RESUMEN
As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution two-dimensional gel electrophoresis study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up- or down-regulated following exposure of the cells to 1 nm TCDD for 8 h. There was an overlap of only nine proteins with those detected as altered by TCDD in our recent study using the non-gel-based isotope-coded protein label method (Sarioglu, H., Brandner, S., Jacobsen, C., Meindl, T., Schmidt, A., Kellermann, J., Lottspeich, F., and Andrae, U. (2006) Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels. Proteomics 6, 2407-2421) indicating a strong complementarity of the two approaches. For the majority of the altered proteins, an effect of TCDD on their abundance or posttranslational modifications had not been known before. Several observations suggest that a sizable fraction of the proteins with altered abundance was induced as an adaptive response to TCDD-induced oxidative stress that was demonstrated using the fluorescent probe dihydrorhodamine 123. A prominent group of these proteins comprised various enzymes for which there is evidence that their expression is regulated via the Keap1/Nrf2/antioxidant response element pathway. Other proteins included several involved in the maintenance of mitochondrial energy production and the regulation of the mitochondrial apoptotic pathway. A particularly intriguing finding was the up-regulation of the mitochondrial outer membrane pore protein, voltage-dependent anion channel-selective protein 2 (VDAC2), which was dependent on the presence of a functional aryl hydrocarbon receptor. The regulatability of VDAC2 protein abundance has not been described previously. In view of the recently discovered central role of VDAC2 as an inhibitor of the activation of the proapoptotic protein BAK and the mitochondrial apoptotic pathway, the present data point to a hitherto unrecognized mechanism by which TCDD may affect cellular homeostasis and survival.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas Mitocondriales/análisis , Dibenzodioxinas Policloradas/toxicidad , Proteoma/análisis , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Animales , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Proteínas Mitocondriales/química , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/química , Estrés Oxidativo/efectos de los fármacos , Proteoma/química , Ratas , Especies Reactivas de Oxígeno/metabolismo , Receptores de Hidrocarburo de Aril/deficiencia , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) is expressed in multiple hormone-dependent cancer cells/tumors. Treatment of ZR-75 breast cancer cells with 17beta-estradiol (E2) induced a greater than fourfold increase of VEGF mRNA levels. ZR-75 breast cancer cells were transfected with pVEGF1, a construct containing a -2018 to +50 VEGF promoter insert, and E2 induced reporter gene (luciferase) activity. Deletion and mutation analysis of the VEGF gene promoter identified a GC-rich region (-66 to -47) which was required for E2-induced transactivation of pVEGF5, a construct containing the minimal promoter (-66 to +54) that exhibited E2-responsiveness. Interactions of nuclear proteins from ZR-75 cells with the proximal GC-rich region of the VEGF gene promoter were investigated by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results demonstrate that both Sp1 and Sp3 proteins bound the GC-rich motif (-66 to -47), and estrogen receptor alpha (ERalpha) interactions were confirmed by chromatin immunoprecipitation. Moreover, E2-dependent activation of constructs containing proximal and distal GC/GT-rich regions of the VEGF promoter was inhibited in ZR-75 cells transfected with small inhibitory RNAs for Sp1 and Sp3. These results were consistent with a mechanism of hormone activation of VEGF through ERalpha/Sp1 and ERalpha/Sp3 interactions with GC-rich motifs.
Asunto(s)
Neoplasias de la Mama/genética , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Estradiol/farmacología , Receptor alfa de Estrógeno , Secuencia Rica en GC , Eliminación de Gen , Genes Reporteros , Humanos , Neoplasias Hormono-Dependientes/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular/genéticaAsunto(s)
Proteínas de Unión al ADN , Antagonistas de Estrógenos/farmacología , Dibenzodioxinas Policloradas/farmacología , Receptor Cross-Talk/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Unión Competitiva , Neoplasias de la Mama/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Cisteína Endopeptidasas/metabolismo , Estradiol/administración & dosificación , Estradiol/metabolismo , Antagonistas de Estrógenos/metabolismo , Receptor alfa de Estrógeno , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Neoplasias de los Genitales Femeninos/metabolismo , Humanos , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Complejo de la Endopetidasa Proteasomal , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/genética , Receptores de Estrógenos/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Útero/metabolismoRESUMEN
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands suppress 17beta-estradiol (E)-induced responses in the rodent uterus and mammary tumors and in human breast cancer cells. Treatment of ZR-75, T47D, and MCF-7 human breast cancer cells with TCDD induces proteasome-dependent degradation of endogenous estrogen receptor alpha (ERalpha). The proteasome inhibitors MG132, PSI, and PSII inhibit the proteasome-dependent effects induced by TCDD, whereas the protease inhibitors EST, calpain inhibitor II, and chloroquine do not affect this response. ERalpha levels in the mouse uterus and breast cancer cells were significantly lower after cotreatment with E plus TCDD than after treatment with E or TCDD alone, and our results indicate that AhR-mediated inhibition of E-induced transactivation is mainly due to limiting levels of ERalpha in cells cotreated with E plus TCDD. TCDD alone or in combination with E increases formation of ubiquitinated forms of ERalpha, and both coimmunoprecipitation and mammalian two-hybrid assays demonstrate that TCDD induces interaction of the AhR with ERalpha in the presence or absence of E. In contrast, E does not induce AhR-ERalpha interactions. Thus, inhibitory AhR-ERalpha cross talk is linked to a novel pathway for degradation of ERalpha in which TCDD initially induces formation of a nuclear AhR complex which coordinately recruits ERalpha and the proteasome complex, resulting in degradation of both receptors.
Asunto(s)
Neoplasias de la Mama/patología , Estradiol/farmacología , Péptido Hidrolasas/metabolismo , Dibenzodioxinas Policloradas/farmacología , Complejo de la Endopetidasa Proteasomal , Receptor Cross-Talk/efectos de los fármacos , Receptores de Hidrocarburo de Aril/fisiología , Receptores de Estrógenos/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Cicloheximida/farmacología , Inhibidores Enzimáticos/farmacología , Receptor alfa de Estrógeno , Femenino , Flavonoides/farmacología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sustancias Macromoleculares , Ratones , Modelos Biológicos , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteínas Quinasas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Activación Transcripcional/efectos de los fármacos , Transfección , Ubiquitina/metabolismoRESUMEN
Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) gamma agonists ciglitazone or 15-deoxy-Delta 12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) alpha proteins, and this was accompanied by decreased cell proliferation and G(1)-G(0)-->S-phase progression. Down-regulation of cyclin D1 and ER alpha by PPARgamma agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-Delta 12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER alpha antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER alpha bands. Thus, PPARgamma-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER alpha), and this pathway may be important for other cancer cell lines.
Asunto(s)
Neoplasias de la Mama/metabolismo , Ciclina D1/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores de Estrógenos/metabolismo , Tiazoles/farmacología , Tiazolidinedionas , Factores de Transcripción/agonistas , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Ciclina D1/genética , Regulación hacia Abajo/efectos de los fármacos , Receptor alfa de Estrógeno , Fase G1/efectos de los fármacos , Humanos , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Fase S/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Ubiquitina/metabolismoRESUMEN
Regulation of estrogen receptor alpha (ERalpha) plays an important role in hormone responsiveness and growth of ER-positive breast cancer cells and tumors. ZR-75 breast cancer cells were grown under conditions of normoxia (21% O(2)) or hypoxia (1% O(2) or cobaltous chloride), and hypoxia significantly increased hypoxia-inducible factor 1alpha protein within 3 h after treatment, whereas ERalpha protein levels were dramatically decreased within 6-12 h, and this response was blocked by the proteasome inhibitor MG-132. In contrast, hypoxia induced only minimal decreases in cellular Sp1 protein and did not affect ERalpha mRNA; however, hypoxic conditions decreased basal and 17beta-estradiol-induced pS2 gene expression (mRNA levels) and estrogen response element-dependent reporter gene activity in ZR-75 cells. Although 17beta-estradiol and hypoxia induce proteasome-dependent degradation of ERalpha, their effects on transactivation are different, and this may have implications for clinical treatment of mammary tumors.
Asunto(s)
Neoplasias de la Mama/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Hipoxia de la Célula , Cobalto/farmacología , Factores de Crecimiento Endotelial/genética , Estradiol/farmacología , Receptor alfa de Estrógeno , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Péptidos y Proteínas de Señalización Intercelular/genética , Leupeptinas/farmacología , Linfocinas/genética , Complejos Multienzimáticos/antagonistas & inhibidores , Regiones Promotoras Genéticas , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , Proteínas/efectos de los fármacos , Proteínas/genética , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/genética , Factor de Transcripción Sp1/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
SNURF is a small RING finger protein that binds the zinc finger region of steroid hormone receptors and enhances Sp1- and androgen receptor-mediated transcription in COS and CV-1 cells. In this study, we show that SNURF coactivates both wild-type estrogen receptor alpha (ERalpha) (4-fold)- and HE19 (ERalpha deletion of activation function 1 (AF1)) (210-fold)-mediated activation of an estrogen-responsive element promoter in ZR-75 cells. In mammalian two-hybrid assays in ZR-75 cells SNURF interactions were estrogen (E2)-dependent and were not observed with the antiestrogen ICI 182,780. ERalpha interacted with multiple regions of SNURF; SNURF interactions with ERalpha were dependent on AF2, and D538N, E542Q, and D545N mutations in helix 12 abrogated both SNURF-ERalpha binding and coactivation. Moreover, peptide fusion proteins that inhibit interactions between helix 12 of ERalpha with LXXLL box-containing proteins also blocked ERalpha coactivation by SNURF. However, cotransfection of SNURF with prototypical steroid receptor coactivators 1, 2, and 3 that contain LXXLL box motifs did not enhance E2 responsiveness, whereas TATA-binding protein (TBP) and SNURF cooperatively coactivated ERalpha-mediated transactivation. The results are consistent with a unique model for cooperative coactivation of ERalpha that requires ligand binding, repositioning of helix 12, recruitment of TBP, and interaction with SNURF, which binds both ERalpha and TBP.