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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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NPC expression is upregulated in breast and ovarian carcinomas.(A) Immunohistochemical analysis of NPC expression was undertaken on a tumor tissue microarray (TMA) containing normal breast epithelial tissue and tumor specimens, including DCIS (ductal carcinoma in situ), IDC (infiltrating ductal carcinoma), and ILC (infiltrating lobular carcinoma). A mouse pan-anti-NPC monoclonal antibody was used for immunostaining. (B) Immunohistochemical staining of NPC on ovarian TMA containing three ovarian serous carcinomas (OvCa) compared to a normal ovarian surface epithelium (left panel). Images were taken at 400× magnification.
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pone-0009295-g005: NPC expression is upregulated in breast and ovarian carcinomas.(A) Immunohistochemical analysis of NPC expression was undertaken on a tumor tissue microarray (TMA) containing normal breast epithelial tissue and tumor specimens, including DCIS (ductal carcinoma in situ), IDC (infiltrating ductal carcinoma), and ILC (infiltrating lobular carcinoma). A mouse pan-anti-NPC monoclonal antibody was used for immunostaining. (B) Immunohistochemical staining of NPC on ovarian TMA containing three ovarian serous carcinomas (OvCa) compared to a normal ovarian surface epithelium (left panel). Images were taken at 400× magnification.

Mentions: Since we observed that the level of NPC is elevated in transformed cells in culture, we next examined whether NPC levels were increased in tumor tissues. Staining of ovarian and breast tumor specimens on several tissue microarrays (TMA) indicated expression of NPC was elevated in most transformed tissues (Figure 5) (see Tables S1, S2, and S3 for information and pathology of ovarian and breast cancers on the TMAs). The majority of breast tumor tissues stained positive for NPC (Figure 5A). In the informative tissue cores, the intensity of NPC staining was typically pervasive (70% of the epithelial component was positive) and strong (intensity was 2–3) in the DCIS (ductal carcinoma in situ) specimens. Normal breast epithelial tissue had weak (0–1) staining in an average 50% (range 40–70%) of the epithelial cells. The more malignant type carcinomas, IDC (infiltrating ductal carcinoma) and ILC (infiltrating lobular carcinoma), had more variable NPC staining (Figure 5A), though in general, the intensity was low to medium (Tables S1, S3). Thus, over-expression of NPC may correlate more closely with DCIS than the other tumor types; however, the sample size is small and would need to be increased substantially to reach a conclusion.


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)

NPC expression is upregulated in breast and ovarian carcinomas.(A) Immunohistochemical analysis of NPC expression was undertaken on a tumor tissue microarray (TMA) containing normal breast epithelial tissue and tumor specimens, including DCIS (ductal carcinoma in situ), IDC (infiltrating ductal carcinoma), and ILC (infiltrating lobular carcinoma). A mouse pan-anti-NPC monoclonal antibody was used for immunostaining. (B) Immunohistochemical staining of NPC on ovarian TMA containing three ovarian serous carcinomas (OvCa) compared to a normal ovarian surface epithelium (left panel). Images were taken at 400× magnification.
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Related In: Results  -  Collection

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

pone-0009295-g005: NPC expression is upregulated in breast and ovarian carcinomas.(A) Immunohistochemical analysis of NPC expression was undertaken on a tumor tissue microarray (TMA) containing normal breast epithelial tissue and tumor specimens, including DCIS (ductal carcinoma in situ), IDC (infiltrating ductal carcinoma), and ILC (infiltrating lobular carcinoma). A mouse pan-anti-NPC monoclonal antibody was used for immunostaining. (B) Immunohistochemical staining of NPC on ovarian TMA containing three ovarian serous carcinomas (OvCa) compared to a normal ovarian surface epithelium (left panel). Images were taken at 400× magnification.
Mentions: Since we observed that the level of NPC is elevated in transformed cells in culture, we next examined whether NPC levels were increased in tumor tissues. Staining of ovarian and breast tumor specimens on several tissue microarrays (TMA) indicated expression of NPC was elevated in most transformed tissues (Figure 5) (see Tables S1, S2, and S3 for information and pathology of ovarian and breast cancers on the TMAs). The majority of breast tumor tissues stained positive for NPC (Figure 5A). In the informative tissue cores, the intensity of NPC staining was typically pervasive (70% of the epithelial component was positive) and strong (intensity was 2–3) in the DCIS (ductal carcinoma in situ) specimens. Normal breast epithelial tissue had weak (0–1) staining in an average 50% (range 40–70%) of the epithelial cells. The more malignant type carcinomas, IDC (infiltrating ductal carcinoma) and ILC (infiltrating lobular carcinoma), had more variable NPC staining (Figure 5A), though in general, the intensity was low to medium (Tables S1, S3). Thus, over-expression of NPC may correlate more closely with DCIS than the other tumor types; however, the sample size is small and would need to be increased substantially to reach a conclusion.

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