Limits...
Nuclear entry of activated MAPK is restricted in primary ovarian and mammary epithelial cells.

Smith ER, Cai KQ, Smedberg JL, Ribeiro MM, Rula ME, Slater C, Godwin AK, Xu XX - PLoS ONE (2010)

Bottom Line: Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription.Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells.ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida, United States of America. esmith@med.miami.edu

ABSTRACT

Background: The MAPK/ERK1/2 serine kinases are primary mediators of the Ras mitogenic signaling pathway. Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription.

Principal findings: Here, however, we observe that in primary cultures of breast and ovarian epithelial cells, phosphorylation and activation of ERK1/2 are disassociated from nuclear translocalization and transcription of downstream targets, such as c-Fos, suggesting that nuclear translocation is limited in primary cells. Accordingly, in import assays in vitro, primary cells showed a lower import activity for ERK1/2 than cancer cells, in which activated MAPK readily translocated into the nucleus and activated c-Fos expression. Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells. Additionally, reduction in expression of nucleoporin 153 by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells.

Conclusion: ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors.

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Related in: MedlinePlus

Immortalization and transformation increase transcription levels of nuclear import proteins.(A,B) The expression of a panel of nuclear import factors was examined by Nanostring nCounter methodology for ovarian (A) and breast epithelial (B) cells. (A, Top Panel) Relative mRNA levels of importin 7 (Imp7) and importin B1 (ImpB1) in normal primary HOSE, HIO, and ovarian cancer cell lines. The numbers on the x-axis correspond to the cells listed to the right of the figure. (A, Middle Panel) Expression profiles for Nup153 and Nup214 for the set of cells listed, as in the top panel. (A, Bottom panel) The expression of a panel of nuclear import factors was examined by Nanostring nCounter methodology. The data represent the mean +/− s.d. for HOSE (n = 7), HIO (n = 1), and carcinoma (n = 7) cell cultures. Differences considered statistically significant (p<0.01), calculated using Student's t-test, are indicated by an “*”. Transcripts were examined for importin 7 (Imp7), importin-alpha6 (KPNA6), importin-beta1 (ImpB1), nucleoporin 153 (Nup153), nucleoporin 214 (Nup214), nucleoporin 62 (Nup62), nucleoporin 88 (Nup88), nuclear transport factor 2 (NUTF2), Ran binding protein 5 (RNBP5), and exportin 1 (XPO1). (B) Nanostring nCounter analyses were performed for 3 HBE, the immortalized MCF10 cells, and 4 breast carcinoma cell lines (MCF7, MDA-MB-231, MDA-MB-468, T47D), as in Figure 3A. (C) Immunoblot analyses were performed for Importin 7, Importin B, and NPC in primary HOSE and HBE cells, human immortalized ovarian epithelial (HIO) cells, MCF7 breast carcinoma cells, and a panel of ovarian carcinoma cells (ES2, UPN251, A1847, A2780, and Ovcar5). NPC proteins were detected with a pan-anti-NPC mouse monoclonal antibody (from Sigma). (D) Primary HOSE and HBE cells and SKOV3 and MCF7 carcinoma cells were fixed and stained for nuclear pores using a pan-anti-NPC mouse monoclonal antibody and an Alexa555-conjugated goat anti-mouse secondary antibody. All images were treated identically using Adobe Photoshop.
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pone-0009295-g003: Immortalization and transformation increase transcription levels of nuclear import proteins.(A,B) The expression of a panel of nuclear import factors was examined by Nanostring nCounter methodology for ovarian (A) and breast epithelial (B) cells. (A, Top Panel) Relative mRNA levels of importin 7 (Imp7) and importin B1 (ImpB1) in normal primary HOSE, HIO, and ovarian cancer cell lines. The numbers on the x-axis correspond to the cells listed to the right of the figure. (A, Middle Panel) Expression profiles for Nup153 and Nup214 for the set of cells listed, as in the top panel. (A, Bottom panel) The expression of a panel of nuclear import factors was examined by Nanostring nCounter methodology. The data represent the mean +/− s.d. for HOSE (n = 7), HIO (n = 1), and carcinoma (n = 7) cell cultures. Differences considered statistically significant (p<0.01), calculated using Student's t-test, are indicated by an “*”. Transcripts were examined for importin 7 (Imp7), importin-alpha6 (KPNA6), importin-beta1 (ImpB1), nucleoporin 153 (Nup153), nucleoporin 214 (Nup214), nucleoporin 62 (Nup62), nucleoporin 88 (Nup88), nuclear transport factor 2 (NUTF2), Ran binding protein 5 (RNBP5), and exportin 1 (XPO1). (B) Nanostring nCounter analyses were performed for 3 HBE, the immortalized MCF10 cells, and 4 breast carcinoma cell lines (MCF7, MDA-MB-231, MDA-MB-468, T47D), as in Figure 3A. (C) Immunoblot analyses were performed for Importin 7, Importin B, and NPC in primary HOSE and HBE cells, human immortalized ovarian epithelial (HIO) cells, MCF7 breast carcinoma cells, and a panel of ovarian carcinoma cells (ES2, UPN251, A1847, A2780, and Ovcar5). NPC proteins were detected with a pan-anti-NPC mouse monoclonal antibody (from Sigma). (D) Primary HOSE and HBE cells and SKOV3 and MCF7 carcinoma cells were fixed and stained for nuclear pores using a pan-anti-NPC mouse monoclonal antibody and an Alexa555-conjugated goat anti-mouse secondary antibody. All images were treated identically using Adobe Photoshop.

Mentions: Nano-String gene expression profiling [28] confirmed that nucleoporins and import transport factors were elevated in ovarian and breast cancer cells at the transcript level (Figure 3). Seven different HOSE cell preparations (for Nano-String profiling) showed similar mRNA levels, whereas the transformed cells consistently had elevated message levels of NPC proteins (Figure 3A, middle and lower panels). ERK has been shown to interact with nucleoporin-214 (Nup214) and nucleoporin-153 (Nup153) through FG-repeat sequences [20], [29]. Moreover, the ovarian cancer cells typically had higher transcript levels of importin 7 (Imp7) and importin B1 (ImpB1, also known as KPNB1), members of the karyopherin/importin-beta family of nuclear transport factors that have been shown in Drosophila to modulate MAPK/ERK nuclear localization [8], [9], [30], [31]. The transcript levels of importin-alpha (KPNA6) and RanBP5 cytosolic transport factors changed minimally. The distinction was apparent even between human immortalized ovarian (HIO) epithelial cells and the primary HOSE cells (Figure 3A, lower panel). Originally generated by transfecting HOSE cells with the SV40 large T antigen, the HIO cells are non-tumorigenic but can be cultured up to 30 passages [32], [33]. We also examined mRNA levels in three HBE, the immortalized MCF10 cell line, and four breast carcinoma cell lines, and observed that gene expression for nucleoporins was significantly elevated (p<0.01) in all breast cancer cells relative to the primary cells (Figure 3B), which followed a trend similar to ovarian cancer and ovarian primary cells. Importin levels were reduced significantly in two of the three primary HBE lines (Figure 3B, top panel), though the average mRNA expression level of the three HBE lines did not appear significantly different from that found in the breast carcinoma cells. Northern blots for importin B1 and Nup153 confirmed that ovarian carcinoma and HIO cells contained increased message levels for these genes compared to normal HOSE cells (not shown). Moreover, immunoblotting confirmed that nucleoporins and import transport factors were elevated at the protein level in the carcinoma cells (Figure 3C). The increased expression of nucleoporins Nup153 and Nup214 and importin-beta factors strongly predicts elevated MAPK nuclear import in tumor tissues.


Nuclear entry of activated MAPK is restricted in primary ovarian and mammary epithelial cells.

Smith ER, Cai KQ, Smedberg JL, Ribeiro MM, Rula ME, Slater C, Godwin AK, Xu XX - PLoS ONE (2010)

Immortalization and transformation increase transcription levels of nuclear import proteins.(A,B) The expression of a panel of nuclear import factors was examined by Nanostring nCounter methodology for ovarian (A) and breast epithelial (B) cells. (A, Top Panel) Relative mRNA levels of importin 7 (Imp7) and importin B1 (ImpB1) in normal primary HOSE, HIO, and ovarian cancer cell lines. The numbers on the x-axis correspond to the cells listed to the right of the figure. (A, Middle Panel) Expression profiles for Nup153 and Nup214 for the set of cells listed, as in the top panel. (A, Bottom panel) The expression of a panel of nuclear import factors was examined by Nanostring nCounter methodology. The data represent the mean +/− s.d. for HOSE (n = 7), HIO (n = 1), and carcinoma (n = 7) cell cultures. Differences considered statistically significant (p<0.01), calculated using Student's t-test, are indicated by an “*”. Transcripts were examined for importin 7 (Imp7), importin-alpha6 (KPNA6), importin-beta1 (ImpB1), nucleoporin 153 (Nup153), nucleoporin 214 (Nup214), nucleoporin 62 (Nup62), nucleoporin 88 (Nup88), nuclear transport factor 2 (NUTF2), Ran binding protein 5 (RNBP5), and exportin 1 (XPO1). (B) Nanostring nCounter analyses were performed for 3 HBE, the immortalized MCF10 cells, and 4 breast carcinoma cell lines (MCF7, MDA-MB-231, MDA-MB-468, T47D), as in Figure 3A. (C) Immunoblot analyses were performed for Importin 7, Importin B, and NPC in primary HOSE and HBE cells, human immortalized ovarian epithelial (HIO) cells, MCF7 breast carcinoma cells, and a panel of ovarian carcinoma cells (ES2, UPN251, A1847, A2780, and Ovcar5). NPC proteins were detected with a pan-anti-NPC mouse monoclonal antibody (from Sigma). (D) Primary HOSE and HBE cells and SKOV3 and MCF7 carcinoma cells were fixed and stained for nuclear pores using a pan-anti-NPC mouse monoclonal antibody and an Alexa555-conjugated goat anti-mouse secondary antibody. All images were treated identically using Adobe Photoshop.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2823791&req=5

pone-0009295-g003: Immortalization and transformation increase transcription levels of nuclear import proteins.(A,B) The expression of a panel of nuclear import factors was examined by Nanostring nCounter methodology for ovarian (A) and breast epithelial (B) cells. (A, Top Panel) Relative mRNA levels of importin 7 (Imp7) and importin B1 (ImpB1) in normal primary HOSE, HIO, and ovarian cancer cell lines. The numbers on the x-axis correspond to the cells listed to the right of the figure. (A, Middle Panel) Expression profiles for Nup153 and Nup214 for the set of cells listed, as in the top panel. (A, Bottom panel) The expression of a panel of nuclear import factors was examined by Nanostring nCounter methodology. The data represent the mean +/− s.d. for HOSE (n = 7), HIO (n = 1), and carcinoma (n = 7) cell cultures. Differences considered statistically significant (p<0.01), calculated using Student's t-test, are indicated by an “*”. Transcripts were examined for importin 7 (Imp7), importin-alpha6 (KPNA6), importin-beta1 (ImpB1), nucleoporin 153 (Nup153), nucleoporin 214 (Nup214), nucleoporin 62 (Nup62), nucleoporin 88 (Nup88), nuclear transport factor 2 (NUTF2), Ran binding protein 5 (RNBP5), and exportin 1 (XPO1). (B) Nanostring nCounter analyses were performed for 3 HBE, the immortalized MCF10 cells, and 4 breast carcinoma cell lines (MCF7, MDA-MB-231, MDA-MB-468, T47D), as in Figure 3A. (C) Immunoblot analyses were performed for Importin 7, Importin B, and NPC in primary HOSE and HBE cells, human immortalized ovarian epithelial (HIO) cells, MCF7 breast carcinoma cells, and a panel of ovarian carcinoma cells (ES2, UPN251, A1847, A2780, and Ovcar5). NPC proteins were detected with a pan-anti-NPC mouse monoclonal antibody (from Sigma). (D) Primary HOSE and HBE cells and SKOV3 and MCF7 carcinoma cells were fixed and stained for nuclear pores using a pan-anti-NPC mouse monoclonal antibody and an Alexa555-conjugated goat anti-mouse secondary antibody. All images were treated identically using Adobe Photoshop.
Mentions: Nano-String gene expression profiling [28] confirmed that nucleoporins and import transport factors were elevated in ovarian and breast cancer cells at the transcript level (Figure 3). Seven different HOSE cell preparations (for Nano-String profiling) showed similar mRNA levels, whereas the transformed cells consistently had elevated message levels of NPC proteins (Figure 3A, middle and lower panels). ERK has been shown to interact with nucleoporin-214 (Nup214) and nucleoporin-153 (Nup153) through FG-repeat sequences [20], [29]. Moreover, the ovarian cancer cells typically had higher transcript levels of importin 7 (Imp7) and importin B1 (ImpB1, also known as KPNB1), members of the karyopherin/importin-beta family of nuclear transport factors that have been shown in Drosophila to modulate MAPK/ERK nuclear localization [8], [9], [30], [31]. The transcript levels of importin-alpha (KPNA6) and RanBP5 cytosolic transport factors changed minimally. The distinction was apparent even between human immortalized ovarian (HIO) epithelial cells and the primary HOSE cells (Figure 3A, lower panel). Originally generated by transfecting HOSE cells with the SV40 large T antigen, the HIO cells are non-tumorigenic but can be cultured up to 30 passages [32], [33]. We also examined mRNA levels in three HBE, the immortalized MCF10 cell line, and four breast carcinoma cell lines, and observed that gene expression for nucleoporins was significantly elevated (p<0.01) in all breast cancer cells relative to the primary cells (Figure 3B), which followed a trend similar to ovarian cancer and ovarian primary cells. Importin levels were reduced significantly in two of the three primary HBE lines (Figure 3B, top panel), though the average mRNA expression level of the three HBE lines did not appear significantly different from that found in the breast carcinoma cells. Northern blots for importin B1 and Nup153 confirmed that ovarian carcinoma and HIO cells contained increased message levels for these genes compared to normal HOSE cells (not shown). Moreover, immunoblotting confirmed that nucleoporins and import transport factors were elevated at the protein level in the carcinoma cells (Figure 3C). The increased expression of nucleoporins Nup153 and Nup214 and importin-beta factors strongly predicts elevated MAPK nuclear import in tumor tissues.

Bottom Line: Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription.Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells.ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida, United States of America. esmith@med.miami.edu

ABSTRACT

Background: The MAPK/ERK1/2 serine kinases are primary mediators of the Ras mitogenic signaling pathway. Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription.

Principal findings: Here, however, we observe that in primary cultures of breast and ovarian epithelial cells, phosphorylation and activation of ERK1/2 are disassociated from nuclear translocalization and transcription of downstream targets, such as c-Fos, suggesting that nuclear translocation is limited in primary cells. Accordingly, in import assays in vitro, primary cells showed a lower import activity for ERK1/2 than cancer cells, in which activated MAPK readily translocated into the nucleus and activated c-Fos expression. Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells. Additionally, reduction in expression of nucleoporin 153 by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells.

Conclusion: ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors.

Show MeSH
Related in: MedlinePlus