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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.

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EGF neither induces E2F4 phosphorylation and nuclear translocation nor G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min, 4 h or 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against phosphorylated ERK1/2, pRb, cyclin D1, cyclin A, p27 and β-actin. B. Cells were also fixed after 24 h stimulation with 3% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and Ki67 were counted in 10 fields. Total cell number was determined using DAPI staining. Ratio of nuclear E2F4 expressing cells and Ki67 positive cells before/after serum, EGF or LPA stimulation are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of an experiment representative of 3. * Significant at p < 0.0001 compared to control cells (no serum) (Student’s t test). C and D. HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min. Cell lysates were separated by 7.5% SDS-PAGE and proteins were analyzed by Western blotting with specific antibodies against E2F4 and β-actin.
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Figure 4: EGF neither induces E2F4 phosphorylation and nuclear translocation nor G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min, 4 h or 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against phosphorylated ERK1/2, pRb, cyclin D1, cyclin A, p27 and β-actin. B. Cells were also fixed after 24 h stimulation with 3% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and Ki67 were counted in 10 fields. Total cell number was determined using DAPI staining. Ratio of nuclear E2F4 expressing cells and Ki67 positive cells before/after serum, EGF or LPA stimulation are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of an experiment representative of 3. * Significant at p < 0.0001 compared to control cells (no serum) (Student’s t test). C and D. HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min. Cell lysates were separated by 7.5% SDS-PAGE and proteins were analyzed by Western blotting with specific antibodies against E2F4 and β-actin.

Mentions: To better understand the mechanisms controlling subcellular E2F4 localization during G1/S phase transition in HIEC, we analyzed the impact of growth factors such as EGF and lysophosphatidic acid (LPA) on these events. EGF is a classical growth factor that can activate many intracellular signaling cascades including the ERK1/2 MAP Kinase cascade [16,17]. In addition, we and others have previously demonstrated that EGF induces proli-feration of rat immortalized intestinal epithelial cells in a MEK-dependent manner [17,18]. In the present series involving human intestinal epithelial cells, although EGF treatment resulted in a rapid and sustained activation of ERK1/2 (at least > 4 h) and in a modest induction of cyclin D1, it was not sufficient to mimic serum-induced cyclin A expression, p27 down-regulation and pRb hyperphosphorylation (Figure 4A). Importantly, in contrast to serum, EGF stimulation did not trigger E2F4 translocation into the nucleus and no Ki67 staining was observed (Figure 4B). Of note, Western blot analysis with an anti-E2F4 antibody revealed three major bands in EGF-stimulated HIEC similar to controls, whereas E2F4 displayed a typical phosphorylated low electrophoretic mobility in serum-stimulated cells (Figure 4C). Neither pRb hyperphosphorylation nor Ki67 staining was observed in the presence of lower and higher EGF concentrations ranging from 2 ng/ml to 1 ug/ml (data not shown), thus eliminating the likelihood of overactivation or underactivation of EGF receptors. Furthermore, HIEC proliferation was not enhanced even after several days of incubation with EGF (data not shown). Hence, these results indicate that EGF by itself is not able to trigger S phase entry and proliferation of normal human non immortalized intestinal epithelial crypt cells.


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)

EGF neither induces E2F4 phosphorylation and nuclear translocation nor G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min, 4 h or 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against phosphorylated ERK1/2, pRb, cyclin D1, cyclin A, p27 and β-actin. B. Cells were also fixed after 24 h stimulation with 3% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and Ki67 were counted in 10 fields. Total cell number was determined using DAPI staining. Ratio of nuclear E2F4 expressing cells and Ki67 positive cells before/after serum, EGF or LPA stimulation are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of an experiment representative of 3. * Significant at p < 0.0001 compared to control cells (no serum) (Student’s t test). C and D. HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min. Cell lysates were separated by 7.5% SDS-PAGE and proteins were analyzed by Western blotting with specific antibodies against E2F4 and β-actin.
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Figure 4: EGF neither induces E2F4 phosphorylation and nuclear translocation nor G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min, 4 h or 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against phosphorylated ERK1/2, pRb, cyclin D1, cyclin A, p27 and β-actin. B. Cells were also fixed after 24 h stimulation with 3% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and Ki67 were counted in 10 fields. Total cell number was determined using DAPI staining. Ratio of nuclear E2F4 expressing cells and Ki67 positive cells before/after serum, EGF or LPA stimulation are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of an experiment representative of 3. * Significant at p < 0.0001 compared to control cells (no serum) (Student’s t test). C and D. HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min. Cell lysates were separated by 7.5% SDS-PAGE and proteins were analyzed by Western blotting with specific antibodies against E2F4 and β-actin.
Mentions: To better understand the mechanisms controlling subcellular E2F4 localization during G1/S phase transition in HIEC, we analyzed the impact of growth factors such as EGF and lysophosphatidic acid (LPA) on these events. EGF is a classical growth factor that can activate many intracellular signaling cascades including the ERK1/2 MAP Kinase cascade [16,17]. In addition, we and others have previously demonstrated that EGF induces proli-feration of rat immortalized intestinal epithelial cells in a MEK-dependent manner [17,18]. In the present series involving human intestinal epithelial cells, although EGF treatment resulted in a rapid and sustained activation of ERK1/2 (at least > 4 h) and in a modest induction of cyclin D1, it was not sufficient to mimic serum-induced cyclin A expression, p27 down-regulation and pRb hyperphosphorylation (Figure 4A). Importantly, in contrast to serum, EGF stimulation did not trigger E2F4 translocation into the nucleus and no Ki67 staining was observed (Figure 4B). Of note, Western blot analysis with an anti-E2F4 antibody revealed three major bands in EGF-stimulated HIEC similar to controls, whereas E2F4 displayed a typical phosphorylated low electrophoretic mobility in serum-stimulated cells (Figure 4C). Neither pRb hyperphosphorylation nor Ki67 staining was observed in the presence of lower and higher EGF concentrations ranging from 2 ng/ml to 1 ug/ml (data not shown), thus eliminating the likelihood of overactivation or underactivation of EGF receptors. Furthermore, HIEC proliferation was not enhanced even after several days of incubation with EGF (data not shown). Hence, these results indicate that EGF by itself is not able to trigger S phase entry and proliferation of normal human non immortalized intestinal epithelial crypt cells.

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