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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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GSK3 inhibition is required for phosphorylation and nuclear translocation of E2F4 as well as G1/S phase entry of HIEC. A. Subconfluent HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min and 24 h. Equal amounts of whole cell lysates were analyzed by Western blotting with specific antibodies against phosphorylated Akt, phosphorylated GSK3β and β-actin. B. Subconfluent HIEC were serum-deprived during for 36 h, stimulated with 5% FBS or 100 ng/ml EGF in presence or absence of 20 μM SB216763 (or DMSO) for 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against pRb, cyclin D1, p27 and β-actin. C. HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF for 30 min with or without prior 10-min 20 μM SB216763 (or DMSO) treatment. Cell lysates were analyzed by Western blotting with specific antibodies against E2F4, ERK2, phosphorylated ERK1/2, phosphorylated glycogen synthase (GS) and β-actin. D. Cells were also fixed after 24 h stimulation and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and positive for Ki67 staining 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 or EGF +/− SB216763 (SB) stimulations are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of 2 separate experiments. * Significant at p < 0.05. ** Significant at p < 0.01. *** Significant at p < 0.005 compared to control cells (no serum) (Student’s t test).
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Figure 5: GSK3 inhibition is required for phosphorylation and nuclear translocation of E2F4 as well as G1/S phase entry of HIEC. A. Subconfluent HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min and 24 h. Equal amounts of whole cell lysates were analyzed by Western blotting with specific antibodies against phosphorylated Akt, phosphorylated GSK3β and β-actin. B. Subconfluent HIEC were serum-deprived during for 36 h, stimulated with 5% FBS or 100 ng/ml EGF in presence or absence of 20 μM SB216763 (or DMSO) for 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against pRb, cyclin D1, p27 and β-actin. C. HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF for 30 min with or without prior 10-min 20 μM SB216763 (or DMSO) treatment. Cell lysates were analyzed by Western blotting with specific antibodies against E2F4, ERK2, phosphorylated ERK1/2, phosphorylated glycogen synthase (GS) and β-actin. D. Cells were also fixed after 24 h stimulation and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and positive for Ki67 staining 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 or EGF +/− SB216763 (SB) stimulations are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of 2 separate experiments. * Significant at p < 0.05. ** Significant at p < 0.01. *** Significant at p < 0.005 compared to control cells (no serum) (Student’s t test).

Mentions: In order to elucidate why EGF, in contrast to LPA and serum, failed to induce G1/S phase transition in HIEC, we analyzed the phosphorylation levels of GSK3β. GSK3β is a constitutively active serine/threonine kinase featured in two signaling pathways that are of particular importance for intestinal epithelial cell proliferation and colorectal cancer: the Wnt/β-catenin pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway [16,20,21]. Indeed, Akt phosphorylates GSK3β on serine 9, leading to inhibition of its constitutive kinase activity. GSK3β is also a component of Wnt signaling, which is thought to block GSK3-mediated β-catenin phosphorylation, leading to the accumulation and nuclear translocation of β-catenin [22]. As shown in Figure 5A, serum markedly induced phosphorylation levels of Akt and GSK3β after both 30 min and 24 h stimulation. By contrast, stimulation of HIEC with EGF only transiently increased phosphorylation of Akt and GSK3β. Interestingly, LPA induced a more sustained Akt and GSK3β phosphorylation (Figure 5A).


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)

GSK3 inhibition is required for phosphorylation and nuclear translocation of E2F4 as well as G1/S phase entry of HIEC. A. Subconfluent HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min and 24 h. Equal amounts of whole cell lysates were analyzed by Western blotting with specific antibodies against phosphorylated Akt, phosphorylated GSK3β and β-actin. B. Subconfluent HIEC were serum-deprived during for 36 h, stimulated with 5% FBS or 100 ng/ml EGF in presence or absence of 20 μM SB216763 (or DMSO) for 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against pRb, cyclin D1, p27 and β-actin. C. HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF for 30 min with or without prior 10-min 20 μM SB216763 (or DMSO) treatment. Cell lysates were analyzed by Western blotting with specific antibodies against E2F4, ERK2, phosphorylated ERK1/2, phosphorylated glycogen synthase (GS) and β-actin. D. Cells were also fixed after 24 h stimulation and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and positive for Ki67 staining 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 or EGF +/− SB216763 (SB) stimulations are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of 2 separate experiments. * Significant at p < 0.05. ** Significant at p < 0.01. *** Significant at p < 0.005 compared to control cells (no serum) (Student’s t test).
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Figure 5: GSK3 inhibition is required for phosphorylation and nuclear translocation of E2F4 as well as G1/S phase entry of HIEC. A. Subconfluent HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min and 24 h. Equal amounts of whole cell lysates were analyzed by Western blotting with specific antibodies against phosphorylated Akt, phosphorylated GSK3β and β-actin. B. Subconfluent HIEC were serum-deprived during for 36 h, stimulated with 5% FBS or 100 ng/ml EGF in presence or absence of 20 μM SB216763 (or DMSO) for 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against pRb, cyclin D1, p27 and β-actin. C. HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF for 30 min with or without prior 10-min 20 μM SB216763 (or DMSO) treatment. Cell lysates were analyzed by Western blotting with specific antibodies against E2F4, ERK2, phosphorylated ERK1/2, phosphorylated glycogen synthase (GS) and β-actin. D. Cells were also fixed after 24 h stimulation and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and positive for Ki67 staining 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 or EGF +/− SB216763 (SB) stimulations are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of 2 separate experiments. * Significant at p < 0.05. ** Significant at p < 0.01. *** Significant at p < 0.005 compared to control cells (no serum) (Student’s t test).
Mentions: In order to elucidate why EGF, in contrast to LPA and serum, failed to induce G1/S phase transition in HIEC, we analyzed the phosphorylation levels of GSK3β. GSK3β is a constitutively active serine/threonine kinase featured in two signaling pathways that are of particular importance for intestinal epithelial cell proliferation and colorectal cancer: the Wnt/β-catenin pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway [16,20,21]. Indeed, Akt phosphorylates GSK3β on serine 9, leading to inhibition of its constitutive kinase activity. GSK3β is also a component of Wnt signaling, which is thought to block GSK3-mediated β-catenin phosphorylation, leading to the accumulation and nuclear translocation of β-catenin [22]. As shown in Figure 5A, serum markedly induced phosphorylation levels of Akt and GSK3β after both 30 min and 24 h stimulation. By contrast, stimulation of HIEC with EGF only transiently increased phosphorylation of Akt and GSK3β. Interestingly, LPA induced a more sustained Akt and GSK3β phosphorylation (Figure 5A).

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