The basic helix-loop-helix transcription factor Nex-1/Math-2 promotes neuronal survival of PC12 cells by modulating the dynamic expression of anti-apoptotic and cell cycle regulators

Abstract

The basic helix-loop-helix transcription factor Nex1/Math-2 belongs to the NeuroD subfamily, which plays a critical role during neuronal differentiation and maintenance of the differentiated state. Previously, we demonstrated that Nex1 is a key regulatory component of the nerve growth factor (NGF) pathway. Further supporting this hypothesis, this study shows that Nex1 has survival-inducing properties similar to NGF, as Nex1-overexpressing PC12 cells survive in the absence of trophic factors. We dissected the molecular mechanism by which Nex1 confers neuroprotection upon serum removal and found that constitutive expression of Nex1 maintained the expression of specific G1 phase cyclin-dependent kinase inhibitors and concomitantly induced a dynamic expression profile of key anti-apoptotic regulators. This study provides the first evidence of the underlying mechanism by which a member of the NeuroD-subfamily promotes an active anti-apoptotic program essential to the survival of neurons. Our results suggest that the survival program may be viewed as an integral component of the intrinsic programming of the differentiated state.

Fig. 1

Overexpression of Nex1 promotes survival of PC12-Nex1 cells upon serum deprivation. (a) Prolonged survival of PC12-Nex1 cells (clones A, B and C) compared with control PC12-bsd and PC12-Nex1-mut1 cells after serum deprivation. Trypan blue exclusion test results are expressed as percentage survival. (b) Overexpression of Nex1 results in increased cell viability, as measured by LDH assay. Values represent the mean ± SEM (n = 9) of three independent experiments.(c) Overexpression of Nex1 prevents DNA fragmentation upon serum deprivation in PC12-Nex1 cells compared with control PC12-bsd cells. Apoptotic DNA was isolated from PC12-bsd and PC12-Nex1 cells at different time points during serum deprivation treatment, according to the manufacturer's recommendations. Equal amounts of DNA were run on a 1.5% agarose gel and stained with ethidium bromide. DNA markers were electrophoresed as a base pair reference. (d) PC12-Nex1 cells (clones A, B, and C) remain metabolically active after a prolonged 15-day serum deprivation treatment. The metabolic activity of PC12-Nex1 cells was measured by a colorimetric WST-1 assay. Data represent mean ± SD of three independent experiments performed in triplicate.

Fig. 2

Expression profile of neuronal markers and cell cycle regulators in serum-deprived PC12-Nex1 cells. (a) Phase-contrast micrographs of serum-deprived PC12-Nex1 cells reveal a maintained neuronal phenotype accompanied by a flattened and wide cell body and significant neurite outgrowth. Serum-grown PC12-Nex1 cells, τ = 0 days; serum-deprived PC12-Nex1 cells, τ = 21 days. (b) Nex1 expression and that of specific neuronal markers remain constant during serum deprivation of PC12-Nex1 cells. PC12-Nex1 cells extracts isolated at successive times after serum deprivation as indicated above the panel were subjected to immunoblot analyses using appropriate antibodies described in Table 1 and corresponding secondary antibodies. The antigen—antibody complexes were detected by chemiluminescence. The membrane was stripped with Restore™ western blot stripping buffer and re-probed with the anti-βIII tubulin, anti-α tubulin, and anti-GAP-43 antibodies separately. (c) Constitutive expression of Nex1 results in sustained expression of specific CDK inhibitors upon serum deprivation. Immunoblot analyses using PC12-Nex1 cell extracts were performed as described above with antibodies described in Table 1. The membrane was stripped with Restore™ stripping buffer and re-probed with the anti-p27^cip1, anti-p16^INK4a and CDC47 antibodies separately. Data shown are representative of at least three independent experiments.

Fig. 3

Expression profile of the anti-apoptotic Bcl2 members upon withdrawal of trophic factors. (a) Constitutive expression of Nex1 induces expression of Bcl-X_L and Bcl-XΔ™ proteins in serum-deprived PC12-Nex1 cells. PC12-Nex1 and PC12-bsd cells were grown in the absence of serum for periods of time indicated above each lane. Whole-cell extracts were prepared and equal amounts of total proteins (40 μg) were electrophoresed and subjected to immunoblot analysis with a polyclonal antibody raised against the 63-amino acid domain specific to the splice variant Bcl-X_L to avoid cross-reaction with Bcl-X_S. Equal loading was verified by staining the membrane with Ponceau S after transfer and by probing with the β-III tubulin antibody. Molecular weight markers are indicated on the left side of each figure. The asterisk indicates a non-specific anti-Bcl-X_L immunoreactive polypeptide migrating more slowly than the Bcl-X_L protein, and serves as an internal control for equal loading. (b) Constitutive expression of Nex1 results in increased expression of Bcl-w upon serum deprivation. Immunoblot analyses were performed as described above after stripping the membrane before probing with an anti-Bcl-w polyclonal antibody that does not cross-react with other Bcl2 proteins. Data shown are representative of at least three independent experiments.

Fig. 4

Overexpression of Nex1 induces the expression of members of the IAP family. (a) XIAP remains expressed in serum-deprived PC12-Nex1 cells. PC12-Nex1 and PC12-bsd cell extracts were prepared at different times during serum deprivation as indicated above each panel and subjected to immunoblot analysis using an anti-XIAP antibody. The antigen—antibody complexes were detected by chemiluminescence. (b) Survivin expression is only triggered upon serum deprivation of PC12-Nex1 cells. PC12-Nex1 and PC12-bsd cell extracts were prepared as described above and survivin expression was examined by western blotting. The antigen—antibody complexes were detected by chemiluminescence. (c) Serum deprivation of PC12-Nex1 cells does not induce PARP cleavage. PC12-Nex1 and PC12-bsd cell extracts were prepared and subjected to immunoblot analysis using a polyclonal antibody that only recognizes the large cleaved fragment of the rat PARP protein. The asterisk indicates a non-specific anti-PARP immunoreactive polypeptide migrating slightly lower than the large cleaved PARP fragment.

Fig. 5

Nex1 promotes long-term survival of serum-deprived Pc12-Nex1 cells, independently of the Akt pathway. (a) Serum deprivation of PC12-Nex1 cells does not lead to phosphorylation of Akt at Ser-437. PC12-Nex1 cells were deprived of serum for the times indicated above the panel and equal amounts of total protein were subjected to immunoblot analysis. Amounts of total and phosphorylated Akt were detected by using anti-Akt and anti-phosphorylated Akt (Ser-437) antibodies respectively. The antigen—antibody complexes were detected by chemiluminescence. The membrane was stripped before probing with the anti-Akt antibody. Molecular weight markers are indicated on the left side of each figure. Data shown are representative of at least three independent experiments. (b) The majority of PTEN protein exists in an inactive phosphorylated form in serum-deprived PC12-Nex1 cells. PC12-Nex1 cells were deprived of serum for times indicated above the panel and whole-cell extracts were analyzed by immunoblotting. Amounts of total and phosphorylated PTEN were detected by using anti-PTEN and anti-phosphorylated PTEN (Ser-380) antibodies respectively. The antigen—antibody complexes were detected by chemiluminescence. The membrane was stripped before probing with the anti-phosphorylated PTEN antibody. Data shown are representative of at least three independent experiments.