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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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Related in: MedlinePlus

Gene expression of cDNA/N2a-m and Δ56LEF-1/N2a-m cells after exposure to estradiol.(A)- Gene expression profile in cDNA/N2a-m and Δ56LEF-1/N2a-m stables cell lines. The upper panel reflects the gene induction of some selected genes, in microarray analysis of RNA collected after a 45 min exposure to estradiol or Wnt3a to detect the early response. Data is expressed as log2R from cDNA/N2a-m cells, that we denoted as group A, and Δ56LEF-1/N2a-m cells, that we denoted as group B. The effect of the treatment was compared between the two stable cell lines (A vs B) (see “Table 1” for a more complete list of the annotated genes). As seen, in the panel we selected some “putative Wnt-regulated genes”, such as Tcf3, Ccnd1 (cyclin D1), GSK3b, Myc and LEF-1, to give some examples of the results in our arrays. We detected changes at the protein level only in Plg, although there were several genes whose expression varied. For example, the levels of plasminogen RNA were much higher in group B than group A (ratio AvsB≥1), and the expression of LEF-1 was higher in Δ56LEF-1 due to the mutant expression (ratio AvsB≤−1). The western blots below are verifications of these differences at the protein level. Among other proteins that did not change between the groups of cells were GSK3 β or myc (see western blots on the right). MMP-2 was tested although it did not display a change in its RNA levels and there was no difference in the total cell extracts. Interestingly, when conditioned medium was prepared, more pro-active MMP-2 (and less active protein) was seen in Δ56LEF-1 cells. [Gene_Symbol: Plg (plasminogen), Tcf3, Ccnd1 (cyclin D1), GSK3b, Myc and LEF1]. (B)- Estradiol induction of N-cadherin and cyclin D2 may be affected by expression of the Δ56LEF-1 protein. Total cell extracts from cDNA/N2a-m cells (group A) and Δ56LEF-1/N2a-m cells (group B) were collected 24 h after estradiol or Wnt3a treatment to analyze several known Wnt or estrogen target genes. As seen in western blots, estradiol upregulated E-cadherin, N-cadherin and cyclin D2 expression in group A cells. N-cadherin and cyclin D2 were also upregulated by Wnt in group A cells. In contrast, E-cadherin expression was not Wnt responsive. The regulatory effects of estradiol on E-cadherin, N-cadherin and cyclin D2 expression were lost when Δ56LEF-1 is expressed, as seen in group B cells. In the case of E-cadherin, the loss of functional LEF-1, which acts as a known gene repressor, implies higher protein levels even without stimulation. In contrast, levels of actin or cyclin D1 remained unchanged.
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pone-0005153-g009: Gene expression of cDNA/N2a-m and Δ56LEF-1/N2a-m cells after exposure to estradiol.(A)- Gene expression profile in cDNA/N2a-m and Δ56LEF-1/N2a-m stables cell lines. The upper panel reflects the gene induction of some selected genes, in microarray analysis of RNA collected after a 45 min exposure to estradiol or Wnt3a to detect the early response. Data is expressed as log2R from cDNA/N2a-m cells, that we denoted as group A, and Δ56LEF-1/N2a-m cells, that we denoted as group B. The effect of the treatment was compared between the two stable cell lines (A vs B) (see “Table 1” for a more complete list of the annotated genes). As seen, in the panel we selected some “putative Wnt-regulated genes”, such as Tcf3, Ccnd1 (cyclin D1), GSK3b, Myc and LEF-1, to give some examples of the results in our arrays. We detected changes at the protein level only in Plg, although there were several genes whose expression varied. For example, the levels of plasminogen RNA were much higher in group B than group A (ratio AvsB≥1), and the expression of LEF-1 was higher in Δ56LEF-1 due to the mutant expression (ratio AvsB≤−1). The western blots below are verifications of these differences at the protein level. Among other proteins that did not change between the groups of cells were GSK3 β or myc (see western blots on the right). MMP-2 was tested although it did not display a change in its RNA levels and there was no difference in the total cell extracts. Interestingly, when conditioned medium was prepared, more pro-active MMP-2 (and less active protein) was seen in Δ56LEF-1 cells. [Gene_Symbol: Plg (plasminogen), Tcf3, Ccnd1 (cyclin D1), GSK3b, Myc and LEF1]. (B)- Estradiol induction of N-cadherin and cyclin D2 may be affected by expression of the Δ56LEF-1 protein. Total cell extracts from cDNA/N2a-m cells (group A) and Δ56LEF-1/N2a-m cells (group B) were collected 24 h after estradiol or Wnt3a treatment to analyze several known Wnt or estrogen target genes. As seen in western blots, estradiol upregulated E-cadherin, N-cadherin and cyclin D2 expression in group A cells. N-cadherin and cyclin D2 were also upregulated by Wnt in group A cells. In contrast, E-cadherin expression was not Wnt responsive. The regulatory effects of estradiol on E-cadherin, N-cadherin and cyclin D2 expression were lost when Δ56LEF-1 is expressed, as seen in group B cells. In the case of E-cadherin, the loss of functional LEF-1, which acts as a known gene repressor, implies higher protein levels even without stimulation. In contrast, levels of actin or cyclin D1 remained unchanged.

Mentions: Genes were normalized with respect to control untreated cells, and divided in different groups. From these groups, we only consider those with differences when compared group A versus group B, for each treatment. From these we selected those with a log2R higher or lower than 1, being 102 genes upregulated and 121 genes downregulated by estradiol; and 137 genes upregulated and 166 genes downregulated by Wnt3a. Table 1 described the list of the annotated genes present in the protein bank with the Ref Seq and the Gene Name. In contrast, the genes modified only after estradiol treatment or only after Wnt3a treatment that presented no significant differences between A and B were discarded. Initially, for the confirmation of the putative deferential response of both cells (A vs B) we selected two genes presented in both arrays that were modified by Wnt as well as by estradiol (Plasminogen and LEF-1). A final validation was performed using specific antibodies against the selected proteins (Figure 9 and 8C). Thus, plasminogen expression in Δ56LEF-1 cells was 2.5 fold higher, although these initial differences were not considerably augmented by treatment with Wnt or estradiol. Similarly, the endogenous LEF-1 was reduced by the presence of the mutant version of this protein (Figure 8). In addition we selected some genes, previously reported by other laboratories as Wnt targets , such as TCF's, Cyclin D, Myc, MMP's (for updated, please check, The Wnt Homepage, at http://www.stanford.edu/˜rnusse/pathways/targets.html).


Estradiol activates beta-catenin dependent transcription in neurons.

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

Gene expression of cDNA/N2a-m and Δ56LEF-1/N2a-m cells after exposure to estradiol.(A)- Gene expression profile in cDNA/N2a-m and Δ56LEF-1/N2a-m stables cell lines. The upper panel reflects the gene induction of some selected genes, in microarray analysis of RNA collected after a 45 min exposure to estradiol or Wnt3a to detect the early response. Data is expressed as log2R from cDNA/N2a-m cells, that we denoted as group A, and Δ56LEF-1/N2a-m cells, that we denoted as group B. The effect of the treatment was compared between the two stable cell lines (A vs B) (see “Table 1” for a more complete list of the annotated genes). As seen, in the panel we selected some “putative Wnt-regulated genes”, such as Tcf3, Ccnd1 (cyclin D1), GSK3b, Myc and LEF-1, to give some examples of the results in our arrays. We detected changes at the protein level only in Plg, although there were several genes whose expression varied. For example, the levels of plasminogen RNA were much higher in group B than group A (ratio AvsB≥1), and the expression of LEF-1 was higher in Δ56LEF-1 due to the mutant expression (ratio AvsB≤−1). The western blots below are verifications of these differences at the protein level. Among other proteins that did not change between the groups of cells were GSK3 β or myc (see western blots on the right). MMP-2 was tested although it did not display a change in its RNA levels and there was no difference in the total cell extracts. Interestingly, when conditioned medium was prepared, more pro-active MMP-2 (and less active protein) was seen in Δ56LEF-1 cells. [Gene_Symbol: Plg (plasminogen), Tcf3, Ccnd1 (cyclin D1), GSK3b, Myc and LEF1]. (B)- Estradiol induction of N-cadherin and cyclin D2 may be affected by expression of the Δ56LEF-1 protein. Total cell extracts from cDNA/N2a-m cells (group A) and Δ56LEF-1/N2a-m cells (group B) were collected 24 h after estradiol or Wnt3a treatment to analyze several known Wnt or estrogen target genes. As seen in western blots, estradiol upregulated E-cadherin, N-cadherin and cyclin D2 expression in group A cells. N-cadherin and cyclin D2 were also upregulated by Wnt in group A cells. In contrast, E-cadherin expression was not Wnt responsive. The regulatory effects of estradiol on E-cadherin, N-cadherin and cyclin D2 expression were lost when Δ56LEF-1 is expressed, as seen in group B cells. In the case of E-cadherin, the loss of functional LEF-1, which acts as a known gene repressor, implies higher protein levels even without stimulation. In contrast, levels of actin or cyclin D1 remained unchanged.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005153-g009: Gene expression of cDNA/N2a-m and Δ56LEF-1/N2a-m cells after exposure to estradiol.(A)- Gene expression profile in cDNA/N2a-m and Δ56LEF-1/N2a-m stables cell lines. The upper panel reflects the gene induction of some selected genes, in microarray analysis of RNA collected after a 45 min exposure to estradiol or Wnt3a to detect the early response. Data is expressed as log2R from cDNA/N2a-m cells, that we denoted as group A, and Δ56LEF-1/N2a-m cells, that we denoted as group B. The effect of the treatment was compared between the two stable cell lines (A vs B) (see “Table 1” for a more complete list of the annotated genes). As seen, in the panel we selected some “putative Wnt-regulated genes”, such as Tcf3, Ccnd1 (cyclin D1), GSK3b, Myc and LEF-1, to give some examples of the results in our arrays. We detected changes at the protein level only in Plg, although there were several genes whose expression varied. For example, the levels of plasminogen RNA were much higher in group B than group A (ratio AvsB≥1), and the expression of LEF-1 was higher in Δ56LEF-1 due to the mutant expression (ratio AvsB≤−1). The western blots below are verifications of these differences at the protein level. Among other proteins that did not change between the groups of cells were GSK3 β or myc (see western blots on the right). MMP-2 was tested although it did not display a change in its RNA levels and there was no difference in the total cell extracts. Interestingly, when conditioned medium was prepared, more pro-active MMP-2 (and less active protein) was seen in Δ56LEF-1 cells. [Gene_Symbol: Plg (plasminogen), Tcf3, Ccnd1 (cyclin D1), GSK3b, Myc and LEF1]. (B)- Estradiol induction of N-cadherin and cyclin D2 may be affected by expression of the Δ56LEF-1 protein. Total cell extracts from cDNA/N2a-m cells (group A) and Δ56LEF-1/N2a-m cells (group B) were collected 24 h after estradiol or Wnt3a treatment to analyze several known Wnt or estrogen target genes. As seen in western blots, estradiol upregulated E-cadherin, N-cadherin and cyclin D2 expression in group A cells. N-cadherin and cyclin D2 were also upregulated by Wnt in group A cells. In contrast, E-cadherin expression was not Wnt responsive. The regulatory effects of estradiol on E-cadherin, N-cadherin and cyclin D2 expression were lost when Δ56LEF-1 is expressed, as seen in group B cells. In the case of E-cadherin, the loss of functional LEF-1, which acts as a known gene repressor, implies higher protein levels even without stimulation. In contrast, levels of actin or cyclin D1 remained unchanged.
Mentions: Genes were normalized with respect to control untreated cells, and divided in different groups. From these groups, we only consider those with differences when compared group A versus group B, for each treatment. From these we selected those with a log2R higher or lower than 1, being 102 genes upregulated and 121 genes downregulated by estradiol; and 137 genes upregulated and 166 genes downregulated by Wnt3a. Table 1 described the list of the annotated genes present in the protein bank with the Ref Seq and the Gene Name. In contrast, the genes modified only after estradiol treatment or only after Wnt3a treatment that presented no significant differences between A and B were discarded. Initially, for the confirmation of the putative deferential response of both cells (A vs B) we selected two genes presented in both arrays that were modified by Wnt as well as by estradiol (Plasminogen and LEF-1). A final validation was performed using specific antibodies against the selected proteins (Figure 9 and 8C). Thus, plasminogen expression in Δ56LEF-1 cells was 2.5 fold higher, although these initial differences were not considerably augmented by treatment with Wnt or estradiol. Similarly, the endogenous LEF-1 was reduced by the presence of the mutant version of this protein (Figure 8). In addition we selected some genes, previously reported by other laboratories as Wnt targets , such as TCF's, Cyclin D, Myc, MMP's (for updated, please check, The Wnt Homepage, at http://www.stanford.edu/˜rnusse/pathways/targets.html).

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