RESUMEN
Non-alcoholic fatty liver disease (NAFLD) is now a public health issue worldwide, but no drug has yet received approval. Genistein, an isoflavonoid derived from soybean, ameliorates high-fat-diet-induced NAFLD in mice, but the molecular underpinnings remain largely elusive. Arachidonic acid (AA) is a major ingredient of animal fats, and the AA cascade has been implicated in chronic inflammation. In this study, we investigated whether genistein was against NAFLD by targeting the AA cascade. Using a mouse model, we showed that genistein supplementation improved high-fat-diet-induced NAFLD by normalizing hepatomegaly, liver steatosis, aminotransferase abnormalities, and glucose tolerance. The thromboxane A2 (TXA2) pathway was aberrantly active in NAFLD, evidenced by an elevation of circulating TXA2 and hepatic thromboxane A2 receptor expression. Mechanistically, we found that genistein directly targeted cyclooxygenase-1 activity as well as its downstream TXA2 biosynthesis, while the TXA2 pathway might mediate NAFLD progression by impairing insulin sensitivity. Taken together, our study revealed a crucial pathophysiological role of the TXA2 pathway in NAFLD and provided an explanation as to how genistein was against NAFLD progression.
Asunto(s)
Genisteína/administración & dosificación , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Tromboxano A2/fisiología , Animales , Aspirina/administración & dosificación , Inhibidores de la Ciclooxigenasa , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Hep G2 , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/análisis , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Receptores de Tromboxano A2 y Prostaglandina H2/fisiología , Tromboxano A2/sangreRESUMEN
Thromboxane A(2) (TxA(2)) is a prostanoid formed by thromboxane synthase using the cyclooxygenase product prostaglandin H(2) as the substrate. Previously, increased expression of thromboxane synthase was found in prostate tumors, and tumor cell motility was attenuated by inhibitors of thromboxane synthase. This study was undertaken to elucidate how tumor motility is regulated by TxA(2). Here, we report that human prostate cancer cells express functional receptors for TxA(2) (TP). Ligand binding assay found that PC-3 cells binded to SQ29548, a high-affinity TP antagonist, in a saturable manner with K(d) of 3.64 nmol/L and B(max) of 120.4 fmol per million cells. Treatment of PC-3 cells by U46619, a TP agonist, induced PC-3 cell contraction, which was blocked by pretreatment with the TP antagonist SQ29548 or pinane TxA(2). The migration of prostate cancer cells was significantly inhibited either by sustained activation of TP or by blockade of TP activation, suggesting that TP activation must be tightly controlled during cell migration. Further studies found that small GTPase RhoA was activated by TP activation, and pretreatment of PC-3 cells with Y27632, a Rho kinase (ROCK) inhibitor, blocked U46619-induced cell contraction. A dominant-negative mutant of RhoA also blocked U46619-induced cell contraction. Taken together, the data suggest that TPs are expressed in prostate cancer and activation of TPs regulates prostate cancer cell motility and cytoskeleton reorganization through activation of Rho.
Asunto(s)
Carcinoma/patología , Movimiento Celular , Neoplasias de la Próstata/patología , Receptores de Tromboxano A2 y Prostaglandina H2/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Amidas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes , Carcinoma/química , Carcinoma/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Ácidos Grasos Insaturados , Humanos , Hidrazinas/farmacología , Ligandos , Masculino , Neoplasias de la Próstata/química , Neoplasias de la Próstata/metabolismo , Piridinas/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/análisis , Receptores de Tromboxano A2 y Prostaglandina H2/efectos de los fármacos , Proteína de Unión al GTP rhoA/análisis , Proteína de Unión al GTP rhoA/antagonistas & inhibidoresRESUMEN
Thromboxane A2 (TXA2) has been thought a potent mediator involved in allergic rhinitis, because TXA2 was recovered from the nasal lavage fluid of allergic rhinitis patients after allergen provocation and TXA2 receptor antagonists relief nasal allergic symptoms. In order to clarify the expression of TXA2 receptor in human nasal mucosa, we investigated TXA2 receptor mRNA expression and its protein localization by polymerase chain reaction (PCR) and immunohistochemistry, respectively. Human turbinates were obtained after turbinectomy from 10 patients with nasal obstruction refractory to medical therapy. RT-PCR analysis of total RNA from nasal mucosa demonstrated the expression of TXA2 receptor alpha mRNA. The immunohistochemical studies revealed that anti-TXA2 receptor alpha antibody labeled vascular smooth muscle cells, vascular endothelial cells, epithelial cells and submucosal glands in the nasal mucosa. The results may have an important clinical implication for understanding the role of TXA2 receptor on upper airway diseases such as allergic rhinitis and non-allergic rhinitis.
Asunto(s)
Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Mucosa Nasal/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/biosíntesis , Adulto , Células Cultivadas , Células Endoteliales/citología , Células Epiteliales/citología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mucosa Nasal/química , Mucosa Nasal/citología , Receptores de Tromboxano A2 y Prostaglandina H2/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In humans, thromboxane (TX) A2 signals through two receptor isoforms, thromboxane receptor (TP)alpha and TPbeta, which are transcriptionally regulated by distinct promoters, Prm1 and Prm3, respectively, within the single TP gene. The aim of the current study was to investigate the ability of the endogenous peroxisome proliferator-activated receptor (PPAR)gamma ligand 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) to regulate expression of the human TP gene and to ascertain its potential effects on the individual TPalpha and TPbeta isoforms. 15d-PGJ2 suppressed Prm3 transcriptional activity and TPbeta mRNA expression in the platelet progenitor megakaryocytic human erythroleukemia (HEL) 92.1.7 cell line but had no effect on Prm1 or Prm2 activity or on TPalpha mRNA expression. 15d-PGJ2 also resulted in reductions in the overall level of TP protein expression and TP-mediated intracellular calcium mobilization in HEL cells. 15d-PGJ2 suppression of Prm3 transcriptional activity and TPbeta mRNA expression was found to occur through a novel mechanism involving direct binding of PPARgamma-retinoic acid X receptor (RXR) heterodimers to a PPARgamma response element (PPRE) composed of two imperfect hexameric direct repeat (DR) sequences centred at -159 and -148, respectively, spaced by five nucleotides (DR5). These data provide direct evidence for the role of PPARgamma in the regulation of human TP gene expression within the vasculature and point to further critical differences in the modes of transcriptional regulation of TPalpha and TPbeta in humans. Moreover, these data highlight a further link between enhanced risk of cardiovascular disease in diabetes mellitus associated with increased synthesis and action of thromboxane A2 (TXA2).