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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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Treatment of estradiol increases Ser9,21-GSK3 phosphorylation and β-catenin accumulation in neuronal cells.(A)-Neuroblastoma N2a-m were treated with different doses of estradiol and for different times (20–120 min with 10–500 nM) and the maximum increase in GSK3β-PSer9 was observed when they were treated for 60 min with 100–200 nM estradiol. (B)- Stabilization of β-catenin. N2a-m cells treated with estradiol showed a clear increase in β-catenin that could be prevented by prior exposure to ICI 182780 (used 100× concentrated in comparison to estradiol), 60 minutes before estradiol treatment (lower panel). (C)-Cortical Primary Neurons (2DIV) were treated with estradiol (100 nM) and the maximum increase in GSK3β-PSer9 and the subsequent stabilization of β-catenin was clearly detected 60–90 min after the onset of exposure. In all cases diagram shows the mean normalized densitometry values and the corresponding standard deviations from at least three independent experiments. Asterisks indicate statistical significance (Student's t-test) ** (P≤0.05), * (P≤0.01). The single * or ** compares data to control whereas the bar between different points shows statistical differences between experimental values.
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pone-0005153-g001: Treatment of estradiol increases Ser9,21-GSK3 phosphorylation and β-catenin accumulation in neuronal cells.(A)-Neuroblastoma N2a-m were treated with different doses of estradiol and for different times (20–120 min with 10–500 nM) and the maximum increase in GSK3β-PSer9 was observed when they were treated for 60 min with 100–200 nM estradiol. (B)- Stabilization of β-catenin. N2a-m cells treated with estradiol showed a clear increase in β-catenin that could be prevented by prior exposure to ICI 182780 (used 100× concentrated in comparison to estradiol), 60 minutes before estradiol treatment (lower panel). (C)-Cortical Primary Neurons (2DIV) were treated with estradiol (100 nM) and the maximum increase in GSK3β-PSer9 and the subsequent stabilization of β-catenin was clearly detected 60–90 min after the onset of exposure. In all cases diagram shows the mean normalized densitometry values and the corresponding standard deviations from at least three independent experiments. Asterisks indicate statistical significance (Student's t-test) ** (P≤0.05), * (P≤0.01). The single * or ** compares data to control whereas the bar between different points shows statistical differences between experimental values.

Mentions: The effect of estradiol on this cell line was examined and we initially determined whether N2a-m cells expressed both α and β estrogen receptors when maintained in serum–free medium (Supplementary Figure S1). When N2a-m cells were exposed to estradiol, ERα-immunoreactivity became more concentrated in the nucleus in a time and dose dependent manner (Supplementary Figure S1). Thus, since these cells respond to estradiol and can be efficiently transfected, we analyzed whether exposure to estradiol could modify GSK3β-PSer using primary cortical neurons as a reference in parallel. Immunoreactivity against GSK3β-PSer was increased in N2a-m cells exposed to estradiol, reaching a maximum (5±1 fold, n = 4) 60–90 minutes after the addition of the hormone (Figure 1A). A similar time-course of GSK3β-PSer immunoreactivity was observed in cortical primary neurons (Figure 1C), reaching a maximum increase (2.2±0.5 fold; n = 3) 90 minutes after exposure to the hormone. The increase in serine phosphorylation of GSK3β was dose-dependent and was maximal in the range of 100 to 200 nM (2.5±0.5 fold; n = 3, Figure 1A).


Estradiol activates beta-catenin dependent transcription in neurons.

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

Treatment of estradiol increases Ser9,21-GSK3 phosphorylation and β-catenin accumulation in neuronal cells.(A)-Neuroblastoma N2a-m were treated with different doses of estradiol and for different times (20–120 min with 10–500 nM) and the maximum increase in GSK3β-PSer9 was observed when they were treated for 60 min with 100–200 nM estradiol. (B)- Stabilization of β-catenin. N2a-m cells treated with estradiol showed a clear increase in β-catenin that could be prevented by prior exposure to ICI 182780 (used 100× concentrated in comparison to estradiol), 60 minutes before estradiol treatment (lower panel). (C)-Cortical Primary Neurons (2DIV) were treated with estradiol (100 nM) and the maximum increase in GSK3β-PSer9 and the subsequent stabilization of β-catenin was clearly detected 60–90 min after the onset of exposure. In all cases diagram shows the mean normalized densitometry values and the corresponding standard deviations from at least three independent experiments. Asterisks indicate statistical significance (Student's t-test) ** (P≤0.05), * (P≤0.01). The single * or ** compares data to control whereas the bar between different points shows statistical differences between experimental values.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664482&req=5

pone-0005153-g001: Treatment of estradiol increases Ser9,21-GSK3 phosphorylation and β-catenin accumulation in neuronal cells.(A)-Neuroblastoma N2a-m were treated with different doses of estradiol and for different times (20–120 min with 10–500 nM) and the maximum increase in GSK3β-PSer9 was observed when they were treated for 60 min with 100–200 nM estradiol. (B)- Stabilization of β-catenin. N2a-m cells treated with estradiol showed a clear increase in β-catenin that could be prevented by prior exposure to ICI 182780 (used 100× concentrated in comparison to estradiol), 60 minutes before estradiol treatment (lower panel). (C)-Cortical Primary Neurons (2DIV) were treated with estradiol (100 nM) and the maximum increase in GSK3β-PSer9 and the subsequent stabilization of β-catenin was clearly detected 60–90 min after the onset of exposure. In all cases diagram shows the mean normalized densitometry values and the corresponding standard deviations from at least three independent experiments. Asterisks indicate statistical significance (Student's t-test) ** (P≤0.05), * (P≤0.01). The single * or ** compares data to control whereas the bar between different points shows statistical differences between experimental values.
Mentions: The effect of estradiol on this cell line was examined and we initially determined whether N2a-m cells expressed both α and β estrogen receptors when maintained in serum–free medium (Supplementary Figure S1). When N2a-m cells were exposed to estradiol, ERα-immunoreactivity became more concentrated in the nucleus in a time and dose dependent manner (Supplementary Figure S1). Thus, since these cells respond to estradiol and can be efficiently transfected, we analyzed whether exposure to estradiol could modify GSK3β-PSer using primary cortical neurons as a reference in parallel. Immunoreactivity against GSK3β-PSer was increased in N2a-m cells exposed to estradiol, reaching a maximum (5±1 fold, n = 4) 60–90 minutes after the addition of the hormone (Figure 1A). A similar time-course of GSK3β-PSer immunoreactivity was observed in cortical primary neurons (Figure 1C), reaching a maximum increase (2.2±0.5 fold; n = 3) 90 minutes after exposure to the hormone. The increase in serine phosphorylation of GSK3β was dose-dependent and was maximal in the range of 100 to 200 nM (2.5±0.5 fold; n = 3, Figure 1A).

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