RESUMEN
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.
Asunto(s)
Expresión Génica , Hipotiroidismo/metabolismo , Lactancia , Receptores de Progesterona/metabolismo , Animales , Femenino , Hipotiroidismo/inducido químicamente , Glándulas Mamarias Animales/metabolismo , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , Coactivador 1 de Receptor Nuclear/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 2 del Receptor Nuclear/genética , Coactivador 2 del Receptor Nuclear/metabolismo , Propiltiouracilo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas Wistar , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Oxitocina/genética , Receptores de Oxitocina/metabolismo , Receptores de Progesterona/genética , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Receptor alfa X Retinoide/genética , Receptor alfa X Retinoide/metabolismoRESUMEN
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.
Asunto(s)
Ciclina D1/metabolismo , Receptores ErbB/metabolismo , Coactivador 1 de Receptor Nuclear/metabolismo , Receptores de Progesterona/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Astrocitoma , Línea Celular Tumoral , Ciclina D1/genética , Receptores ErbB/genética , Técnicas de Silenciamiento del Gen , Antagonistas de Hormonas/farmacología , Humanos , Mifepristona/farmacología , Coactivador 1 de Receptor Nuclear/genética , Progesterona/farmacología , Progestinas/farmacología , ARN Mensajero/metabolismo , Receptores de Progesterona/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
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.
Asunto(s)
Astrocitoma/fisiopatología , Neoplasias Encefálicas/fisiopatología , Línea Celular Tumoral , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 3 de Receptor Nuclear/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/fisiología , Ciclina D1/genética , Ciclina D1/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptor alfa de Estrógeno/genética , Humanos , Coactivador 1 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
Astrocitoma/metabolismo , Estradiol/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 3 de Receptor Nuclear/metabolismo , Progesterona/metabolismo , Western Blotting , Línea Celular Tumoral , Humanos , Coactivador 1 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores de Estradiol/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
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.