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Estradiol activates beta-catenin dependent transcription in neurons.

Varea O, Garrido JJ, Dopazo A, Mendez P, Garcia-Segura LM, Wandosell F - PLoS ONE (2009)

Bottom Line: 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.These findings may reveal a set of new physiological roles for estrogens, at least in the Central Nervous System (CNS).

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Centro de Biología Molecular "Severo Ochoa", CSIC-UAM, Madrid, Spain.

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 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 demonstrated that estrogen receptor alpha associates with beta-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 beta-catenin transcription in neuroblastoma cells and in primary cortical neurons. This activation is time and concentration-dependent, and it may be abolished by the estrogen receptor antagonist ICI 182780. The transcriptional activation of beta-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 enhanced by estradiol in a similar fashion to that produced by Wnt3a. In addition, activation of a luciferase reporter driven by the engrailed 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 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 demonstrate the existence 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).

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Estradiol increase TCF/LEF-dependent transcription in cortical neurons.(A)-Cortical neurons from E18 embryos were nucleofected with TOPFlash or FOPFlash reporter plasmids and luciferase activity was analyzed after 2DIV. Estradiol treatment (60 min, 100 nM) selectively increased transcription from the TOPFlash reporter plasmid compared to FOPFlash, which shows no activity. Insulin treatment (5 µg/ml) was used as a control of induction. (B–C)- Expression of the LacZ gene in response to estradiol in transgenic mice. The scheme represents the lacZ transgene under the control of three consensus TCF/LEF-binding motifs upstream of the c-fos promoter, as described in Materials and Methods. (B)-Upper panel. Total extracts of cortical neurons (2DIV) were obtained from a TCF/LEF-lacZ transgenic mouse (see Materials and Methods) and the β-galactosidase (β-gal) expression was assessed in western blots after estradiol treatment (100–200 nM) for 3 h. Wnt3a (20 ng/ml) was used as a control of TCF-mediated induction. A slight increase in β-gal protein was observed after exposure to estradiol, as with recombinant Wnt3a protein. (B), Lower panel. Basal expression of β-galactosidase in neurons from transgenic mice was assessed by immunocytochemistry using specific antibodies against β galactosidase (green) and Phalloidin-labelled with Alexas 549. (C), Alternatively, after treatment with estradiol or Wnt3a, total Lac Z expression was quantified by RT-PCR using specific β-gal oligonucleotides and using actin (a housekeeping gene) as an internal standard (see Methods). The amplification of both genes was analyzed on agarose gels and the graph represents the normalized data obtained from the Lightcycler analysis. Both treatments clearly increase transcriptional activity when compared to controls.
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pone-0005153-g004: Estradiol increase TCF/LEF-dependent transcription in cortical neurons.(A)-Cortical neurons from E18 embryos were nucleofected with TOPFlash or FOPFlash reporter plasmids and luciferase activity was analyzed after 2DIV. Estradiol treatment (60 min, 100 nM) selectively increased transcription from the TOPFlash reporter plasmid compared to FOPFlash, which shows no activity. Insulin treatment (5 µg/ml) was used as a control of induction. (B–C)- Expression of the LacZ gene in response to estradiol in transgenic mice. The scheme represents the lacZ transgene under the control of three consensus TCF/LEF-binding motifs upstream of the c-fos promoter, as described in Materials and Methods. (B)-Upper panel. Total extracts of cortical neurons (2DIV) were obtained from a TCF/LEF-lacZ transgenic mouse (see Materials and Methods) and the β-galactosidase (β-gal) expression was assessed in western blots after estradiol treatment (100–200 nM) for 3 h. Wnt3a (20 ng/ml) was used as a control of TCF-mediated induction. A slight increase in β-gal protein was observed after exposure to estradiol, as with recombinant Wnt3a protein. (B), Lower panel. Basal expression of β-galactosidase in neurons from transgenic mice was assessed by immunocytochemistry using specific antibodies against β galactosidase (green) and Phalloidin-labelled with Alexas 549. (C), Alternatively, after treatment with estradiol or Wnt3a, total Lac Z expression was quantified by RT-PCR using specific β-gal oligonucleotides and using actin (a housekeeping gene) as an internal standard (see Methods). The amplification of both genes was analyzed on agarose gels and the graph represents the normalized data obtained from the Lightcycler analysis. Both treatments clearly increase transcriptional activity when compared to controls.

Mentions: To determine whether estradiol can regulate the expression of a reporter controlled by TCF in primary neurons, we used two complementary approaches. First, we transfected TOPFlash or FOPFlash into primary neurons (Figure 4A) and after TOPFlash transfection, we detected an increase in luciferase activity (4.8±1.2 folds; n = 3) in the presence of estradiol (100 nM), which was not induced after FOPFlash transfection (Figure 4A). High concentrations of insulin (ITS supplements) also produced a 10-fold increase in these cells as a positive control (Figure 4A). In a second approach, we obtained primary cultures of cortical neurons from TCF-βgal transgenic mice, a colony designed to analyze the activation of the Wnt pathway in vivo [21]. We detected β-galactosidase expression in almost all cortical neurons of TCF-βgal transgenic mice (Figure 4B) and thus, we analyzed the amount of β-galactosidase after exposure to either estradiol or Wnt3a. Both estradiol and Wnt3a produced a similar moderate increase in β-galactosidase, assessed in western blots (Figure 4B). This finding was confirmed by quantitative RT-PCR in parallel experiments. Hence, in total RNA obtained from cultured cortical neurons, a similar increase in the expression of the LacZ-transgene was detected by RealTime RT-PCR after exposure to Wnt3a or estradiol when normalized to actin mRNA expression (Figure 4C).


Estradiol activates beta-catenin dependent transcription in neurons.

Varea O, Garrido JJ, Dopazo A, Mendez P, Garcia-Segura LM, Wandosell F - PLoS ONE (2009)

Estradiol increase TCF/LEF-dependent transcription in cortical neurons.(A)-Cortical neurons from E18 embryos were nucleofected with TOPFlash or FOPFlash reporter plasmids and luciferase activity was analyzed after 2DIV. Estradiol treatment (60 min, 100 nM) selectively increased transcription from the TOPFlash reporter plasmid compared to FOPFlash, which shows no activity. Insulin treatment (5 µg/ml) was used as a control of induction. (B–C)- Expression of the LacZ gene in response to estradiol in transgenic mice. The scheme represents the lacZ transgene under the control of three consensus TCF/LEF-binding motifs upstream of the c-fos promoter, as described in Materials and Methods. (B)-Upper panel. Total extracts of cortical neurons (2DIV) were obtained from a TCF/LEF-lacZ transgenic mouse (see Materials and Methods) and the β-galactosidase (β-gal) expression was assessed in western blots after estradiol treatment (100–200 nM) for 3 h. Wnt3a (20 ng/ml) was used as a control of TCF-mediated induction. A slight increase in β-gal protein was observed after exposure to estradiol, as with recombinant Wnt3a protein. (B), Lower panel. Basal expression of β-galactosidase in neurons from transgenic mice was assessed by immunocytochemistry using specific antibodies against β galactosidase (green) and Phalloidin-labelled with Alexas 549. (C), Alternatively, after treatment with estradiol or Wnt3a, total Lac Z expression was quantified by RT-PCR using specific β-gal oligonucleotides and using actin (a housekeeping gene) as an internal standard (see Methods). The amplification of both genes was analyzed on agarose gels and the graph represents the normalized data obtained from the Lightcycler analysis. Both treatments clearly increase transcriptional activity when compared to controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664482&req=5

pone-0005153-g004: Estradiol increase TCF/LEF-dependent transcription in cortical neurons.(A)-Cortical neurons from E18 embryos were nucleofected with TOPFlash or FOPFlash reporter plasmids and luciferase activity was analyzed after 2DIV. Estradiol treatment (60 min, 100 nM) selectively increased transcription from the TOPFlash reporter plasmid compared to FOPFlash, which shows no activity. Insulin treatment (5 µg/ml) was used as a control of induction. (B–C)- Expression of the LacZ gene in response to estradiol in transgenic mice. The scheme represents the lacZ transgene under the control of three consensus TCF/LEF-binding motifs upstream of the c-fos promoter, as described in Materials and Methods. (B)-Upper panel. Total extracts of cortical neurons (2DIV) were obtained from a TCF/LEF-lacZ transgenic mouse (see Materials and Methods) and the β-galactosidase (β-gal) expression was assessed in western blots after estradiol treatment (100–200 nM) for 3 h. Wnt3a (20 ng/ml) was used as a control of TCF-mediated induction. A slight increase in β-gal protein was observed after exposure to estradiol, as with recombinant Wnt3a protein. (B), Lower panel. Basal expression of β-galactosidase in neurons from transgenic mice was assessed by immunocytochemistry using specific antibodies against β galactosidase (green) and Phalloidin-labelled with Alexas 549. (C), Alternatively, after treatment with estradiol or Wnt3a, total Lac Z expression was quantified by RT-PCR using specific β-gal oligonucleotides and using actin (a housekeeping gene) as an internal standard (see Methods). The amplification of both genes was analyzed on agarose gels and the graph represents the normalized data obtained from the Lightcycler analysis. Both treatments clearly increase transcriptional activity when compared to controls.
Mentions: To determine whether estradiol can regulate the expression of a reporter controlled by TCF in primary neurons, we used two complementary approaches. First, we transfected TOPFlash or FOPFlash into primary neurons (Figure 4A) and after TOPFlash transfection, we detected an increase in luciferase activity (4.8±1.2 folds; n = 3) in the presence of estradiol (100 nM), which was not induced after FOPFlash transfection (Figure 4A). High concentrations of insulin (ITS supplements) also produced a 10-fold increase in these cells as a positive control (Figure 4A). In a second approach, we obtained primary cultures of cortical neurons from TCF-βgal transgenic mice, a colony designed to analyze the activation of the Wnt pathway in vivo [21]. We detected β-galactosidase expression in almost all cortical neurons of TCF-βgal transgenic mice (Figure 4B) and thus, we analyzed the amount of β-galactosidase after exposure to either estradiol or Wnt3a. Both estradiol and Wnt3a produced a similar moderate increase in β-galactosidase, assessed in western blots (Figure 4B). This finding was confirmed by quantitative RT-PCR in parallel experiments. Hence, in total RNA obtained from cultured cortical neurons, a similar increase in the expression of the LacZ-transgene was detected by RealTime RT-PCR after exposure to Wnt3a or estradiol when normalized to actin mRNA expression (Figure 4C).

Bottom Line: 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.These findings may reveal a set of new physiological roles for estrogens, at least in the Central Nervous System (CNS).

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Centro de Biología Molecular "Severo Ochoa", CSIC-UAM, Madrid, Spain.

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 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 demonstrated that estrogen receptor alpha associates with beta-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 beta-catenin transcription in neuroblastoma cells and in primary cortical neurons. This activation is time and concentration-dependent, and it may be abolished by the estrogen receptor antagonist ICI 182780. The transcriptional activation of beta-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 enhanced by estradiol in a similar fashion to that produced by Wnt3a. In addition, activation of a luciferase reporter driven by the engrailed 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 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 demonstrate the existence 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).

Show MeSH
Related in: MedlinePlus