated targets. DGKθ was also required for bile-acid-dependent decreased glucose production. Taken together, our results establish DGKθ as a key mediator of bile-acid-stimulated modulation of mTORC2 (mTOR complex 2), the Akt pathway and glucose homoeostasis.


Subject(s)
Chenodeoxycholic Acid/metabolism , Diacylglycerol Kinase/metabolism , Glucose/metabolism , Hepatocytes/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Cells, Cultured , Diacylglycerol Kinase/antagonists & inhibitors , Diacylglycerol Kinase/chemistry , Diacylglycerol Kinase/genetics , Gene Expression Regulation/drug effects , Gene Silencing , Genes, Reporter , Hep G2 Cells , Hepatocytes/cytology , Hepatocytes/drug effects , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Isoxazoles/pharmacology , Mechanistic Target of Rapamycin Complex 2 , Multiprotein Complexes/metabolism , Mutation , Phosphatidic Acids/metabolism , Phosphorylation/drug effects , Promoter Regions, Genetic/drug effects , Protein Processing, Post-Translational/drug effects , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/genetics , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism
5.
J Lipid Res ; 54(8): 2121-2132, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23610160

ABSTRACT

Diacylglycerol kinase (DGK)θ is a lipid kinase that phosphorylates diacylglycerol to form phosphatidic acid (PA). We have previously shown that PA is a ligand for the nuclear receptor steroidogenic factor 1 (SF1) and that cAMP-stimulated expression of SF1 target genes requires DGKθ. In this study, we sought to investigate the role of cAMP signaling in regulating DGKθ gene expression. Real time RT-PCR and Western blot analysis revealed that dibutyryl cAMP (Bt2cAMP) increased the mRNA and protein expression, respectively, of DGKθ in H295R human adrenocortical cells. SF1 and sterol regulatory element binding protein 1 (SREBP1) increased the transcriptional activity of a reporter plasmid containing 1.5 kb of the DGKθ promoter fused to the luciferase gene. Mutation of putative cAMP responsive sequences abolished SF1- and SREBP-dependent DGKθ reporter gene activation. Consistent with this finding, chromatin immunoprecipitation assay demonstrated that Bt2cAMP signaling increased the recruitment of SF1 and SREBP1 to the DGKθ promoter. Coimmunoprecipitation assay revealed that SF1 and SREBP1 interact, suggesting that the two transcription factors form a complex on the DGKθ promoter. Finally, silencing SF1 and SREBP1 abolished cAMP-stimulated DGKθ expression. Taken together, we demonstrate that SF1 and SREBP1 activate DGKθ transcription in a cAMP-dependent manner in human adrenocortical cells.


Subject(s)
Cyclic AMP/pharmacology , Diacylglycerol Kinase/genetics , Steroidogenic Factor 1/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , Transcription, Genetic/drug effects , Cyclic AMP/metabolism , Humans , Tumor Cells, Cultured
6.
Mol Biol Cell ; 24(6): 848-57, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23325789

ABSTRACT

Diaphanous homologue 1 (DIAPH1) is a Rho effector protein that coordinates cellular dynamics by regulating microfilament and microtubule function. We previously showed that DIAPH1 plays an integral role in regulating the production of cortisol by controlling the rate of mitochondrial movement, by which activation of the adrenocorticotropin (ACTH)/cAMP signaling pathway stimulates mitochondrial trafficking and promotes the interaction between RhoA and DIAPH1. In the present study we use mass spectrometry to identify DIAPH1 binding partners and find that DIAPH1 interacts with several proteins, including RhoA, dynamin-1, kinesin, ß-tubulin, ß-actin, oxysterol-binding protein (OSBP)-related protein 2 (ORP2), and ORP10. Moreover, DIAPH1 is phosphorylated in response to dibutyryl cAMP (Bt2cAMP) at Thr-759 via a pathway that requires extracellular signal-related kinase (ERK). Alanine substitution of Thr-759 renders DIAPH1 more stable and attenuates the interaction between DIAPH1 and kinesin, ORP2, and actin but has no effect on the ability of the protein to interact with RhoA or ß-tubulin. Finally, overexpression of a DIAPH1 T759A mutant significantly decreases the rate of Bt2cAMP-stimulated mitochondrial movement. Taken together, our findings establish a key role for phosphorylation in regulating the stability and function of DIAPH1.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adrenal Cortex/metabolism , Cyclic AMP/metabolism , Microtubules/metabolism , Actins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Cell Line , Dynamin I/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Formins , Humans , Kinesins/metabolism , Mitochondria/metabolism , Mutation , Phosphorylation , Protein Binding , Protein Stability , Receptors, Steroid/metabolism , Signal Transduction , Tubulin/metabolism , rhoA GTP-Binding Protein/metabolism
7.
Mol Cell Endocrinol ; 371(1-2): 79-86, 2013 May 22.
Article in English | MEDLINE | ID: mdl-23186810

ABSTRACT

The production of glucocorticoids and aldosterone in the adrenal cortex is regulated at multiple levels. Biosynthesis of these hormones is initiated when cholesterol, the substrate, enters the inner mitochondrial membrane for conversion to pregnenolone. Unlike most metabolic pathways, the biosynthesis of adrenocortical steroid hormones is unique because some of the enzymes are localized in mitochondria and others in the endoplasmic reticulum (ER). Although much is known about the factors that control the transcription and activities of the proteins that are required for steroid hormone production, the parameters that govern the exchange of substrates between the ER and mitochondria are less well understood. This short review summarizes studies that have begun to provide insight into the role of the cytoskeleton, mitochondrial transport, and the physical interaction of the ER and mitochondria in the production of adrenocortical steroid hormones.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adrenal Cortex Hormones/biosynthesis , Adrenal Cortex/metabolism , Cytoskeleton/metabolism , Microtubules/metabolism , Biological Transport , Cholesterol/metabolism , Endoplasmic Reticulum/metabolism , Formins , Humans , Mitochondria/metabolism , Signal Transduction
8.
Mol Cell Biol ; 32(21): 4419-31, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22927646

ABSTRACT

Adrenocorticotropin (ACTH) signaling increases glucocorticoid production by promoting the interaction of transcription factors and coactivator proteins with the promoter of steroidogenic genes. The nuclear receptor steroidogenic factor 1 (SF-1) is essential for steroidogenic gene transcription. Sphingosine (SPH) is a ligand for SF-1. Moreover, suppression of expression of acid ceramidase (ASAH1), an enzyme that produces SPH, increases the transcription of multiple steroidogenic genes. Given that SF-1 is a nuclear protein, we sought to define the molecular mechanisms by which ASAH1 regulates SF-1 function. We show that ASAH1 is localized in the nuclei of H295R adrenocortical cells and that cyclic AMP (cAMP) signaling promotes nuclear sphingolipid metabolism in an ASAH1-dependent manner. ASAH1 suppresses SF-1 activity by directly interacting with the receptor. Chromatin immunoprecipitation (ChIP) assays revealed that ASAH1 is recruited to the promoter of various SF-1 target genes and that ASAH1 and SF-1 colocalize on the same promoter region of the CYP17A1 and steroidogenic acute regulatory protein (StAR) genes. Taken together, these results demonstrate that ASAH1 is a novel coregulatory protein that represses SF-1 function by directly binding to the receptor on SF-1 target gene promoters and identify a key role for nuclear lipid metabolism in regulating gene transcription.


Subject(s)
Acid Ceramidase/metabolism , Adrenal Cortex/metabolism , Lipid Metabolism , Steroidogenic Factor 1/metabolism , Transcription, Genetic , Adrenal Cortex/cytology , Adrenal Cortex/enzymology , Adrenocorticotropic Hormone/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Chromatin Immunoprecipitation , Cyclic AMP/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Haplorhini , Humans , Mice , Phosphoproteins/genetics , Promoter Regions, Genetic , RNA Interference , RNA, Small Interfering , Regulatory Sequences, Nucleic Acid , Signal Transduction/genetics , Sphingolipids/metabolism , Sphingosine/metabolism , Steroid 17-alpha-Hydroxylase/genetics , Steroidogenic Factor 1/genetics
9.
Endocrinology ; 153(7): 3258-68, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22585829

ABSTRACT

In the human adrenal cortex, cortisol is synthesized from cholesterol by members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenases. Both the first and last steps of cortisol biosynthesis occur in mitochondria. Based on our previous findings that activation of ACTH signaling changes the ratio of nicotinamide adenine dinucleotide (NAD) phosphate to reduced NAD phosphate in adrenocortical cells, we hypothesized that pyridine nucleotide metabolism may regulate the activity of the mitochondrial NAD(+)-dependent sirtuin (SIRT) deacetylases. We show that resveratrol increases the protein expression and half-life of P450 side chain cleavage enzyme (P450scc). The effects of resveratrol on P450scc protein levels and acetylation status are dependent on SIRT3 and SIRT5 expression. Stable overexpression of SIRT3 abrogates the cellular content of acetylated P450scc, concomitant with an increase in P450scc protein expression and cortisol secretion. Mutation of K148 and K149 to alanine stabilizes the expression of P450scc and results in a 1.5-fold increase in pregnenolone biosynthesis. Finally, resveratrol also increases the protein expression of P450 11ß, another mitochondrial enzyme required for cortisol biosynthesis. Collectively, this study identifies a role for NAD(+)-dependent SIRT deacetylase activity in regulating the expression of mitochondrial steroidogenic P450.


Subject(s)
Cholesterol Side-Chain Cleavage Enzyme/metabolism , Gene Expression Regulation, Neoplastic , Hydrocortisone/metabolism , Sirtuin 1/metabolism , Stilbenes/pharmacology , Antioxidants/pharmacology , Humans , Lysine/metabolism , Mitochondria/metabolism , Mutagenesis , Mutation , Protein Processing, Post-Translational , RNA Interference , Resveratrol , Sirtuin 3/metabolism , Sirtuins/metabolism
10.
Mol Endocrinol ; 26(2): 228-43, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22261821

ABSTRACT

In H295R human adrenocortical cells, ACTH rapidly activates ceramide (Cer) and sphingosine (SPH) turnover with a concomitant increase in SPH-1-phosphate secretion. These bioactive lipids modulate adrenocortical steroidogenesis, primarily by acting as second messengers in the protein kinase A/cAMP-dependent pathway. Acid ceramidase (ASAH1) directly regulates the intracellular balance of Cer, SPH, and SPH-1-phosphate by catalyzing the hydrolysis of Cer into SPH. ACTH/cAMP signaling stimulates ASAH1 transcription and activity, supporting a role for this enzyme in glucocorticoid production. Here, the role of ASAH1 in regulating steroidogenic capacity was examined using a tetracycline-inducible ASAH1 short hairpin RNA H295R human adrenocortical stable cell line. We show that ASAH1 suppression increases the transcription of multiple steroidogenic genes, including Cytochrome P450 monooxygenase (CYP)17A1, CYP11B1/2, CYP21A2, steroidogenic acute regulatory protein, hormone-sensitive lipase, 18-kDa translocator protein, and the melanocortin-2 receptor. Induced gene expression positively correlated with enhanced histone H3 acetylation at target promoters. Repression of ASAH1 expression also induced the expression of members of the nuclear receptor nuclear receptor subfamily 4 (NR4A) family while concomitantly suppressing the expression of dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1. ASAH1 knockdown altered the expression of genes involved in sphingolipid metabolism and changed the cellular amounts of distinct sphingolipid species. Finally, ASAH1 silencing increased basal and cAMP-dependent cortisol and dehydroepiandrosterone secretion, establishing ASAH1 as a pivotal regulator of steroidogenic capacity in the human adrenal cortex.


Subject(s)
Acid Ceramidase/physiology , Adrenal Cortex/metabolism , Gene Expression Regulation , Steroid Hydroxylases/genetics , Steroids/biosynthesis , Acetylation , Acid Ceramidase/genetics , Acid Ceramidase/metabolism , Adrenocorticotropic Hormone/pharmacology , Biosynthetic Pathways/genetics , Cell Line, Tumor , Cell Proliferation , Ceramides/metabolism , Cyclic AMP/metabolism , Cyclin B2/metabolism , Dehydroepiandrosterone/metabolism , Gene Knockdown Techniques , Histones/metabolism , Humans , Hydrocortisone/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Promoter Regions, Genetic , RNA Interference , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Sphingolipids/genetics , Sphingolipids/metabolism , Steroid Hydroxylases/metabolism , Transcription, Genetic , beta Catenin/metabolism
11.
Annu Rev Physiol ; 74: 131-51, 2012.
Article in English | MEDLINE | ID: mdl-21888508

ABSTRACT

Nuclear lipid metabolism is implicated in various processes, including transcription, splicing, and DNA repair. Sphingolipids play roles in numerous cellular functions, and an emerging body of literature has identified roles for these lipid mediators in distinct nuclear processes. Different sphingolipid species are localized in various subnuclear domains, including chromatin, the nuclear matrix, and the nuclear envelope, where sphingolipids exert specific regulatory and structural functions. Sphingomyelin, the most abundant nuclear sphingolipid, plays both structural and regulatory roles in chromatin assembly and dynamics in addition to being an integral component of the nuclear matrix. Sphingosine-1-phosphate modulates histone acetylation, sphingosine is a ligand for steroidogenic factor 1, and nuclear accumulation of ceramide has been implicated in apoptosis. Finally, nuclear membrane-associated ganglioside GM1 plays a pivotal role in Ca(2+) homeostasis. This review highlights research on the factors that control nuclear sphingolipid metabolism and summarizes the roles of these lipids in various nuclear processes.


Subject(s)
Cell Nucleus/metabolism , Sphingolipids/metabolism , Animals , Calcium/metabolism , Ceramides/metabolism , G(M1) Ganglioside/metabolism , Gangliosides/metabolism , Homeostasis , Humans , Lysophospholipids/metabolism , Sphingolipids/biosynthesis , Sphingolipids/chemistry , Sphingomyelins/metabolism , Sphingosine/analogs & derivatives , Sphingosine/metabolism
12.
Mol Cell Endocrinol ; 348(1): 165-75, 2012 Jan 02.
Article in English | MEDLINE | ID: mdl-21864647

ABSTRACT

In the acute phase of adrenocortical steroidogenesis, adrenocorticotrophin (ACTH) activates a cAMP/PKA-signaling pathway that promotes the transport of free cholesterol to the inner mitochondrial membrane. We have previously shown that ACTH rapidly stimulates the metabolism of sphingolipids and the secretion of sphingosine-1-phosphate (S1P) in H295R cells. In this study, we examined the effect of S1P on genes involved in the acute phase of steroidogenesis. We show that S1P increases the expression of steroidogenic acute regulatory protein (StAR), 18-kDa translocator protein (TSPO), low-density lipoprotein receptor (LDLR), and scavenger receptor class B type I (SR-BI). S1P-induced StAR mRNA expression requires Gα(i) signaling, phospholipase C (PLC), Ca(2+)/calmodulin-dependent kinase II (CamKII), and ERK1/2 activation. S1P also increases intracellular Ca(2+), the phosphorylation of hormone sensitive lipase (HSL) at Ser(563), and cortisol secretion. Collectively, these findings identify multiple roles for S1P in the regulation of glucocorticoid biosynthesis.


Subject(s)
Adrenal Cortex/cytology , Cholesterol/metabolism , Gene Expression Regulation , Hydrocortisone/biosynthesis , Lysophospholipids/physiology , Sphingosine/analogs & derivatives , Bucladesine/pharmacology , Calcium Signaling , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Line , Dehydroepiandrosterone/metabolism , Enzyme Activation , Humans , Lysophospholipids/pharmacology , MAP Kinase Signaling System , Pertussis Toxin/pharmacology , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Receptors, GABA/genetics , Receptors, GABA/metabolism , Receptors, LDL/genetics , Receptors, LDL/metabolism , Scavenger Receptors, Class B/genetics , Scavenger Receptors, Class B/metabolism , Sphingosine/pharmacology , Sphingosine/physiology , Sterol Esterase/genetics , Sterol Esterase/metabolism , Type C Phospholipases/metabolism
13.
J Biol Chem ; 286(22): 19399-409, 2011 Jun 03.
Article in English | MEDLINE | ID: mdl-21493710

ABSTRACT

Sphingolipid metabolites, such as ceramide (Cer), sphingosine (SPH), and sphingosine 1-phosphate (S1P), contribute to multiple aspects of carcinogenesis including cell proliferation, migration, angiogenesis, and tumor resistance. The cellular balance between Cer and S1P levels, for example, is an important determinant of cell fate, with the former inducing apoptosis and the later mitogenesis. Acid ceramidase (ASAH1) plays a pivotal role in regulating the intracellular concentration of these two metabolites by hydrolyzing Cer into SPH, which is rapidly phosphorylated to form S1P. Genistein is a phytoestrogen isoflavone that exerts agonist and antagonist effects on the proliferation of estrogen-dependent MCF-7 cells in a dose-dependent manner, primarily as a ligand for estrogen receptors. Genistein can also activate signaling through GPR30, a G-protein-coupled cell surface receptor. Based on the relationship between bioactive sphingolipids and tumorigenesis, we sought to determine the effect of genistein on ASAH1 transcription in MCF-7 breast cancer cells. We show herein that nanomolar concentrations of genistein induce ASAH1 transcription through a GPR30-dependent, pertussis toxin-sensitive pathway that requires the activation of c-Src and extracellular signal regulated kinase 1/2 (ERK1/2). Activation of this pathway promotes histone acetylation and recruitment of phospho-estrogen receptor α and specificity protein-1 to the ASAH1 promoter, ultimately culminating in increased ceramidase activity. Finally, we show that genistein stimulates cyclin B2 expression and cell proliferation in an ASAH1-dependent manner. Collectively, these data identify a mechanism through which genistein promotes sphingolipid metabolism and support a role for ASAH1 in breast cancer cell growth.


Subject(s)
Acid Ceramidase/biosynthesis , Anticarcinogenic Agents/pharmacology , Breast Neoplasms/enzymology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genistein/pharmacology , Acetylation/drug effects , Acid Ceramidase/genetics , Breast Neoplasms/genetics , CSK Tyrosine-Protein Kinase , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Neoplastic/genetics , Histones/genetics , Histones/metabolism , Humans , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptors, Estrogen , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Sphingolipids/genetics , Sphingolipids/metabolism , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , src-Family Kinases
14.
Endocrinology ; 151(9): 4313-23, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20591975

ABSTRACT

Steroid hormones are formed by the successive action of enzymes that are localized in mitochondria and the endoplasmic reticulum (ER). Compartmentalization of these enzymes in different subcellular organelles dictates the need for efficient transfer of intermediary metabolites between the mitochondrion and ER; however, the molecular determinants that regulate interorganelle substrate exchange are unknown. The objective of this study was to define the molecular mechanism by which adrenocorticotropin (ACTH) signaling regulates communication between mitochondria and the ER during steroidogenesis. Using live cell video confocal microscopy, we found that ACTH and dibutyryl cAMP rapidly increased the rate of mitochondrial movement. Inhibiting tubulin polymerization prevented both basal and ACTH/cAMP-stimulated mitochondrial trafficking and decreased cortisol secretion. This decrease in cortisol secretion evoked by microtubule inhibition was paralleled by an increase in dehydroepiandrosterone production. In contrast, treatment with paclitaxel to stabilize microtubules or latrunculin B to inhibit actin polymerization and disrupt microfilament organization increased both mitochondrial trafficking and cortisol biosynthesis. ACTH-stimulated mitochondrial movement was dependent on RhoA and the RhoA effector, diaphanous-related homolog 1 (DIAPH1). ACTH signaling temporally increased the cellular concentrations of GTP-bound and Ser-188 phosphorylated RhoA, which promoted interaction with DIAPH1. Expression of a dominant-negative RhoA mutant or silencing DIAPH1 impaired mitochondrial trafficking and cortisol biosynthesis and concomitantly increased the secretion of adrenal androgens. We conclude that ACTH regulates cortisol production by facilitating interorganelle substrate transfer via a process that is mediated by RhoA and DIAPH1, which act to coordinate the dynamic trafficking of mitochondria.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adrenocorticotropic Hormone/pharmacology , Hydrocortisone/biosynthesis , Mitochondria/metabolism , Adaptor Proteins, Signal Transducing/genetics , Biological Transport/drug effects , Bucladesine/pharmacology , Cell Line, Tumor , Colchicine/pharmacology , Endoplasmic Reticulum/metabolism , Formins , Humans , Microscopy, Fluorescence/methods , Microscopy, Video/methods , Microtubules/metabolism , Mutation , Protein Binding/drug effects , RNA Interference , Time Factors , Tubulin Modulators/pharmacology , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism
15.
Steroids ; 75(6): 390-9, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20138078

ABSTRACT

Steroid hormones regulate various physiological processes including development, reproduction, and metabolism. These regulatory molecules are synthesized from cholesterol in endocrine organs - such as the adrenal glands and gonads - via a multi-step enzymatic process that is catalyzed by the cytochrome P450 superfamily of monooxygenases and hydroxysteroid dehydrogenases. Steroidogenesis is induced by trophic peptide hormones primarily via the activation of a cAMP/protein kinase A (PKA)-dependent pathway. However, other signaling molecules, including cytokines and growth factors, control the steroid hormone biosynthetic pathway. More recently, sphingolipids, including ceramide, sphingosine-1-phosphate, and sphingosine, have been found to modulate steroid hormone secretion at multiple levels. In this review, we provide a brief overview of the mechanisms by which sphingolipids regulate steroidogenesis. In addition, we discuss how steroid hormones control sphingolipid metabolism. Finally, we outline evidence supporting the emerging role of bioactive sphingolipids in various nuclear processes and discuss a role for nuclear sphingolipid metabolism in the control of gene transcription.


Subject(s)
Sphingolipids/metabolism , Steroids/metabolism , Cell Communication/physiology , Molecular Structure , Signal Transduction/physiology , Sphingolipids/chemistry , Steroids/chemistry , Transcription, Genetic
16.
Biochim Biophys Acta ; 1791(8): 706-13, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19298866

ABSTRACT

Acid ceramidase (encoded by ASAH1) is a lipid hydrolase that catalyzes the conversion of ceramide (cer) into sphingosine (SPH) and a free fatty acid. Adrenocortical steroidogenesis is regulated by the trophic peptide hormone adrenocorticotropin (ACTH), which induces the expression of steroidogenic genes in the human adrenal cortex primarily via a cAMP/protein kinase A (PKA)-dependent pathway. ACTH also stimulates sphingolipid metabolism in H295R adrenocortical cells leading to changes in steroidogenic gene expression. Based on our previous data identifying SPH as an antagonist for the nuclear receptor steroidogenic factor 1 (SF-1) and the role of ACTH-stimulated changes in sphingolipid metabolism on steroidogenic gene transcription, the aim of the current study was to determine the role of ACTH signaling in regulating the expression of the ASAH1 gene in H295R cells. We show that activation of the ACTH signaling pathway induces ASAH1 gene expression by stimulating the binding of the cAMP-responsive element binding protein (CREB) to multiple regions of the ASAH1 promoter. CREB binding promotes the recruitment of the coactivators CREB binding protein (CBP) and p300 to the CREB-responsive regions of the promoter. Consistent with transcriptional activation, we show that cAMP signaling increases the trimethylation of Lys 4 on histone H3 (H3K4) along the ASAH1 promoter. Finally, RNA interference (RNAi) experiments demonstrate that CREB is indispensable for cAMP-induced ASAH1 transcription. These data identify the ACTH/cAMP signaling pathway and CREB as transcriptional regulators of the ASAH1 gene in the human adrenal cortex.


Subject(s)
Acid Ceramidase/genetics , Acid Ceramidase/metabolism , Adrenal Cortex/cytology , Adrenal Cortex/enzymology , Cyclic AMP Response Element-Binding Protein/metabolism , Gene Expression Regulation, Enzymologic , Cell Line , Cyclic AMP/pharmacology , Cyclic AMP Response Element-Binding Protein/genetics , Gene Expression Regulation, Enzymologic/drug effects , Gene Silencing/drug effects , Genes, Dominant , Genes, Reporter , Humans , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , p300-CBP Transcription Factors/metabolism
17.
Mol Cell Endocrinol ; 300(1-2): 109-14, 2009 Mar 05.
Article in English | MEDLINE | ID: mdl-19007851

ABSTRACT

Optimal steroid hormone biosynthesis occurs via the integration of multiple regulatory processes, one of which entails a coordinate increase in the transcription of all genes required for steroidogenesis. In the human adrenal cortex adrenocorticotropin (ACTH) activates a signaling cascade that promotes the dynamic assembly of protein complexes on the promoters of steroidogenic genes. For CYP17, multiple transcription factors, including steroidogenic factor-1 (SF-1), GATA-6, and sterol regulatory binding protein 1 (SREBP1), are recruited to the promoter during activated transcription. The ability of these factors to increase CYP17 mRNA expression requires the formation of higher order coregulatory complexes, many of which contain enzymatic activities that post-translationally modify both the transcription factors and histones. We discuss the mechanisms by which transcription factors and coregulatory proteins regulate CYP17 transcription and summarize the role of kinases, phosphatases, acetyltransferases, and histone deacetylases in controlling CYP17 mRNA expression.


Subject(s)
Gene Expression Regulation, Enzymologic , Promoter Regions, Genetic , Steroid 17-alpha-Hydroxylase/genetics , Adrenal Glands/cytology , Adrenal Glands/enzymology , Humans , Steroid 17-alpha-Hydroxylase/metabolism , Transcription Factors/metabolism
18.
Lipids ; 43(12): 1109-15, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18726632

ABSTRACT

Steroid hormones are synthesized in response to signaling cascades initiated by the trophic peptide hormones derived from the anterior pituitary. The mechanisms by which these peptide hormones regulate steroid hormone production are multifaceted and include controlling the transcription of steroidogenic genes, regulating cholesterol (substrate) uptake and transport, modulating steroidogenic enzyme activity, and controlling electron availability. Cytoskeletal polymers such as microfilaments and microtubules have also been implicated in regulating steroidogenesis. Of note, steroidogenesis is a multi-step process that occurs in two organelles, the endoplasmic reticulum (ER) and the mitochondrion. However, the precise mechanism by which substrates are delivered back and forth between these two organelles is unknown. In this review we will discuss the role of components of the cytoskeleton in conferring optimal steroidogenic potential. Finally, we present data that identifying a novel mechanism by which sphingosine-1-phosphate induces mitochondrial trafficking to promote steroidogenesis.


Subject(s)
Cytoskeleton/metabolism , Steroids/biosynthesis , Cells, Cultured , Cholesterol/metabolism , Colchicine/pharmacology , Cytoskeleton/drug effects , Humans , Organelles/metabolism , Tubulin Modulators/pharmacology
19.
Subcell Biochem ; 49: 387-412, 2008.
Article in English | MEDLINE | ID: mdl-18751920

ABSTRACT

Steroid hormones are essential regulators of a vast number of physiological processes. The biosynthesis of these chemical messengers occurs in specialized steroidogenic tissues via a multi-step process that is catalyzed by members of the cytochrome P450 superfamily of monooxygenases and hydroxysteroid dehydrogenases. Though numerous signaling mediators, including cytokines and growth factors control steroidogenesis, trophic peptide hormones are the primary regulators of steroid hormone production. These peptide hormones activate a cAMP/cAMP-dependent kinase (PKA) signaling pathway, however, studies have shown that crosstalk between multiple signal transduction pathways and signaling molecules modulates optimal steroidogenic capacity. Sphingolipids such as ceramide, sphingosine, sphingosine-1-phosphate, sphingomyelin, and gangliosides have been shown to control the steroid hormone biosynthetic pathway at multiple levels, including regulating steroidogenic gene expression and activity as well as acting as second messengers in signaling cascades. In this review, we provide an overview of recent studies that have investigated the role of sphingolipids in adrenal, gonadal, and neural steroidogenesis.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Gonadal Steroid Hormones/biosynthesis , Sphingolipids/physiology , Adrenal Glands/metabolism , Animals , Ceramides/physiology , Cyclic AMP/physiology , Cyclic AMP-Dependent Protein Kinases/physiology , Female , Gangliosides/physiology , Humans , Lysophospholipids/physiology , Male , Metabolic Networks and Pathways , Nervous System/metabolism , Ovary/metabolism , Signal Transduction , Sphingosine/analogs & derivatives , Sphingosine/physiology , Testis/metabolism
20.
Mol Cell Endocrinol ; 283(1-2): 104-13, 2008 Feb 13.
Article in English | MEDLINE | ID: mdl-18191017

ABSTRACT

In this study, we examined the role of sphingosine-1-phosphate (S1P) in regulating the transcription of the liver receptor homologue-1 (LRH-1) in breast cancer cells. We show that S1P induces LRH-1 mRNA expression in MCF-7 cells in a prostaglandin E2 (PGE2)-dependent manner. Both S1P and PGE2 stimulate the recruitment of LRH-1, cAMP response element binding protein (CREB), CCAAT/enhancer binding proteins (C/EBP), and RNA Polymerase II (Pol II) to the LRH-1 promoter, as well as increase acetylation of histone H3 in this region of chromatin. S1P and PGE2 promote the direct interaction of CREB and LRH-1, which is potentiated by C/EBPdelta and the coactivators CREB-binding protein (CBP), and steroid receptor coactivator-3 (SRC-3). CREB and LRH-1 synergistically increase LRH-1 transcription, suggesting an integral role for LRH-1 in regulating the transcription of its own gene.


Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic/drug effects , Lysophospholipids/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Sphingosine/analogs & derivatives , Transcription Factors/genetics , Acetylation/drug effects , Animals , Base Sequence , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclooxygenase 2/metabolism , Dinoprostone/biosynthesis , Genes, Reporter , Histones/metabolism , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sphingosine/pharmacology , Transcription, Genetic/drug effects , Two-Hybrid System Techniques
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