Limits...
ERK-associated changes in E2F4 phosphorylation, localization and transcriptional activity during mitogenic stimulation in human intestinal epithelial crypt cells.

Paquin MC, Cagnol S, Carrier JC, Leblanc C, Rivard N - BMC Cell Biol. (2013)

Bottom Line: Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition.The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC.This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département d'Anatomie et Biologie Cellulaire, Cancer Research Pavillon, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3201, Jean-Mignault, Sherbrooke, J1E4K8, QC, Canada.

ABSTRACT

Background: The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.

Results: Treatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3β phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in APC and KRAS or BRAF genes, known to deregulate GSK3/β-catenin and MEK/ERK signaling, respectively.

Conclusions: The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

Show MeSH

Related in: MedlinePlus

E2F4 is overexpressed, phosphorylated and nuclear localized in epithelial cells of human colorectal adenomas. A. E2F4 and β-actin protein levels were determined in six paired specimens of advanced adenomas by Western blot (resection margins (M) and primary tumors). B. E2F4 expression levels were normalized to β-actin levels. E2F4 expression levels were also normalized to a mean of E2F4 expression levels in every resection margins, resulting in a fold induction value. E2F4 protein contents in adenoma tissues relative to their corresponding normal samples were analyzed by paired t-test. **, Significant at p < 0.01. C. Representative immunohistochemistry of E2F4 from frozen tissue sections of resection margin and corresponding adenoma. Bars: 100 μm. Higher magnifications are shown on the left panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750237&req=5

Figure 6: E2F4 is overexpressed, phosphorylated and nuclear localized in epithelial cells of human colorectal adenomas. A. E2F4 and β-actin protein levels were determined in six paired specimens of advanced adenomas by Western blot (resection margins (M) and primary tumors). B. E2F4 expression levels were normalized to β-actin levels. E2F4 expression levels were also normalized to a mean of E2F4 expression levels in every resection margins, resulting in a fold induction value. E2F4 protein contents in adenoma tissues relative to their corresponding normal samples were analyzed by paired t-test. **, Significant at p < 0.01. C. Representative immunohistochemistry of E2F4 from frozen tissue sections of resection margin and corresponding adenoma. Bars: 100 μm. Higher magnifications are shown on the left panels.

Mentions: Both MEK/ERK and GSK3 signaling pathways are thought to be affected in early stages of colorectal cancer formation due to frequent mutations in KRAS/BRAF and APC genes respectively [23]. We therefore verified the protein status of E2F4 in human colorectal adenomas. As shown in Figure 6A and B, adenomas displayed significantly higher expression levels of E2F4 in comparison to their corresponding benign epithelium (margin). More importantly, all normal specimens analyzed (resection margins, M) presented hypophosphorylated forms of E2F4 (arrowhead) whereas all adenoma samples (A) exhibited hyperphosphorylated forms of E2F4 (arrows) (Figure 6B). Furthermore, immunohistochemical analysis demonstrated that E2F4 protein was especially overexpressed in the nucleus of all epithelial cells in colorectal adenomas (Figure 6C, lower panels, arrows) while only localized in the nucleus of certain cells along the colonic crypt, presumably in proliferative cells (Figure 6C, right panel, arrows) as previously demonstrated [9]. Of note, all of the adenomas analyzed exhibited APC inactivating mutations (exon 15) in combination with KRAS (G12D, G13D, Q61H) or BRAF (V600E) activating mutations. Hence, these results emphasize the close correlation between the phosphorylation of E2F4 and its nuclear localization.


ERK-associated changes in E2F4 phosphorylation, localization and transcriptional activity during mitogenic stimulation in human intestinal epithelial crypt cells.

Paquin MC, Cagnol S, Carrier JC, Leblanc C, Rivard N - BMC Cell Biol. (2013)

E2F4 is overexpressed, phosphorylated and nuclear localized in epithelial cells of human colorectal adenomas. A. E2F4 and β-actin protein levels were determined in six paired specimens of advanced adenomas by Western blot (resection margins (M) and primary tumors). B. E2F4 expression levels were normalized to β-actin levels. E2F4 expression levels were also normalized to a mean of E2F4 expression levels in every resection margins, resulting in a fold induction value. E2F4 protein contents in adenoma tissues relative to their corresponding normal samples were analyzed by paired t-test. **, Significant at p < 0.01. C. Representative immunohistochemistry of E2F4 from frozen tissue sections of resection margin and corresponding adenoma. Bars: 100 μm. Higher magnifications are shown on the left panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750237&req=5

Figure 6: E2F4 is overexpressed, phosphorylated and nuclear localized in epithelial cells of human colorectal adenomas. A. E2F4 and β-actin protein levels were determined in six paired specimens of advanced adenomas by Western blot (resection margins (M) and primary tumors). B. E2F4 expression levels were normalized to β-actin levels. E2F4 expression levels were also normalized to a mean of E2F4 expression levels in every resection margins, resulting in a fold induction value. E2F4 protein contents in adenoma tissues relative to their corresponding normal samples were analyzed by paired t-test. **, Significant at p < 0.01. C. Representative immunohistochemistry of E2F4 from frozen tissue sections of resection margin and corresponding adenoma. Bars: 100 μm. Higher magnifications are shown on the left panels.
Mentions: Both MEK/ERK and GSK3 signaling pathways are thought to be affected in early stages of colorectal cancer formation due to frequent mutations in KRAS/BRAF and APC genes respectively [23]. We therefore verified the protein status of E2F4 in human colorectal adenomas. As shown in Figure 6A and B, adenomas displayed significantly higher expression levels of E2F4 in comparison to their corresponding benign epithelium (margin). More importantly, all normal specimens analyzed (resection margins, M) presented hypophosphorylated forms of E2F4 (arrowhead) whereas all adenoma samples (A) exhibited hyperphosphorylated forms of E2F4 (arrows) (Figure 6B). Furthermore, immunohistochemical analysis demonstrated that E2F4 protein was especially overexpressed in the nucleus of all epithelial cells in colorectal adenomas (Figure 6C, lower panels, arrows) while only localized in the nucleus of certain cells along the colonic crypt, presumably in proliferative cells (Figure 6C, right panel, arrows) as previously demonstrated [9]. Of note, all of the adenomas analyzed exhibited APC inactivating mutations (exon 15) in combination with KRAS (G12D, G13D, Q61H) or BRAF (V600E) activating mutations. Hence, these results emphasize the close correlation between the phosphorylation of E2F4 and its nuclear localization.

Bottom Line: Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition.The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC.This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département d'Anatomie et Biologie Cellulaire, Cancer Research Pavillon, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3201, Jean-Mignault, Sherbrooke, J1E4K8, QC, Canada.

ABSTRACT

Background: The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.

Results: Treatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3β phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in APC and KRAS or BRAF genes, known to deregulate GSK3/β-catenin and MEK/ERK signaling, respectively.

Conclusions: The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

Show MeSH
Related in: MedlinePlus