RESUMEN
3Beta-hydroxysteroid dehydrogenase (3beta-HSD) activity is essential for the synthesis of all classes of steroid hormones, converting various delta5-3beta-hydroxysteroids into hormonally active delta4-3-ketosteroids in NAD+ -dependent reactions. Certain 3beta-HSD isoforms have been reported to exhibit additional dehydrogenase character (e.g., 17-hydroxysteroid dehydrogenase/reductase). We have investigated whether mouse type I (adrenal/gonadal) and type VI 3beta-HSDs (uterine/embryonic) display significant 17beta-HSD-like activity. Nonsteroidogenic HEK 293T cells were transiently transfected with pCMV-based expression vectors containing mouse type I and type VI 3beta-HSDs. Transfected cells expressing either mouse type I or type VI 3beta-HSD converted testosterone to androstenedione, albeit at rates one-tenth of those of pregnenolone to progesterone in similarly transfected 293T cells. Our findings demonstrate that the mouse 3beta-HSD I and VI isoforms can inactivate testosterone within an intact cell milieu. These findings are important not only in establishment of structure-function relationships, but also whenever murine systems are used for developmental/reproductive paradigms associated with human disorders.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Testosterona/metabolismo , Androstenodiona/biosíntesis , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Humanos , Isoenzimas/metabolismo , Ratones , RatasRESUMEN
Thermally assisted infrared multiphoton photodissociation (TA-IRMPD) provides an effective means to dissociate ions in the quadrupole ion trap mass spectrometer (QITMS) without detrimentally affecting the performance of the instrument. IRMPD can offer advantages over collision-induced dissociation (CID). However, collisions with the QITMS bath gas at the standard pressure and ambient temperature cause IR-irradiated ions to lose energy faster than photons can be absorbed to induce dissociation. The low pressure required for IRMPD (< or = 10(-5) Torr) is not that required for optimal performance of the QITMS (10(-3) Torr), and sensitivity and resolution suffer. TA-IRMPD is performed with the bath gas at an elevated temperature. The higher temperature of the bath gas results in less energy lost in collisions of the IR-excited ions with the bath gas. Thermal assistance allows IRMPD to be used at or near optimal pressures, which results in an approximately 1 order of magnitude increase in signal intensity. Unlike CID, IRMPD allows small product ions, those less than about one-third the m/z of the parent ion, to be observed. IRMPD should also be more easily paired with fluctuating ion sources, as the corresponding fluctuations in resonant frequencies do not affect IRMPD. Finally, while IR irradiation nonselectively causes dissociation of all ions, TA-IRMPD can be made selective by using axial expansion to move ions away from the path of the laser beam.
Asunto(s)
Rayos Infrarrojos , Fotones , Espectrometría de MasasRESUMEN
Dissociation pathways of alkali-cationized peptides have been studied using multiple stages of mass spectrometry (MSx) with a quadrupole ion trap mass spectrometer. Over 100 peptide ions ranging from 2 to 10 residues in length and containing each of the 20 common amino acids have been examined. The formation of the [b(n-1) + Na + OH]+ product ion is the predominant dissociation pathway for the majority of the common amino acids. This product corresponds to a sodium-cationized peptide one residue shorter in length than the original peptide. In a few cases, product ions such as [b(n-1) + Na - H]+ and those formed by loss, or partial loss, of a sidechain are observed. Both [b(n-1) + Na + OH]+ and [b(n-1) + Na - H]+ product ions can be selected as parent ions for a subsequent stage of tandem mass spectrometry (MS/MS). It is shown that these dissociation patterns provide opportunities for peptide sequencing by successive dissociation from the C-terminus of alkali-cationized peptides. Up to seven stages of MS/MS have been performed on a series of [b + Na + OH]+ ions to provide sequence information from the C-terminus. This method is analogous to Edman degradation except that the cleavage occurs from the C-terminus instead of the N-terminus, making it more attractive for N-terminal blocked peptides. The isomers leucine and isoleucine cannot be differentiated by this method but the isobars lysine and glutamine can.
Asunto(s)
Álcalis/química , Aminoácidos/análisis , Oligopéptidos/química , Análisis de Secuencia de Proteína , Sodio/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Glutamina/química , Isoleucina/química , Isomerismo , Leucina/química , Lisina/química , Espectrometría de Masas , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
MS/MS has been used to sequence peptides and small proteins for a number of years. This method allows one to isolate the peptide of interest, which makes it possible to analyze impure samples and unseparated mixtures, such as protein digests. Collision-induced dissociation (CID) of the selected peptide ion generates the product ions that provide sequence information. However, often the MS/MS spectrum does not provide adequate information for complete sequence determination. The quadrupole ion trap has the capability to do multiple stages of mass spectrometry, MSn, which can increase the information available to determine the peptide sequence. A regular and predictable dissociation pattern for peptides further simplifies this analysis. By forming predominantly one type of ion, ambiguity is removed as to whether the ion is N- or C-terminal. This pattern can also be advantageous in that ion intensity remains concentrated for the next stage of MS/MS. In this work, a method to take advantage of the MSn capabilities of the quadrupole ion trap by controlling the dissociation pathways is explored. Dissociation is altered by acetylating the N-terminus of the peptide. MSn of a variety of acetylated peptides is used to determine the effects of the identity of the C-terminal residue and the length of the peptide on the dissociation pathways observed.
Asunto(s)
Péptidos/análisis , Secuencia de Aminoácidos , Grupo Citocromo c/química , Espectrometría de Masas , Datos de Secuencia MolecularRESUMEN
The ontogeny and functional role of steroidogenesis during early gestation in rodents is poorly understood. In previous studies, we have shown that expression of messenger RNAs (mRNAs) encoding two key enzymes indispensable for de novo synthesis of steroid hormones, i.e. cholesterol side chain cleavage cytochrome P450 (P450scc) and a newly identified isoform of murine 3beta-hydroxysteroid dehydrogenase/isomerase type VI (3betaHSD VI), is initiated upon decidualization of the uterine wall induced by implantation. In situ hybridization and immunohistochemical visualization of 3betaHSD VI mRNA and protein shows high expression of this enzyme in the antimesometrial cells of the decidua of days 6.5-7.5 post coitum (p.c.). Thereafter, expression of 3betaHSD VI in the decidual zones disappears and is replaced by a high expression of mRNA and protein in the embryonal giant trophoblast cells. At the peak of their development on day 9.5 p.c., the mouse giant trophoblast cells also express Steroidogenic Acute Regulatory (StAR) protein, which is required for steroidogenesis in the gonads and adrenal cortex. Our findings also suggest that the declining levels of P450scc, 3betaHSD VI, and StAR proteins between days 10.5-14.5 p.c. in the developing placenta is consistent with previous reports that the mouse placenta is not involved in de novo synthesis of steroids during the second half of pregnancy. Collectively, the results of the present study suggest that, during early phases of pregnancy, local progesterone synthesis in the maternal decidua and the trophoblast layers surrounding the embryonal cavity is important for successful implantation and/or maintenance of pregnancy. We propose that the local production of progesterone acts as an immunosuppressant at the fetal maternal interface preventing the rejection of the fetal allograft.
Asunto(s)
Expresión Génica , Progesterona/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Femenino , Edad Gestacional , Isoenzimas/genética , Ratones , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Fosfoproteínas/genética , Placenta/metabolismo , Embarazo , Progesterona Reductasa/genética , Ratas , Ratas Sprague-Dawley , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide Isomerasas/genéticaRESUMEN
Müllerian-inhibiting substance (MIS) is a hormone produced by Sertoli cells of the fetal testes that causes regression of the Müllerian ducts, the precursors to female reproductive tract structures that are present in the bipotential urogenital ridge. MIS is also produced in the adult gonads of both males and females, albeit at much lower levels than those measured during the fetal and perinatal periods. Adult transgenic mice chronically overexpressing MIS exhibit severe gonadal abnormalities and, in males, dramatically reduced levels of testosterone, which might lead to the incomplete virilization observed in some of the males. To understand the roles played by MIS in the adult gonad, we performed Northern analyses to show that the MIS type II receptor is expressed in purified Leydig cells and in two rodent Leydig cell lines, R2C and MA-10. Addition of purified recombinant human MIS to cultures of both R2C and MA-10 cells reduced steroid production. With MA-10 cells, the reduction of testosterone secretion into the medium was reduced to 1/10th of that in the control culture, which provided us with a means to study the molecular mechanisms underlying MIS-mediated suppression of testosterone synthesis. Northern analysis revealed that after stimulation with cAMP, the expression of messenger RNA for P450c17 hydroxylaselyase, the enzyme that catalyzes the conversion of progesterone to androstenedione, was reduced to background levels in the presence of MIS. Addition of cycloheximide, a protein synthesis inhibitor, did not prevent the effect of MIS, indicating a direct effect of MIS signal transduction on the expression of P450c17. Analysis of the transcriptional activity of Cyp17, the gene for murine P450c17, with Cyp17 promoter/luciferase reporter constructs shows that MIS regulates the transcription of Cyp17 in a concentration- and time-dependent manner. From our results, we conclude that MIS might play a physiological role in maintaining testosterone homeostasis. These findings will allow us in the future to use the transcriptional regulation of Cyp17 as a model to uncover the signal transduction pathways of MIS and the molecular mechanisms of its suppression of androgen synthesis.
Asunto(s)
Andrógenos/biosíntesis , Andrógenos/genética , Glicoproteínas , Inhibidores de Crecimiento/fisiología , Hormonas Testiculares/fisiología , Transcripción Genética/fisiología , Animales , Hormona Antimülleriana , Expresión Génica/fisiología , Inhibidores de Crecimiento/farmacología , Humanos , Células Intersticiales del Testículo/metabolismo , Masculino , Isoformas de Proteínas/metabolismo , Ratas , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta , Proteínas Recombinantes , Esteroide 17-alfa-Hidroxilasa/genética , Esteroides/antagonistas & inhibidores , Esteroides/biosíntesis , Hormonas Testiculares/farmacología , Células Tumorales CultivadasRESUMEN
Six isoforms of the enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) have been identified in the mouse, each the product of a distinct gene. Two of these isoforms (type I and type VI) are detectable in the adult testis but changes in their expression during development are unknown. In this study we have examined changes in testicular expression and localization of mRNA encoding the type I and type VI isoforms of 3betaHSD. Total 3betaHSD (type I plus type VI) mRNA was measured by reverse transcription-polymerase chain reaction and showed a peak of expression at day 5 after birth followed by a decline and then a further rise after day 10 that continued up to adulthood. When each isoform was measured individually it was clear that the type I isoform was expressed at all ages from embryonic day 13 to adulthood. In contrast, the type VI isoform was only expressed at significant levels during fetal life on embryonic day 13 and then not again until after day 10 postnatally. Expression of the type VI isoform mRNA increased markedly after day 10 so that by adulthood it was the predominant 3betaHSD isoform present in the testis. Closer examination of the timing of type VI expression showed that the isoform mRNA was first detectable at a significant level on day 11. In-situ hybridization confirmed that the type I isoform is the only one expressed in the fetal/neonatal animal and showed that expression was limited to the interstitial tissue. In the adult, both type I and type VI expression was within the interstitial tissue. The timing of 3betaHSD type VI mRNA expression suggests, strongly, that this isoform is expressed only by adult-type Leydig cells in the mouse testis and that this development starts shortly before day 11. The limited expression of the type VI isoform means that it will be a useful marker in studies of adult Leydig cell development.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/biosíntesis , Isoenzimas/biosíntesis , Testículo/enzimología , Testículo/crecimiento & desarrollo , 3-Hidroxiesteroide Deshidrogenasas/genética , Animales , Femenino , Hibridación in Situ , Técnicas In Vitro , Isoenzimas/genética , Células Intersticiales del Testículo/enzimología , Masculino , Ratones , ARN Mensajero/biosíntesis , Testículo/citologíaRESUMEN
The enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme in the biosynthesis of steroid hormones. To date, this laboratory has isolated and characterized five distinct 3beta-HSD complementary DNAs (cDNAs) in the mouse (3beta-HSD I through V). These different forms are expressed in a tissue- and developmentally-specific manner and fall into two functionally distinct enzymes. 3beta-HSD I and III, and most likely II, function as dehydrogenase/isomerases, whereas 3beta-HSD IV and V function as 3-ketosteroid reductases. This study describes the isolation, characterization, and tissue-specific expression of a sixth member of this gene family, 3beta-HSD VI. This new isoform functions as an NAD+-dependent dehydrogenase/isomerase exhibiting very low Michaelis-Menten constant (Km) values for pregnenolone (approximately 0.035 microM) and dehydroepiandrosterone (approximately 0.12 microM). 3beta-HSD VI is the earliest isoform to be expressed during embryogenesis in cells of embryonic origin at 7 and 9.5 days postcoitum (pc), and is the major isoform expressed in uterine tissue at the time of implantation (4.5 days pc) and continues to be expressed in uterine tissue at 6.5, 7.5, and 9.5 days pc. 3beta-HSD VI is expressed in giant trophoblasts at 9.5 days pc and is expressed in the placenta through day 15.5 pc. In the adult mouse, 3beta-HSD VI appears to be the only isoform expressed in the skin and also is expressed in the testis, but to a lesser extent than 3beta-HSD I. Mouse 3beta-HSD VI cDNA is orthologous to human 3beta-HSD I cDNA. Human type I 3beta-HSD has been shown to be the only isoform expressed in the placenta and skin. The demonstration that mouse 3beta-HSD VI functions as a dehydrogenase/isomerase and is the predominant isoform expressed during the first half of pregnancy in uterine tissue and in embryonic cells suggests that this isoform may be involved in local production of progesterone, which is needed for successful implantation of the blastocyst and/or maintenance of early pregnancy.
Asunto(s)
Isoenzimas/aislamiento & purificación , Progesterona Reductasa/aislamiento & purificación , Glándulas Suprarrenales/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Western Blotting , Células COS , Deshidroepiandrosterona/metabolismo , Femenino , Gónadas/química , Humanos , Isoenzimas/química , Masculino , Ratones , Ratones Endogámicos C57BL/embriología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Embarazo , Pregnenolona/metabolismo , Progesterona Reductasa/química , Útero/enzimologíaRESUMEN
Steroid 5alpha-reductase is a system of two isozymes (5alphaR-1 and 5alphaR-2) which catalyzes the NADPH-dependent reduction of testosterone to dihydrotestosterone in many androgen sensitive tissues and which is related to several human endocrine diseases such as benign prostatic hyperplasia (BPH), prostatic cancer, acne, alopecia, pattern baldness in men and hirsutism in women. The discovery of new potent and selective 5alphaR inhibitors is thus of great interest for pharmaceutical treatment of these diseases. The synthesis of a novel class of inhibitors for human 5alphaR-1 and 5alphaR-2, having the 19-nor-10-azasteroid skeleton, is described. The inhibitory potency of the 19-nor-10-azasteroids was determined in homogenates of human hypertrophic prostates toward 5alphaR-2 and in DU-145 human prostatic adenocarcinoma cells toward 5alphaR-1, in comparison with finasteride (IC50 = 3 nM for 5alphaR-2 and approximately 42 nM for 5alphaR-1), a drug which is currently used for BPH treatment. The inhibition potency was dependent on the type of substituent at position 17 and on the presence and position of the unsaturation in the A and C rings. delta9(11)-19-Nor-10-azaandrost-4-ene-3,17-dione (or 10-azaestra-4,9(11)-diene-3,17-dione) (4a) and 19-nor-10-azaandrost-4-ene-3,17-dione (5) were weak inhibitors of 5alphaR-2 (IC50 = 4.6 and 4.4 microM, respectively) but more potent inhibitors of 5alphaR-1 (IC50 = 263 and 299 nM, respectively), whereas 19-nor-10-aza-5alpha-androstane-3,17-dione (7) was inactive for both the isoenzymes. The best result was achieved with the 9:1 mixture of delta9(11)- and delta8(9)-17beta-(N-tert-butylcarbamoyl)-19-nor-10-aza-4- androsten-3-one (10a,b) which was a good inhibitor of 5alphaR-1 and 5alphaR-2 (IC50 = 127 and 122 nM, respectively), with a potency very close to that of finasteride. The results of ab initio calculations suggest that the inhibition potency of 19-nor-10-azasteroids could be directly related to the nucleophilicity of the carbonyl group in the 3-position.
Asunto(s)
Inhibidores de 5-alfa-Reductasa , Azaesteroides/farmacología , Inhibidores Enzimáticos/farmacología , Azaesteroides/química , Femenino , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Neoplasias de la Próstata/enzimología , Células Tumorales CultivadasRESUMEN
The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) is essential for the biosynthesis of all active steroid hormones. To date five distinct isoforms have been identified in the mouse. The different isoforms are indicated by roman numerals (I-V) in the chronological order in which they have been isolated. The different isoforms are expressed in a tissue- and developmentally specific manner and fall into two functionally distinct groups. 3 beta-HSD I, II, and III function as NAD(+)-dependent dehydrogenaselisomerases, and IV and V function as NADPH-dependent 3-keto steroid reductases. These latter two isoforms, therefore, are not involved in the biosynthesis of steroid hormones, but most likely in the inactivation of steroid hormones. In the adult mouse 3 beta-HSD I is expressed in the classical steroidogenic tissues, the adrenal glands and the gonads. 3 beta-HSD II and III are expressed in the liver and kidney, with III being the major isoform expressed in the adult liver. 3 beta-HSD IV is expressed almost exclusively in the kidney of both sexes, and expression of 3 beta-HSD V is observed only in the male liver starting late in puberty. In the fetal liver of both sexes, 3 beta-HSD I is the major or only isoform expressed at 13.5 days postconception and remains the major isoform until the day of birth, after which 3 beta-HSD III becomes the major isoform. Expression of 3 beta-HSD I in the liver decreases after birth and ceases by day 20 postnatally. Thus the liver expresses four distinct isoforms of 3 beta-HSD, I, II, III, and V, at different times during development. The mouse 3 beta-HSD genes, Hsd3b, have been mapped to a small region on mouse chromosome 3. Analysis of two yeast artificial chromosome (YAC) libraries identified one clone that contains the entire Hsd3b locus within a 1400-kb insert. Hybridization by Southern blot analysis of restriction-enzyme-digested YAC DNA using an 18-base oligonucleotide that hybridizes without mismatch to all known Hsd3b sequences indicates that there are a total of seven Hsd3b genes or pseudogenes in the mouse genome. Further analysis of mouse genomic DNA by pulse field gel electrophoresis suggests that all of the Hsd3b gene family is found within a 400-kb fragment.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glándulas Suprarrenales/enzimología , Factores de Edad , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Femenino , Isoenzimas , Hígado/enzimología , Masculino , Ratones , Datos de Secuencia Molecular , Placenta/enzimología , Embarazo , Ratas , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Distribución TisularRESUMEN
In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) protein and the accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated mechanism. To examine the mechanism by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene, constructs containing 5'-flanking sequences of the mouse Cyp17 linked to the chloramphenicol acetyltransferase (CAT) reporter gene were cotransfected into MA-10 tumor Leydig cells with a mouse AR expression plasmid. In the presence of dihydrotestosterone, the cAMP-induced expression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestosterone was observed when the -1021 bp Cyp17-CAT construct was cotransfected with a human AR expression plasmid missing the second zinc finger of the DNA-binding domain, indicating that DNA binding is involved in AR-mediated repression of Cyp17 expression. Analysis of deletions -346 bp of 5'-flanking region of the mouse Cyp17 promoter are sufficient to confer androgen repression of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footprinting analysis indicated that the AR interacts with sequences between -330. and -278 bp of the Cyp17 promoter. This region overlaps with the previously identified cAMP-responsive region located between -346 and -245 bp of the Cyp17 promoter. These results suggest that AR-mediated repression involves binding of the AR to sequences in the cAMP-responsive region of the Cyp17 promoter, possibly interfering with the binding of the protein(s) that mediate cAMP induction of Cyp17.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Dihidrotestosterona/farmacología , Receptores Androgénicos/fisiología , Proteínas Represoras/fisiología , Esteroide 17-alfa-Hidroxilasa/biosíntesis , Animales , AMP Cíclico/fisiología , Sistema Enzimático del Citocromo P-450/genética , Huella de ADN , Inducción Enzimática/efectos de los fármacos , Genes Reporteros , Humanos , Tumor de Células de Leydig/patología , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Sistemas de Mensajero Secundario , Esteroide 17-alfa-Hidroxilasa/genética , Neoplasias Testiculares/patología , Testosterona/biosíntesis , Transfección , Células Tumorales CultivadasRESUMEN
The enzyme 3 beta-hydroxysteroid dehydrogenase (3 betaHSD) is essential for all steroid hormone biosynthesis. Six distinct 3 betaHSD cDNAs in the mouse (3 betaHSD I-VI) have been isolated previously. This study reports the isolation of genes or partial genes encoding the 3 betaHSDI, II, III, and IV isoforms. Characterization of the genes revealed that they consist of four exons, the same structure that has been observed for characterized human 3 betaHSD genes. Primer extension and nuclease S1 analysis identified the start sites of transcription of Hsd3b -1 and -4. The proximal promoter regions of Hsd3b-1 and -4 were sequenced and putative cis-acting sequences were determined. Previously, we reported that the then known 3 betaHSD genes (3 betaHSD I-IV) were located in a small region of mouse chromosome 3. To analyze this locus further, six yeast artificial chromosome clones containing the 3 betaHSD sequence were identified. One clone appears to contain the complete 3 betaHSD locus within its 1,400-kbp insert. Further analysis of this YAC, along with analysis of mouse genomic DNA by pulsed-field gel electrophoresis, suggests all members of the 3 betaHSD gene family may be contained on a 400-kbp fragment.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , Mapeo Cromosómico , Familia de Multigenes/genética , Animales , Secuencia de Bases , Cromosomas Artificiales de Levadura/genética , Clonación Molecular , ADN/análisis , Exones/genética , Femenino , Genes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Análisis de Secuencia de ADN , Transcripción Genética/genéticaRESUMEN
The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) is essential for the biosynthesis of all steroid hormones. To date this laboratory has isolated and characterized five distinct 3 beta HSD cDNAs in the mouse (3 beta HSD I-V). The different isoforms fall into two functionally distinct groups. 3 beta HSD I and III function as dehydrogenase/isomerases and 3 beta HSD IV and V function as 3-ketosteroid reductases. Previously it was shown that the liver of the adult mouse expresses 3 beta HSD II, III and V, with 3 beta HSD III being the major isoform. This study examines the expression of the different forms of 3 beta HSD mRNAs and proteins in the livers of male and female mice during fetal and postnatal development. 3 beta HSD I, which in the adult mouse is expressed only in the gonads and adrenal glands, is the major isoform expressed in both male and female livers during fetal development until the first postnatal (pn) day after which time 3 beta HSD III becomes the major isoform. Expression of 3 beta HSD I mRNA and protein completely ceases after day 20 pn. The expression of 3 beta HSD V is first detected at day 40 pn and is observed only in the male. Very low expression of 3 beta HSD II mRNA is detected throughout development. Previous characterization of enzymatic activity of the expressed proteins showed that 3 beta HSD I exhibits lower Km values for the delta 5-3 beta-hydroxysteroids than 3 beta HSD III, indicating that 3 beta HSD I functions as a more efficient 3 beta-hydroxysteroid dehydrogenase/isomerase than 3 beta HSD III. The results of this study suggest that the liver may play an important role in the biosynthesis of steroid hormones during murine fetal development.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , Expresión Génica , Isoenzimas/genética , Hígado/enzimología , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Secuencia de Bases , Femenino , Hidroxiesteroides/metabolismo , Isoenzimas/metabolismo , Cetosteroides/metabolismo , Hígado/embriología , Hígado/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Caracteres Sexuales , Especificidad por SustratoRESUMEN
The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) plays an essential role in the biosynthesis of all steroid hormones. We previously reported the isolation, characterization, and tissue-specific expression of four distinct but highly homologous 3 beta HSD cDNAs (forms I, II, III, and IV). Enzymatic characterization of three of these isoforms demonstrated that mouse 3 beta HSD I and III function as dehydrogenase/isomerases, but 3 beta HSD IV functions exclusively as a 3-ketosteroid reductase. We now report the isolation and characterization of an additional distinct mouse 3 beta HSD cDNA, 3 beta HSD V, which is expressed in the liver of male mice beginning in late puberty. Similar to 3 beta HSD IV, 3 beta HSD V functions exclusively as a 3-ketosteroid reductase converting an active androgen, dihydrotestosterone (DHT), into an inactive androgen, 5 alpha-androstane-3 beta,17 beta-diol. Expressed 3 beta HSD V, however, exhibits a considerably lower apparent Michaelis-Menten constant (Km) value for DHT than 3 beta HSD IV (0.47 microM vs. 2.2 microM, respectively). The complete predicted amino acid sequence of 3 beta HSD II is also reported. The predicted amino acid sequence of mouse 3 beta HSD V reveals that this new form is more closely related to the 3-ketosteroid reductases, mouse 3 beta HSD IV and rat III (93 and 84% identity, respectively), than to the other rodent isoforms that share less than 75% identity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , Familia de Multigenes , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Dihidrotestosterona/metabolismo , Cinética , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , NAD/farmacología , NADP/farmacología , Ratas , Maduración Sexual , Especificidad por Sustrato , TransfecciónRESUMEN
Testosterone biosynthesis in Leydig cells is dependent on the action of 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 (P450c17), which is encoded by the Cyp17 gene. Tumor necrosis factor-alpha (TNF alpha), a proinflammatory cytokine, inhibits cAMP-stimulated testosterone production in mouse Leydig cells. The inhibition of testosterone production is parallel to the inhibition of P450c17 messenger RNA and protein levels. To examine the mechanism of TNF alpha-mediated inhibition of steroidogenesis, the effect of TNF alpha on cAMP-stimulated induction of Cyp17 expression was investigated. To determine whether the protein kinase C (PKC) signaling pathway is involved in TNF alpha inhibition of steroidogenesis, the effects of the PKC activator, phorbol 12-myristate 13-acetate (PMA), and the PKC inhibitor, calphostin C, were examined. Treatment of normal mouse Leydig cells in primary culture with 50 microM 8-bromo-cAMP (cAMP) plus 1 ng/ml TNF alpha or 10 nM PMA caused a similar (approximately 90%) decrease in testosterone accumulation and cAMP-stimulated P450c17 messenger RNA levels compared to those after treatment with cAMP alone. To determine whether TNF alpha inhibits the cAMP-induced expression of the Cyp17 gene, plasmids containing two different size fragments of the 5'-flanking region of the Cyp17 gene upstream of the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into MA-10 tumor Leydig cells, and the effect of TNF alpha on cAMP-induced CAT activity was determined. Treatment of cells, transfected with either plasmid, with 500 microM cAMP plus increasing concentrations (0.1, 1.0, and 10 ng/ml) of TNF alpha resulted in a dose-dependent repression of cAMP-stimulated CAT activity. Higher concentrations of TNF alpha (up to 100 ng/ml) did not result in greater inhibition. Treatment of transfected cells with 10 nM PMA resulted in a 51 +/- 6.6% inhibition of cAMP-stimulated CAT activity. Calphostin C (1 microM) completely reversed the inhibitory effect of TNF alpha or PMA. Calphostin C alone had no effect on promoter activity. TNF alpha-stimulated PKC alpha translocation was quantitated by Western blot. After treatment for 3 h, the distribution of immunoreactive PKC alpha in cytosol vs. nucleus was 55%/45%, 60%/40%, and 29%/71% in control, cAMP-treated, and TNF alpha-treated cells, respectively. TNF alpha-stimulated PKC alpha translocation was further demonstrated by indirect immunofluorescence assay. PMA, a known activator of PKC, and TNF alpha had a similar inhibitory effect on P450c17 expression, testosterone production, and Cyp17-CAT activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Aldehído-Liasas/genética , Sistema Enzimático del Citocromo P-450/genética , Expresión Génica/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/metabolismo , Animales , Transporte Biológico , Células Cultivadas , AMP Cíclico/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Células Intersticiales del Testículo/fisiología , Masculino , Ratones , Ratones Endogámicos , Naftalenos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Esteroide 17-alfa-Hidroxilasa , Testosterona/biosíntesis , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The classical form of the enzyme 5-ene-3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3 beta-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3 beta HSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3 beta HSD I and III function as 3 beta-hydroxysteroid dehydrogenases and 5-en-->4-en isomerases using NAD+ as a cofactor. The enzymatic function of 3 beta HSD II has not been completely characterized. Mouse 3 beta HSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3 beta HSD I is expressed in the gonads and adrenal glands of the adult mouse. 3 beta HSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Animales , Evolución Biológica , Mapeo Cromosómico , Genes , Humanos , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , NAD/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Esteroides/biosíntesis , Especificidad por Sustrato , Distribución TisularRESUMEN
The Leydig cell of the testis is the only cell in the male that has the capacity to synthesize testosterone from cholesterol. Testosterone is critical during fetal development for male sexual differentiation, and postnatally for initiation and maintenance of spermatogenesis and the expression of the male secondary sex characteristics. The biosynthesis of testosterone requires the activities of four enzymes, cholesterol side-chain cleavage enzyme (P450scc), 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD), 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha)), and 17-ketosteroid reductase. The expression of these enzymes appears to be regulated by different mechanisms. The recent isolation of the mouse cDNAs and structural genes that encode these enzymes has enabled us to begin to investigate the regulation of their expression at the molecular level. This review discusses the regulation by cAMP and steroids of three enzymes in Leydig cells, P450scc, P450(17 alpha), and 3 beta HSD, as well as characterization of the promoters of the mouse genes that encode P450scc and P450(17 alpha).
Asunto(s)
Regulación de la Expresión Génica , Células Intersticiales del Testículo/enzimología , Testosterona/biosíntesis , 17-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/genética , Animales , Secuencia de Bases , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Humanos , Masculino , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Esteroide 17-alfa-Hidroxilasa/genética , Testículo/embriología , Testículo/enzimologíaRESUMEN
It is well known that fetal androgens are required for male sexual differentiation, and it is thought that fetal ovaries are not steroidogenically active. However, molecular details, such as which steroidogenic enzymes are present in fetal testes and which enzymes are absent in fetal ovaries, have not been established. The pattern of expression of the genes that encode four of the steroidogenic enzymes necessary for androgen and estrogen production was examined during fetal development in mouse gonads. Messenger RNA (mRNA) expression for cholesterol side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD), P450 17 alpha-hydroxylase/C17-20 lyase (P450c17), and P450 aromatase (P450arom) was determined before ovaries and testes were distinguishable (13 days postconception) and during sexual differentiation (15, 17, and 20 days postconception) using reverse transcriptase-polymerase chain reactions (RT-PCR). A PCR assay for Sry was used to determine gender on day 13. P450scc, 3 beta HSD, and P450c17 transcripts were detected at all ages in fetal testes, indicating that mRNAs for the steroidogenic enzymes that are required to convert cholesterol to androgens are present in the male gonad even before sexual differentiation. P450arom mRNA was detected in several fetal testes on day 17, but consistently observed on day 20. The expression of P450arom suggests the potential of fetal and neonatal testes to convert androgens to estrogens. In contrast, although 3 beta HSD mRNA was detected in several of the ovaries examined, the detection of P450scc, P450c17, and P450arom transcripts was rare. These data suggest that the absence of fetal ovarian steroid hormone production is the result of lack of expression of at least three of the steroidogenic enzymes, P450scc, P450c17, and P450arom.
Asunto(s)
Enzimas/genética , Feto/metabolismo , Regulación de la Expresión Génica , Gónadas/embriología , Esteroides/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/genética , Animales , Aromatasa/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Femenino , Feto/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Análisis para Determinación del Sexo , Esteroide 17-alfa-Hidroxilasa/genéticaRESUMEN
The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is essential for the biosynthesis of all steroid hormones. The enzyme catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. We report the isolation of a novel mouse 3 beta HSD cDNA, 3 beta HSD IV, and describe the tissue-specific expression of its mRNA, the enzyme characteristics of the 3 beta HSD IV protein, and the structural and functional relationships it has to other 3 beta HSD isoforms previously characterized in the mouse and rat. The predicted amino acid sequence of mouse 3 beta HSD IV is 77% and 73% identical to that of mouse 3 beta HSD I and III, respectively. Comparison of the nucleotide and amino acid sequences of the four isoforms characterized to date show that 3 beta HSD IV is more distantly related to I, II, and III than these three forms are to each other. 3 beta HSD IV mRNA is only expressed in mouse kidney. In situ hybridization of mouse kidney indicates that expression is found only in the cortex and appears to be associated with the proximal tubules. Transiently expressed 3 beta HSD IV protein could not convert the delta 5-3 beta-hydroxysteroids, pregnenolone or dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone or androstenedione, nor did it have the capacity to convert 5 alpha-androstane-3 beta, 17 beta-diol to dihydrotestosterone, characteristic enzymatic activities of expressed mouse 3 beta HSD I and III. 3 beta HSD IV could only catalyze the conversion of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADPH. Thus, 3 beta HSD IV is a 3-ketosteroid reductase rather than a 3 beta-hydroxysteroid dehydrogenase/isomerase despite its homology to the other members of the 3 beta HSD family. Mouse 3 beta HSD IV is functionally and structurally most closely related to rat 3 beta HSD III, an isoform expressed predominantly in male rat liver.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , Corteza Renal/enzimología , Ratones/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Femenino , Genes , Humanos , Túbulos Renales Proximales/enzimología , Masculino , Ratones/metabolismo , Datos de Secuencia Molecular , Filogenia , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Esteroides/metabolismo , Especificidad por SustratoRESUMEN
The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.