RESUMO
Mutations in the EIF2AK3 gene have been identified in patients with Wolcott-Rallison syndrome - a rare autosomal recessive disorder associated with permanent neonatal insulin-dependent diabetes. Despite the fact that different mutations have been observed in every single unrelated case reported so far, most patients presented with similar characteristics, such as osteopenia, epiphyseal dysplasia as well as hepatic and/or renal dysfunction. The EIF2AK3 gene was analyzed using a PCR-based sequencing approach in two Wolcott-Rallison patients and their parents. We report two cases from different families carrying the same and novel truncating nonsense mutation in the EIF2AK3 gene that encodes the pancreatic eukaryotic initiation factor 2alpha kinase 3. This mutation clearly displays different clinical characteristics in the two patients we examined. Remarkably, the onset of diabetes was different for the two patients, and there was also heterogeneity in other clinical manifestations. These cases illustrate the important role of alternative pathways that could, to some extent, take over or supplement a defective metabolic pathway. This supports the idea that there is no simple relationship among clinical manifestations and EIF2AK3 mutations.
Assuntos
Códon sem Sentido , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/genética , Osteocondrodisplasias/enzimologia , Osteocondrodisplasias/genética , eIF-2 Quinase/genética , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , DNA/genética , Éxons , Feminino , Genes Recessivos , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Humanos , Recém-Nascido , Masculino , Fenótipo , SíndromeRESUMO
Genes that are regulated by androgens in the human prostate are believed to play an essential role in prostate physiology and they may also be involved in the proliferative response of prostate cancer cells to androgens. We used a cDNA subtraction approach to identify novel androgen-regulated transcripts in LNCaP cells that were exposed to 0.1 nM R1881 for 24 h. We report here that SPAK, a recently identified STE20/SPS1-related kinase that modulates p38 MAP kinase activity, exhibited increased expression in androgen-treated LNCaP cells. Androgen regulation of SPAK was both dose- and time-dependent. R1881-induced SPAK expression was completely abrogated by the antiandrogen casodex and by actinomycin D indicating that androgen induction of SPAK requires the androgen receptor and transcription. Cycloheximide caused a partial inhibition of R1881-induced SPAK expression which suggests that androgen induction of SPAK expression may require synthesis of additional proteins. Northern blot and ribonuclease protection assays demonstrated that SPAK is expressed at high levels in normal human testes and prostate, as well as in a number of breast and prostate cancer cell lines. These results identify SPAK, a member of a key cell signalling pathway, as an androgen-responsive gene in LNCaP cells. We hypothesize that SPAK may mediate androgen action in the normal and cancerous prostate gland.
Assuntos
Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Congêneres da Testosterona/farmacologia , Regulação para Cima/efeitos dos fármacos , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Neoplasias da Mama/metabolismo , Clonagem Molecular , Cicloeximida/farmacologia , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Metribolona/farmacologia , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The cyclin-dependent kinase (CDK) inhibitor p18 blocks progression of the cell cycle by associating with the cyclin D-dependent kinases CDK6 and CDK4. To better understand the regulation of p18 gene expression, we isolated full-length cDNA clones from a human BT-20 breast cancer cell cDNA library. These clones were then used to isolate the human gene from a human genomic DNA library. The human p18 gene spans at least 7.5 kb and is composed of three exons, two of which encode the p18 protein. The genomic clone we isolated contained 5 kb of putative promotor sequence which directed expression of the luciferase reporter gene in transient transfection experiments. The longest cDNA that we isolated from BT-20 cells contained 2103 nucleotides which corresponds to the size of the major RNA transcript detected by Northern analysis in these cells. Transcription start sites mapping to the 5' end of the putative full-length cDNA were identified by ribonuclease protection assays. A novel polymorphism was identified in the 3' untranslated region of BT-20 cell cDNA clones that contained the previously described codon 72 mutation. The codon 72 mutation was also detected in 3 of 35 breast tumors analyzed using a mismatch PCR/RFLP strategy.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Sequência de Bases , Neoplasias da Mama/química , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p18 , Quinases Ciclina-Dependentes/genética , Análise Mutacional de DNA , DNA Complementar/química , Éxons , Genes Neoplásicos , Humanos , Íntrons , Dados de Sequência Molecular , RNA Mensageiro/química , Células Tumorais CultivadasRESUMO
Progression from G1 to the S-phase of the cell cycle is controlled by a family of low molecular weight cyclin-dependent kinase (CDK) inhibitors. The importance of these proteins in cell growth control is underscored by the observation that some members of this family are deleted or mutated in human cancers. For example, the gene encoding the CDK inhibitor p18 is located on a segment of chromosome 1 that is often abnormal in human breast tumors. We have identified an alanine to proline substitution at codon 72 of the p18 gene in BT-20 human breast cancer cells. This mutation abrogates the ability of p18 to interact with CDK6 and renders p18 deficient in suppressing cell growth in a colony formation assay. Our results suggest that p18 inactivation by point mutations may contribute to deregulated growth control in certain cell lines and/or tumors.
Assuntos
Neoplasias da Mama/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Inibidores Enzimáticos , Genes Supressores de Tumor/genética , Mutação Puntual/genética , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Inibidor de Quinase Dependente de Ciclina p18 , Quinases Ciclina-Dependentes/metabolismo , Glutationa Transferase/metabolismo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/química , RNA Mensageiro/genética , Células Tumorais CultivadasAssuntos
3-Hidroxiesteroide Desidrogenases/genética , Esteroide Isomerases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Isoenzimas , Macaca , Camundongos , Dados de Sequência Molecular , Ratos , Alinhamento de Sequência , Esteroide Isomerases/metabolismo , Relação Estrutura-Atividade , TrutaRESUMO
A quantitative in situ hybridization study was carried out to determine the precise localization and androgen regulation of the flank organ regulated (FAR-17A) mRNA expression in the different cellular components of the hamster flank organs. Although FAR-17A mRNA was highly expressed in the epithelial cells of the sebaceous glands, it was also found in the outer root sheath of the hair follicles and in melanocytes. The changes in FAR-17A mRNA levels, in the size of the flank organ and sebaceous gland areas as well as in the weight of the seminal vesicles and prostate, were compared following castration and after 5alpha-dihydrotestosterone treatment. FAR-17A mRNA levels were already significantly decreased 1 d after castration, in parallel with a concomitant decrease in the number of labeled cells with the FAR-17A probe. A maximal decrease was found 7 d after castration. The other parameters were significantly reduced later. After 7 d of treatment with dihydrotestosterone, all values returned to those found in intact animals. Similar stimulatory effects on these parameters were observed after treatment with the adrenal sex steroid precursor dehydroepiandrosterone. These data show that all of the components of the flank organs (sebaceous glands, hair follicles, and melanocytes) express the flank organ regulated (17A) type gene (FAR-17A) gene and that its expression is stimulated by treatment with either dihydrotestosterone or dehydroepiandrosterone. Moreover, FAR-17A mRNA levels respond to androgen stimulation more rapidly than the standard morphologic parameters, revealing that the FAR-17A gene could be a more sensitive and cell specific marker to study the mechanisms of androgen action in the skin.
Assuntos
Cricetinae/genética , Regulação da Expressão Gênica , Expressão Gênica , Glândulas Sebáceas/fisiologia , Androgênios/fisiologia , Animais , Sequência de Bases , DNA Complementar/genética , Hibridização In Situ , Masculino , Mesocricetus , Sondas Moleculares/genética , Dados de Sequência Molecular , Orquiectomia , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The isoenzymes of the 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3 beta-HSD) gene family catalyse the transformation of all 5-ene-3 beta-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. The two human 3 beta-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3 beta-HSD isoenzymes prefer NAD+ to NADP+ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr36 in the rat type III and of Phe36 in mouse type IV enzymes instead of Asp36 found in other 3 beta-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3 beta-HSD proteins possess an intrinsic 17 beta-HSD activity specific to 5 alpha-androstane 17 beta-ol steroids, thus suggesting that such "secondary" activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3 beta-HSD deficiency, the structures of the types I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (approximately 1%). Mutations found in nonsalt-loser patients have some residual activity ranging from approximately 1 to approximately 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3 beta-HSD superfamily.
Assuntos
3-Hidroxiesteroide Desidrogenases/deficiência , Isoenzimas/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Progesterona Redutase/genética , Progesterona Redutase/metabolismo , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Mapeamento Cromossômico , Doenças do Sistema Endócrino/enzimologia , Feminino , Genes , Humanos , Isoenzimas/metabolismo , Cinética , Masculino , Camundongos , Dados de Sequência Molecular , Mutação Puntual , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , TrutaRESUMO
The isozymes of the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) gene family are responsible for the formation of the 17 beta-hydroxysteroids delta 5-androstene-3 beta,17 beta-diol, testosterone, 17 beta-estradiol, and dihydrotestosterone from their corresponding 17-ketosteroid precursors, thus playing a pivotal role in the formation of active sex steroids in both steroidogenic and peripheral target tissues. To clone the type II 17 beta-HSD gene, the full-length cDNA type II 17 beta-HSD was used as probe to screen a human leukocyte genomic DNA library. The type II 17 beta-HSD gene contains seven exons and spans > 40 kbp. The type II 17 beta-HSD gene encodes two alternatively spliced mRNAs that give rise to the previously identified type IIA 17 beta-HSD protein of 387 amino acids, as well as to a related 291-amino-acid type IIB 17 beta-HSD protein of unknown function. RNA blot analysis revealed the presence of a major 1.45-kb transcript that is abundant in placenta and endometrium. The mRNA cap site has been localized in a region between 179 and 167 nucleotides upstream of the ATG start codon by RNase protection and S1 nuclease mapping analyses. Cloning of the 17 beta-HSD type II gene provides us with the tools to study its transcriptional expression.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Genes , Isoenzimas/genética , Splicing de RNA , Sequência de Aminoácidos , Sequência de Bases , Endométrio/enzimologia , Éxons , Feminino , Biblioteca Gênica , Vetores Genéticos , Hormônios Esteroides Gonadais/biossíntese , Humanos , Dados de Sequência Molecular , Placenta/enzimologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
We have investigated 17 alpha-hydroxylase and C17,20-lyase activities and the presence of cytochrome P450c17 mRNA in the esophagus, stomach, duodenum, and colon of adult rats of both sexes. All tissues converted [4-14C]pregnenolone mainly to dehydroepiandrosterone (DHEA) through the 5-ene-3 beta-hydroxysteroid route as opposed to the 4-ene-3-ketosteroid pathway in a control testicular incubate. Synthesis of dehydroepiandrosterone was particularly high in the duodenum and was found to be lower in the stomach, colon and esophagus, in decreasing order. 20 alpha-Hydroxypregnenolone and progesterone were also formed primarily by the esophagus and colon, respectively. P450c17 mRNA was demonstrated by ribonuclease protection assay in the stomach and duodenum, but not in esophagus and colon. However, a 335 bp-long cDNA fragment, whose sequence corresponded to that of rat P450c17 cDNA, was amplified by reverse transcription (RT) and polymerase chain reaction (PCR) from the poly(A)+ RNAs of all four tissues. This result was further confirmed by Southern blotting using a 794-bp testicular probe. The complete sequence of P450c17 cDNA in the stomach and duodenum was identical to that reported for rat testis P450c17 cDNA. No amplification and no positive signal in Southern blotting were observed with the total RNAs from adult male adrenal and spleen, which were taken as negative controls since they had been previously found unable to form androgens from pregnenolone. Although the levels of transcription in gonads, duodenum and stomach were found to be equivalent, as indicated by the RNase protection assay and semiquantitative RT-PCR assay, P450c17 enzyme activity was much higher in the testis, pointing at a possible dissimilarity in the respective rates of mRNA translation. Thus, P450c17 is differentially expressed in the rat gastrointestinal tract, where it leads to the synthesis of the sex steroid precursor DHEA, especially in the duodenum and stomach.
Assuntos
Desidroepiandrosterona/biossíntese , Sistema Digestório/enzimologia , RNA Mensageiro/metabolismo , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Aldeído Liases/metabolismo , Animais , Sequência de Bases , Colo/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/química , Duodeno/enzimologia , Esôfago/enzimologia , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Pregnenolona/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Estômago/enzimologiaRESUMO
The enzymes of the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) gene family are responsible for a key step in the formation and degradation of androgens and estrogens: catalyzing the interconversion of 17-ketosteroids and their active 17 beta-hydroxysteroid counterparts. The structure of human type II 17 beta-HSD cDNA was recently reported. This enzyme catalyzes the interconversion of delta 4-androstenedione and testosterone, androstanedione and dihydrotestosterone, and estrone and 17 beta-estradiol, whereas type I 17 beta-HSD catalyzes exclusively the interconversion of estrogens. To locate the HSD17B2 gene, the novel dinucleotide CA repeat sequence found 571 bp downstream from the end of exon 1 was genotyped into eight CEPH reference families by PCR. Two-point linkage analysis was performed between the latter polymorphism and the 2066 microsatellite markers of Généthon. The maximal pairwise lod score (Zmax = 33.3) with a maximal recombination fraction (theta max) of 0.008 was obtained with the marker D16S422 located on 16q24.1-q24.2. To define further the localization of the HSD17B2 gene, we constructed a high-resolution genetic map of the region flanking the polymorphic HSD17B2 gene including eight Généthon markers. The order of the HSD17B2 gene and markers is qter-D16S516-D16S504-D16S507-D16S505-D16S511+ ++-[HSD17B2-D16S422]-D16S520- D16S413-tel.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Cromossomos Humanos Par 16 , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Humanos , Dados de Sequência MolecularRESUMO
In rat skin, type IV is the major 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) isoenzyme expressed. Although types I and II 3 beta-HSD mRNAs are also present in the skin, their level of expression is about two orders of magnitude lower than that of type IV. In this study, we have investigated the control of type IV 3 beta-HSD mRNA levels as well as 3 beta-HSD enzymatic activity in hypophysectomized adult rats of both sexes. Skin 3 beta-HSD activity was measured by the conversion of [14C]-dehydroepiandrosterone into [14C]-androstenedione, whereas ribonuclease protection assay using a specific type IV cRNA probe was used to assess mRNA levels. Intact male and female rats show a similar level of skin 3 beta-HSD activity, although hypophysectomy caused opposite effects, a decrease being observed in males while an increase was observed in hypophysectomized female animals. We next studied the effects of hyperprolactinemia, corticosterone and 1-thyroxine in hypophysectomized animals. L-thyroxine was found to stimulate 3 beta-HSD expression and activity in male rats whereas no significant effect was observed on the already elevated levels in hypophysectomized female rats. Corticosterone caused an inhibition of type IV 3 beta-HSD mRNA levels and activity in both male and female animals. Hyperprolactinemia achieved by pituitary implants inserted under the kidney capsule stimulated the expression of type IV mRNA as well as 3 beta-HSD enzymatic activity in hypophysectomized male and female animals. The present data demonstrate the multihormonal regulation of 3 beta-HSD/isomerase expression and activity in the rat skin.
Assuntos
Corticosterona/farmacologia , Isomerases/fisiologia , Complexos Multienzimáticos/fisiologia , Progesterona Redutase/fisiologia , Prolactina/farmacologia , Pele/enzimologia , Esteroide Isomerases/fisiologia , Animais , Feminino , Regulação Enzimológica da Expressão Gênica , Hiperprolactinemia/sangue , Isomerases/análise , Isomerases/genética , Masculino , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/genética , Progesterona Redutase/análise , Progesterona Redutase/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Pele/química , Esteroide Isomerases/análise , Esteroide Isomerases/genética , Tiroxina/farmacologiaRESUMO
Hydroxysteroid sulfotransferases (DHEA ST) represent a family of enzymes that catalyze the conversion of dehydroepiandrosterone and other 3 beta-hydroxysteroids into more hydrophilic water-soluble sulfate conjugates. The present study was designed to investigate the regulation of hepatic DHEA ST expression by sex steroids and pituitary hormones, namely GH and PRL, in both male and female rats. DHEA ST mRNA levels were measured by dot blot hybridization using a 332-basepair fragment of rat DHEA ST (ST-20) cDNA as a probe. Hepatic DHEA ST mRNA levels were 2.8-fold higher in females than in males. A 15-day gonadectomy did not affect DHEA ST mRNA levels in the male liver; in females, a 35% decrease in DHEA ST mRNA levels compared with those in intact controls was observed. Administration of 17 beta-estradiol (E2; 1 microgram/kg, twice daily) alone had no effect on the accumulation of DHEA ST mRNA, but the same treatment completely reversed the marked inhibitory effect of dihydrotestosterone (DHT; 400 micrograms/kg, twice daily) on this parameter in both gonadectomized males and females. In female rats, 24-day hypophysectomy (HYPOX) decreased DHEA ST mRNA levels by 62%, whereas no change was detected in males. In HYPOX animals, treatment with E2 or DHT for 9 days starting 15 days after surgery had no effect on hepatic DHEA ST mRNA levels in rats of both sexes. In intact males, the presence of pituitary implants under the kidney capsule increased DHEA ST mRNA levels by 190% (above the control), which reached levels similar to those in intact females, whereas pituitary implants exerted no effect on this parameter in intact females. However, treatment of HYPOX rats with ovine PRL (oPRL; 4 mg/kg, twice daily) had no significant effect on the accumulation of DHEA ST mRNA in male and female animals. In HYPOX females, administration of rat GH (80 micrograms/kg, twice daily) had no effect on DHEA ST mRNA levels, whereas continuous infusion of rat GH (3.6 micrograms/h), using osmotic minipumps to mimic the female GH secretory pattern, increased this parameter by 290% (above the control value), thus completely reversing the effect of hypophysectomy. In HYPOX males, both modes of GH administration increased DHEA ST mRNA levels by 70-90% (above the control). The present study demonstrates that in both gonadectomized male and female rats, administration of E2 completely blocks the marked inhibitory effect of DHT on hepatic DHEA ST mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Hormônios/fisiologia , Fígado/metabolismo , RNA Mensageiro/metabolismo , Sulfotransferases/genética , Animais , Sequência de Bases , Castração , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Feminino , Hipofisectomia , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , RNA Mensageiro/antagonistas & inibidores , Ratos , Ratos Sprague-DawleyRESUMO
In order to better understand the role of prolactin (PRL) and luteinizing hormone (LH) on progesterone biosynthesis in the ovary, we have investigated the time course (1-9 days) of the effect of PRL and human chorionic gonadotropin (hCG) on ovarian 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) expression in the hypophysectomized rat. As evaluated by quantitative in situ hybridization using a 35S labelled type I 3 beta-HSD cDNA probe, the administration of hCG for 2, 3 and 9 days induced increases of 63%, 145% and 146% above control, respectively, in 3 beta-HSD mRNA levels in ovarian interstitial cells. The absence of apparent effect of the gonadotropin in other ovarian cell types could explain the small modulation of ovarian 3 beta-HSD protein content and enzymatic activity observed in total ovarian tissue. On the other hand, treatment with PRL caused a rapid decrease in 3 beta-HSD mRNA levels in corpus luteum by 23%, 63%, 76% and 78% (P < 0.01) following 1, 2, 5 and 9 days of treatment, respectively. The short-term inhibitory effect of PRL was also observed on ovarian immunoreactive 3 beta-HSD protein, as measured by Western blot analysis, and on 3 beta-HSD activity measured by the conversion of [14C]dehydroepiandrosterone into [14C]androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Gonadotropina Coriônica/farmacologia , Complexos Multienzimáticos/biossíntese , Ovário/efeitos dos fármacos , Progesterona Redutase/biossíntese , Prolactina/farmacologia , Esteroide Isomerases/biossíntese , Animais , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/enzimologia , DNA Complementar/genética , Indução Enzimática/efeitos dos fármacos , Feminino , Hipofisectomia , Hibridização In Situ , Complexos Multienzimáticos/genética , Tamanho do Órgão/efeitos dos fármacos , Ovário/anatomia & histologia , Ovário/enzimologia , Pregnenolona/sangue , Progesterona/sangue , Progesterona Redutase/genética , Ratos , Ratos Sprague-Dawley , Esteroide Isomerases/genéticaRESUMO
Human skin has been shown to contain a high level of 5 alpha-reductase activity, the enzyme that catalyses the conversion of the weak androgen testosterone into dihydrotestosterone, the most potent androgen. Because two types of 5 alpha-reductase genes have been characterized in humans, we have cloned 5 alpha-reductase cDNAs from adult human keratinocyte and skin fibroblast cDNA libraries to identify and gain better knowledge of the 5 alpha-reductase expressed in normal human skin. Nucleotide sequence analysis shows that the clones obtained correspond to the type I 5 alpha-reductase. RNase protection analysis using (poly A)+ RNA obtained from human skin and prostate also confirms that type I 5 alpha-reductase is the predominant type expressed in normal skin, whereas type II 5 alpha-reductase is the major form found in the prostate. Following polymerase chain reaction amplification of human keratinocyte and skin fibroblast cDNA, a low level of type II 5 alpha-reductase cDNA has been detected. Using antipeptide antibodies raised in rabbits against the peptide sequence covering amino acids 227 -240 to perform immunohistochemical localization of 5 alpha-reductase, we have found that 5 alpha-reductase is distributed in sweat and sebaceous glands, as well as in the epidermal cell layers, thus providing the basis for the important role of androgens in human skin and its appendages.
Assuntos
Oxirredutases/análise , Pele/enzimologia , Sequência de Bases , Células Cultivadas , Colestenona 5 alfa-Redutase , DNA/análise , DNA/genética , Fibroblastos/química , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Immunoblotting , Imuno-Histoquímica , Queratinócitos/química , Queratinócitos/citologia , Queratinócitos/enzimologia , Masculino , Dados de Sequência Molecular , Oxirredutases/genética , Reação em Cadeia da Polimerase , Próstata/química , Próstata/enzimologia , Glândulas Sebáceas/enzimologia , Pele/química , Pele/citologia , Glândulas Sudoríparas/enzimologiaRESUMO
Structures of cDNA clones encoding three members of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family were characterized. To search for potential new types of 3 beta-HSD, rat types I and II 3 beta-HSD cDNAs were used as probes to screen a rat genomic DNA library. Among the clones isolated, one encodes a novel predicted rat 3 beta-HSD isoenzyme, chronologically designated type IV. The corresponding full-length cDNA was thereafter isolated by selective polymerase chain reaction amplification from rat ovary and day-15 placenta cDNA libraries. The rat type IV 3 beta-HSD cDNA encodes a predicted 372-amino acid protein of 41,854 daltons, which shares 90.9, 87.9, and 78.8% sequence identity with rat types I, II, and III proteins, respectively. Ribonuclease protection assay reveals that type IV 3 beta-HSD is the sole 3 beta-HSD mRNA species detectable in the skin and represents the predominant species in the placenta while being also detectable in the ovary and, to a lower degree, in the adrenal gland. Transient expression of type IV cDNA in SW-13 cells indicates 3 beta-HSD activity similar to that of rat type I 3 beta-HSD. The presence of multiple 3 beta-HSD genes should permit differential and tissue-specific regulation of this rate-limiting enzymatic activity essential in the biosynthesis of all classes of steroid hormones in both classical steroidogenic and intracrine peripheral tissues.
Assuntos
Regulação Enzimológica da Expressão Gênica , Isoenzimas/biossíntese , Isoenzimas/genética , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Progesterona Redutase/biossíntese , Progesterona Redutase/genética , Pele/enzimologia , Esteroide Isomerases/biossíntese , Esteroide Isomerases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Feminino , Humanos , Isoenzimas/metabolismo , Cinética , Masculino , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/metabolismo , Oligodesoxirribonucleotídeos , Especificidade de Órgãos , Poli A/genética , Poli A/isolamento & purificação , Progesterona Redutase/metabolismo , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Esteroide Isomerases/metabolismoRESUMO
We have recently characterized three types of complementary DNA clones encoding predicted isoenzymes of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family. Transient expression in nonsteroidogenic cells reveals that the type III isoenzyme specific for male liver does not display oxidative activity for classical substrates of 3 beta-HSD, in contrast to the two other 3 beta-HSD isoenzymes, thus showing exclusively 3-ketosteroid reductase (3-KSR) activity. In order to better understand the sex-specific control of 3 beta-HSD activity and type III 3-KSR gene expression in rat liver, we have studied in adult animals of both sexes the effect of sex steroids and hypophysectomy, pituitary implants, PRL, and GH on type III 3-KSR messenger RNA (mRNA) levels and 3 beta-HSD/delta 5-delta 4 isomerase activity as measured by the conversion of [14C]dehydroepiandrosterone into [14C] delta 4-androstenedione. Ribonuclease protection assay using types I-, II-, and III-specific complementary RNA probes reveals that type III transcripts are the only species detectable in liver RNA extracted from intact males, whereas no hybridization signal was detectable with any of the three probes in intact female liver RNA. In males, 15 days after castration, liver type III 3-KSR mRNA levels decreased by 80% compared to intact controls, whereas 3 beta-HSD activity was reduced by 48%. Administration of dihydrotestosterone (DHT) increased by 8.25-fold type III 3-KSR mRNA concentration and completely reversed the inhibitory effect of orchiectomy on 3 beta-HSD activity. In ovariectomized animals, treatment with DHT markedly increased type III 3-KSR mRNA accumulation and 3 beta-HSD activity, thus leading to values similar to those measured in intact males. Simultaneous treatment with 17 beta-estradiol almost completely abolished the stimulatory effect of DHT in female rats, whereas no significant effect was seen in males. Twenty-four days after hypophysectomy, type III 3-KSR mRNA levels were decreased by 50-55% in males, whereas in females these transcripts markedly increased from undetectable to 28-36% of the value measured in intact male rats. Treatment with DHT or 17 beta-estradiol for a period of 9 days starting 15 days after hypophysectomy had no effect in male and female rats. On the other hand, treatment with ovine PRL (1 mg, twice daily) had no effect in males but completely blocked the elevation of type III 3-KSR mRNA levels and 3 beta-HSD activity observed after hypophysectomy in females.(ABSTRACT TRUNCATED AT 400 WORDS)