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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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Constitutively active forms of PKB prevent the cell death induced by CaM antagonists in NT-maintained cultures. PC12 cells (A and B) or MTNs (C) were transiently cotransfected with pEGFP and pCMV5-HA-PKBT308D/S473D (PKBDD) or pSG5-Gag-PKB or the empty vector. (A and B) PC12 cells were serum starved and treated with NGF (10 ng/ml), W13 (30 μM), or left untreated as indicated. After 15 h, cells were fixed and stained with Hoechst 33258. The percentage of apoptotic cells scored into the EGFP-positive cell population was evaluated and represented as the mean ± SEM of three independent experiments. (C) MTNs were treated with BDNF (10 ng/ml), W13 (30 μM), LY294002 (20 μM), or left untreated as indicated. After 24 h, EGFP-positive cells were counted, and survival was expressed as the percentage remaining of EGFP-positive cells in each treatment. The graph shows the mean ± SEM of three independent experiments. **P value using the Student's t test was <0.01 when comparing the cultures transfected with pCMV5-HA-PKBT308D/S473D or pSG5-Gag-PKB with those transfected with the empty vector in each treatment (A–C).
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fig4: Constitutively active forms of PKB prevent the cell death induced by CaM antagonists in NT-maintained cultures. PC12 cells (A and B) or MTNs (C) were transiently cotransfected with pEGFP and pCMV5-HA-PKBT308D/S473D (PKBDD) or pSG5-Gag-PKB or the empty vector. (A and B) PC12 cells were serum starved and treated with NGF (10 ng/ml), W13 (30 μM), or left untreated as indicated. After 15 h, cells were fixed and stained with Hoechst 33258. The percentage of apoptotic cells scored into the EGFP-positive cell population was evaluated and represented as the mean ± SEM of three independent experiments. (C) MTNs were treated with BDNF (10 ng/ml), W13 (30 μM), LY294002 (20 μM), or left untreated as indicated. After 24 h, EGFP-positive cells were counted, and survival was expressed as the percentage remaining of EGFP-positive cells in each treatment. The graph shows the mean ± SEM of three independent experiments. **P value using the Student's t test was <0.01 when comparing the cultures transfected with pCMV5-HA-PKBT308D/S473D or pSG5-Gag-PKB with those transfected with the empty vector in each treatment (A–C).

Mentions: The results above show a good correlation between inhibition of PKB and inhibition of cell survival induced by CaM antagonists. To test whether the prevention of cell survival triggered by these inhibitors was due to the inhibition of PKB, we transfected PC12 cells and MTNs with one of the two types of constitutive active forms: Gag-PKB, a constitutive plasma membrane-bound protein, and HA-PKBT308D/S473D, a tagged form of the protein that carries a mutational acidic charge in addition to its main regulatory phosphorylation sites (Burgering and Coffer, 1995; Alessi et al., 1996). The transfected cultures were then treated with the corresponding NT plus W13, and at the end of the treatments cell survival was evaluated and compared with control cultures transfected with the empty vector. Results showed that Gag-PKB protected both PC12 cells and MTNs from the cell death induced by trophic factor withdrawal (Fig. 4, A and C , untreated cultures). Similar effects displayed HA-PKBT308D/S473D in serum-starved PC12 cells (Fig. 4 B, untreated cultures). Interestingly, Gag-PKB and HA-PKBT308D/S473D were also able to prevent the cell death triggered by W13 in cultures of PC12 maintained with NGF (Fig. 4, A and B, respectively). Accordingly, the cell death induced by W13 in BDNF-maintained MTNs was strongly reduced in Gag-PKB– (Fig. 4 C) and in HA-PKBT308D/S473D-transfected cultures (unpublished data) when compared with the empty vector–transfected cultures. These results indicate that CaM antagonists exert their effect on NT-induced survival, mainly inhibiting the activation of PKB.


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)

Constitutively active forms of PKB prevent the cell death induced by CaM antagonists in NT-maintained cultures. PC12 cells (A and B) or MTNs (C) were transiently cotransfected with pEGFP and pCMV5-HA-PKBT308D/S473D (PKBDD) or pSG5-Gag-PKB or the empty vector. (A and B) PC12 cells were serum starved and treated with NGF (10 ng/ml), W13 (30 μM), or left untreated as indicated. After 15 h, cells were fixed and stained with Hoechst 33258. The percentage of apoptotic cells scored into the EGFP-positive cell population was evaluated and represented as the mean ± SEM of three independent experiments. (C) MTNs were treated with BDNF (10 ng/ml), W13 (30 μM), LY294002 (20 μM), or left untreated as indicated. After 24 h, EGFP-positive cells were counted, and survival was expressed as the percentage remaining of EGFP-positive cells in each treatment. The graph shows the mean ± SEM of three independent experiments. **P value using the Student's t test was <0.01 when comparing the cultures transfected with pCMV5-HA-PKBT308D/S473D or pSG5-Gag-PKB with those transfected with the empty vector in each treatment (A–C).
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Related In: Results  -  Collection

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fig4: Constitutively active forms of PKB prevent the cell death induced by CaM antagonists in NT-maintained cultures. PC12 cells (A and B) or MTNs (C) were transiently cotransfected with pEGFP and pCMV5-HA-PKBT308D/S473D (PKBDD) or pSG5-Gag-PKB or the empty vector. (A and B) PC12 cells were serum starved and treated with NGF (10 ng/ml), W13 (30 μM), or left untreated as indicated. After 15 h, cells were fixed and stained with Hoechst 33258. The percentage of apoptotic cells scored into the EGFP-positive cell population was evaluated and represented as the mean ± SEM of three independent experiments. (C) MTNs were treated with BDNF (10 ng/ml), W13 (30 μM), LY294002 (20 μM), or left untreated as indicated. After 24 h, EGFP-positive cells were counted, and survival was expressed as the percentage remaining of EGFP-positive cells in each treatment. The graph shows the mean ± SEM of three independent experiments. **P value using the Student's t test was <0.01 when comparing the cultures transfected with pCMV5-HA-PKBT308D/S473D or pSG5-Gag-PKB with those transfected with the empty vector in each treatment (A–C).
Mentions: The results above show a good correlation between inhibition of PKB and inhibition of cell survival induced by CaM antagonists. To test whether the prevention of cell survival triggered by these inhibitors was due to the inhibition of PKB, we transfected PC12 cells and MTNs with one of the two types of constitutive active forms: Gag-PKB, a constitutive plasma membrane-bound protein, and HA-PKBT308D/S473D, a tagged form of the protein that carries a mutational acidic charge in addition to its main regulatory phosphorylation sites (Burgering and Coffer, 1995; Alessi et al., 1996). The transfected cultures were then treated with the corresponding NT plus W13, and at the end of the treatments cell survival was evaluated and compared with control cultures transfected with the empty vector. Results showed that Gag-PKB protected both PC12 cells and MTNs from the cell death induced by trophic factor withdrawal (Fig. 4, A and C , untreated cultures). Similar effects displayed HA-PKBT308D/S473D in serum-starved PC12 cells (Fig. 4 B, untreated cultures). Interestingly, Gag-PKB and HA-PKBT308D/S473D were also able to prevent the cell death triggered by W13 in cultures of PC12 maintained with NGF (Fig. 4, A and B, respectively). Accordingly, the cell death induced by W13 in BDNF-maintained MTNs was strongly reduced in Gag-PKB– (Fig. 4 C) and in HA-PKBT308D/S473D-transfected cultures (unpublished data) when compared with the empty vector–transfected cultures. These results indicate that CaM antagonists exert their effect on NT-induced survival, mainly inhibiting the activation of PKB.

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