RESUMEN
The expression of the 17 beta-hydroxysteroid dehydrogenase (17-HSD) gene in a series of human breast cancer cell lines was studied by Northern blot hybridization with a cDNA probe and by a time-resolved immunofluorometric assay using polyclonal antibodies against the enzyme protein. The 17-HSD enzyme protein concentration was measured in the 800 x g cell extract. A high concentration was measured in the BT-20 cell line, corresponding to one-fourth of the average concentration in placental tissue. Western blot analysis indicated that the antigen corresponded to a single Mr 35,000 band. In 2 other cell lines (MDA-MB-361 and T-47D), the 17-HSD protein concentration was much lower, but still measurable, whereas in the remaining 5 cell lines (HBL-100, MCF-7, MDA-MB-231, MDA-MB-468, and ZR-75-1) it was below the detection limit of the assay. Treatment of the cells for 5 days with the synthetic progestin, ORG2058, resulted in an increase of the 17-HSD protein concentration only in the T-47D cell line. By Northern blot analysis, a low level of 2.3-kilobase mRNA transcripts was detected in all 8 cell lines. In addition, a 1.3-kilobase 17-HSD mRNA was present in the samples from the 3 cell lines containing measurable amounts of 17-HSD protein in the cell extract, and the band intensities were proportional to the amount of protein measured with the immunofluorometric assay. Only in the T-47D cell line did progestin treatment correspond to an increased amount of the 17-HSD 1.3-kilobase mRNA. These results suggest that the 1.3-kilobase mRNA for 17-HSD is the form most closely associated with protein expression and is also the only form responding to the progestin induction of the 17-HSD gene.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , Neoplasias de la Mama/genética , Pregnenodionas/farmacología , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Neoplasias de la Mama/enzimología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/genética , Placenta/enzimología , Congéneres de la Progesterona/farmacología , ARN Mensajero/genética , ARN Neoplásico/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Células Tumorales CultivadasRESUMEN
The radioimmunoassay of human prostate-specific acid phosphatase and the measurement of the catalytic activity of acid phosphatase using p-nitrophenyl phosphate as substrate were compared in the diagnosis and follow-up of therapy of prostatic cancer patients. We monitored 17 patients without metastases and eight patients with metastases for 12 months. We detected elevation of the catalytic activity of acid phosphatase [the upper limit for the reference range was mean + 2 (S.D.)] in 24% of the sera of all these patients (n = 25), and the concentration of prostate-specific acid phosphatase measured by radioimmunoassay [the upper limit for the reference range was mean + 3 (S.D.)] was elevated in 80% of these samples before therapy. The radioimmunological measurement of prostate-specific acid phosphatase was therefore more efficient in detecting prostatic cancer than was measurement of the catalytic activity. Favorable effects of the various forms of endocrine treatment were detected more clearly by the measurement of immunoassayable prostatic acid phosphatase than by the measurement of catalytic activity. Activation of the disease during various forms of endocrine treatment of prostatic carcinoma is possibly more efficiently signaled by radioimmunoassay than by measurement of catalytic activity.
Asunto(s)
Fosfatasa Ácida/metabolismo , Isoenzimas/metabolismo , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/enzimología , Humanos , Masculino , Neoplasias de la Próstata/terapia , RadioinmunoensayoRESUMEN
Concentrations of cytosol estrogen (ERC) and cytosol progestin (PRC) receptors were assayed in malignant tissue specimens of 230 patients with endometrial cancer, and those of nuclear estrogen and nuclear progestin receptors, and 17 beta-hydroxysteroid dehydrogenase activities in about 100 specimens. Endometrial cancer was at an early stage in 205 and advanced in 25 patients. As a supplement to surgical and radiation therapy, all patients received p.o. medroxyprogesterone acetate (100 mg a day) for 2 years. The follow-up time varied from 12 to 96 months (median, 42 months). Concentrations of ERC, PRC, nuclear estrogen, and nuclear progestin receptors in endometrial cancer tissue were significantly lower in clinical stages III-IV than in clinical stage I. In clinical stage I, ERC and PRC appeared in significantly lower concentrations in anaplastic than in moderately and well differentiated malignancies. The concentrations of these receptors were increased in obese patients, and the activity of 17 beta-hydroxysteroid dehydrogenase was increased in patients younger than 50 years, suggesting that endogenous female steroid hormones modify the pattern of female steroid receptors in malignant endometrium. In clinical stage I, 13 of 153 patients with adequate therapy contracted a recurrent disease. Poor prognosis was predicted by anaplastic structure of the malignancy (P less than 0.001), low tissue concentrations (0-30 fmol/mg protein) of ERC alone (P = 0.006), PRC alone (P = 0.010), and ERC and PRC simultaneously (P = 0.004). All 101 patients who simultaneously had ERC and PRC in concentrations higher than 30 fmol/mg protein remained disease free for 2 years, whereas all recurrences in patients with receptor-poor tumors appeared during the 2 years of medroxyprogesterone acetate treatment. In clinical stage II, with 30 patients, no prognosis indicators predicted the clinical outcome, whereas in clinical stages III + IV, with 25 patients, low ERC concentrations were associated with a worsened prognosis (P = 0.045). Conclusively, cytosol and nuclear estrogen and nuclear progestin receptor concentrations and 17 beta-hydroxysteroid dehydrogenase activity give valuable information about the endocrine associations in endometrial cancer. Cytosol estrogen and cytosol progestin receptors appeared to be useful predictors of recurrent disease. They also have the potential to distinguish between patients expected to benefit from adjuvant progestin therapy and those expected to be unresponsive to the same treatment.
Asunto(s)
Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Neoplasias Uterinas/análisis , Anciano , Femenino , Humanos , Medroxiprogesterona/análogos & derivados , Medroxiprogesterona/uso terapéutico , Acetato de Medroxiprogesterona , Persona de Mediana Edad , Miometrio/patología , Pronóstico , Neoplasias Uterinas/mortalidad , Neoplasias Uterinas/patologíaRESUMEN
Several studies have indicated that in prostate cancer, frequent aberrations take place in several genomic regions. In the present study, we have analyzed allelic losses in chromosome 16 region q in 50 prostate cancer specimens of various histological grades. The most frequently deleted region was located at 16q23-16q24.2 between loci D16S504 and D16S422. The highest percentage of loss of heterozygosity (LOH) at 16q was also found within this area at loci HSD17B2 and D16S422 located at 16q24.1-q24.2. The LOH at 16q24.1-q24.2 was significantly associated with clinically aggressive behavior of the disease, metastatic disease, and higher tumor grade. Of the metastatic diseases, 83% showed LOH, whereas only 40% of the nonmetastatic diseases were found to show it. Similarly, LOH was found in 76% of the clinically aggressive diseases and in 33% of the nonaggressive diseases. The data suggest that a potentially important gene associated with prostate cancer progression is located close to 16q24.1-q24.2.
Asunto(s)
Cromosomas Humanos Par 16/genética , Eliminación de Gen , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Mapeo Cromosómico , Humanos , Masculino , Repeticiones de Microsatélite , Metástasis de la Neoplasia/genéticaRESUMEN
The therapeutic efficacy, plasma levels, and psychomotor effects of tryptophan (L-tryptophan), clomipramine hydrochloride, and doxepin were investigate in "neurotically" depressed outpatients. The tricyclic antidepressants were significantly more efficacious than tryptophan in inducing remission. The alleviation of depression was preceded by an improvement of the initially slow information-processing rates in the depressed patients. The plasma levels of the tricyclics that were associated with a therapeutic response were significantly lower than those reported in "endogenously" depressed inpatients.
Asunto(s)
Clomipramina/uso terapéutico , Trastorno Depresivo/tratamiento farmacológico , Doxepina/uso terapéutico , Adulto , Clomipramina/sangre , Doxepina/sangre , Femenino , Humanos , Masculino , Tiempo de Reacción/efectos de los fármacos , Triptófano/sangre , Triptófano/uso terapéuticoRESUMEN
The inhibitory effects of recombinant porcine interferon-gamma (IFN gamma) on human CG (hCG)-stimulated testosterone production, and on mRNA concentrations of cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20lyase (P450c 17) were investigated using porcine primary Leydig cell culture as a model. After preincubation of Leydig cells for 24 h with 1000 pM IFN gamma, hCG-stimulated (10 ng/ml, 2 h) testosterone production was inhibited by 50%, whereas no significant changes were seen in hCG-stimulated cAMP production. Incubation with 10 microM 5-cholestene-3 beta,22(R)-diol or 10 microM 5-cholestene-3 beta,20 alpha-diol together with hCG (10 ng/ml, 2 h) reversed most of the inhibitory effect of IFN gamma, suggesting that IFN gamma inhibits P450scc activity, possibly by inhibiting the substrate (cholesterol) availability for P450scc. Incubation with IFN gamma also decreased basal concentrations of P450scc (45%) and P450c 17 (35%) mRNA, although these changes probably did not contribute to the decreased testosterone production. Long-term treatment with hCG (100 ng/ml, 24 h) increased P450scc mRNA (3- to 4-fold) and P450c 17 mRNA (4- to 5-fold) concentrations. Simultaneous treatment with IFN gamma attenuated these hCG-induced increases in P450scc mRNA (50%) and P450c 17 mRNA (40-100%) concentrations, as well as in testosterone production (77%). This inhibition of testosterone production could only be partly reversed by the hydroxylated cholesterol derivatives. This suggests that in addition to possible suppression of cholesterol availability, decreased P450scc and/or P450c 17 activities (through decreased mRNA concentrations) were also involved in the IFN gamma suppressed steroidogenic capacity of porcine Leydig cells during long-term hCG stimulation.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/antagonistas & inhibidores , Interferón gamma/farmacología , Células Intersticiales del Testículo/metabolismo , ARN Mensajero/metabolismo , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Esteroide Hidroxilasas/antagonistas & inhibidores , Testosterona/biosíntesis , Animales , Células Cultivadas , Gonadotropina Coriónica/farmacología , AMP Cíclico/biosíntesis , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Proteínas Recombinantes/farmacología , PorcinosRESUMEN
17 beta-Hydroxysteroid dehydrogenases/17-ketosteroid reductases (17HSDs) modulate the biological activity of certain estrogens and androgens by catalyzing reductase or dehydrogenase reactions between 17-keto- and 17 beta-hydroxysteroids. In the present study, we demonstrate expression cloning of a novel type of 17HSD, chronologically named 17HSD type 7, from the HC11 cell line derived from mouse mammary gland. The cloned cDNA, 1.7 kb in size, encodes a protein of 334 amino acids with a calculated molecular mass of 37,317 Da. The primary structure contains segments characteristic of enzymes belonging to the short-chain dehydrogenase/reductase superfamily. Strikingly, mouse 17HSD type 7 (m17HSD7) shows 89% identity with a recently cloned rat protein called PRL receptor-associated protein (PRAP). The function of PRAP has not yet been demonstrated. The enzymatic characteristics of m17HSD7 and RT-PCR-cloned rat PRAP (rPRAP) were analyzed in cultured HEK-293 cells, where both of the enzymes efficiently catalyzed conversion of estrone (E1) to estradiol (E2). With other substrates tested no detectable 17HSD or 20 alpha-hydroxysteroid dehydrogenase activities were found. Kinetic parameters for m17HSD7 further indicate that E1 is a preferred substrate for this enzyme. Relative catalytic efficiencies (Vmax/K(m) values) for E1 and E2 are 244 and 48, respectively. As it is the case with rPRAP, m17HSD7 is most abundantly expressed in the ovaries of pregnant animals. Further studies show that the rat enzyme is primarily expressed in the middle and second half of pregnancy, in parallel with E2 secretion from the corpus luteum. The mRNA for m17HSD7 is also apparent in the placenta, and a slight signal for m17HSD7 is found in the ovaries of adult nonpregnant mice, in the mammary gland, liver, kidney, and testis. Altogether, because of their similar primary structures, enzymatic characteristics, and the tissue distribution of m17HSD7 and rPRAP, we suggest that rPRAP is rat 17HSD type 7. Furthermore, the results indicate that 17HSD7 is an enzyme of E2 biosynthesis, which is predominantly expressed in the corpus luteum of the pregnant animal.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , Clonación Molecular , Fosfoproteínas , 17-Hidroxiesteroide Deshidrogenasas/química , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/química , Femenino , Expresión Génica , Glándulas Mamarias Animales/enzimología , Ratones , Datos de Secuencia Molecular , Peso Molecular , Especificidad de Órganos , Ovario/enzimología , Embarazo , ARN Mensajero/análisis , RatasRESUMEN
Human 17 beta-hydroxysteroid dehydrogenase (17-HSD) type 1 catalyzes the conversion of the low activity estrogen, estrone, into highly active estradiol, both in the gonads and in target tissues. The present study was carried out to characterize the dimerization, microheterogeneity, and phosphorylation of human 17-HSD type 1 and to evaluate the current model of hydride transfer and substrate recognition of the enzyme, based on its x-ray structure. 17-HSD type 1 is a homodimer consisting of noncovalently bound subunits, and the data in the present study indicate an exceptionally strong association between the monomers [dissociation constant (Kd) < 5 pmol/monomers liter]. Furthermore, substitutions constructed at the hydrophobic dimer interface always resulted in inactive aggregates of the protein. The enzyme was shown to be phosphorylated by protein kinase A exclusively at Ser134 only in vitro. However, in contrast to previous suggestions, phosphorylation of Ser134 was shown to play no role in the activity or microheterogeneity of human 17-HSD type 1. The presence of microheterogeneity in the recombinant enzyme also indicates that it does not result from the frequent protein polymorphism previously found for the enzyme. In line with the x-ray structure and the proposed catalytic mechanism of the enzyme, our results indicate that Ser142, Tyr155, and Lys159 are all critical for hydride transfer in human 17-HSD type 1. In contrast, the proposed interaction between His221, Glu282, and the 3-OH group of the steroid at the substrate recognition helix could not be shown to exist. Neither of these residues plays a critical role in the catalytic action of the enzyme in cultured cells.
Asunto(s)
Estradiol Deshidrogenasas/química , Conformación Proteica , Sitios de Unión , Catálisis , Dimerización , Estradiol/metabolismo , Estradiol Deshidrogenasas/genética , Estradiol Deshidrogenasas/metabolismo , Estrona/metabolismo , Humanos , Enlace de Hidrógeno , Focalización Isoeléctrica , Mutagénesis Sitio-Dirigida , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Serina/fisiología , Relación Estructura-ActividadRESUMEN
Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes primarily the reductive reaction of estrone to the biologically more active form, estradiol. The enzyme is highly expressed in the human placenta and the ovary and, in addition, in certain estrogen target cells, such as breast epithelial cells. To elucidate the transcriptional control of the EDH17B2 gene, the gene encoding 17HSD type 1, we fused a series of 5'-deletion mutants of the EDH17B2 gene into chloramphenicol acetyl transferase reporter gene vectors. An enhancer region was identified within the bases -661 to -392 and it increased, in both orientations, thymidine kinase promoter activity more than 200-fold in JEG-3 choriocarcinoma cells. This enhancer region was also functional in another choriocarcinoma cell line, JAR, although to a lesser extent. In BT-20 and T-47D breast cancer cells the enhancer region increased thymidine kinase promoter activity to some degree but not as efficiently as expected on the basis of endogenous enzyme expression. No such enhancer activity was observed in 17HSD type 1 nonexpressing cell lines. The retinoic acid responsive element, which was located between bases -503 and -487 in the EDH17B2 enhancer, bound retinoid acid receptor alpha retinoid X receptor alpha complex and transmitted retinoic acid induction on transcription in JEG-3 and T-47D cells. Finally, a silencer, functional in all the cell lines tested, was localized in the region from -392 to -78. Deletion of the region lad to a 4-fold increase in reporter gene expression. Altogether, our findings suggest that transcriptional control of the EDH17B2 gene is coordinated by the cell-specific enhancer and the silencer.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , Elementos de Facilitación Genéticos , Regulación Enzimológica de la Expresión Génica , Transcripción Genética , Tretinoina/farmacología , Secuencia de Bases , Neoplasias de la Mama , Cloranfenicol O-Acetiltransferasa/genética , Coriocarcinoma , Femenino , Eliminación de Gen , Humanos , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Neoplasias de la Próstata , Proteínas Recombinantes de Fusión , Transfección , Células Tumorales CultivadasRESUMEN
The metabolism of estrogens catalyzed by human placental 17 beta-hydroxysteroid dehydrogenase (17HSD) transiently expressed in COS-m6 cells was studied, and the properties of the enzyme were compared with those of an endogenous hydroxysteroid dehydrogenase (HSD) expressed in the cells. In cultured cells, the endogenous HSD had almost exclusively oxidative activity, converting estradiol to estrone (oxidative and reductive activity, 0.84 +/- 0.164 and 0.034 +/- 0.01 nmol/mg protein.h, respectively). This was, nevertheless, opposed to the activity of the transiently expressed human placental 17HSD, as a high reductive activity (0.86 +/- 0.30 nmol/mg protein.h) appeared in the cells after transfection, whereas oxidative activity was not significantly induced. In the different transfections, the reductive activity was induced 13- to 34-fold, and the oxidative activity in the 17HSD-transfected cells was 65-162% of that in the mock-transfected cells. Thus, in cultured cells, these two enzymes preferentially catalyze opposite reactions. When the metabolism of the estrogens was followed up to 20 h, the two enzymes were found to regulate the proportion of estrone to estradiol in the culture medium. The different properties found for the enzymes show that the endogenous HSD expressed in the COS-m6 cells is an additional member of the family of 17HSD enzymes. It is suggested that different 17HSD enzymes exist, with differential estrogen substrate specificities in cultured cells. Thus, in addition to cofactor and substrate availability, the biological activity of estrogens in different cell types may be regulated by the expression of different forms of 17HSD enzymes, resulting in the dominance of either estradiol or estrone production.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , Estradiol/farmacología , Hidroxiesteroide Deshidrogenasas/metabolismo , Placenta/enzimología , Northern Blotting , Línea Celular Transformada , Humanos , Oxidación-Reducción , Especificidad por Sustrato , Factores de Tiempo , TransfecciónRESUMEN
17 beta-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) is a steroidogenic enzyme that catalyzes the reversible interconversion of estrone and estradiol. In this study, we investigated the roles of epidermal growth factor (EGF) and tumor growth factor-alpha (TGF alpha) in the regulation of 17HSD type 1 gene expression and catalytic activity in cultured JAR, JEG-3, and BeWo choriocarcinoma cells. EGF and TGF alpha increased 17HSD type 1 protein concentrations in JAR and JEG-3 cells, as measured by time-resolved immunofluorometric assay, and 17HSD catalytic activity, as determined by production of estradiol from estrone. These increases were accompanied by parallel increases in concentrations of the 1.3-kilobase messenger RNA coding for 17HSD type 1 in these cells. EGF receptor tyrosine kinase activity inhibitors, tyrphostins, inhibited EGF action in JEG-3 cells, indicating that tyrosine kinase activity is needed for stimulation of the 17HSD type 1 gene. Treatment with 8-bromo-cAMP or phorbol 12-myristate 13-acetate increased the amount of 17HSD type 1 protein. Furthermore, phorbol 12-myristate 13-acetate potentiated the stimulatory effect of EGF. These results suggest that EGF and/or TGF alpha may play an important role in 17HSD type 1 regulation and, consequently, in estrogen production in the human placenta.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , Coriocarcinoma/enzimología , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento Transformador alfa/farmacología , Tirfostinos , 17-Hidroxiesteroide Deshidrogenasas/clasificación , 17-Hidroxiesteroide Deshidrogenasas/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Catecoles/farmacología , Coriocarcinoma/patología , Humanos , Nitrilos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Ovarian estradiol (E2) production is regulated by complex interaction of different hormones, such as gonadotropins, steroids, and growth factors. Despite the key role of 17 beta-hydroxysteroid dehydrogenase (17HSD) in E2 biosynthesis, little is known about its regulation in the ovary. Recently, we have characterized the structure of rat 17HSD type 1 and demonstrated that its expression is regulated by gonadotropins and diethylstilbestrol (DES) in rat ovary in vivo. In the present study, the hormonal regulation of 17HSD type 1, and the expression of cytochrome P450 aromatase were examined in parallel in cultured granulosa cells obtained from DES-primed immature rats. Under these conditions, the cells show high expression of 17HSD type 1. Both the enzyme activity and 17HSD type 1 messenger RNA expression in these cells decreased over 2 days of culture in serum-free medium. However, recombinant FSH (recFSH) partially prevented the decreases in enzyme activity and messenger RNA expression in a dose-dependent manner. This effect appears to be mediated by a cAMP-dependent pathway. In contrast to recFSH, neither estrogens (DES or E2) nor androgens (testosterone or dihydrotestosterone) alone affected expression of the enzyme in the cultured cells. However, both estrogens and androgens clearly enhanced the effect of recFSH on 17HSD type 1 expression and 17HSD activity in a dose-dependent manner. Among the growth factors, epidermal growth factor (EGF) has previously been shown to decrease the expression of cytochrome P450 aromatase and E2 biosynthesis in granulosa cells. In the present study, we found that treatment with EGF caused a marked decrease in the effect of recFSH on 17HSD type 1 expression and 17HSD activity. The fact that 17HSD type 1 expression and 17HSD activity always behaved in parallel suggests that 17HSD type 1 is the major 17HSD enzyme involved in estradiol biosynthesis in rat granulosa cells. In conclusion, these data indicate that expression of 17HSD type 1 in rat granulosa cells is under multihormonal regulation. The enzyme is regulated by FSH, via cAMP, and the effect of FSH is modulated by estrogens, androgens, and EGF.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/fisiología , Andrógenos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Estrógenos/farmacología , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/citología , Células de la Granulosa/enzimología , 17-Hidroxiesteroide Deshidrogenasas/análisis , Animales , Northern Blotting , Células Cultivadas , Relación Dosis-Respuesta a Droga , Estradiol/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Células de la Granulosa/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacologíaRESUMEN
17 beta-Hydroxysteroid dehydrogenases (17HSDs) are enzymes catalyzing the conversion between 17 beta-hydroxy- and 17-ketosteroids. Both estrogens and androgens possess their highest activity in the 17 beta-hydroxy form, and the enzymes, therefore, regulate the biological activity of sex hormones. In this study, we have characterized the complementary DNA (cDNA) for rat 17HSD type 2. The cDNA encodes a protein with a predicted mol wt of 42,010 Da. The protein has 77% similarity and 62% identity with the human 17HSD type 2 enzyme. Furthermore, the hydropathicity profiles of the enzymes are very similar. The two isozymes contain a putative transmembrane region close to the N-terminus. However, the rat isozyme lacks the two lysine-rich amino acid cluster present at the N- and C-terminals of human 17HSD type 2. The tissue distribution of the rat 17HSD type 1 and type 2 enzymes is very similar to that of the human enzymes. The highest expression of 17HSD type 2 was detected in the placenta. In addition, a 1.5-kilobase messenger RNA for the enzyme was detected in the small intestine, liver, and kidney of both sexes. The two messenger RNAs for rat 17HSD type 1 (1.4 and 1.7 kilobases) were highly expressed only in the ovary, and at very low concentrations in the kidney of both sexes. Transiently expressed rat 17HSD type 2 showed oxidative activity almost exclusively in cultured human embryonic kidney 293 cells, converting estradiol into estrone and testosterone into androstenedione, whereas the opposite was observed for the rat type 1 enzyme. The data suggest that similarly to the corresponding human isoforms, rat 17HSD type 2 is mostly involved in the oxidation of 17 beta-hydroxysteroids into their relatively inactive keto derivative in peripheral tissues, whereas rat 17HSD type 1 is mainly involved in the glandular biosynthesis of estradiol.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Clonación Molecular , Isoenzimas/genética , Isoenzimas/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , ADN Complementario/química , ADN Complementario/genética , Femenino , Humanos , Intestino Delgado/enzimología , Isoenzimas/análisis , Riñón/enzimología , Hígado/enzimología , Masculino , Datos de Secuencia Molecular , Placenta/enzimología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) primarily catalyzes the reduction of low activity estrone to high activity estradiol in ovarian granulosa cells and placental trophoblasts 17HSD type 1 is also present in certain peripheral tissues, such as breast tissue. In the present study we investigated the effects of retinoic acids (RAs) together with other stimuli known to modulate estradiol production and/or cell growth on expression of 17HSD type 1 in JEG-3 choriocarcinoma cells and estrogen-responsive T47D breast cancer cells. Treatment of cultured JEG-3 and T47D cells with all-trans-RA and 9-cis-RA increased reductive 17HSD activity and 17HSD type 1 messenger RNA expression severalfold in both cell lines. On the other hand, epidermal growth factor (EGF), Ca ionophore, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA), and cAMP elevated 17HSD type 1 expression only in JEG-3 cells. Correspondingly, the effects of RAs were potentiated by EGF, TPA, and cAMP in JEG-3 cells, whereas no such phenomenon was observed in T47D cells. In JEG-3 cells, simultaneous administration of RAs with TPA and EGF maximally resulted in approximately 40- and 20-fold increases in 17HSD type 1 messenger RNA expression, respectively. The present data indicate that RAs may stimulate estradiol biosynthesis by regulating 17HSD type 1 expression in certain breast cancer and choriocarcinoma cells. The results suggest that interaction of multiple regulatory pathways is involved in maintaining high 17HSD type 1 expression in the placenta. In addition, regulation of 17HSD type 1 expression may be different in trophoblast cells from that in breast epithelial cells.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , Neoplasias de la Mama/metabolismo , Coriocarcinoma/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Isoenzimas/metabolismo , Tretinoina/farmacología , Neoplasias Uterinas/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/genética , Neoplasias de la Mama/patología , Coriocarcinoma/patología , AMP Cíclico/farmacología , Sinergismo Farmacológico , Femenino , Humanos , Embarazo , ARN Mensajero/metabolismo , Estimulación Química , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Neoplasias Uterinas/patologíaRESUMEN
The 5'-flanking region from -78 to +9 in the HSD17B1 gene serves as a promoter, and an HSD17B1 silencer element is located in position -113 to -78. In the present studies, we have characterized three regulatory elements in the proximal 5'-flanking regions of the gene, using electrophoretic mobility shift assays and reporter gene analysis. First, nuclear factors recognized by antibodies against Sp1 and Sp3 were found to bind the Sp1 motif in the region from -52 to -43. Mutation of the Sp1-binding site decreased the promoter activity to 30% in JEG-3 cells and to 60% in JAR cells, suggesting that binding to the Sp1 motif has a substantial role in the complete functioning of the HSD17B1 promoter. Second, the binding of AP-2 to its motif in the region from -62 to -53 led to reduced binding of Sp1 and Sp3, and furthermore, mutation of the AP-2 element increased promoter activity to 260% in JEG-3 cells. The data thus implied that AP-2 can repress the function of the HSD17B1 promoter by preventing binding to the Sp1 motif. Finally, GATA factors, GATA-3 in particular, were demonstrated to bind their cognate sequence in the HSD17B1 silencer region, and mutations introduced into the GATA-binding site increased transcriptional activity to the level seen in constructs not containing the silencer element. Thus, GATA-3 seems to prevent transcription in the constructs, and hence, the GATA motif also may operate as a negative control element for HSD17B1 transcription.
Asunto(s)
Coriocarcinoma/genética , Proteínas de Unión al ADN/genética , Hidroxiesteroide Deshidrogenasas/genética , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Factor de Transcripción Sp1/genética , Transactivadores/genética , Factores de Transcripción/genética , Secuencia de Bases , Coriocarcinoma/patología , Coriocarcinoma/fisiopatología , ADN de Neoplasias/análisis , ADN de Neoplasias/química , ADN de Neoplasias/genética , Factor de Transcripción GATA2 , Factor de Transcripción GATA3 , Eliminación de Gen , Genes Reporteros/genética , Humanos , Plásmidos , Reacción en Cadena de la Polimerasa , Factor de Transcripción AP-2 , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Human 17beta-hydroxysteroid dehydrogenase (17-HSD) type 1 predominantly catalyzes the 17beta-reduction of estrone to estradiol. The present results, however, show that rat 17-HSD type 1 equally uses both estrone and androstenedione as substrates. Analyzing the activity of various rat/human chimeric enzymes indicated that the region between amino acids 148 and 268 is responsible for the difference in substrate specificity, which is in line with the structural data showing that the recognition end of the active site is primarily at residues 185-230. The enzymes are highly conserved between amino acids 148-191, and the data indicate that in this region Asn152HisAsp153Glu and Pro187Ala variations are most closely related to the differential steroid specificity. The structural analyses furthermore suggested that the presence of His instead of Asn at position 152 of the human enzyme might result in considerable rearrangement of the loop located close to the beta-face of the A- and B-rings of the bound substrate, and that the Pro187Ala variation could modify the flexible region involved in substrate recognition and access of the substrate to the active site. Altogether, our results indicate that the Asn152His and Pro187Ala variations, together with several amino acid variations at the recognition end of the catalytic cleft built by residues 190-230, alter the structure of the active site of rat 17-HSD type 1 to one more favorable to an androgenic substrate.
Asunto(s)
Hidroxiesteroide Deshidrogenasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Secuencia de Bases , Línea Celular , Sondas de ADN/análisis , Sondas de ADN/química , Sondas de ADN/genética , ADN Complementario/análisis , ADN Complementario/química , ADN Complementario/genética , Estradiol/metabolismo , Estrona/metabolismo , Humanos , Hidroxiesteroide Deshidrogenasas/química , Hidroxiesteroide Deshidrogenasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Spodoptera/citología , Especificidad por Sustrato , Factores de TiempoRESUMEN
In the present study, we evaluated the expression and regulation of 17beta-hydroxysteroid dehydrogenase (17HSD) type 1 and type 2, cytochrome P450 aromatase (P450arom), and 20alpha-hydroxysteroid dehydrogenase (20HSD) in mature and pregnant rats. Immunohistochemical analysis of rat 17HSD type 1 showed that the enzyme is exclusively expressed in the granulosa cells of developing, healthy, primary, secondary, and tertiary follicles at all stages of the estrous cycle and pregnancy, and is not detected in the corpora lutea. The data showed that the amount of the enzyme expressed in the follicle increases as follicular maturation progresses and is highest in tertiary and Graafian follicles. However, Northern blot analysis of total RNA from whole ovaries showed a rather constitutive expression of the 17HSD type 1 enzyme. It is evident that compared with P450arom, 17HSD type 1 is more widely expressed in the follicles during the various maturational stages of folliculogenesis. Hence, the data indicate distinct localization, expression, and regulation patterns for 17HSD type 1 and P450arom during the rat estrous cycle and pregnancy. Furthermore, compared with the two estradiol biosynthetic enzymes, a different expression pattern was detected for 20HSD messenger RNA. During the estrous cycle the enzyme was detected in the ovaries throughout the cycle, and in the ovaries of pregnant animals the enzyme showed an expression pattern the opposite of that observed for P450arom. Rat 17HSD type 2, not detected in the ovaries, was constitutively expressed in both female and male liver and small intestine in 21-day-old fetuses up to 6-week-old mature animals. Similarly, in these tissues the enzyme was constitutively expressed in normal cycling and pregnant animals, but it showed increasing expression in the placenta as pregnancy advanced. The relatively constitutive expression of the enzyme at all physiological stages of the animals suggests a general role for the enzyme in the inactivation of circulating sex steroids.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , 20-Hidroxiesteroide Deshidrogenasas/metabolismo , Envejecimiento/metabolismo , Aromatasa/metabolismo , Isoenzimas/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/genética , 20-Hidroxiesteroide Deshidrogenasas/genética , 20-alfa-Hidroxiesteroide Deshidrogenasa , Animales , Aromatasa/genética , Estradiol/metabolismo , Estrona/metabolismo , Femenino , Isoenzimas/genética , Masculino , Ovario/enzimología , Placenta/enzimología , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
17 beta-Hydroxysteroid dehydrogenase (17HSD) catalyzes the reversible conversion of estrone into estradiol. The complementary DNA (cDNA) coding for rat 17HSD type 1 was cloned from a commercial rat ovarian cDNA library, using human 17HSD type 1 cDNA as a probe. The nucleotide sequence extends for 1160 basepairs (bp), including 1035 bp of open reading frame, a stop codon, and 125 bp of 3'-untranslated sequence. The cDNA encodes a protein of 344 amino acids, with a calculated molecular mass of 36,967 daltons. The overall amino acid identity and similarity between rat and human 17HSD type 1 enzymes are 68% and 80%, respectively. Immunohistochemistry and in situ and Northern hybridizations were used to study regulation of the enzyme in rat ovary in vivo. Enzyme expression was detected in granulosa cells only, whereas no expression was observed in stromal or thecal cells. The enzyme was almost undetectable in ovaries from immature hypophysectomized rats. After 2-day treatment with recombinant FSH (recFSH), an induction of 17HSD type 1 expression was observed in granulosa cells of growing antral follicles. During 5 days of diethylstilbestrol (DES) treatment, a time-dependent increase in developing follicles was observed, showing strong expression of 17HSD type 1 in granulosa cells. Treatment with recFSH for 2 days in DES-primed animals resulted in down-regulation of ovarian enzyme expression. This reduction of enzyme expression was associated with luteinization of the follicles. hCG treatment of recFSH- or DES- plus recFSH-primed animals further induced luteinization, resulting in strong down-regulation of 17HSD type 1 expression. The enzyme was not detected in corpora lutea. The data show that 17HSD type 1 expression in rat ovary is regulated by gonadotropins and estrogens. The results suggest that expression of 17HSD type 1 and that of cytochrome P450 aromatase are regulated by distinct mechanisms, and 17HSD type 1 may be down-regulated earlier than P450 aromatase during luteinization, limiting estradiol biosynthesis in luteinizing granulosa cells in rat ovary.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/química , 17-Hidroxiesteroide Deshidrogenasas/genética , Dietilestilbestrol/farmacología , Gonadotropinas/farmacología , Ovario/enzimología , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Animales , Aromatasa/genética , Aromatasa/metabolismo , Aromatasa/fisiología , Secuencia de Bases , Northern Blotting , Gonadotropina Coriónica/farmacología , ADN/análisis , ADN/genética , Activación Enzimática , Femenino , Hormona Folículo Estimulante/farmacología , Regulación Enzimológica de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Ovario/efectos de los fármacos , Ovario/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Factores de TiempoRESUMEN
The function of the gene encoding human 17beta-hydroxysteroid dehydrogenase (17HSD) type 1, the hHSD17B1 gene, is regulated by a cell-specific enhancer at position -662 to -392. The adjacent hHSD17BP1 gene, whose function is not known, contains an analogous region in its 5'-flanking region. The identity between the hHSD17B1 enhancer and the hHSD17BP1 equivalent is as high as 98%, i.e. they differ by only five nucleotides. Results from reporter gene analyses showed that the hHSD17BP1 analog, a pseudoenhancer, has only 10% the activity of the hHSD17B1 enhancer. Furthermore, the results indicate that the reduced function of the pseudoenhancer is a consequence of the presence of G and A at positions -480 and -486, whereas the hHSD17B1 enhancer contains -480C and -486G. In addition, three protected areas were localized to regions -495/-485 (FP1), -544/-528 (FP2), and -589/-571 (FP3) in deoxyribonuclease I footprinting analysis of the hHSD17B1 enhancer. Replacement of the footprinted regions with a nonsense sequence demonstrated that the FP2 region is the most critical for enhancer activity. Mutations of FP2 or a short palindromic region within it led to almost complete abolishment of enhancer activity. We have identified several subelements that are essential for appropriate function of the hHSD17B1 enhancer. The results also show that the hHSD17B1 and hHSD17BP1 genes operate differently despite the high homology between them.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , Elementos de Facilitación Genéticos , 17-Hidroxiesteroide Deshidrogenasas/biosíntesis , Secuencia de Bases , Neoplasias de la Mama , Cloranfenicol O-Acetiltransferasa/genética , Coriocarcinoma , Femenino , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Plásmidos , Embarazo , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Neoplasias UterinasRESUMEN
Human 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 is a widely distributed enzyme that primarily converts the highly active 17beta-hydroxysteroids to their inactive keto forms. In the present study, full-length human 17HSD type 2 was localized in the endoplasmic reticulum using a double immunofluorescence labeling technique. As a consequence of its strong membrane interaction, full-length human 17HSD type 2 could not be solubilized as a biologically active form in vitro. However, by deleting the first 29 amino acids from the N-terminus, we were able to purify a catalytically active enzyme from the cytosolic fraction of Sf9 insect cells. Biochemical and catalytic properties of the purified truncated human 17HSD type 2 protein confirm its suitability for structure-function analyses of the enzyme. Both intact and truncated 17HSD type 2 enzymes efficiently catalyzed the oxidation of estradiol, testosterone, dihydrotestosterone, androstenediol, and 20alpha-dihydroprogesterone. The oxidation of estradiol brought about by human 17HSD type 2 was effectively inhibited by several other steroidal compounds, such as 2-hydroxyestradiol, 5beta-androstan-3alpha,17beta-diol, 5alpha-androstan-3alpha,17beta-diol, and 5alpha-androstan-3beta,17beta-diol. The broad substrate specificity of human 17HSD type 2 together with its predominant oxidative activity and intracellular location, as observed in this study, indicate the physiological role of the enzyme to be primarily an inactivator of highly active 17beta-hydroxysteroids.