RESUMO
Cell types with specialized functions fundamentally regulate animal behaviour, and yet the genetic mechanisms that underlie the emergence of novel cell types and their consequences for behaviour are not well understood1. Here we show that the monogamous oldfield mouse (Peromyscus polionotus) has recently evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone. We then demonstrate that 20α-hydroxyprogesterone is more abundant in oldfield mice, where it induces monogamous-typical parental behaviours, than in the closely related promiscuous deer mice (Peromyscus maniculatus). Using quantitative trait locus mapping in a cross between these species, we ultimately find interspecific genetic variation that drives expression of the nuclear protein GADD45A and the glycoprotein tenascin N, which contribute to the emergence and function of this cell type in oldfield mice. Our results provide an example by which the recent evolution of a new cell type in a gland outside the brain contributes to the evolution of social behaviour.
Assuntos
Glândulas Suprarrenais , Evolução Biológica , Comportamento Paterno , Peromyscus , Animais , Feminino , Masculino , 20-alfa-Di-Hidroprogesterona/metabolismo , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/enzimologia , Glândulas Suprarrenais/metabolismo , Estradiol Desidrogenases/genética , Estradiol Desidrogenases/metabolismo , Proteínas GADD45/genética , Variação Genética , Hibridização Genética , Peromyscus/classificação , Peromyscus/genética , Peromyscus/fisiologia , Progesterona/metabolismo , Locos de Características Quantitativas , Comportamento Social , Tenascina/genéticaRESUMO
OBJECTIVE: Progesterone metabolites 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αP) have opposite effects on proliferation, apoptosis and metastasis in the breast. Evidence regarding their influence on ductal carcinoma in situ (DCIS) lesions is lacking. METHODS: MCF10DCIS.com cells were cultured in a 3D culture system and treated with 5αP or 3αP. After 5 and 12 days of treatment, polymerase chain reaction (PCR) of proliferation, invasion/metastasis, anti-apoptotic or other markers was performed. Cells treated with the tumor-promoting 5αP were observed under the light and confocal microscopes to reveal possible morphological changes that could indicate a transition from an in situ to an invasive phenotype. As a control, the morphology of the MDA-MB-231 invasive cell line was examined. The invasive potential after exposure to 5αP was also assessed using a detachment assay. RESULTS: The PCR analysis of the chosen markers showed no statistically significant difference between naive cells and cells treated with 5αP or 3αP. DCIS spheroids retained their in situ morphology after treatment with 5αP. The detachment assay showed no increased potential for invasion after exposure to 5αP. Progesterone metabolites 5αP and 3αP do not facilitate or prohibit tumor promotion/invasion in MCF10DCIS.com cells, respectively. CONCLUSION: As oral micronized progesterone has been proved effective for hot flushes in postmenopausal women, first in vitro data propose that progesterone-only therapy could possibly be considered for women after DCIS suffering from hot flushes.
Assuntos
20-alfa-Di-Hidroprogesterona , Carcinoma Intraductal não Infiltrante , Feminino , Humanos , 20-alfa-Di-Hidroprogesterona/metabolismo , Progesterona/farmacologia , Progesterona/metabolismo , Carcinoma Intraductal não Infiltrante/tratamento farmacológico , Pós-Menopausa , Linhagem Celular TumoralRESUMO
Lipedema is an often underdiagnosed chronic disorder that affects subcutaneous adipose tissue almost exclusively in women, which leads to disproportionate fat accumulation in the lower and upper body extremities. Common comorbidities include anxiety, depression, and pain. The correlation between mood disorder and subcutaneous fat deposition suggests the involvement of steroids metabolism and neurohormones signaling, however no clear association has been established so far. In this study, we report on a family with three patients affected by sex-limited autosomal dominant nonsyndromic lipedema. They had been screened by whole exome sequencing (WES) which led to the discovery of a missense variant p.(Leu213Gln) in AKR1C1, the gene encoding for an aldo-keto reductase catalyzing the reduction of progesterone to its inactive form, 20-α-hydroxyprogesterone. Comparative molecular dynamics simulations of the wild-type vs. variant enzyme, corroborated by a thorough structural and functional bioinformatic analysis, suggest a partial loss-of-function of the variant. This would result in a slower and less efficient reduction of progesterone to hydroxyprogesterone and an increased subcutaneous fat deposition in variant carriers. Overall, our results suggest that AKR1C1 is the first candidate gene associated with nonsyndromic lipedema.
Assuntos
20-Hidroxiesteroide Desidrogenases/genética , Sequenciamento do Exoma/métodos , Lipedema/genética , Mutação de Sentido Incorreto , 20-Hidroxiesteroide Desidrogenases/química , 20-Hidroxiesteroide Desidrogenases/metabolismo , 20-alfa-Di-Hidroprogesterona/metabolismo , Adulto , Idoso , Feminino , Humanos , Lipedema/metabolismo , Mutação com Perda de Função , Pessoa de Meia-Idade , Modelos Moleculares , Simulação de Dinâmica Molecular , Linhagem , Progesterona/metabolismo , Conformação ProteicaRESUMO
Both systemic and local production contribute to the concentration of steroids measured in the brain. This idea was originally based on rodent studies and was later extended to other species, including humans and birds. In quail, a widely used model in behavioural neuroendocrinology, it was demonstrated that all enzymes needed to produce sex steroids from cholesterol are expressed and active in the brain, although the actual concentrations of steroids produced were never investigated. We carried out a steroid profiling in multiple brain regions and serum of sexually mature male and female quail by gas chromatography coupled with mass spectrometry. The concentrations of some steroids (eg, corticosterone, progesterone and testosterone) were in equilibrium between the brain and periphery, whereas other steroids (eg, pregnenolone (PREG), 5α/ß-dihydroprogesterone and oestrogens) were more concentrated in the brain. In the brain regions investigated, PREG sulphate, progesterone and oestrogen concentrations were higher in the hypothalamus-preoptic area. Progesterone and its metabolites were more concentrated in the female than the male brain, whereas testosterone, its metabolites and dehydroepiandrosterone were more concentrated in males, suggesting that sex steroids present in quail brain mainly depend on their specific steroidogenic pathways in the ovaries and testes. However, the results of castration experiments suggested that sex steroids could also be produced in the brain independently of the peripheral source. Treatment with testosterone or oestradiol restored the concentrations of most androgens or oestrogens, respectively, although penetration of oestradiol in the brain appeared to be more limited. These studies illustrate the complex interaction between local brain synthesis and the supply from the periphery for the steroids present in the brain that are either directly active or represent the substrate of centrally located enzymes.
Assuntos
Encéfalo/metabolismo , Codorniz/fisiologia , Caracteres Sexuais , Esteroides/sangue , Esteroides/metabolismo , 20-alfa-Di-Hidroprogesterona/sangue , 20-alfa-Di-Hidroprogesterona/metabolismo , 5-alfa-Di-Hidroprogesterona/sangue , 5-alfa-Di-Hidroprogesterona/metabolismo , Animais , Castração , Corticosterona/sangue , Corticosterona/metabolismo , Estrogênios/sangue , Estrogênios/metabolismo , Feminino , Hipotálamo/metabolismo , Masculino , Pregnenolona/sangue , Pregnenolona/metabolismo , Área Pré-Óptica/metabolismo , Testosterona/sangue , Testosterona/metabolismoRESUMO
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (n=9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6-20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all Δ4 steroids. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (Δ5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus Δ4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5α-pregnanes) marked the luteo-placental shift, Δ5 steroids defined fetal gonadal growth and 5α-reduced metabolites of DHP dominated the steroid profile in mid- to late-gestation. Comprehensive LC-MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development.
Assuntos
Corpo Lúteo/metabolismo , Placenta/metabolismo , Prenhez , Esteroides/metabolismo , Espectrometria de Massas em Tandem/métodos , 20-alfa-Di-Hidroprogesterona/metabolismo , Animais , Biomarcadores/metabolismo , Cromatografia Líquida , Feminino , Cavalos , Gravidez , Pregnanolona/metabolismo , Pregnenolona/metabolismo , Progesterona/metabolismoRESUMO
RATIONALE: Progesterone and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP), have actions in the ventral tegmental area (VTA) that are required for lordosis, a characteristic mating posture of female rodents. 17ß-estradiol (estradiol) co-varies with progestogens over natural cycles, enhances production of 3α,5α-THP, and is required for successful reproductive behavior. OBJECTIVES: A question of interest is the role of pregnane xenobiotic receptor (PXR), a nuclear receptor that regulates enzymes needed for the production of 3α,5α-THP, for estradiol-mediated lordosis. The hypothesis tested was that if PXR is involved in estradiol-mediated biosynthesis of 3α,5α-THP and reproductive behavior, knocking down expression of PXR in the VTA of estradiol-primed, but not vehicle-primed, rats should decrease lordosis and midbrain 3α,5α-THP; effects may be attenuated by 3α,5α-THP administered to the VTA. METHODS: Ovariectomized rats were administered subcutaneous injections of oil vehicle or estradiol. Rats were then administered PXR antisense oligonucleotides (PXR AS-ODNs; which are expected to locally knock down expression of PXR), or control (saline), infusions to the VTA. Rats were administered 3α,5α-THP or vehicle via infusions to the VTA. Reproductive behavior (paced mating task) of rats was determined in addition to exploratory (open field), affective (elevated plus maze), and pro-social (social interaction task) behavior. RESULTS: Reproductive behavior (i.e., increased lordosis) was enhanced with estradiol-priming and infusions of 3α,5α-THP to the VTA. Infusions of PXR AS-ODNs to the VTA attenuated responses in estradiol-, but not vehicle-, primed rats, compared to control infusions. CONCLUSIONS: PXR may be involved in a neuroregulatory response involving biosynthesis of 3α,5α-THP in the midbrain VTA of estradiol-primed rats.
Assuntos
Estradiol/farmacologia , Pregnanolona/farmacologia , Receptores de Esteroides/efeitos dos fármacos , Comportamento Sexual Animal/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , 20-alfa-Di-Hidroprogesterona/metabolismo , Animais , Ansiedade/psicologia , Feminino , Relações Interpessoais , Atividade Motora/efeitos dos fármacos , Receptor de Pregnano X , Progesterona/metabolismo , Ratos , Ratos Long-Evans , Área Tegmentar Ventral/metabolismoRESUMO
At middle-age, the reproductive capacity of female rats begins to decline. Whether there are consequences for social and reproductive behaviors related to changes in estradiol (E(2)), progesterone (P(4)) and its 5α-reduced metabolites, dihydroprogesterone (DHP) and 5α-pregnan-3α-ol-20-one (3α,5α-THP), is of interest. In Experiment 1, 1-year-old female breeder rats that had "maintained their reproductive status" (having 4-5 days estrous cycles, > 60% successful pregnancies after mating, > 10 pups/litter) or their age-matched counterparts with "declining reproductive status" were assessed in social interaction, standard mating, and paced mating when in proestrus. Rats that maintained reproductive status tended to have higher levels of proceptivity, and significantly reduced aggression, towards males, compared to rats with declining reproductive status. Basal midbrain E(2) and DHP levels accounted for a significant proportion of variance in lordosis. In Experiment 2, 1-year-old, age-matched, female breeders that had maintained reproductive status or were in reproductive decline were compared to three-month old, nulliparous females that had regular (4-5 days) or irregular estrous cycles. Age did not influence paced mating but younger rats had greater diencephalon E(2) than did middle-aged rats. After mating, rats with declining/irregular reproductive status had higher P(4) and DHP levels in midbrain than did rats with maintaining/regular reproductive status, albeit differences in midbrain 3α,5α-THP were not seen. Middle-aged rats that maintained reproductive function had greater 3α,5α-THP formation in diencephalon compared to other groups. Thus, age-related changes in central progestogen formation in midbrain or diencephalon may contribute to some variability in expression of reproductive behaviors.
Assuntos
20-alfa-Di-Hidroprogesterona/metabolismo , 5-alfa-Di-Hidroprogesterona/antagonistas & inibidores , 5-alfa-Di-Hidroprogesterona/metabolismo , Mesencéfalo/metabolismo , Progesterona/metabolismo , Comportamento Sexual Animal/fisiologia , Comportamento Social , 20-alfa-Di-Hidroprogesterona/biossíntese , 5-alfa-Di-Hidroprogesterona/biossíntese , Fatores Etários , Animais , Feminino , Masculino , Mesencéfalo/fisiologia , Progesterona/antagonistas & inibidores , Progesterona/fisiologia , Ratos , Ratos Long-EvansRESUMO
While phase I and phase II drug metabolites are important for drug development and toxicity studies, e.g. in the context of metabolites in safety testing (MIST), they are often not commercially available and their classical chemical synthesis can be cumbersome. Therefore, a biotechnological production of drug metabolites using microorganisms that recombinantly express human enzymes has been established in recent years. However, no whole-cell biotransformations that make use of human aldo-keto reductases (AKRs) have yet been reported. In this study, we have functionally expressed human AKR1C1 (20α-hydroxysteroid dehydrogenase) in the fission yeast Schizosaccharomyces pombe and demonstrate the ability of the resulting yeast strain to efficiently catalyze the reduction of progesterone or dydrogesterone to 20α-dihydroprogesterone (20α-DHP) and 20α-dihydrodydrogesterone (20α-DHD), respectively. The formation of any by-products or the occurrence of a back reaction were not detected. Seven other steroids with a 20-keto group (pregnenolone, 17α-hydroxyprogesterone, 11-deoxycortisol, cortisol, 11-deoxycorticosterone, corticosterone, and aldosterone) were not reduced by this system. At shaking flask scale we obtained conversion rates of 90 (±26) µM/d 20α-DHP and 244 (±93) µM/d 20α-dihydrodydrogesterone (20α-DHD), respectively. In a fed-batch fermentation under optimized reaction conditions an average 20α-DHP production rate of 300 µM/d was determined for a total biotransformation time of 72 h. We thus established an AKR-dependent whole-cell biotransformation process that can be used for production of human AKR metabolites on a large scale.
Assuntos
20-Hidroxiesteroide Desidrogenases/metabolismo , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , 20-Hidroxiesteroide Desidrogenases/química , 20-Hidroxiesteroide Desidrogenases/genética , 20-alfa-Di-Hidroprogesterona/análise , 20-alfa-Di-Hidroprogesterona/química , 20-alfa-Di-Hidroprogesterona/metabolismo , Biotransformação , Contagem de Células , Clonagem Molecular , Fermentação , Glucose/metabolismo , Humanos , Progesterona/química , Progesterona/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Schizosaccharomyces/genéticaRESUMO
The mechanisms by which progestogens influence affective behaviors in females are poorly understood despite clear changes in mood/affect that are associated with their decline during menopause. Conjugated equine estrogens (CEE), with or without medroxyprogesterone acetate (MPA), are commonly prescribed hormone-replacement, but there is heterogeneity in responses to these pharmacotherapies. One way in which these compounds differ is in their capacity to potentiate metabolism of progesterone to its 5alpha-reduced products, dihydroprogesterone and 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP). This study investigated whether responses to CEE and MPA may be related to the capacity to metabolize progesterone. Middle-aged female rats that had maintained reproductive status, or those that had a decline, were administered vehicle, CEE and/or MPA. Effects on anxiety-like (open field, elevated plus maze) and social behaviors (social interaction test), and plasma and hippocampus steroid levels were determined. We hypothesized that CEE, but not MPA, would decrease anxiety-like behavior coincident with increased hippocampal metabolism of progesterone. CEE, or CEE+MPA, increased central entries in the open field and time spent on the open arms of the plus maze, but did not alter social interaction of rats that had maintained reproductive status. CEE and/or CEE+MPA increased E2 and 3alpha,5alpha-THP in plasma and/or hippocampus of rats, but MPA increased levels of dihydroprogesterone in the hippocampus of rats with declining reproductive status. Simple regressions showed that hippocampus 3alpha,5alpha-THP levels accounted for a significant proportion of the variance in anxiety-like behavior. Therefore, effects of CEE to reduce anxiety-like behavior of middle-aged rats may be owing, in part, to its capacity to enhance levels of 3alpha,5alpha-THP in the hippocampus.
Assuntos
20-alfa-Di-Hidroprogesterona/metabolismo , Desoxicorticosterona/análogos & derivados , Estrogênios Conjugados (USP)/farmacologia , Acetato de Medroxiprogesterona/farmacologia , Animais , Ansiedade/tratamento farmacológico , Comportamento Animal/efeitos dos fármacos , Desoxicorticosterona/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Estrogênios Conjugados (USP)/administração & dosagem , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Acetato de Medroxiprogesterona/administração & dosagem , Progesterona/metabolismo , Ratos , Ratos Long-EvansRESUMO
Aldo-keto reductase (AKR) 1C3 (type 5 17beta-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD(2) to 9alpha,11beta-PGF(2), Delta(4)-androstenedione to testosterone, progesterone to 20alpha-hydroxyprogesterone, and to a lesser extent, estrone to 17beta-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1muM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17beta-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor alpha induced proliferation. MCF-7-AKR1C3 cells also reduced PGD(2), limiting its dehydration to form PGJ(2) products. The AKR1C3 product was confirmed as 9alpha,11beta-PGF(2) and quantified with a stereospecific stable isotope dilution liquid chromatography-mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD(2) on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor gamma (PPARgamma) signaling by reducing formation of 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)).
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Hormônios Esteroides Gonadais/metabolismo , Hidroxiprostaglandina Desidrogenases/metabolismo , Prostaglandinas/metabolismo , Regulação para Cima , 20-alfa-Di-Hidroprogesterona/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 5-alfa-Di-Hidroprogesterona/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Androstenodiona/metabolismo , Androsterona/metabolismo , Biocatálise , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidrotestosterona/metabolismo , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Estradiol/metabolismo , Estradiol/farmacologia , Estrona/metabolismo , Estrona/farmacologia , Etiocolanolona/análogos & derivados , Etiocolanolona/metabolismo , Feminino , Hormônios Esteroides Gonadais/farmacologia , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Cetosteroides/metabolismo , Cinética , Progesterona/análogos & derivados , Progesterona/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacologia , Prostaglandinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testosterona/metabolismo , TransfecçãoRESUMO
Previous studies have shown that breast tissues and breast cell lines convert progesterone (P) to 5alpha-dihydroprogesterone (5alphaP) and 3alpha-dihydroprogesterone (3alphaHP) and that 3alphaHP suppresses, whereas 5alphaP promotes, cell proliferation and detachment. The objectives of the current studies were to determine if the 5alphaP- and 3alphaHP-induced changes in cell numbers are due to altered rates of mitosis and/or apoptosis, and if 3alphaHP and 5alphaP act on tumorigenic and non-tumorigenic cells, regardless of estrogen (E) and P receptor status. The studies were conducted on tumorigenic (MCF-7, MDA-MB-231, T47D) and non-tumorigenic (MCF-10A) human breast cell lines, employing several methods to assess the effects of the hormones on cell proliferation, mitosis, apoptosis and expression of Bcl-2, Bax and p21. In all four cell lines, 5alphaP increased, whereas 3alphaHP decreased cell numbers, [(3)H]thymidine uptake and mitotic index. Apoptosis was stimulated by 3alphaHP and suppressed by 5alphaP. 5alphaP resulted in increases in Bcl-2/Bax ratio, indicating decreased apoptosis; 3alphaHP resulted in decreases in Bcl-2/Bax ratio, indicating increased apoptosis. The effects of either 3alphaHP or 5alphaP on cell numbers, [(3)H]thymidine uptake, mitosis, apoptosis, and Bcl-2/Bax ratio, were abrogated when cells were treated simultaneously with both hormones. The expression of p21 was increased by 3alphaHP, and was unaffected by 5alphaP. The results provide the first evidence that 5alphaP stimulates mitosis and suppresses apoptosis, whereas 3alphaHP inhibits mitosis and stimulates apoptosis. The opposing effects of 5alphaP and 3alphaHP were observed in all four breast cell lines examined and the data suggest that all breast cancers (estrogen-responsive and unresponsive) might be suppressed by blocking 5alphaP formation and/or increasing 3alphaHP. The findings further support the hypothesis that progesterone metabolites are key regulatory hormones and that changes in their relative concentrations in the breast microenvironment determine whether breast tissues remain normal or become cancerous.
Assuntos
20-alfa-Di-Hidroprogesterona/análogos & derivados , 5-alfa-Di-Hidroprogesterona/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mitose/efeitos dos fármacos , 20-alfa-Di-Hidroprogesterona/metabolismo , 20-alfa-Di-Hidroprogesterona/farmacologia , 5-alfa-Di-Hidroprogesterona/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Glândulas Mamárias Humanas , Progesterona/análogos & derivados , Progesterona/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
BACKGROUND: Progesterone is metabolized in the normal breast mainly into 4-ene-pregnenes (e.g. 20alpha-dihydroprogesterone, 20alphaDHP) but, in contrast, in breast cancer tissue the 5alpha-dihydropregnanes (e.g. 5alpha-dihydroprogesterone, 5alphaDHP) are prevalent. In the present study the effect of progesterone and its main metabolites 20alphaDHP and 5alphaDHP on the aromatase activity in a stable aromatase-expressing estrogen receptor-positive human breast cancer cell line, MCF-7aro, was explored. MATERIALS AND METHODS: The MCF-7aro cells were stripped of endogenous steroids and incubated with physiological concentrations of [3H]-testosterone ([3H]-testos: 5 x 10(-9)M) alone or in the presence of progesterone, 20alphaDHP or 5alphaDHP (5 x 10(-6) or 5 x 10(-8)M) for 24 h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [3H]-Estradiol (E2), [3H]-estrone (E1) and [3H]-testos were characterized by thin layer chromatography and quantified using the corresponding standard. RESULTS: Aromatase activity was present at a high level in the MCF-7aro cells after incubation with [3H]-testos when the concentration of [3H]-E2 was 3.70 pmol/mg DNA; 20alphaDHP at concentrations of 5 x 10(-6)M or 5 x 10(-8)M significantly inhibited this conversion by 50.3% and 36.5%, respectively. No significant effect was found with the metabolite 5alphaDHP or the parent hormone, progesterone. CONCLUSION: The MCF-7aro cell line shows high detectable aromatase activity. The present data indicate that the progesterone metabolite 20alphaDHP, found mainly in normal breast tissue, can act as an anti-aromatase agent.
Assuntos
20-alfa-Di-Hidroprogesterona/farmacologia , 5-alfa-Di-Hidroprogesterona/farmacologia , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , 20-alfa-Di-Hidroprogesterona/metabolismo , 5-alfa-Di-Hidroprogesterona/metabolismo , Aromatase/metabolismo , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Estradiol/metabolismo , Humanos , Progesterona/metabolismo , Progesterona/farmacologia , Testosterona/metabolismo , TrítioRESUMO
Sequential actions of 17beta-estradiol (E(2)) and progesterone (P(4)) in the hypothalamus and the P(4) metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP), in the midbrain ventral tegmental area (VTA) respectively mediate the initiation and intensity of lordosis of female rats and may also modulate anxiety and social behaviors, through actions in these, and/or other brain regions. Biosynthesis of E(2), P(4), and 3alpha,5alpha-THP can also occur in brain, independent of peripheral gland secretion, in response to environmental/behavioral stimuli. The extent to which engaging in tasks related to reproductive behaviors and/or mating increased E(2) or progestin concentrations in brain was investigated. In Experiment 1, proestrous rats were randomly assigned to be tested in individual tasks, including the open field, elevated plus maze, partner preference, social interaction, or no test control, in conjunction with paced mating or no mating. Engaging in paced mating, but not other behaviors, significantly increased dihydroprogesterone (DHP) and 3alpha,5alpha-THP levels in midbrain, hippocampus, striatum, and cortex. In Experiment 2, proestrous rats were tested in the combinations of the above tasks (open field and elevated plus maze, partner preference, and social interaction) with or without paced mating. As in Experiment 1, only engaging in paced mating increased DHP and 3alpha,5alpha-THP concentrations in midbrain, hippocampus, striatum, and cortex. Thus, paced mating enhances concentrations of 5alpha-reduced progestins in brain areas associated with reproduction (midbrain), as well as exploration/anxiety (hippocampus and striatum) and social behavior (cortex).
Assuntos
Ansiedade , Encéfalo/metabolismo , Comportamento Exploratório , Progestinas/metabolismo , Comportamento Sexual Animal , Comportamento Social , 20-alfa-Di-Hidroprogesterona/análise , 20-alfa-Di-Hidroprogesterona/metabolismo , Animais , Córtex Cerebral/química , Córtex Cerebral/metabolismo , Corpo Estriado/química , Corpo Estriado/metabolismo , Estradiol/análise , Estradiol/metabolismo , Feminino , Hipocampo/química , Hipocampo/metabolismo , Mesencéfalo/química , Mesencéfalo/metabolismo , Pregnanolona/análise , Pregnanolona/metabolismo , Radioimunoensaio , Distribuição Aleatória , Ratos , Ratos EndogâmicosRESUMO
Progesterone is an endogenous immunomodulator and can suppress T-cell activation during pregnancy. We have previously shown that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress cellular genomic responses to mitogens. This study aimed to show that acidification is due to a non-genomic inhibition of Na(+)/H(+)-exchange 1 (NHE1) by progesterone and correlate this with immunosuppressive phytohemagglutinin (PHA)-induced T-cell proliferation. The presence of amiloride-sensitive NHE 1 was identified in T cells. The activity of NHE1 was inhibited by progesterone but not by 20alpha-hydroxyprogesterone (20alpha-OHP). Furthermore, 20alpha-OHP was able to compete with progesterone and release the inhibitory effect on the NHE1. The inhibition of NHE1 activity by progesterone-BSA demonstrated non-genomic action via plasma membrane sites. Finally, co-stimulation with PHA and progesterone or amiloride, (5-(N, N-dimethyl)-amiloride, DMA), inhibited PHA-induced T-cell proliferation, but this inhibition did not occur with 20alpha-OHP and PHA co-stimulation. However, when DMA was applied 72 h after PHA stimulation, it was able to suppress PHA-induced T-cell proliferation. This is the first study to show that progesterone causes a rapid non-genomic inhibition of plasma membrane NHE1 activity in T cells within minutes which is released by 20alpha-OHP. The inhibition of NHE1 leads to immunosuppressive T-cell proliferation and suggests that progesterone might exert a major rapid non-genomic suppressive effect on NHE1 activity at the maternal-fetal interface in vivo and that 20alpha-OHP may possibly be able to quickly release the suppression when T cells circulated away from the interface.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Fatores Imunológicos/metabolismo , Ativação Linfocitária , Progesterona/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Linfócitos T/metabolismo , 20-alfa-Di-Hidroprogesterona/metabolismo , Adulto , Amilorida/análogos & derivados , Amilorida/farmacologia , Ligação Competitiva , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Concentração de Íons de Hidrogênio , Fatores Imunológicos/farmacologia , Líquido Intracelular/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Mitógenos/farmacologia , Fito-Hemaglutininas/farmacologia , Progesterona/farmacologia , RNA Mensageiro/análise , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/genética , Linfócitos T/efeitos dos fármacos , Fatores de TempoRESUMO
Previous studies have shown that the progesterone metabolite, 5alpha-pregnane-3,20-dione (5alphaP), exhibits mitogenic and metastatic activity in breast cell lines and that specific, high affinity receptors for 5alphaP are located in the plasma membrane fractions of tumorigenic (ER/PR-positive) MCF-7 cells. The aim of this study was to determine the effects of the mitogenic (estradiol; 5alphaP) and anti-mitogenic (3alpha-hydroxy-4-pregnen-20-one, 3alphaHP; 20alpha-hydroxy-4-pregnen-3-one, 20alphaHP) endogenous steroid hormones on 5alphaP receptor (5alphaP-R) numbers and on cell proliferation and adhesion of MCF-7 and MCF-10A cells. Exposure of MCF-7 cells for 24h to estradiol or 5alphaP resulted in significant (p < 0.05-0.001) dose-dependent increases in 5alphaP-R levels. Conversely, treatment with 3alphaHP or 20alphaHP resulted in significant (p < 0.05-0.01) dose-dependent decreases in 5alphaP-R levels. Treatment with one mitogenic and one anti-mitogenic hormone resulted in inhibition of the mitogen-induced increases, whereas treatment with two mitogenic or two anti-mitogenic hormones resulted in additive effects on 5alphaP-R numbers. Treatments with cycloheximide and actinomycin D indicate that changes in 5alphaP-R levels depend upon transcription and translation. The non-tumorigenic breast cell line, MCF-10A, was also shown to posses specific, high affinity plasma membrane receptors for 5alphaP that were up-regulated by estradiol and 5alphaP and down-regulated by 3alphaHP. Estradiol binding was demonstrated in MCF-10A cell membrane fractions and may explain the estradiol action in these cells that lack intracellular ER. In both MCF-7 and MCF-10A cells, the increases in 5alphaP-R due to estradiol or 5alphaP, and decreases due to 3alphaHP or 20alphaHP correlate with respective increases and decreases in cell proliferation as well as detachment. These results show distribution of 5alphaP-R in several cell types and they provide further evidence of the significance of progesterone metabolites and their novel membrane-associated receptors in breast cancer stimulation and control. The findings that 3alphaHP and 20alphaHP down-regulate 5alphaP-R and suppress mitogenic and metastatic activity suggest that these endogenous anti-mitogenic progesterone metabolites deserve considerations in designing new breast cancer therapeutic agents.
Assuntos
5-alfa-Di-Hidroprogesterona/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Progesterona/farmacologia , Receptores de Progesterona/metabolismo , 20-alfa-Di-Hidroprogesterona/análogos & derivados , 20-alfa-Di-Hidroprogesterona/metabolismo , 20-alfa-Di-Hidroprogesterona/farmacologia , 5-alfa-Di-Hidroprogesterona/metabolismo , Adesão Celular/efeitos dos fármacos , Fracionamento Celular , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação para Baixo , Estradiol/metabolismo , Estradiol/farmacologia , Feminino , Humanos , Progesterona/metabolismo , Receptores de Estradiol/efeitos dos fármacos , Regulação para CimaRESUMO
The molecular mechanisms controlling the initiation of parturition remain largely undefined. We report a new animal model in which parturition does not occur. A line of mice expressing a human apolipoprotein B (APOB) gene fail to deliver their young if the transgene is present in homozygous (Tg/Tg), but not in heterozygous (Tg/Wt), form. Cloning and mapping of the transgene insertion locus indicate that 10 copies of the 80-kilobase APOB genomic fragment inserted into mouse chromosome 6 result in a small, 390-base pair deletion of mouse genomic DNA. Nine other lines expressing the transgene have normal labor, suggesting that transgene insertion in this mutant line disrupted a mouse gene crucial for successful parturition. The pathophysiology of parturition failure in these animals was defined using physiological, endocrinological, and morphological techniques. Results indicate that luteolysis occurs in Tg/Tg mice but is delayed by 1 day. Delivery did not occur in mutant mice at term after spontaneous luteolysis or even after removal of progesterone action by ovariectomy or antiprogestin treatment. Biomechanical and functional studies of the uterus and cervix revealed that the primary cause of failed parturition in Tg/Tg mice was not inadequate uterine contractions of labor but, rather, a rigid, inelastic cervix at term that was abnormally rich in neutrophils and tissue monocytes. Characterization of the transgene insertional mutant, Tg/Tg, indicates that progesterone withdrawal is insufficient to complete parturition in the presence of inadequate cervical ripening at term.
Assuntos
Colo do Útero/fisiopatologia , Cromossomos , Camundongos Transgênicos/genética , Parto/genética , 20-alfa-Di-Hidroprogesterona/metabolismo , Animais , Colo do Útero/metabolismo , Feminino , Perfilação da Expressão Gênica , Homozigoto , Camundongos , Ocitocina/metabolismo , Parto/fisiologia , Gravidez , Progesterona/metabolismo , Transdução de Sinais , Transcrição Gênica , Contração Uterina/genéticaRESUMO
Previous studies indicate that steroid hormones may be protective for Schwann cells and promote the expression of myelin proteins in the sciatic nerve of adult rats. In this study, we have evaluated the effect of progesterone (P), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT) and 5alpha-androstan-3alpha, 17beta-diol (3alpha-diol) on the morphological alterations of myelinated fibers in the sciatic nerve of 22-24-month-old male rats. The sciatic nerves of untreated old male rats, showed a general disorganization and a significant reduction in the density of myelinated fibers, compared to nerves from 3-month-old male rats. The effect of aging was particularly evident in myelinated fibers of small caliber (<5 microm in diameter). In addition, the sciatic nerves of old rats showed a significant increase in the number of fibers with myelin infoldings in the axoplasm and in the number of fibers with irregular shapes. Treatments of old rats with P, DHP and THP resulted in a significant increase in the number of myelinated fibers of small caliber, a significant reduction in the frequency of myelin abnormalities and a significant increase in the g ratio of small myelinated fibers. Furthermore, P treatment significantly reduced the frequency of myelinated fibers with irregular shapes. In contrast, treatments with T, DHT or 3alpha-diol did not significantly affect any of the morphological parameters examined. In conclusion, our data indicate that P, and its derivatives DHP and THP, are able to reduce aging-associated morphological abnormalities of myelin and aging-associated myelin fiber loss in the sciatic nerve. These data suggest that P, DHP and THP may represent useful therapeutic alternatives to maintain peripheral nerve integrity in aged animals.
Assuntos
Envelhecimento/fisiologia , Hormônios Esteroides Gonadais/metabolismo , Fibras Nervosas Mielinizadas/fisiologia , Nervo Isquiático/anatomia & histologia , Nervo Isquiático/fisiologia , 20-alfa-Di-Hidroprogesterona/metabolismo , Androstano-3,17-diol/metabolismo , Animais , Di-Hidrotestosterona/metabolismo , Masculino , Degeneração Neural/metabolismo , Progesterona/metabolismo , Ratos , Ratos Sprague-Dawley , Testosterona/metabolismoRESUMO
The subependymal layer (SEL) of the adult mammalian brain provides a continuous supply of newborn cells that migrate to the olfactory bulb (OB) where they differentiate into interneurons. These newly generated cells migrate tangentially to the OB within a dense meshwork of astrocytic cells, organized to form tangentially oriented channels (glial tubes). The central nervous system is able to synthesize a variety of steroids. Among these, we analyzed the effects of progesterone (P) and its neuroactive metabolites dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), administered by intraventricular injection, on the SEL of the adult rat. We found that THP and DHP, but not their precursor P, modify glial tubes organization and decrease immunoreactivity for glial associated proteins in SEL astrocytes. Moreover P metabolites reduce the proliferative activity within the SEL.
Assuntos
Epêndima/citologia , Epêndima/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Progesterona/farmacologia , 20-alfa-Di-Hidroprogesterona/metabolismo , 20-alfa-Di-Hidroprogesterona/farmacologia , Animais , Epêndima/metabolismo , Neurônios/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/efeitos dos fármacos , Bulbo Olfatório/metabolismo , Progesterona/metabolismo , Prosencéfalo/citologia , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , RatosRESUMO
The C21 steroids, progesterone and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-DHP) play pivotal roles in the initiation, timing and maintenance of ovulatory function and pregnancy in female mammals. They also have growth factor and central nervous system (CNS) effects; some of these are non-genomic effects mediated through 5 alpha-reduced and 3 alpha-hydroxylated derivatives. These studies examined the in vivo uptake and conversion of 20 alpha-DHP in selected CNS sites and peripheral tissues after injection of [(3)H]-20 alpha-DHP. The effects of steroid mass, time after injection, and ovariectomy, adrenalectomy and estradiol treatment were assessed in the pineal gland, preoptic area of the hypothalamus (POA), medial basal hypothalamus (MBH), midbrain, cerebellum, cerebral cortex, anterior pituitary (AP), uterus and skeletal muscle. Tissue extracts were analyzed by scintillation counting and chromatography to quantify and localize 20 alpha-DHP and its 5 alpha-reduced derivatives. Injection of increasing mass of [(3)H]-20 alpha-DHP to ovariectomized/adrenalectomized (ovx/adx) rats results in a linear increase in (3)H-steroid 10 min post injection in all tissues. (3)H-steroid content increases with time over 1 h post injection in the pineal, AP and uterus. Tissue differences in (3)H-steroid level are observed with higher levels in pineal, MBH, POA, AP and midbrain than in cerebral cortex and cerebellum, and in uterus, ovary and adrenal than in muscle. Ovariectomy, adrenalectomy and estradiol treatment affect (3)H-steroid levels in a tissue dependent manner, and the metabolites of 20 alpha-DHP in MBH and AP differ between groups. The findings demonstrate that target tissues, including areas of the CNS, are able to selectively take up and retain 20 alpha-DHP, and also support a physiological role for this progestin and its metabolites in modulation of CNS and reproductive functions.
Assuntos
20-alfa-Di-Hidroprogesterona/metabolismo , 20-alfa-Di-Hidroprogesterona/farmacocinética , Animais , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Feminino , Modelos Químicos , Ovário/fisiologia , Progestinas/metabolismo , Ratos , Fatores de Tempo , Distribuição TecidualRESUMO
We have investigated the potential of autonomous hormonal steroidogenesis in liver and small intestine of male and female frogs, Rana esculenta, during the recovery phase. After incubation of mitochondrial fractions with [4-14C]cholesterol, female liver and intestine formed pregnenolone at a rate of 0.63 and 2.3 pmol/mg protein/h, respectively, whereas conversion by male organs was only c. 0.03 pmol/mg protein/h. Minced tissues preparations transformed [4-14C]pregnenolone into progesterone and 17alpha-hydroxypregnenolone, the former prevailing in the liver, the latter in the intestine. Moreover, both tissues produced 20alpha-dihydropregnenolone, 20alpha-dihydroprogesterone and dehydroepiandrosterone. From incubates with [4-14C]dehydroepiandrosterone, androstenedione and androst-5-ene-3beta, 17beta-diol were identified, the former being more abundant in the liver, the latter in the intestine. These results indicate that both liver and intestine in frog can be independent sources of hormonally active steroids in both sexes.