RESUMO
Thyroid hormones (TH) regulate mammary function. Hypothyroidism (HypoT) has deleterious effects on lactation, litter growth and survival. We analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT in the expression of nuclear receptors, co-regulators and oxytocin receptor (OTR) on lactation (L) days 2, 7 and 14. TH receptors (TRs) were increased on L7 at mRNA and protein levels, except TRα protein, that fell on L14. HypoT decreased TRα2 mRNA on L7 and TRα1 protein on L2, while TRß1 protein increased on L14. HypoT increased estrogen receptor ß (ERß) mRNA on L7 but decreased its protein levels on L14. Progesterone receptor A (PRA) mRNA decreased from L2 to L14 while PRB increased, and at protein levels PRA levels showed a nadir on L7, while PRB peaked. HypoT decreased PRA mRNA and protein and increased PRB mRNA at L14. Nuclear receptor co-activator (NCOA) 1 and RXRα mRNA showed an opposite pattern to the TRs, while NCOA2 increased at L14; HypoT blocked the variations in NCOA1 and NCOA2. HypoT increased NCOR1 on L2 and decreased OTR at L2 and circulating estradiol and NCOR2 at L14. In controls the most notable changes occurred on L7, suggesting it is a key inflection point in mammary metabolism. The low levels of TRα1, NCOA1 and OTR, and increased NCOR1 produced by HypoT on L2 may hinder the mammary ability to achieve normal milk synthesis and ejection, leading to defective lactation. Later on, altered ER and PR expression may impair further mammary function.
Assuntos
Expressão Gênica , Hipotireoidismo/metabolismo , Lactação , Receptores de Progesterona/metabolismo , Animais , Feminino , Hipotireoidismo/induzido quimicamente , Glândulas Mamárias Animais/metabolismo , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 2 de Receptor Nuclear/genética , Coativador 2 de Receptor Nuclear/metabolismo , Propiltiouracila , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos Wistar , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Progesterona/genética , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismoRESUMO
Astrocytomas are the most common primary brain tumors in humans. It has been reported that vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), cyclin D1 and progesterone receptor (PR) expression levels are elevated in patients with high-grade astrocytomas. Progesterone (P) regulates astrocytomas growth through its interaction with PR, which recruits coregulatory proteins such as steroid receptor coactivator-1 (SRC-1) that are required for efficient transcriptional activation. The regulation of VEGF, EGFR and cyclin D1 expression by P in human astrocytoma cells is not known. We studied the role of PR and SRC-1 in the expression of VEGF, EGFR and cyclin D1 mediated by P in human astrocytoma cell lines grade III (U373) and IV (D54). P significantly increased VEGF and EGFR mRNA expression after 12h of treatment in D54 cells that was reflected at protein level 24h after treatment. This effect was blocked by the PR antagonist, RU 486. In U373 cells cyclin D1 mRNA and protein expression was induced by P after 6 and 8h of treatment, respectively, and this effect was blocked with RU 486. Transfection with short hairpin RNA targeting coactivator SRC-1 significantly reduced VEGF expression after 24h of treatment. Collectively, our results indicate that P regulates VEGF and EGFR expression in D54 cells and cyclin D1 expression in U373 through PR, and that SRC-1 participates in the regulation of VEGF expression.
Assuntos
Ciclina D1/metabolismo , Receptores ErbB/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Receptores de Progesterona/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Astrocitoma , Linhagem Celular Tumoral , Ciclina D1/genética , Receptores ErbB/genética , Técnicas de Silenciamento de Genes , Antagonistas de Hormônios/farmacologia , Humanos , Mifepristona/farmacologia , Coativador 1 de Receptor Nuclear/genética , Progesterona/farmacologia , Progestinas/farmacologia , RNA Mensageiro/metabolismo , Receptores de Progesterona/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERß, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERß agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.
Assuntos
Astrocitoma/fisiopatologia , Neoplasias Encefálicas/fisiopatologia , Linhagem Celular Tumoral , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/fisiologia , Ciclina D1/genética , Ciclina D1/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/genética , Humanos , Coativador 1 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The disruption of stromal cell signals in prostate tissue microenvironment influences the development of prostate cancer to androgen independence. 1α,25-Dihydroxyvitamin D(3) (1,25D(3)) and glucocorticoids, either alone or in combination, have been investigated as alternatives for the treatment of advanced prostate cancers that fails androgen therapies. The effects of glucocorticoids are mediated by the intracellular glucocorticoid receptor (GR). Similarly, the effect of 1,25D(3) is mediated by the 1,25D(3) nuclear receptor (VDR). In this study, fibroblasts from benign- (BAS) and carcinoma-associated stroma (CAS) were isolated from human prostates to characterize VDR and GR function as transcription factors in prostate stroma. The VDR-mediated transcriptional activity assessed using the CYP24-luciferase reporter was limited to 3-fold induction by 1,25D(3) in 9 out of 13 CAS (70%), as compared to >10-fold induction in the BAS clinical sample pair. Expression of His-tagged VDR (Ad-his-VDR) failed to recover the low transcriptional activity of the luciferase reporter in 7 out of 9 CAS. Interestingly, expression of Ad-his-VDR successfully recovered receptor-mediated induction in 2 out of the 9 CAS analyzed, suggesting that changes in the receptor protein itself was responsible for decreased response and resistance to 1,25D(3) action. Conversely, VDR-mediated transcriptional activity was more efficient in 4 out of 13 CAS (30%), as compared to the BAS sample pair. Consistent with the reduced response to 1,25D(3) observed in CAS, chromatin immunoprecipitation (ChIP) assays indicated decreased recruitment of coactivators SRC-1/CBP, without major changes in the recruitment of VDR to the CYP24 promoter. In addition, we observed that GR-mediated transcriptional activity was also altered in CAS, as compared to BAS. Disruption of coactivators SRC-1/CBP recruitment may promote hormone resistance in CaP, and highlights the relevance of molecular diagnosis and drug design in tumor cell microenvironment.
Assuntos
Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Receptores de Calcitriol/metabolismo , Receptores de Glucocorticoides/metabolismo , Microambiente Tumoral/genética , Humanos , Masculino , Coativador 1 de Receptor Nuclear/metabolismo , Neoplasias da Próstata/metabolismo , Receptores de Calcitriol/genética , Receptores de Glucocorticoides/genética , Células Estromais/metabolismo , Células Tumorais CultivadasRESUMO
Progesterone (P(4)) and estradiol (E(2)) regulate many cell functions through their interaction with specific intracellular receptors, which require the participation of coactivators such as SRC-1 and SRC-3 for enhancing their transcriptional activity. Coactivator expression is altered in many cancers and in some of them their expression is regulated by P(4) and E(2). In this study, we determined progesterone and estrogen receptor isoform expression in two human astrocytoma cell lines with different evolution grade (U373, grade III; and D54, grade IV) by Western Blot. We studied the role of P(4) and E(2) on SRC-1 and SRC-3 expression in U373 and D54 cell lines by RT-PCR and Western blot. In U373 cells, P(4) did not modify SRC-1 expression, but in D54 cells it increased SRC-1 mRNA expression after 12 h of treatment without significant changes after 24 h. P(4) also increased SRC-1 protein content after 24 h, but reduced it after 48 h. E(2) did not change SRC-1 expression in any cell line. SRC-3 expression was not regulated by either E(2) or P(4). Our data suggest that SRC-1 and SRC-3 expression is differentially regulated by sex steroid hormones in astrocytomas and that P(4) regulates SRC-1 expression depending on the evolution grade of human astrocytoma cells.
Assuntos
Astrocitoma/metabolismo , Estradiol/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Progesterona/metabolismo , Western Blotting , Linhagem Celular Tumoral , Humanos , Coativador 1 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptores de Estradiol/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Estrogen receptors (ER), members of the nuclear steroid receptor superfamily, act to activate transcription through ligand-dependent recruitment of coregulators and chromatin modifications. A series of synthetic A-ring reduced 19-nortestosterone-derived progestins has the capacity to selectively bind ERalpha for activated transcription, and to recruit coregulatory factors. In this study, we have analyzed the ability of synthetic 19-nortestosterone derivatives to visibly alter the configuration of ER-target gene chromatin using a novel mammalian promoter transcriptional biosensor (PRL-array) stably transfected into the genome of HeLa cells (PRL-HeLa cells). Results from synthetic steroid-treated cells expressing functional GFP-ERalpha or YFP-ERbeta chimeras were compared to those obtained with estradiol (E(2)) and the antiestrogen tamoxifen. In the presence of synthetic ligands or E(2) a concentration-dependent increase in area of the biosensor array was observed in GFP-ERalpha-expressing PRL-HeLa cells. No significant differences were found between the effects obtained with natural and synthetic steroids. Similarly, E(2) or synthetic steroids-treated PRL-HeLa cells also resulted in similar colocalization of SRC-1- and RNAPII-immunofluorescence at the array. YFP-ERbeta-expressing PRL-HeLa cells treated with E(2) showed increases in array area that were similar to ERalpha; however, treatment of YFP-ERbeta-expressing cells with synthetic ligands was indistinguishable from vehicle controls. These data indicate that A-ring reduced 19-nortestosterone derivatives have an estrogen-like effect on chromatin, including recruitment of transcription factors through selective interactions with ERalpha.
Assuntos
Técnicas Biossensoriais , Cromatina/metabolismo , Receptores de Estrogênio/metabolismo , Cromatina/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Células HeLa , Humanos , Ligantes , Nandrolona/farmacologia , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismo , Receptores de Estrogênio/genética , Transcrição GênicaRESUMO
Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERá and hERâ and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the â-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the â-galactosidase activity (EC50) of the tuber extracts in relation to 17â-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERá and hERâ, respectively, with a higher relative estrogenic potency with hERâ than with hERá. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17â-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.
Assuntos
Animais , Ratos , Receptor alfa de Estrogênio/análise , Receptor beta de Estrogênio/análise , Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pueraria/química , Bioensaio , Cromatografia Líquida de Alta Pressão , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Isoflavonas/análise , Isoflavonas/metabolismo , Fígado/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , /metabolismo , beta-Galactosidase/análise , beta-Galactosidase/antagonistas & inibidoresRESUMO
Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERalpha and hERbeta and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the beta-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the beta-galactosidase activity (EC(50)) of the tuber extracts in relation to 17beta-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERalpha and hERbeta, respectively, with a higher relative estrogenic potency with hERbeta than with hERalpha. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17beta-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.
Assuntos
Receptor alfa de Estrogênio/análise , Receptor beta de Estrogênio/análise , Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pueraria/química , Animais , Bioensaio , Cromatografia Líquida de Alta Pressão , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Isoflavonas/análise , Isoflavonas/metabolismo , Fígado/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Ratos , beta-Galactosidase/análise , beta-Galactosidase/antagonistas & inibidoresRESUMO
Binding of 1alpha,25-dihydroxy vitamin D(3) to the C-terminal ligand-binding domain (LBD) of its receptor (VDR) induces a conformational change that enables interaction of VDR with transcriptional coactivators such as members of the p160/SRC family or the DRIP (vitamin D receptor-interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. The p160/SRC members contain intrinsic histone acetyl transferase (HAT) activities that remodel chromatin at promoter regulatory regions, and the DRIP/Mediator complex may establish a molecular bridge between the VDR complex and the basal transcription machinery. Here, we have analyzed the rate of recruitment of these coactivators to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy vitamin D3. We report that in intact osteoblastic cells VDR, in association with SRC-1, rapidly binds to the OC promoter in response to the ligand. The recruitment of SRC-1 correlates with maximal transcriptional enhancement of the OC gene at 4 h and with increased histone acetylation at the OC promoter. In contrast to other 1alpha,25-dihydroxy vitamin D3-enhanced genes, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter only after several hours of incubation with 1alpha,25-dihydroxy vitamin D(3), concomitant with the release of SRC-1. Together, our results support a model where VDR preferentially recruits SRC-1 to enhance bone-specific OC gene transcription.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Osteocalcina/genética , Regiões Promotoras Genéticas/genética , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Vitamina D/análogos & derivados , Animais , Subunidade 1 do Complexo Mediador , Modelos Genéticos , Coativador 1 de Receptor Nuclear , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologiaRESUMO
Binding of 1alpha,25-dihydroxy Vitamin D3 to the C-terminal domain (LBD) of its receptor (VDR), induces a conformational change that enables interaction of VDR with transcriptional coactivators such as the members of the p160/SRC family or the DRIP (Vitamin D interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. Recent reports indicate that nuclear receptors, including VDR, interact with p160/SRC members and the DRIP/Mediator complex in a sequential, cyclical, and mutually exclusive manner when bound to a target promoter, exhibiting also a high exchange rate. Here, we present an overview of how these coactivators are recruited to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy Vitamin D3. We find that in intact osteoblastic cells VDR and SRC-1 rapidly bind to the OC promoter in response to the ligand. This recruitment correlates with transcriptional enhancement of the OC gene and with increased histone acetylation at the OC promoter. In contrast, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter after several hours of incubation with 1alpha,25-dihydroxy Vitamin D3. Together, our results indicate that VDR preferentially recruits SRC-1 to enhance basal bone-specific OC gene transcription. We propose a model where specific protein-DNA and protein-protein interactions that occur within the context of the OC gene promoter in osteoblastic cells stabilize the preferential association of the VDR-SRC-1 complex.
Assuntos
Histona Acetiltransferases/metabolismo , Osteocalcina/genética , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Animais , Modelos Biológicos , Coativador 1 de Receptor Nuclear , Regiões Promotoras Genéticas/genética , RatosRESUMO
The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-X(L) (antiapoptotic)/bcl-X(S) (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-X(L)/bcl-X(S) favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4.
Assuntos
Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Linfócitos/metabolismo , Regiões Promotoras Genéticas/genética , Fator de Transcrição STAT5/metabolismo , Timo/metabolismo , Proteína bcl-X/metabolismo , Acetilação , Animais , Apoptose , Células COS , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Antagonistas de Hormônios/farmacologia , Linfócitos/citologia , Masculino , Camundongos , Mifepristona/farmacologia , Coativador 1 de Receptor Nuclear , Plasmídeos/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/citologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genéticaRESUMO
The binding of estradiol (E(2)) to estrogen receptors (ER) is followed by conformational changes resulting in coactivator or corepressor recruitment that influences gene transcription. A series of synthetic A-ring reduced 19-nortestosterone-derived progestins has the capacity to selectively bind and activate transcription through the ERalpha. Herein, the molecular mechanisms involved in ER subtype-selective interactions of these compounds as assessed by their effects upon both ERalpha and ERbeta structural conformation and their ability to induce recruitment of steroid receptor coactivator-1 (SRC-1) to ERalpha were investigated. The results demonstrated that all synthetic A-ring 3beta,5alpha-tetrahydro-reduced derivatives of 19-nortestosterone induced an ERalpha trypsin digestion pattern similar to that seen with E(2), without effects upon ERbeta. In addition, these compounds had the ability to recruit SRC-1 to the ligand-binding domain of ERalpha similar to E(2). Our data indicate that A-ring 3beta,5alpha-tetrahydro-reduced 19-nortestosterone-derived progestins behave as selective ERalpha agonists with ligand-receptor structural and functional responses similar to those induced with natural E(2).
Assuntos
Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Nandrolona/análogos & derivados , Fatores de Transcrição/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/metabolismo , Fulvestranto , Células HeLa , Histona Acetiltransferases , Humanos , Mifepristona/farmacologia , Nandrolona/química , Nandrolona/farmacologia , Coativador 1 de Receptor Nuclear , Progesterona/farmacologia , Progestinas/farmacologia , Conformação Proteica , Receptores de Progesterona/agonistas , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Transfecção , Tripsina/metabolismoRESUMO
In this work we have determined the role of the 26S proteasome in the regulation of the content of progesterone receptors (PR-A and PR-B), estrogen receptors (ER-alpha and ER-beta), the coactivator SRC-1 and the corepressor SMRT in the rat brain during the estrous cycle. The 26S proteasome inhibitor MG132 was injected once into the lateral ventricle on proestrous day; and 24h later, on estrous day we evaluated the content of PR and ER isoforms, SRC-1 and SMRT in the hypothalamus, the preoptic area and the hippocampus by Western blot. A significant increase in the content of both PR isoforms, ER-beta and SRC-1 was observed after the administration of MG132 in the three studied cerebral regions. SMRT content was increased in the hypothalamus and the preoptic area and a significant increase in ER-alpha content was only observed in the preoptic area. These results suggest that essential proteins that participate in progesterone and estrogen actions in the brain should be regulated by the 26S proteasome in a tissue-specific manner in physiological conditions.
Assuntos
Encéfalo/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Ciclo Estral/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Progesterona/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Receptores de Estrogênio/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting/métodos , Encéfalo/metabolismo , Ciclo Estral/fisiologia , Feminino , Histona Acetiltransferases , Correpressor 2 de Receptor Nuclear , Coativador 1 de Receptor Nuclear , Ratos , Ratos WistarRESUMO
Progesterone and estradiol play an important role in the regulation of lung functions such as ventilation and vasoconstriction. The genomic actions of progesterone and estradiol are mediated by their nuclear receptors: progesterone receptors (PR) and estrogen receptors (ER). These actions are linked to interactions between steroid receptors and some cofactors that function as coactivators or corepressors. In this work we determined the content of PR isoforms, ER-beta, one coactivator (SRC-1), and one corepressor (SMRT) in the lung of both female rats during the estrous cycle and intact males by Western blot. The rat lung presented a higher content of PR-A than that of PR-B during the estrous cycle. The highest content of both PR isoforms was observed on the day of proestrus whereas the lowest one was found on the day of estrus. In contrast, the content of ER-beta was the lowest on the day of proestrus and it increased at estrus. The content of SRC-1 and SMRT increased on the day of diestrus. SRC-1 content decreased at proestrus and estrus, while SMRT content decreased at proestrus and increased again at estrus. In the lung of adult male rats the content of PR isoforms, ER-beta and SMRT was lower than in that of females, whereas the content of SRC-1 was similar in both sexes. Our results suggest a sexual dimorphism in the content of PR, ER-beta, and SMRT in the rat lung as well as a relation of their content to the physiological levels of progesterone and estradiol.