Limits...
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.

Show MeSH

Related in: MedlinePlus

FKHR nuclear export: regulation by estradiol and a role in hormone-induced DNA synthesis in MCF-7 cells. Quiescent MCF-7 cells on coverslips were used. (a) Cells were transfected with the indicated plasmids then left unstimulated or stimulated for 24 h with 10 nM estradiol. After in vivo pulse with BrdU, DNA synthesis was analyzed by immunofluorescence and BrdU incorporation was calculated as in Fig. 4. (b) Cells were transfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for the indicated times. Endogenous ERα localization as well as expression of GFP, GFP-FKHR wt, or GFP-FKHR AAA mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively ERα nuclear fluorescence were scored, and data were expressed as a percentage of transfected cells. (c) Cells were cotransfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for 60 min. Localization of GFP-FKHR wt, Myc-HEG0, or Myc-HEGIL mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively FKHR nuclear fluorescence were scored and the data were expressed as a percentage of cotransfected cells. For each experiment in panels a–c, data were derived from at least 500 scored cells. The results of several independent experiments were averaged; n represents the number of experiments. (d) Images from one experiment in b or c are shown. They represent the staining of endogenous ERα (red) in MCF-7 cells expressing GFP-FKHR wt (left, green) or the mutant, GFP-FKHR AAA (middle, green), and treated for 60 min with estradiol. (right) The staining of Myc-tagged NES-ERα mutant (red) in MCF-7 cells coexpressing GFP-FKHR wt (green) and treated for 60 min with estradiol. Merged images are also shown on the bottom. Bar, 5 μm. (e) The cells were cotransfected with ERα siRNA (ERα siRNA) or nontargeting siRNA (nt siRNA) and GFP-FKHR wt. The cells were then left unstimulated or stimulated with 10 nM estradiol for the indicated times. GFP-FKHR wt localization was monitored by confocal microscopy. Cells that fell into the category of exclusively GFP-FKHR wt nuclear fluorescence were scored and data were expressed as a percentage of transfected cells. Data were derived from at least 200 scored cells. The results of two independent experiments were averaged. The blot in panel e confirms the silencing of ERα in MCF-7 cells transfected with ERα siRNA (top). The bottom shows the blot of loading proteins revealed using the anti-tubulin antibody. (a, b, c, and e) Means and SEM are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2483513&req=5

fig5: FKHR nuclear export: regulation by estradiol and a role in hormone-induced DNA synthesis in MCF-7 cells. Quiescent MCF-7 cells on coverslips were used. (a) Cells were transfected with the indicated plasmids then left unstimulated or stimulated for 24 h with 10 nM estradiol. After in vivo pulse with BrdU, DNA synthesis was analyzed by immunofluorescence and BrdU incorporation was calculated as in Fig. 4. (b) Cells were transfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for the indicated times. Endogenous ERα localization as well as expression of GFP, GFP-FKHR wt, or GFP-FKHR AAA mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively ERα nuclear fluorescence were scored, and data were expressed as a percentage of transfected cells. (c) Cells were cotransfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for 60 min. Localization of GFP-FKHR wt, Myc-HEG0, or Myc-HEGIL mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively FKHR nuclear fluorescence were scored and the data were expressed as a percentage of cotransfected cells. For each experiment in panels a–c, data were derived from at least 500 scored cells. The results of several independent experiments were averaged; n represents the number of experiments. (d) Images from one experiment in b or c are shown. They represent the staining of endogenous ERα (red) in MCF-7 cells expressing GFP-FKHR wt (left, green) or the mutant, GFP-FKHR AAA (middle, green), and treated for 60 min with estradiol. (right) The staining of Myc-tagged NES-ERα mutant (red) in MCF-7 cells coexpressing GFP-FKHR wt (green) and treated for 60 min with estradiol. Merged images are also shown on the bottom. Bar, 5 μm. (e) The cells were cotransfected with ERα siRNA (ERα siRNA) or nontargeting siRNA (nt siRNA) and GFP-FKHR wt. The cells were then left unstimulated or stimulated with 10 nM estradiol for the indicated times. GFP-FKHR wt localization was monitored by confocal microscopy. Cells that fell into the category of exclusively GFP-FKHR wt nuclear fluorescence were scored and data were expressed as a percentage of transfected cells. Data were derived from at least 200 scored cells. The results of two independent experiments were averaged. The blot in panel e confirms the silencing of ERα in MCF-7 cells transfected with ERα siRNA (top). The bottom shows the blot of loading proteins revealed using the anti-tubulin antibody. (a, b, c, and e) Means and SEM are shown.

Mentions: Estradiol activation of PI3K is required to drive MCF-7 cells into S phase (Castoria et al., 2001). Activation of the PI3K–AKT pathway by estradiol regulates ERα nuclear export (Fig. 1), and FKHR nuclear export depends on its phosphorylation by AKT (Biggs et al., 1999). In addition, previous studies reported that an estradiol-dependent interaction between ERα and FKHR occurs in vitro (Schuur et al., 2001). Thus, we hypothesized a role for FKHR in both estradiol-regulated ERα nuclear export and cell cycle arrest mediated by the NES-ERα mutant. To analyze the role of FKHR in estradiol-induced DNA synthesis and ERα nuclear export, quiescent MCF-7 cells were transiently transfected with wt FKHR (GFP-FKHR wt) or a mutant containing a triple alanine substitution (GFP-FKHR AAA) that localizes in nuclei and blocks FKHR phosphorylation by AKT, thereby inducing G1 arrest of cells (Nakamura et al., 2000). Fig. 5 a shows that expression of this mutant reduced estradiol-triggered BrdU incorporation in MCF-7 cells, whereas expression of GFP-FKHR wt did not. We then used confocal microscopy to analyze the role of FKHR in the estradiol-regulated subcellular distribution of ERα. Although ectopically expressed GFP-FKHR wt or GFP alone did not modify the 60-min estradiol-induced nuclear export of ERα, the mutant GFP-FKHR AAA sequestered ERα in the nuclear compartment at that time (Fig. 5, b and d). Conversely, overexpression of the tagged NES-ERα mutant, Myc-ERα IL, resulted in retention of GFP-FKHR wt in the nuclear compartment 60 min after estradiol treatment of MCF-7 cells (Fig. 5, c and d). Thus, estradiol simultaneously regulates ERα- and FKHR-associated nuclear export.


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)

FKHR nuclear export: regulation by estradiol and a role in hormone-induced DNA synthesis in MCF-7 cells. Quiescent MCF-7 cells on coverslips were used. (a) Cells were transfected with the indicated plasmids then left unstimulated or stimulated for 24 h with 10 nM estradiol. After in vivo pulse with BrdU, DNA synthesis was analyzed by immunofluorescence and BrdU incorporation was calculated as in Fig. 4. (b) Cells were transfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for the indicated times. Endogenous ERα localization as well as expression of GFP, GFP-FKHR wt, or GFP-FKHR AAA mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively ERα nuclear fluorescence were scored, and data were expressed as a percentage of transfected cells. (c) Cells were cotransfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for 60 min. Localization of GFP-FKHR wt, Myc-HEG0, or Myc-HEGIL mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively FKHR nuclear fluorescence were scored and the data were expressed as a percentage of cotransfected cells. For each experiment in panels a–c, data were derived from at least 500 scored cells. The results of several independent experiments were averaged; n represents the number of experiments. (d) Images from one experiment in b or c are shown. They represent the staining of endogenous ERα (red) in MCF-7 cells expressing GFP-FKHR wt (left, green) or the mutant, GFP-FKHR AAA (middle, green), and treated for 60 min with estradiol. (right) The staining of Myc-tagged NES-ERα mutant (red) in MCF-7 cells coexpressing GFP-FKHR wt (green) and treated for 60 min with estradiol. Merged images are also shown on the bottom. Bar, 5 μm. (e) The cells were cotransfected with ERα siRNA (ERα siRNA) or nontargeting siRNA (nt siRNA) and GFP-FKHR wt. The cells were then left unstimulated or stimulated with 10 nM estradiol for the indicated times. GFP-FKHR wt localization was monitored by confocal microscopy. Cells that fell into the category of exclusively GFP-FKHR wt nuclear fluorescence were scored and data were expressed as a percentage of transfected cells. Data were derived from at least 200 scored cells. The results of two independent experiments were averaged. The blot in panel e confirms the silencing of ERα in MCF-7 cells transfected with ERα siRNA (top). The bottom shows the blot of loading proteins revealed using the anti-tubulin antibody. (a, b, c, and e) Means and SEM are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483513&req=5

fig5: FKHR nuclear export: regulation by estradiol and a role in hormone-induced DNA synthesis in MCF-7 cells. Quiescent MCF-7 cells on coverslips were used. (a) Cells were transfected with the indicated plasmids then left unstimulated or stimulated for 24 h with 10 nM estradiol. After in vivo pulse with BrdU, DNA synthesis was analyzed by immunofluorescence and BrdU incorporation was calculated as in Fig. 4. (b) Cells were transfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for the indicated times. Endogenous ERα localization as well as expression of GFP, GFP-FKHR wt, or GFP-FKHR AAA mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively ERα nuclear fluorescence were scored, and data were expressed as a percentage of transfected cells. (c) Cells were cotransfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for 60 min. Localization of GFP-FKHR wt, Myc-HEG0, or Myc-HEGIL mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively FKHR nuclear fluorescence were scored and the data were expressed as a percentage of cotransfected cells. For each experiment in panels a–c, data were derived from at least 500 scored cells. The results of several independent experiments were averaged; n represents the number of experiments. (d) Images from one experiment in b or c are shown. They represent the staining of endogenous ERα (red) in MCF-7 cells expressing GFP-FKHR wt (left, green) or the mutant, GFP-FKHR AAA (middle, green), and treated for 60 min with estradiol. (right) The staining of Myc-tagged NES-ERα mutant (red) in MCF-7 cells coexpressing GFP-FKHR wt (green) and treated for 60 min with estradiol. Merged images are also shown on the bottom. Bar, 5 μm. (e) The cells were cotransfected with ERα siRNA (ERα siRNA) or nontargeting siRNA (nt siRNA) and GFP-FKHR wt. The cells were then left unstimulated or stimulated with 10 nM estradiol for the indicated times. GFP-FKHR wt localization was monitored by confocal microscopy. Cells that fell into the category of exclusively GFP-FKHR wt nuclear fluorescence were scored and data were expressed as a percentage of transfected cells. Data were derived from at least 200 scored cells. The results of two independent experiments were averaged. The blot in panel e confirms the silencing of ERα in MCF-7 cells transfected with ERα siRNA (top). The bottom shows the blot of loading proteins revealed using the anti-tubulin antibody. (a, b, c, and e) Means and SEM are shown.
Mentions: Estradiol activation of PI3K is required to drive MCF-7 cells into S phase (Castoria et al., 2001). Activation of the PI3K–AKT pathway by estradiol regulates ERα nuclear export (Fig. 1), and FKHR nuclear export depends on its phosphorylation by AKT (Biggs et al., 1999). In addition, previous studies reported that an estradiol-dependent interaction between ERα and FKHR occurs in vitro (Schuur et al., 2001). Thus, we hypothesized a role for FKHR in both estradiol-regulated ERα nuclear export and cell cycle arrest mediated by the NES-ERα mutant. To analyze the role of FKHR in estradiol-induced DNA synthesis and ERα nuclear export, quiescent MCF-7 cells were transiently transfected with wt FKHR (GFP-FKHR wt) or a mutant containing a triple alanine substitution (GFP-FKHR AAA) that localizes in nuclei and blocks FKHR phosphorylation by AKT, thereby inducing G1 arrest of cells (Nakamura et al., 2000). Fig. 5 a shows that expression of this mutant reduced estradiol-triggered BrdU incorporation in MCF-7 cells, whereas expression of GFP-FKHR wt did not. We then used confocal microscopy to analyze the role of FKHR in the estradiol-regulated subcellular distribution of ERα. Although ectopically expressed GFP-FKHR wt or GFP alone did not modify the 60-min estradiol-induced nuclear export of ERα, the mutant GFP-FKHR AAA sequestered ERα in the nuclear compartment at that time (Fig. 5, b and d). Conversely, overexpression of the tagged NES-ERα mutant, Myc-ERα IL, resulted in retention of GFP-FKHR wt in the nuclear compartment 60 min after estradiol treatment of MCF-7 cells (Fig. 5, c and d). Thus, estradiol simultaneously regulates ERα- and FKHR-associated nuclear export.

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