Luciferase activity was calculated at different concentrations of Wnt3a protein, as indicated above, and Wnt3a produced a significant increase in -catenin/TCF dependent transcription

Luciferase activity was calculated at different concentrations of Wnt3a protein, as indicated above, and Wnt3a produced a significant increase in -catenin/TCF dependent transcription. both isoforms in total cell extracts from control cells.(9.68 MB EPS) pone.0005153.s001.eps (9.2M) GUID:?59685E69-FBF4-42F3-9908-176E0CE7F5BE Physique S2: Distribution of -catenin after exposure of N2a-m cells to estradiol does not reveal the translocation of -catenin to the nucleus. Cell fractions corresponding to the soluble, membrane and nuclear fractions were analyzed in control and estradiol treated cells. Neither 30 nor 60 min exposure to estradiol provoked the nuclear accumulation of -catenin, statistically significant. In contrast, recruitment of -catenin to the membrane fraction was more evident. We also checked the movement of ER as a control of the assay, which clearly accumulated in the nuclear fraction (see Physique 8).(2.36 MB EPS) pone.0005153.s002.eps (2.2M) GUID:?846F46A4-B524-43BF-ACB9-A497939C2DFC Physique S3: N2a-m cells are responsive to Wnt3a protein. (A)- N2a-m cells respond to Wnt3a (20 ng/ml) with the accumulation of -catenin and with a maximal effect observed after 90C120 minutes. (B)- The transcriptional activity of -catenin was analyzed with the TCF-luc reporter. Luciferase activity was calculated at different concentrations of Wnt3a protein, as indicated above, and Wnt3a produced a significant increase in -catenin/TCF dependent transcription. Asterisk represents P value from Student’s t-test: * (P0.05), when compared the two Wnt concentrations; and ** (P0.01) when compared with control.(1.61 MB EPS) pone.0005153.s003.eps (1.5M) GUID:?473B7277-25F2-49F3-9BD9-E71B3B76D9E8 Figure S4: Neither ER nor TCF-3 antibodies modify the migration of nuclear protein extracts from N2a-m cells exposed to estradiol or Wnt3a. Nuclear protein extracts were obtained from control or estradiol/Wnt3a treated cells as indicated in each lane. The incubation with specific antibodies against ER or TCF3 did not produce a higher molecular weight band that migrated more slowly than that previously identified as a TCF-DNA complex, nor did they prevent the formation of the complex.(2.50 MB EPS) pone.0005153.s004.eps (2.3M) GUID:?55D8FE8D-B47C-4A3E-AFE3-BEB436BEB5FA Abstract Estradiol may fulfill a plethora of functions in neurons, in which much of its activity is associated with its capacity to directly bind and dimerize estrogen receptors. This hormone-protein complex can either bind directly to estrogen response elements (ERE’s) in gene promoters, or it may act as a NPS-2143 (SB-262470) cofactor at non-ERE sites interacting with other DNA-binding elements such as AP-1 or c-Jun. Many of the neuroprotective effects described for estrogen have been associated with this mode of action. However, recent evidence suggests that in addition to these genomic effects, estrogen may also act as a more general trophic factor triggering cytoplasmic signals and extending the potential activity of this hormone. We exhibited that estrogen receptor alpha associates with -catenin and glycogen synthase kinase 3 in the brain and in neurons, which has since been confirmed by others. Here, we show that the action of estradiol activates -catenin transcription NPS-2143 (SB-262470) in neuroblastoma cells and in primary cortical neurons. This activation is usually time and concentration-dependent, and it may be KR1_HHV11 antibody abolished by the estrogen receptor antagonist ICI 182780. The transcriptional activation of -catenin is dependent on lymphoid enhancer binding factor-1 (LEF-1) and a truncated-mutant of LEF-1 almost completely blocks estradiol TCF-mediated transcription. Transcription of a TCF-reporter in a transgenic mouse model is usually enhanced by estradiol in a similar fashion to that produced by Wnt3a. In addition, activation of a luciferase reporter driven by the promoter with three LEF-1 repeats was mediated by estradiol. We established a cell line that constitutively expresses a dominant-negative LEF-1 and it was NPS-2143 (SB-262470) used in a gene expression microarray analysis. In this way, genes that respond to estradiol or Wnt3a, sensitive to LEF-1, could be identified and validated. Together, these data NPS-2143 (SB-262470) demonstrate the presence of a new signaling pathway controlled by estradiol in neurons. This pathway shares some elements of the insulin-like growth factor-1/Insulin and Wnt signaling pathways, however, our data strongly suggest that it is different from that of both these ligands. These findings may reveal a set of new physiological roles for estrogens, at least in the Central Nervous System (CNS). Introduction Estrogens fulfill a wide range of functions during development and differentiation in mammals of both sexes. In addition to these functions, they are also thought to play an important role in neuroprotection [1]C[3]. The actions of estrogens have been classified as either genomic actions or non-genomic, rapid actions. The genomic actions are based on the capacity of the estrogen receptors (ERs) to bind to co-activators or co-repressors in order to enhance or inhibit the transcription of target genes, and it has been reported in many cell types (reviewed in [4]). This activity involves the dimerization of two receptor molecules mediated by the presence of the hormone and the generation of a macromolecular complex with.

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