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First published online June 20, 2006
doi: 10.1242/10.1242/jcs.03012

Journal of Cell Science 119, 2739-2748 (2006)
Published by The Company of Biologists 2006
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Modulation of the PI 3-kinase–Akt signalling pathway by IGF-I and PTEN regulates the differentiation of neural stem/precursor cells

Gaizka Otaegi1, María J. Yusta-Boyo1, Eva Vergaño-Vera1,2, Héctor R. Méndez-Gómez2, Ana C. Carrera3, José L. Abad4, Manuel González4, Enrique J. de la Rosa1, Carlos Vicario-Abejón1,2,*,{ddagger} and Flora de Pablo1,*,{ddagger}

1 Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, Madrid 28040, Spain
2 Instituto Cajal, CSIC, Avenue Dr. Arce 37, Madrid 28002, Spain
3 Centro Nacional de Biotecnología, CSIC, Universidad Autónoma, Cantoblanco, Madrid 28049, Spain
4 Genetrix S.L., Marconi 1, Tres Cantos, Madrid 28760, Spain


Figure 1
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  		Fig. 1. IGF-I stimulates Akt-phosphorylation in cultured OB neural stem cells. OBSCs derived from E14.5 embryos were treated with 100 ng/ml IGF-I or with BSA for 30 minutes in conditions of proliferation (presence of EGF and FGF-2) and differentiation (in the absence of mitogens). After lysing the cultured cells, proteins were analysed by immunoblotting with specific antibodies against P-AktSer473, P-AktThr308, Akt, P-Erk1/2 and Erk1/2. The very low basal levels of P-Akt were markedly increased by the presence of IGF-I and maintained for up to 24 hours (not shown). Similar results were obtained in samples from six experiments.

 

Figure 2
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  		Fig. 2. Absence of endogenous IGF-I reduces P-Akt levels in the OB in vivo. (A) OBs from E16.5-E18.5 Igf-I+/+ and Igf-I–/– mouse embryos from the same litters were homogenised and the proteins were analysed by immunoblotting using specific antibodies against P-AktSer473 and Akt. (B) The optical density of the specific bands in immunoblotted membranes was measured by densitometry using Quantity One software. The levels of P-Akt were normalised to the levels of Akt and expressed in arbitrary units. (*P<0.05; Igf-I+/+, n=8; Igf-I–/–, n=9.)

 

Figure 3
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  		Fig. 3. Infection of OBSCs with retroviral vectors expressing PTEN and GFP. (A-F) OBSCs derived from E14.5 embryos were infected with a retroviral construct expressing GFP (B,D,F) or they were mocked infected (A,C,E). The GFP-infected cells (B) proliferated with the same pattern as mocked infected ones (A). On average, 90% of the OBSCs were infected with the GFP-expressing vector as determined by flow cytometry (E,F). No detectable GFP was observed in the mock-infected cells (C,E). (G) OBSC cultures were infected in parallel with a vector expressing PTEN-GFP or with a vector expressing GFP. Immunoblotting with an antibody against the HA-tag confirmed that PTEN was overexpressed in infected cells. The average relative levels of PTEN were 2.2-fold greater in the PTEN-GFP infected cells than in control GFP cells (n=3). (H) OBSC cultures were infected in parallel with a vector expressing PTEN-C/S-GFP or with a vector expressing GFP. Immunoblotting with an antibody against PTEN confirmed that mutant PTEN was overexpressed in infected cells. PTEN-C/S-GFP levels were 3.1-fold those of endogenous PTEN (n=2). (I-Q). GFP infected cells were induced to differentiate by mitogen withdrawal, they were then fixed 48 hours later and the co-expression of TuJ1 and GFP (I-K) or GFAP and GFP (L-N) or O4 and GFP (O-Q) was evaluated in the cultures by double immunostaining with specific antibodies. Bar, 30 µm.

 

Figure 4
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  		Fig. 4. PTEN overexpression reduces the differentiation of OBSCs into neurons and astrocytes. OBSC cultures were infected in parallel with a vector expressing PTEN-GFP or with a vector expressing GFP alone. Three days later, the cells were passaged and cultured for 2-3 days in conditions of proliferation (with EGF and FGF-2) or differentiation (in the absence of mitogens), in the presence or absence of insulin/IGF-I (10 µg/ml of insulin had similar potency as 100 ng/ml IGF-I to stimulate OBSC differentiation). (A) Proliferative cultures were incubated with 5 µM BrdU for 22 hours before fixation. (A-D) The proportions of TuJ1-positive, GFAP-positive and O4-positive cells co-labelled for GFP were calculated. The data obtained in PTEN-GFP infected cultures was expressed as the percentage of the data obtained in GFP-infected cultures, which were considered to be 100% (dotted line). The results are expressed as the average ± s.e.m. of data of 4-8 cultures from 2-5 experiments. Statistical analysis comparing values obtained with insulin (+INS) and without insulin (–INS) was performed using Student's t-test. PTEN overexpression significantly reduced the proportions of TuJ1-positive and GFAP-positive cells in the absence of insulin (*P<0.05) whereas the proportion of O4-positive cells showed a tendency to increase. (E) The proportions of TUNEL-positive cells were not affected by PTEN overexpression under any of the growth conditions tested.

 

Figure 5
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  		Fig. 5. PTEN overexpression reduces basal levels of Akt phosphorylation at residues Ser473 and Thr308. (A) OBSCs derived from E14.5 CD1 embryos were infected with the PTEN-GFP or GFP construct and they were then stimulated with 100 ng/ml IGF-I or BSA (basal level) for 30 minutes under conditions of differentiation. Whereas IGF-I greatly stimulated P-Akt levels in both PTEN-GFP- and GFP-infected cells, the overexpression of PTEN reduced P-Akt basal levels. Similar results were obtained in samples from three experiments. (B) OBSCs derived from E14.5. Igf-I–/– embryos were infected with the PTEN-GFP or GFP construct and were then stimulated with 10-100 ng/ml IGF-I or BSA (basal level) for 30 minutes under conditions of differentiation. The overexpression of PTEN reduced IGF-I-stimulated P-Akt levels only when 10 ng/ml of the growth factor was used. In B, to observe the signals corresponding to P-Akt basal levels, the films were developed for a long time, saturating for signals corresponding to IGF-I-stimulated P-Akt. For this reason, they are presented separately. Similar results were obtained in samples from two experiments.

 

Figure 6
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  		Fig. 6. Overexpression of a catalytically inactive mutant form of PTEN promotes neuron and astrocyte differentiation. OBSC cultures from Igf-I–/– embryos were infected in parallel with a vector expressing PTEN-C/S-GFP or with a vector expressing GFP alone. Three days later, the cells were passaged and cultured for 2-3 days in conditions of differentiation (in the absence of mitogens), in the presence or absence of 100 ng/ml IGF-I. (A,B) Of the total number of GFP-positive cells, the proportion that co-expressed TuJ1 or GFAP was calculated. The data obtained in PTEN-C/S-GFP-infected cultures was expressed as the percentage of the data obtained in GFP-infected cultures that were considered to be 100% (dotted line). The results are expressed as the average ± s.e.m. of data of 4-8 cultures from two experiments. PTEN-C/S-GFP overexpression increased the proportions of TuJ1-positive and GFAP-positive cells by 52% and 42%, respectively, in the absence of IGF-I. (*P<0.05; Student's t-test.)

 

Figure 7
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  		Fig. 7. Effects of PI 3-kinase inhibition on OBSC proliferation, cell death and OBSC number. Cultures of OBSCs derived from E14.5 embryos were plated for 48 hours in conditions of proliferation, in the presence or absence of 100 ng/ml IGF-I, and they were treated with 30 µM LY294002 for the last 24 hours. (A) P-AktSer473 phosphorylation was strongly inhibited by LY294002 while the reduction of P-AktThr308 was moderate, without any change in the levels of P-Erk1/2. One representative immunoblot out of three experiments is shown. (B,E,F) Proliferating cultures were incubated with 5 µM BrdU for 22 hours before fixation. The addition of LY294002 significantly decreased the percentages of BrdU-positive cells in the presence of BSA or IGF-I. (C) LY294002 caused a significant reduction in the total number of cells growing in IGF-I and augmented the percentage of TUNEL-positive cells in the presence of BSA or IGF-I (D,G,H). Results are the average ± s.e.m. from 4-6 cultures. Statistical analysis was performed using Student's t test. (*P<0.05 **P<0.01.) Bar, 30 µm.

 

Figure 8
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  		Fig. 8. Effects of PI 3-kinase inhibition by LY294002 on OBSC neuronal and astrocyte differentiation. OBSCs derived from E14.5 embryos were cultured for 48 hours in differentiation conditions in the presence or absence of 100 ng/ml IGF-I, and they were treated with 30 µM LY294002 for the last 24 hours. (A) P-AktSer473 phosphorylation was strongly inhibited by LY294002, whereas the reduction of P-AktThr308 was moderate. LY294002 produced a small effect on the levels of P-Erk1 (but not on P-Erk2) only in conditions of differentiation. One representative immunoblot out of three experiments is shown. (B) The addition of LY294002 significantly decreased the percentages of TuJ-1-positive cells in the presence of IGF-I and it significantly decreased the percentages of GFAP-positive cells in the presence of BSA and IGF-I (C). (D) Exposure to LY294002 produced a slight but significant increase in the number of TUNEL-positive cells, only in the presence of IGF-I. The results are the average ± s.e.m. from 4-6 cultures. Representative fields of neuronal (TuJ-1; E,F) and astrocyte (GFAP; G,H) morphologies are shown. The number of TUNEL-positive cells was only slightly higher in cultures treated with LY294002 (I,J). Statistical analysis was performed using Student's t test. (*P<0.05, **P<0.01.) Bar, 25 µm.

 

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© The Company of Biologists Ltd 2006