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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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MEK/ERK pathway is required for E2F4 nuclear translocation and G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min or 24 h with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE for Western blot analysis of the expression of ERK2, pRb, cyclin D1, p27, phosphorylated ERK1/2 and β-actin. B. HIEC grown on coverslips were serum-deprived during 36 h and then stimulated with 5% FBS with or without 20 μM U0126 (or DMSO). After 24 h, cells were fixed using 3% paraformaldehyde, permeabilized and analyzed by immunofluorescence for E2F4 expression.
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Figure 1: MEK/ERK pathway is required for E2F4 nuclear translocation and G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min or 24 h with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE for Western blot analysis of the expression of ERK2, pRb, cyclin D1, p27, phosphorylated ERK1/2 and β-actin. B. HIEC grown on coverslips were serum-deprived during 36 h and then stimulated with 5% FBS with or without 20 μM U0126 (or DMSO). After 24 h, cells were fixed using 3% paraformaldehyde, permeabilized and analyzed by immunofluorescence for E2F4 expression.

Mentions: We have previously shown that E2F4 is required for proper expression of many cell cycle regulatory proteins controlling G1/S phase transition and for proliferation of normal human intestinal epithelial cells (HIEC) [10]. In contrast to E2F1, which is constitutively localized in the nucleus, E2F4 has a diffuse cytoplasmic localization in quiescent HIEC and a nuclear localization in proliferative cells suggesting that its localization is regulated by signaling pathways activated by mitogens [9]. In light of the above, we analyzed the signaling pathways that could be involved in serum-induced E2F4 nuclear translocation and G1/S phase transition in HIEC. We first verified the involvement of the MEK/ERK pathway given that we had previously demonstrated that sustained activation of ERK1/2 is required for intestinal epithelial cells to enter S-phase [15]. Among physiological events relevant for G1/S phase transition, there is the phosphorylation of the retinoblastoma gene product pRb by cyclin D/Cdk4,6 and cyclin E/Cdk2 complexes, which causes the release and activation of E2F/DP transcription factors [1]. E2F4 localization and hyperphosphorylation of pRb were therefore analyzed following treatment of HIEC with serum in absence or presence of U0126, a potent inhibitor of MEK1/2 (the upstream activators of ERK1/2). As expected, addition of 20 μM U0126 to HIEC potently inhibited serum-induced ERK1/2 phosphorylation (Figure 1A) without affecting phosphorylation of other signaling kinases such as ERK5 and AKT (Additional file 1: Figure S1). Of note, the stimulatory effect of serum on cyclin D1 expression, p27 down-regulation and pRb hyperphosphorylation was also abolished by U0126. Furthermore, U0126 treatment totally prevented nuclear translocation of E2F4 in response to serum (Figure 1B).


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)

MEK/ERK pathway is required for E2F4 nuclear translocation and G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min or 24 h with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE for Western blot analysis of the expression of ERK2, pRb, cyclin D1, p27, phosphorylated ERK1/2 and β-actin. B. HIEC grown on coverslips were serum-deprived during 36 h and then stimulated with 5% FBS with or without 20 μM U0126 (or DMSO). After 24 h, cells were fixed using 3% paraformaldehyde, permeabilized and analyzed by immunofluorescence for E2F4 expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: MEK/ERK pathway is required for E2F4 nuclear translocation and G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min or 24 h with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE for Western blot analysis of the expression of ERK2, pRb, cyclin D1, p27, phosphorylated ERK1/2 and β-actin. B. HIEC grown on coverslips were serum-deprived during 36 h and then stimulated with 5% FBS with or without 20 μM U0126 (or DMSO). After 24 h, cells were fixed using 3% paraformaldehyde, permeabilized and analyzed by immunofluorescence for E2F4 expression.
Mentions: We have previously shown that E2F4 is required for proper expression of many cell cycle regulatory proteins controlling G1/S phase transition and for proliferation of normal human intestinal epithelial cells (HIEC) [10]. In contrast to E2F1, which is constitutively localized in the nucleus, E2F4 has a diffuse cytoplasmic localization in quiescent HIEC and a nuclear localization in proliferative cells suggesting that its localization is regulated by signaling pathways activated by mitogens [9]. In light of the above, we analyzed the signaling pathways that could be involved in serum-induced E2F4 nuclear translocation and G1/S phase transition in HIEC. We first verified the involvement of the MEK/ERK pathway given that we had previously demonstrated that sustained activation of ERK1/2 is required for intestinal epithelial cells to enter S-phase [15]. Among physiological events relevant for G1/S phase transition, there is the phosphorylation of the retinoblastoma gene product pRb by cyclin D/Cdk4,6 and cyclin E/Cdk2 complexes, which causes the release and activation of E2F/DP transcription factors [1]. E2F4 localization and hyperphosphorylation of pRb were therefore analyzed following treatment of HIEC with serum in absence or presence of U0126, a potent inhibitor of MEK1/2 (the upstream activators of ERK1/2). As expected, addition of 20 μM U0126 to HIEC potently inhibited serum-induced ERK1/2 phosphorylation (Figure 1A) without affecting phosphorylation of other signaling kinases such as ERK5 and AKT (Additional file 1: Figure S1). Of note, the stimulatory effect of serum on cyclin D1 expression, p27 down-regulation and pRb hyperphosphorylation was also abolished by U0126. Furthermore, U0126 treatment totally prevented nuclear translocation of E2F4 in response to serum (Figure 1B).

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