RESUMEN
Recently we and other groups have shown that molecular iodine (I(2)) exhibits potent antiproliferative and apoptotic effects in mammary cancer models. In the human breast cancer cell line MCF-7, I(2) treatment generates iodine-containing lipids similar to 6-iodo-5-hydroxy-eicosatrienoic acid and the 6-iodolactone (6-IL) derivative of arachidonic acid (AA), and it significantly decreases cellular proliferation and induces caspase-dependent apoptosis. Several studies have shown that AA is a natural ligand of the peroxisome proliferator-activated receptors (PPARs), which are nuclear transcription factors thought to participate in regulating cancer cell proliferation. Our results show that in MCF-7 cells: (1) 6-IL binds specifically and with high affinity to PPAR proteins (EMSA assays), (2) 6-IL activates both transfected (by transactivation assays) and endogenous (by lipid accumulation) peroxisome proliferator response elements, and (3) 6-IL supplementation increases PPAR gamma and decreases PPAR alpha expression. These results implicate PPARs in a molecular mechanism by which I(2), through formation of 6-IL, inhibits the growth of human breast cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Araquidónicos/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Yodo/farmacología , PPAR gamma/metabolismo , Animales , Antineoplásicos/uso terapéutico , Ácido Araquidónico/análisis , Ácido Araquidónico/química , Ácidos Araquidónicos/análisis , Ácidos Araquidónicos/química , Línea Celular Tumoral , Biología Computacional , Ensayo de Cambio de Movilidad Electroforética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Yodo/uso terapéutico , Radioisótopos de Yodo/química , PPAR alfa/metabolismo , PPAR gamma/química , PPAR gamma/genética , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Elementos de Respuesta , Receptores X Retinoide/química , Receptores X Retinoide/metabolismo , Coloración y EtiquetadoRESUMEN
The physiological role played by thyroid hormones (TH) in hydro-osmotic homeostasis in fish remains a controversial issue. Previous studies have shown that in Fundulus heteroclitus (killifish) hypo-osmotic stress increases liver iodothyronine deiodinase type 2 (D2) mRNA and D2 activity. In this study we identified two conserved osmotic response element (ORE) motifs in the promoter region of the killifish D2 gene (FhDio2) and examined their possible role in the transcriptional regulation of FhDio2 during hypo-osmotic stress. As assessed by the electrophoretic mobility shift assay, results from in vivo and in vitro experiments demonstrate that exposure to an abrupt hyposmotic challenge triggers in the liver of killifish a strong nuclear recruitment of a putative osmotic response element binding protein (OREBP). This protein-DNA binding is time-dependent, attains a maximum within 2-8 h after the osmotic stress, and is followed by a significant increase in D2 activity. Furthermore, protein-DNA binding and the subsequent elevation in enzyme activity were blocked by the tyrosine kinase inhibitor genistein. Thus, during hypo-osmotic stress, a putative OREBP kinase-activated pathway stimulates FhDio2 transcription and enzymatic activity. These data and the fact that D2 is the major enzyme providing local intracellular T(3) suggest that TH plays a direct role in osmoregulation in fish, possibly by participating in hepatic ammonia metabolism. This study provides important insight into the physiological role of TH in hydro-osmotic homeostasis in fish.
Asunto(s)
Proteínas de Peces/genética , Fundulidae/genética , Yoduro Peroxidasa/genética , Presión Osmótica , Elementos de Respuesta , Animales , Secuencia de Bases , Secuencia Conservada , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Peces/química , Regulación Enzimológica de la Expresión Génica , Homeostasis , Yoduro Peroxidasa/química , Factores de Transcripción NFATC/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/metabolismo , Hormonas Tiroideas/fisiologíaRESUMEN
Until recently, 3,5-diiodothyronine (3,5-T(2)) has been considered an inactive by-product of triiodothyronine (T(3)) deiodination. However, studies from several laboratories have shown that 3,5-T(2) has specific, nongenomic effects on mitochondrial oxidative capacity and respiration rate that are distinct from those due to T(3). Nevertheless, little is known about the putative genomic effects of 3,5-T(2). We have previously shown that hyperthyroidism induced by supraphysiological doses of 3,5-T(2) inhibits hepatic iodothyronine deiodinase type 2 (D2) activity and lowers mRNA levels in the killifish in the same manner as T(3) and T(4), suggesting a pretranslational effect of 3,5-T(2) (Garcia-G C, Jeziorski MC, Valverde-R C, Orozco A. Gen Comp Endocrinol 135: 201-209, 2004). The question remains as to whether 3,5-T(2) would have effects under conditions similar to those that are physiological for T(3). To this end, intact killifish were rendered hypothyroid by administering methimazole. Groups of hypothyroid animals simultaneously received 30 nM of either T(3), reverse T(3), or 3,5-T(2). Under these conditions, we expected that, if it were bioactive, 3,5-T(2) would mimic T(3) and thus reverse the compensatory upregulation of D2 and tyroid receptor beta1 and downregulation of growth hormone that characterize hypothyroidism. Our results demonstrate that 3,5-T(2) is indeed bioactive, reversing both hepatic D2 and growth hormone responses during a hypothyroidal state. Furthermore, we observed that 3,5-T(2) and T(3) recruit two distinct populations of transcription factors to typical palindromic and DR4 thyroid hormone response elements. Taken together, these results add further evidence to support the notion that 3,5-T(2) is a bioactive iodothyronine.
Asunto(s)
Diyodotironinas/farmacología , Fundulidae/fisiología , Hormona del Crecimiento/sangre , Yoduro Peroxidasa/metabolismo , Glándula Tiroides/fisiología , Receptores beta de Hormona Tiroidea/metabolismo , Animales , Diyodotironinas/sangre , Regulación Enzimológica de la Expresión Génica/fisiología , Hipertiroidismo/metabolismo , Hipertiroidismo/fisiopatología , Hipotiroidismo/metabolismo , Hipotiroidismo/fisiopatología , Yoduro Peroxidasa/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , ARN Mensajero/metabolismo , Elementos de Respuesta/fisiología , Glándula Tiroides/efectos de los fármacos , Regulación hacia Arriba/fisiología , Yodotironina Deyodinasa Tipo IIRESUMEN
OBJECTIVE AND METHODS: Over-expression of the immune response can lead to pathological conditions such as septic shock or chronic inflammation. Endothelial cell activation by pro-inflammatory products of activated macrophages plays a key role in these conditions. Here we examine the response of primary human endothelial cells (HUVEC) to conditioned media (CM) obtained from LPS-activated macrophages. We further characterized the translocation of NF-kappaB in the presence of CM by studying the degradation rate of individual IkappaB isoforms. RESULTS: We show that, as expected, CM induced NF-kappaB translocation, as well as adhesion capacity in HUVEC. We further show that this response is critically dependent on TNF-alpha and IL1beta naturally present in the CM. However, both the amplitude of NF-kappaB translocation and adhesiveness observed with CM were well beyond the saturation levels attained after the sole stimulation with recombinant TNF-alpha and IL-1beta, either separately or together. Our results show that CM induced a faster degradation of the IkappaB-beta and IkappaB-epsilon isoforms than the recombinant cytokines, leading to an enhanced recruitment of NF-kappaB activity. CONCLUSIONS: The above results suggest that the physiological context of factors co-secreted by LPS-activated macrophages enhances TNF-alpha mediated endothelial activation.