RESUMEN
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.
Asunto(s)
Colesterol/metabolismo , Hidrocortisona/biosíntesis , Receptores de Esteroides/fisiología , Corteza Suprarrenal/metabolismo , Línea Celular , Deshidroepiandrosterona/biosíntesis , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Homeostasis/efectos de los fármacos , Humanos , Receptores de Esteroides/genética , Esteroides/metabolismoRESUMEN
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.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , AMP Cíclico/metabolismo , Glucocorticoides/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Línea Celular , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Silenciador del Gen , Glucocorticoides/genética , Humanos , Hidrocortisona/genética , Hidrocortisona/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Factores de Transcripción de Octámeros/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteolisis , Proteínas de Unión al ARN/genéticaRESUMEN
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.
Asunto(s)
Corteza Suprarrenal/metabolismo , Colesterol/metabolismo , Deshidroepiandrosterona/biosíntesis , Diacilglicerol Quinasa/metabolismo , Hidrocortisona/biosíntesis , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Diacilglicerol Quinasa/antagonistas & inhibidores , Diacilglicerol Quinasa/genética , Regulación de la Expresión Génica , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Fosforilación , Regiones Promotoras Genéticas , Factores de Empalme de ARN , Transducción de Señal/efectos de los fármacos , Proteínas de Unión a los Elementos Reguladores de Esteroles/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Ácido Quenodesoxicólico/metabolismo , Diacilglicerol Quinasa/metabolismo , Glucosa/metabolismo , Hepatocitos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Células Cultivadas , Diacilglicerol Quinasa/antagonistas & inhibidores , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/genética , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Genes Reporteros , Células Hep G2 , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoxazoles/farmacología , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/metabolismo , Mutación , Ácidos Fosfatidicos/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
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.
Asunto(s)
AMP Cíclico/farmacología , Diacilglicerol Quinasa/genética , Factor Esteroidogénico 1/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Transcripción Genética/efectos de los fármacos , AMP Cíclico/metabolismo , Humanos , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Corteza Suprarrenal/metabolismo , AMP Cíclico/metabolismo , Microtúbulos/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Dinamina I/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Forminas , Humanos , Cinesinas/metabolismo , Mitocondrias/metabolismo , Mutación , Fosforilación , Unión Proteica , Estabilidad Proteica , Receptores de Esteroides/metabolismo , Transducción de Señal , Tubulina (Proteína)/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Corticoesteroides/biosíntesis , Corteza Suprarrenal/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Transporte Biológico , Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Forminas , Humanos , Mitocondrias/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Ceramidasa Ácida/metabolismo , Corteza Suprarrenal/metabolismo , Metabolismo de los Lípidos , Factor Esteroidogénico 1/metabolismo , Transcripción Genética , Corteza Suprarrenal/citología , Corteza Suprarrenal/enzimología , Hormona Adrenocorticotrópica/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Haplorrinos , Humanos , Ratones , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal/genética , Esfingolípidos/metabolismo , Esfingosina/metabolismo , Esteroide 17-alfa-Hidroxilasa/genética , Factor Esteroidogénico 1/genéticaRESUMEN
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.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Regulación Neoplásica de la Expresión Génica , Hidrocortisona/metabolismo , Sirtuina 1/metabolismo , Estilbenos/farmacología , Antioxidantes/farmacología , Humanos , Lisina/metabolismo , Mitocondrias/metabolismo , Mutagénesis , Mutación , Procesamiento Proteico-Postraduccional , Interferencia de ARN , Resveratrol , Sirtuina 3/metabolismo , Sirtuinas/metabolismoRESUMEN
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.
Asunto(s)
Ceramidasa Ácida/fisiología , Corteza Suprarrenal/metabolismo , Regulación de la Expresión Génica , Esteroide Hidroxilasas/genética , Esteroides/biosíntesis , Acetilación , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Hormona Adrenocorticotrópica/farmacología , Vías Biosintéticas/genética , Línea Celular Tumoral , Proliferación Celular , Ceramidas/metabolismo , AMP Cíclico/metabolismo , Ciclina B2/metabolismo , Deshidroepiandrosterona/metabolismo , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Humanos , Hidrocortisona/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Esfingolípidos/genética , Esfingolípidos/metabolismo , Esteroide Hidroxilasas/metabolismo , Transcripción Genética , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Corteza Suprarrenal/citología , Colesterol/metabolismo , Regulación de la Expresión Génica , Hidrocortisona/biosíntesis , Lisofosfolípidos/fisiología , Esfingosina/análogos & derivados , Bucladesina/farmacología , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , Deshidroepiandrosterona/metabolismo , Activación Enzimática , Humanos , Lisofosfolípidos/farmacología , Sistema de Señalización de MAP Quinasas , Toxina del Pertussis/farmacología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Receptores de GABA/genética , Receptores de GABA/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Esfingosina/farmacología , Esfingosina/fisiología , Esterol Esterasa/genética , Esterol Esterasa/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Esfingolípidos/metabolismo , Animales , Calcio/metabolismo , Ceramidas/metabolismo , Gangliósido G(M1)/metabolismo , Gangliósidos/metabolismo , Homeostasis , Humanos , Lisofosfolípidos/metabolismo , Esfingolípidos/biosíntesis , Esfingolípidos/química , Esfingomielinas/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
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.
Asunto(s)
Ceramidasa Ácida/biosíntesis , Anticarcinógenos/farmacología , Neoplasias de la Mama/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genisteína/farmacología , Acetilación/efectos de los fármacos , Ceramidasa Ácida/genética , Neoplasias de la Mama/genética , Proteína Tirosina Quinasa CSK , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Histonas/genética , Histonas/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Estrógenos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Esfingolípidos/genética , Esfingolípidos/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Familia-src QuinasasRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hormona Adrenocorticotrópica/farmacología , Hidrocortisona/biosíntesis , Mitocondrias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Transporte Biológico/efectos de los fármacos , Bucladesina/farmacología , Línea Celular Tumoral , Colchicina/farmacología , Retículo Endoplásmico/metabolismo , Forminas , Humanos , Microscopía Fluorescente/métodos , Microscopía por Video/métodos , Microtúbulos/metabolismo , Mutación , Unión Proteica/efectos de los fármacos , Interferencia de ARN , Factores de Tiempo , Moduladores de Tubulina/farmacología , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
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.
Asunto(s)
Esfingolípidos/metabolismo , Esteroides/metabolismo , Comunicación Celular/fisiología , Estructura Molecular , Transducción de Señal/fisiología , Esfingolípidos/química , Esteroides/química , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Corteza Suprarrenal/citología , Corteza Suprarrenal/enzimología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación Enzimológica de la Expresión Génica , Línea Celular , AMP Cíclico/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Genes Dominantes , Genes Reporteros , Humanos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
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.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Regiones Promotoras Genéticas , Esteroide 17-alfa-Hidroxilasa/genética , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/enzimología , Humanos , Esteroide 17-alfa-Hidroxilasa/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hormonas Esteroides Gonadales/biosíntesis , Esfingolípidos/fisiología , Glándulas Suprarrenales/metabolismo , Animales , Ceramidas/fisiología , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Femenino , Gangliósidos/fisiología , Humanos , Lisofosfolípidos/fisiología , Masculino , Redes y Vías Metabólicas , Sistema Nervioso/metabolismo , Ovario/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/fisiología , Testículo/metabolismoRESUMEN
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.
Asunto(s)
Citoesqueleto/metabolismo , Esteroides/biosíntesis , Células Cultivadas , Colesterol/metabolismo , Colchicina/farmacología , Citoesqueleto/efectos de los fármacos , Humanos , Orgánulos/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
In the human adrenal cortex, the peptide hormone adrenocorticotropin (ACTH) directs cortisol and adrenal androgen biosynthesis by activating a cAMP/cAMP-dependent protein kinase (PKA) pathway. Carboxyl-terminal binding protein 1 (CtBP1) is a corepressor that regulates transcription of the CYP17 gene by periodically interacting with steroidogenic factor-1 in response to ACTH signaling. Given that CtBP1 function is regulated by NADH binding, we hypothesized that ACTH-stimulated changes in cellular pyridine nucleotide concentrations modulate the ability of CtBP1 to repress CYP17 transcription. Further, we postulated that PKA evokes changes in the phosphorylation status of CtBP1 that control the ability of the protein to bind to steroidogenic factor-1 and the coactivator GCN5 (general control nonderepressed 5) and repress CYP17 gene expression. We show that ACTH alters pyridine nucleotide redox state and identify amino acid residues in CtBP1 that are targeted by PKA and PAK6. Both ACTH/cAMP signaling and NADH/NAD+ ratio stimulate nuclear-cytoplasmic oscillation of both CtBP proteins. We provide evidence that PKA 1) induces metabolic changes in the adrenal cortex and 2) phosphorylates CtBP proteins, particularly CtBP1 at T144, resulting in CtBP protein partnering and ACTH-dependent CYP17 transcription.