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Neuronal survival induced by neurotrophins requires calmodulin.

Egea J, Espinet C, Soler RM, Dolcet X, Yuste VJ, Encinas M, Iglesias M, Rocamora N, Comella JX - J. Cell Biol. (2001)

Bottom Line: We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells.This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB.We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

View Article: PubMed Central - PubMed

Affiliation: Grup de Neurobiologia Molecular, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Catalonia, Spain.

ABSTRACT
It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

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NGF requires CaM to promote cell survival in PC12 cells. PC12 cells were serum starved and treated with NGF (10 ng/ml), LY294002 (20 μM), W13 (30 μM), or left untreated as indicated. After 15 h, cells were fixed, stained with Hoechst 33258, or subjected to a TUNEL assay. (A) Percentage of cells displaying typical nuclear apoptotic morphology. The values represent the mean ± SEM of three independent experiments. **P value using the Student's t test was <0.01 relative to the treatment with NGF alone. (B) Representative photomicrographs showing the morphology of the nuclei of the cells in the different treatments. Arrowheads indicate the apoptotic nuclei. (C) Representative phase–contrast micrographs and TUNEL reaction of the same field of the cultures treated above. (D) PC12 cells were treated with W13 (30 μM) and then stimulated for the indicated times with NGF (10 ng/ml). Phosphorylation of the residues Thr308 (top panel) and Ser473 (middle panel) of PKB was analyzed by Western blot using specific phospho-antibodies. Protein loading was checked, reprobing the filters with a specific antibody against α-tubulin (bottom panel). Bars: (B) 10 μm; (C) 20 μm.
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fig2: NGF requires CaM to promote cell survival in PC12 cells. PC12 cells were serum starved and treated with NGF (10 ng/ml), LY294002 (20 μM), W13 (30 μM), or left untreated as indicated. After 15 h, cells were fixed, stained with Hoechst 33258, or subjected to a TUNEL assay. (A) Percentage of cells displaying typical nuclear apoptotic morphology. The values represent the mean ± SEM of three independent experiments. **P value using the Student's t test was <0.01 relative to the treatment with NGF alone. (B) Representative photomicrographs showing the morphology of the nuclei of the cells in the different treatments. Arrowheads indicate the apoptotic nuclei. (C) Representative phase–contrast micrographs and TUNEL reaction of the same field of the cultures treated above. (D) PC12 cells were treated with W13 (30 μM) and then stimulated for the indicated times with NGF (10 ng/ml). Phosphorylation of the residues Thr308 (top panel) and Ser473 (middle panel) of PKB was analyzed by Western blot using specific phospho-antibodies. Protein loading was checked, reprobing the filters with a specific antibody against α-tubulin (bottom panel). Bars: (B) 10 μm; (C) 20 μm.

Mentions: PKB activity is mainly induced by phosphorylation of the residues Thr308 and Ser473 (Alessi et al., 1996). We used specific phospho-antibodies against each of these two residues to check the phosphorylation of PKB upon NGF stimulation in the presence of Ca2+ chelators or CaM antagonists. According to the experiments of kinase activity shown above, BAPTA (Fig. 1 C) and W13 but not W12 (Fig. 1 D) blocked the phosphorylation of both residues. Moreover, the inhibition exerted by W13 was sustained over the time of treatment (Fig. 2 D). Other CaM inhibitors, such as W7 (100 μM) or the W13 structurally unrelated trifluoperazine dimaleate (TFP; 50 μM), displayed similar effects as those observed with W13 (Fig. 1 E). In these experiments, we also included the CaM inhibitor W5 as a control of the unspecific effects of W7 (W7IC50 = 28 μM versus W5IC50 = 240 μM; Hidaka and Tanaka, 1983). As shown in Fig. 1 E, W5 did not significantly affect PKB phosphorylation, confirming the specificity of W7 effects. Finally, as expected from the kinase activity assay shown in Fig. 1 A, EGTA did not modify the phosphorylation of the PKB induced by NGF (unpublished data).


Neuronal survival induced by neurotrophins requires calmodulin.

Egea J, Espinet C, Soler RM, Dolcet X, Yuste VJ, Encinas M, Iglesias M, Rocamora N, Comella JX - J. Cell Biol. (2001)

NGF requires CaM to promote cell survival in PC12 cells. PC12 cells were serum starved and treated with NGF (10 ng/ml), LY294002 (20 μM), W13 (30 μM), or left untreated as indicated. After 15 h, cells were fixed, stained with Hoechst 33258, or subjected to a TUNEL assay. (A) Percentage of cells displaying typical nuclear apoptotic morphology. The values represent the mean ± SEM of three independent experiments. **P value using the Student's t test was <0.01 relative to the treatment with NGF alone. (B) Representative photomicrographs showing the morphology of the nuclei of the cells in the different treatments. Arrowheads indicate the apoptotic nuclei. (C) Representative phase–contrast micrographs and TUNEL reaction of the same field of the cultures treated above. (D) PC12 cells were treated with W13 (30 μM) and then stimulated for the indicated times with NGF (10 ng/ml). Phosphorylation of the residues Thr308 (top panel) and Ser473 (middle panel) of PKB was analyzed by Western blot using specific phospho-antibodies. Protein loading was checked, reprobing the filters with a specific antibody against α-tubulin (bottom panel). Bars: (B) 10 μm; (C) 20 μm.
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Related In: Results  -  Collection

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fig2: NGF requires CaM to promote cell survival in PC12 cells. PC12 cells were serum starved and treated with NGF (10 ng/ml), LY294002 (20 μM), W13 (30 μM), or left untreated as indicated. After 15 h, cells were fixed, stained with Hoechst 33258, or subjected to a TUNEL assay. (A) Percentage of cells displaying typical nuclear apoptotic morphology. The values represent the mean ± SEM of three independent experiments. **P value using the Student's t test was <0.01 relative to the treatment with NGF alone. (B) Representative photomicrographs showing the morphology of the nuclei of the cells in the different treatments. Arrowheads indicate the apoptotic nuclei. (C) Representative phase–contrast micrographs and TUNEL reaction of the same field of the cultures treated above. (D) PC12 cells were treated with W13 (30 μM) and then stimulated for the indicated times with NGF (10 ng/ml). Phosphorylation of the residues Thr308 (top panel) and Ser473 (middle panel) of PKB was analyzed by Western blot using specific phospho-antibodies. Protein loading was checked, reprobing the filters with a specific antibody against α-tubulin (bottom panel). Bars: (B) 10 μm; (C) 20 μm.
Mentions: PKB activity is mainly induced by phosphorylation of the residues Thr308 and Ser473 (Alessi et al., 1996). We used specific phospho-antibodies against each of these two residues to check the phosphorylation of PKB upon NGF stimulation in the presence of Ca2+ chelators or CaM antagonists. According to the experiments of kinase activity shown above, BAPTA (Fig. 1 C) and W13 but not W12 (Fig. 1 D) blocked the phosphorylation of both residues. Moreover, the inhibition exerted by W13 was sustained over the time of treatment (Fig. 2 D). Other CaM inhibitors, such as W7 (100 μM) or the W13 structurally unrelated trifluoperazine dimaleate (TFP; 50 μM), displayed similar effects as those observed with W13 (Fig. 1 E). In these experiments, we also included the CaM inhibitor W5 as a control of the unspecific effects of W7 (W7IC50 = 28 μM versus W5IC50 = 240 μM; Hidaka and Tanaka, 1983). As shown in Fig. 1 E, W5 did not significantly affect PKB phosphorylation, confirming the specificity of W7 effects. Finally, as expected from the kinase activity assay shown in Fig. 1 A, EGTA did not modify the phosphorylation of the PKB induced by NGF (unpublished data).

Bottom Line: We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells.This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB.We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

View Article: PubMed Central - PubMed

Affiliation: Grup de Neurobiologia Molecular, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Catalonia, Spain.

ABSTRACT
It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

Show MeSH
Related in: MedlinePlus