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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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The engrailed 1-luciferase construct responds to estradiol.The scheme represents the structure of the 2.8 kB of construct containing the proximal region of endogenous engrailed 1 promoter bind to luciferase reporter. N2a-m cells were transfected with the pENP1-luciferase reporter plasmid (250, 200 and 750 ng) (represented as 0.25, 0.5, 0.75), which contains three LEF-1 sites (see upper panel). The Lower panel shows a comparison of the induction of TOPFlash (750 ng) and pENP-luc in this cell line. Although basal levels of luciferase activity are lower in the pENP1-luc reporter plasmid, estradiol induces this activity to 3-fold that of the control levels, as shown in the right panel. The graph in B shows the normalized luciferase activity from at least three independent experiments. The P value from the Student's t-test was ** (P≤0.01) when compared with control data.
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pone-0005153-g005: The engrailed 1-luciferase construct responds to estradiol.The scheme represents the structure of the 2.8 kB of construct containing the proximal region of endogenous engrailed 1 promoter bind to luciferase reporter. N2a-m cells were transfected with the pENP1-luciferase reporter plasmid (250, 200 and 750 ng) (represented as 0.25, 0.5, 0.75), which contains three LEF-1 sites (see upper panel). The Lower panel shows a comparison of the induction of TOPFlash (750 ng) and pENP-luc in this cell line. Although basal levels of luciferase activity are lower in the pENP1-luc reporter plasmid, estradiol induces this activity to 3-fold that of the control levels, as shown in the right panel. The graph in B shows the normalized luciferase activity from at least three independent experiments. The P value from the Student's t-test was ** (P≤0.01) when compared with control data.

Mentions: As a complementary alternative we try to analyze whether an endogenous promoter may be regulated by estradiol in a similar fashion as Wnt3a. For that purpose we used a construct containing a 2.8 Kb region of the Engrailed-1 promoter that has three LEF-1 sites situated upstream of a luciferase gene pENP1Luc [22] (see scheme in Figure 5A). Again luciferase expression was clearly driven by estradiol, although at identical amounts of cDNA the response of TOPFlash was more potent (Figure 5B).


Estradiol activates beta-catenin dependent transcription in neurons.

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

The engrailed 1-luciferase construct responds to estradiol.The scheme represents the structure of the 2.8 kB of construct containing the proximal region of endogenous engrailed 1 promoter bind to luciferase reporter. N2a-m cells were transfected with the pENP1-luciferase reporter plasmid (250, 200 and 750 ng) (represented as 0.25, 0.5, 0.75), which contains three LEF-1 sites (see upper panel). The Lower panel shows a comparison of the induction of TOPFlash (750 ng) and pENP-luc in this cell line. Although basal levels of luciferase activity are lower in the pENP1-luc reporter plasmid, estradiol induces this activity to 3-fold that of the control levels, as shown in the right panel. The graph in B shows the normalized luciferase activity from at least three independent experiments. The P value from the Student's t-test was ** (P≤0.01) when compared with control data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664482&req=5

pone-0005153-g005: The engrailed 1-luciferase construct responds to estradiol.The scheme represents the structure of the 2.8 kB of construct containing the proximal region of endogenous engrailed 1 promoter bind to luciferase reporter. N2a-m cells were transfected with the pENP1-luciferase reporter plasmid (250, 200 and 750 ng) (represented as 0.25, 0.5, 0.75), which contains three LEF-1 sites (see upper panel). The Lower panel shows a comparison of the induction of TOPFlash (750 ng) and pENP-luc in this cell line. Although basal levels of luciferase activity are lower in the pENP1-luc reporter plasmid, estradiol induces this activity to 3-fold that of the control levels, as shown in the right panel. The graph in B shows the normalized luciferase activity from at least three independent experiments. The P value from the Student's t-test was ** (P≤0.01) when compared with control data.
Mentions: As a complementary alternative we try to analyze whether an endogenous promoter may be regulated by estradiol in a similar fashion as Wnt3a. For that purpose we used a construct containing a 2.8 Kb region of the Engrailed-1 promoter that has three LEF-1 sites situated upstream of a luciferase gene pENP1Luc [22] (see scheme in Figure 5A). Again luciferase expression was clearly driven by estradiol, although at identical amounts of cDNA the response of TOPFlash was more potent (Figure 5B).

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