RESUMEN
Triptolide (TP), a primary bioactive ingredient isolated from the traditional Chinese herbal medicine Tripterygium wilfordii Hook. F. (TWHF), has attracted great interest for its therapeutic biological activities in inflammation and autoimmune disease. However, its clinical use is limited by severe testicular toxicity, and the underlying mechanism has not been elucidated. Our preliminary evidence demonstrated that TP disrupted glucose metabolism and caused testicular toxicity. During spermatogenesis, Sertoli cells (SCs) provide lactate as an energy source to germ cells by glycolysis. The transcription factors GATA-binding protein 4 (GATA4) and specificity protein 1 (Sp1) can regulate glycolysis. Based on this evidence, we speculate that TP causes abnormal glycolysis in SCs by influencing the expression of the transcription factors GATA4 and Sp1. The mechanism of TP-induced testicular toxicity was investigated in vitro and in vivo. The data indicated that TP decreased glucose consumption, lactate production, and the mRNA levels of glycolysis-related transporters and enzymes. TP also downregulated the protein expression of the transcription factors GATA4 and Sp1, as well as the glycolytic enzyme phosphofructokinase platelet (PFKP). Phosphorylated GATA4 and nuclear GATA4 protein levels were reduced in a dose- and time-dependent manner after TP incubation. Similar effects were observed in shGata4-treated TM4 cells and BALB/c mice administered 0.4 mg/kg TP for 28 days, and glycolysis was also inhibited. Gata4 knockdown downregulated Sp1 and PFKP expression. Furthermore, the Sp1 inhibitor plicamycin inhibited PFKP protein levels in TM4 cells. In conclusion, TP inhibited GATA4-mediated glycolysis by suppressing Sp1-dependent PFKP expression in SCs and caused testicular toxicity.
Asunto(s)
Diterpenos/farmacología , Factor de Transcripción GATA4/metabolismo , Glucólisis/efectos de los fármacos , Fenantrenos/farmacología , Fosfofructoquinasa-1 Tipo C/metabolismo , Células de Sertoli/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Animales , Línea Celular , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Compuestos Epoxi/farmacología , Factor de Transcripción GATA4/efectos de los fármacos , Factor de Transcripción GATA4/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Fosfofructoquinasa-1 Tipo C/efectos de los fármacos , Fosfofructoquinasa-1 Tipo C/genética , Células de Sertoli/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/efectos de los fármacos , Factor de Transcripción Sp1/genéticaRESUMEN
The increasing incidence of reproductive anomalies, described as testicular dysgenesis syndrome, is thought to be related to the exposure of the population to chemicals in the environment. Bisphenol A (BPA) and di(2-ethylhexyl)phthalate (DEHP), which have hormonal and antihormonal activity, have attracted public attention due to their presence in consumer products. The present study investigated the effects of BPA and DEHP on reproductive development. Timed-pregnant female rats were exposed to BPA and DEHP by gavage from gestational days 12 to 21. Results showed that prenatal exposures to test chemicals exerted variable effects on steroidogenic factor 1 and GATA binding protein 4 protein expression and increased (P < .05) sex-determining region Y-box 9 and antimüllerian hormone protein in the infantile rat testis compared with levels in the control unexposed animals. Pituitary LHß and FSHß subunit protein expression was increased (P < .05) in BPA- and DEHP-exposed prepubertal male rats but were decreased (P < .05) in adult animals relative to control. Exposure to both BPA and DEHP in utero inhibited (P < .05) global DNA hydroxymethylation in the adult testis in association with altered DNA methyltransferase protein expression. Together the present data suggest that altered developmental programming in the testes associated with chemical exposures are related to the disruption of sexual differentiation events and DNA methylation patterns. The chemical-induced effects impact the development of steroidogenic capacity in the adult testis.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Dietilhexil Ftalato/farmacología , Contaminantes Ambientales/farmacología , Estrógenos no Esteroides/farmacología , Fenoles/farmacología , Plastificantes/farmacología , Diferenciación Sexual/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Hormona Antimülleriana/metabolismo , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/efectos de los fármacos , Metilasas de Modificación del ADN/metabolismo , Disruptores Endocrinos/farmacología , Femenino , Hormona Folículo Estimulante de Subunidad beta/efectos de los fármacos , Hormona Folículo Estimulante de Subunidad beta/metabolismo , Factor de Transcripción GATA4/efectos de los fármacos , Factor de Transcripción GATA4/metabolismo , Disgenesia Gonadal , Hormona Luteinizante de Subunidad beta/efectos de los fármacos , Hormona Luteinizante de Subunidad beta/metabolismo , Masculino , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Proteína de la Región Y Determinante del Sexo/efectos de los fármacos , Proteína de la Región Y Determinante del Sexo/metabolismo , Factor Esteroidogénico 1/efectos de los fármacos , Factor Esteroidogénico 1/metabolismo , Enfermedades Testiculares , Testículo/metabolismoRESUMEN
Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. Bone morphogenetic protein 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about 10 ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.
Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Endodermo/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína Morfogenética Ósea 4/fisiología , Diferenciación Celular/genética , Línea Celular , Gonadotropina Coriónica/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Células Madre Embrionarias/citología , Endodermo/fisiología , Factor de Transcripción GATA4/efectos de los fármacos , Factor de Transcripción GATA4/metabolismo , Factor de Transcripción GATA6/efectos de los fármacos , Factor de Transcripción GATA6/metabolismo , Proteínas de Homeodominio/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Humanos , Laminina/efectos de los fármacos , Laminina/metabolismo , Macaca fascicularis , Factores de Transcripción SOXB1/efectos de los fármacos , Factores de Transcripción SOXB1/metabolismo , Trofoblastos/citología , Trofoblastos/fisiología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: The retinoic acid receptors (RARs) alpha, beta2, and gamma regulate specific subsets of target genes during all-trans retinoic acid (RA) induced differentiation of F9 teratocarcinoma stem cells. The Tie1 gene exhibited reduced expression in RA-treated F9 RARgamma-/- cells as compared to wild-type (WT) by microarray analysis. Our goal was to analyze the Tie1 gene, which encodes a surface receptor tyrosine kinase expressed in the hematovascular system. MATERIALS AND METHODS: We assessed Tie1, Tie2, Flk1, Runx1, Peg/Mest2, and angiopoietin-1 and 2 mRNA levels and Tie1 promoter activity. RESULTS: We showed that RARgamma, but not RARalpha or RARbeta2, is required for Tie1 promoter activation by RA. Treatment with a RARgamma selective agonist plus a retinoid X receptor agonist (LGD1069) increased Tie1 mRNA levels by 11- +/- 2.5-fold 48 hours after RA addition in F9 WT, but not in F9 RARgamma-/- cells, by quantitative reverse transcription polymerase chain reaction. Multiple putative GATA elements were identified in the Tie1 proximal promoter. RA increased GATA4 transcripts by 12- +/- 1-fold in F9 WT at 48 hours, but not in F9 RARgamma-/- cells. In addition, transfection of a GATA4 expression vector increased Tie1 promoter/luciferase activity in both RA-treated F9 WT and RARgamma-/- cells. Tie1 promoter deletion analyses indicated that a region of the promoter that possessed multiple GATA sites mediated the RA-associated Tie1 transcriptional increase. CONCLUSIONS: Our results indicate that GATA4 plays a role in the RA/RARgamma-associated transcriptional activation of the Tie1 promoter. An understanding of RAR specificity in RA signaling should result in insights into hematopoietic stem cell signaling and potentially in improved therapies for several human diseases.