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Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells.

Lombardi M, Castoria G, Migliaccio A, Barone MV, Di Stasio R, Ciociola A, Bottero D, Yamaguchi H, Appella E, Auricchio F - J. Cell Biol. (2008)

Bottom Line: NES-ERalpha mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERalpha-dependent transcription is normal.ERalpha is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins.A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERalpha and is trapped in the nucleus, blocking S phase entry.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Patologia Generale, Il Università di Napoli, 80138 Naples, Italy.

ABSTRACT
In breast cancer cells, cytoplasmic localization of the estradiol receptor alpha (ERalpha) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERalpha and show that its export is dependent on both estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERalpha NES disrupts ERalpha-CRM1 interaction and prevents nuclear export of ERalpha- and estradiol-induced DNA synthesis. NES-ERalpha mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERalpha-dependent transcription is normal. ERalpha is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERalpha knockdown or ERalpha NES mutations prevent ERalpha and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERalpha and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERalpha, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry.

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Related in: MedlinePlus

The ERα 444–456 sequence restores export activity of the NES-deficient REV1.4-GFP. Growing MCF-7 cells were used. (a and b) Cells were transfected with the indicated constructs. After transfection, the cells were left untreated (no drug) or treated with actinomycin D (ActD) at 5 μg/ml, alone or together with 5 ng/ml LMB. The subcellular distribution of GFP proteins was determined by fluorescence microscopy, and cells that fell into the category of exclusively nuclear fluorescence were scored. Data are expressed as a percentage of transfected cells, with mean values taken from at least three experiments. For each experiment, at least 600 cells were scored. (b) Images of one experiment in panel a. (c) The wt ERα 444–456 sequence (NES ERα wt) as well as its mutated version (NES ERα mutant). The putative NES-ERα sequence is indicated by the underlined amino acids, which were substituted with alanine residues in the mutant sequence. The NES-ERα wt as well as the NES-ERα mutant subcloned into the Rev mutant were transfected (d and e) in growing MCF-7 cells. After transfection, the cells were left untreated (−) or treated with 5 μg/ml Act D (+). The percentage of cells with nuclear GFP protein was determined by fluorescence microscopy and graphically shown in panel d. Data were derived from at least 600 scored cells. The results of several independent experiments were averaged. (a and d) Means and SEM are shown. n represents the number of experiments. (e) Images of one experiment in panel d. Bars, 5 μm
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fig2: The ERα 444–456 sequence restores export activity of the NES-deficient REV1.4-GFP. Growing MCF-7 cells were used. (a and b) Cells were transfected with the indicated constructs. After transfection, the cells were left untreated (no drug) or treated with actinomycin D (ActD) at 5 μg/ml, alone or together with 5 ng/ml LMB. The subcellular distribution of GFP proteins was determined by fluorescence microscopy, and cells that fell into the category of exclusively nuclear fluorescence were scored. Data are expressed as a percentage of transfected cells, with mean values taken from at least three experiments. For each experiment, at least 600 cells were scored. (b) Images of one experiment in panel a. (c) The wt ERα 444–456 sequence (NES ERα wt) as well as its mutated version (NES ERα mutant). The putative NES-ERα sequence is indicated by the underlined amino acids, which were substituted with alanine residues in the mutant sequence. The NES-ERα wt as well as the NES-ERα mutant subcloned into the Rev mutant were transfected (d and e) in growing MCF-7 cells. After transfection, the cells were left untreated (−) or treated with 5 μg/ml Act D (+). The percentage of cells with nuclear GFP protein was determined by fluorescence microscopy and graphically shown in panel d. Data were derived from at least 600 scored cells. The results of several independent experiments were averaged. (a and d) Means and SEM are shown. n represents the number of experiments. (e) Images of one experiment in panel d. Bars, 5 μm

Mentions: It has been found that NLSs of the PgR are responsible for its nuclear export (Guiochon-Mantel et al., 1994). Therefore, we studied whether NLSs are involved in ERα export using in vivo export assay (Henderson and Eleftheriou, 2000). In this assay, nuclear export sequences are identified by their ability to restore export activity of the NES-deficient REV1.4-GFP (Rev mutant) to levels similar to those observed with the wild-type (wt) pREV-GFP or the REV1.4-GFP NES (Rev positive control), in which the NES is the canonical export sequence of the REV protein. The NLS sequences of ERα were subcloned into the Rev mutant and expressed in MCF-7 cells. After transfection, cells were incubated in the absence or presence of actinomycin D because it causes cytoplasmic accumulation of the putative NES-containing REV protein by preventing nuclear import of REV (Henderson, 2000). Irrespective of experimental conditions, the Rev mutant NLS localizes in nuclei of MCF-7 cells (Fig. 2, a and b). In the same experiment, the Rev-positive control completely shifted to the cytoplasm in the presence of actinomycin D, whereas the Rev mutant NLS sequences from the ERα showed nuclear, sometimes nucleolar, staining. These data indicate that the NLS sequences of ERα are inactive in this export assay.


Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells.

Lombardi M, Castoria G, Migliaccio A, Barone MV, Di Stasio R, Ciociola A, Bottero D, Yamaguchi H, Appella E, Auricchio F - J. Cell Biol. (2008)

The ERα 444–456 sequence restores export activity of the NES-deficient REV1.4-GFP. Growing MCF-7 cells were used. (a and b) Cells were transfected with the indicated constructs. After transfection, the cells were left untreated (no drug) or treated with actinomycin D (ActD) at 5 μg/ml, alone or together with 5 ng/ml LMB. The subcellular distribution of GFP proteins was determined by fluorescence microscopy, and cells that fell into the category of exclusively nuclear fluorescence were scored. Data are expressed as a percentage of transfected cells, with mean values taken from at least three experiments. For each experiment, at least 600 cells were scored. (b) Images of one experiment in panel a. (c) The wt ERα 444–456 sequence (NES ERα wt) as well as its mutated version (NES ERα mutant). The putative NES-ERα sequence is indicated by the underlined amino acids, which were substituted with alanine residues in the mutant sequence. The NES-ERα wt as well as the NES-ERα mutant subcloned into the Rev mutant were transfected (d and e) in growing MCF-7 cells. After transfection, the cells were left untreated (−) or treated with 5 μg/ml Act D (+). The percentage of cells with nuclear GFP protein was determined by fluorescence microscopy and graphically shown in panel d. Data were derived from at least 600 scored cells. The results of several independent experiments were averaged. (a and d) Means and SEM are shown. n represents the number of experiments. (e) Images of one experiment in panel d. Bars, 5 μm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483513&req=5

fig2: The ERα 444–456 sequence restores export activity of the NES-deficient REV1.4-GFP. Growing MCF-7 cells were used. (a and b) Cells were transfected with the indicated constructs. After transfection, the cells were left untreated (no drug) or treated with actinomycin D (ActD) at 5 μg/ml, alone or together with 5 ng/ml LMB. The subcellular distribution of GFP proteins was determined by fluorescence microscopy, and cells that fell into the category of exclusively nuclear fluorescence were scored. Data are expressed as a percentage of transfected cells, with mean values taken from at least three experiments. For each experiment, at least 600 cells were scored. (b) Images of one experiment in panel a. (c) The wt ERα 444–456 sequence (NES ERα wt) as well as its mutated version (NES ERα mutant). The putative NES-ERα sequence is indicated by the underlined amino acids, which were substituted with alanine residues in the mutant sequence. The NES-ERα wt as well as the NES-ERα mutant subcloned into the Rev mutant were transfected (d and e) in growing MCF-7 cells. After transfection, the cells were left untreated (−) or treated with 5 μg/ml Act D (+). The percentage of cells with nuclear GFP protein was determined by fluorescence microscopy and graphically shown in panel d. Data were derived from at least 600 scored cells. The results of several independent experiments were averaged. (a and d) Means and SEM are shown. n represents the number of experiments. (e) Images of one experiment in panel d. Bars, 5 μm
Mentions: It has been found that NLSs of the PgR are responsible for its nuclear export (Guiochon-Mantel et al., 1994). Therefore, we studied whether NLSs are involved in ERα export using in vivo export assay (Henderson and Eleftheriou, 2000). In this assay, nuclear export sequences are identified by their ability to restore export activity of the NES-deficient REV1.4-GFP (Rev mutant) to levels similar to those observed with the wild-type (wt) pREV-GFP or the REV1.4-GFP NES (Rev positive control), in which the NES is the canonical export sequence of the REV protein. The NLS sequences of ERα were subcloned into the Rev mutant and expressed in MCF-7 cells. After transfection, cells were incubated in the absence or presence of actinomycin D because it causes cytoplasmic accumulation of the putative NES-containing REV protein by preventing nuclear import of REV (Henderson, 2000). Irrespective of experimental conditions, the Rev mutant NLS localizes in nuclei of MCF-7 cells (Fig. 2, a and b). In the same experiment, the Rev-positive control completely shifted to the cytoplasm in the presence of actinomycin D, whereas the Rev mutant NLS sequences from the ERα showed nuclear, sometimes nucleolar, staining. These data indicate that the NLS sequences of ERα are inactive in this export assay.

Bottom Line: NES-ERalpha mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERalpha-dependent transcription is normal.ERalpha is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins.A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERalpha and is trapped in the nucleus, blocking S phase entry.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Patologia Generale, Il Università di Napoli, 80138 Naples, Italy.

ABSTRACT
In breast cancer cells, cytoplasmic localization of the estradiol receptor alpha (ERalpha) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERalpha and show that its export is dependent on both estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERalpha NES disrupts ERalpha-CRM1 interaction and prevents nuclear export of ERalpha- and estradiol-induced DNA synthesis. NES-ERalpha mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERalpha-dependent transcription is normal. ERalpha is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERalpha knockdown or ERalpha NES mutations prevent ERalpha and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERalpha and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERalpha, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry.

Show MeSH
Related in: MedlinePlus