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Constitutive overexpression of the basic helix-loop-helix Nex1/MATH-2 transcription factor promotes neuronal differentiation of PC12 cells and neurite regeneration - PubMed

  • ️Tue Jan 01 2002

Constitutive overexpression of the basic helix-loop-helix Nex1/MATH-2 transcription factor promotes neuronal differentiation of PC12 cells and neurite regeneration

Martine Uittenbogaard et al. J Neurosci Res. 2002.

Abstract

Elucidation of the intricate transcriptional pathways leading to neural differentiation and the establishment of neuronal identity is critical to the understanding and design of therapeutic approaches. Among the important players, the basic helix-loop-helix (bHLH) transcription factors have been found to be pivotal regulators of neurogenesis. In this study, we investigate the role of the bHLH differentiation factor Nex1/MATH-2 in conjunction with the nerve growth factor (NGF) signaling pathway using the rat phenochromocytoma PC12 cell line. We report that the expression of Nex1 protein is induced after 5 hr of NGF treatment and reaches maximal levels at 24 hr, when very few PC12 cells have begun extending neurites and ceased cell division. Furthermore, our study demonstrates that Nex1 has the ability to trigger neuronal differentiation of PC12 cells in the absence of neurotrophic factor. We show that Nex1 plays an important role in neurite outgrowth and has the capacity to regenerate neurite outgrowth in the absence of NGF. These results are corroborated by the fact that Nex1 targets a repertoire of distinct types of genes associated with neuronal differentiation, such as GAP-43, betaIII-tubulin, and NeuroD. In addition, our findings show that Nex1 up-regulates the expression of the mitotic inhibitor p21(WAF1), thus linking neuronal differentiation to cell cycle withdrawal. Finally, our studies show that overexpression of a Nex1 mutant has the ability to block the execution of NGF-induced differentiation program, suggesting that Nex1 may be an important effector of the NGF signaling pathway.

Copyright 2002 Wiley-Liss, Inc.

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Figures

Fig. 1
Fig. 1

Bacterial expression of the truncated Nex1-Ag protein and NGF-induced expression of full-length Nex1 expression in PC12 cells. A: Coomassie blue staining of SDS-PAGE-separated E. coli whole-cell extracts expressing the truncated recombinant Nex1 protein Nex1-Ag. Bacterial whole-cell extracts were prepared before and after induction with IPTG as indicated at the top. Molecular weights (kDa) are indicated on the left, and the overexpressed truncated Nex1-Ag protein is indicated by an arrow. A schematic representation of the full-length Nex1 protein is shown at the bottom, to illustrate the C-terminal portion of Nex1 (Nex1-Ag) used to raise the anti-Nex1 rabbit polyclonal antibody. B: Specificity of the anti-Nex1 polyclonal antibody. Overexpressing truncated Nex1-Ag bacterial whole-cell extracts before and after IPTG induction were analyzed by western blot using the generated anti-Nex1 polyclonal antibody. The antigen–antibody complexes were detected by chemiluminescence. Truncated Nex1 protein is detected prior to IPTG induction because of leaky expression of the T7 RNA polymerase in the pLysS strain, resulting in a basal level of Nex1 expression. Among the many thousands proteins present in the uninduced and induced whole-cell extracts, only a single band at the expected MW is detected by the anti-Nex1 antibody. C: Timing of full-length Nex1 expression in untreated and NGF-treated PC12 cells. PC12 cells were grown in the absence or presence of NGF (100 ng/ml) for specific periods of time as indicated at the top. Nex1 protein was detected by Western assay using the anti-Nex1 polyclonal antibody. Nex1 protein is detected after 5 hr of NGF treatment and remains highly expressed after 5 days of NGF exposure.

Fig. 2
Fig. 2

Constitutive expression of Nex1 in PC12 cells generates several clusters displaying an extreme differentiation phenotype. PC12 cells were stably transfected with either the eukaryotic expression vector pcDNA6/Nex or the control vector pcDNA/OOF, which contains a stop codon right after the Nex1 first amino acid. On the transformed plate, a dozen PC12–Nex1-E clusters established an extensive neurite network independently of NGF induction. In contrast, PC12 cells and control pC12-OOF cells failed to display such a differentiation phenotype. Four of these PC12–Nex1-E clusters are illustrated.

Fig. 3
Fig. 3

Overexpression of Nex1 induces a substantial spontaneous neurite outgrowth in untreated PC12–Nex1-M clones. The expression level of exogenous Nex1 protein was assessed by Western blot analysis using equal amounts of total protein from control PC12 cells and three stable PC12–Nex1-M(A), PC12–Nex1-M(B), and PC12–Nex1-M(C) clones. The three PC12–Nex1-M clones express similar amounts of the Nex1 protein, whereas control PC12 cells do not express Nex1 protein. Nex1 was detected with either our polyclonal antibody (A) or the anti-Xpress antibody raised against the N-terminal tag epitope (B). C: The clones PC12–Nex1-M(A), PC12–Nex1-M(B), and PC12–Nex1-M(C) show a significant increase in spontaneous neurite projections compared with PC12 cells. This is a representative graph from a triplicate experiment that was repeated four times. The error is the standard deviation of the mean. D–F: The phase-contrast micrograph of PC12–Nex1-M cells reveals their moderate differentiation phenotype. Cells from the three stably transfected PC12–Nex1-M clones are characterized by a flattened cell body and spontaneous neurite extensions.

Fig. 4
Fig. 4

Constitutive expression of Nex1 accelerates the initial neurite outgrowth in NGF-treated PC12–Nex1-M cells. PC12 and PC12–Nex1-M(B) cells were grown on collagen I-coated plates and treated with NGF (50 ng/ml) for 6, 24, or 48 hr as indicated and neurite outgrowth was determined as described in Materials and Methods. A: Quantitation of the degree of neurite outgrowth observed in the NGF-treated PC12 and PC12–Nex1-M cells. The percentage of cells bearing neurites is consistently higher upon constitutive expression of Nex1 during the NGF treatment. PC12 cells are represented by open bars, whereas PC12–Nex1-M(B) cells are indicated by shaded bars. This is a representative graph from a triplicate experiment that was repeated four times. The error is the standard deviation of the mean. B: PC12–Nex1-M(B) cells already extend neurites after 6 hr of exposure to NGF, whereas PC12 cells require at least 24 hr of NGF treatment to extend neurites. Similar results were obtained with the two other PC12–Nex1-M clones. Micrographs of NGF-treated PC12–Nex1-M cells at t = 24 and 48 hr were taken at low magnification to reveal the appearing neurite network.

Fig. 5
Fig. 5

Ectopic expression of Nex1 promotes the neurite regeneration of NGF-differentiated cells in the absence of an NGF boost. PC12 cells and PC12–Nex1-M(B) cells were differentiated for 7 days in the presence of NGF (50 ng/ml), after which the cells were thoroughly washed with NGF-free medium. The neurites were mechanically sheared as described in Materials and Methods. The cells were replated on collagen I-coated plates in the absence or presence of NGF (50 ng/ml). The regeneration process was analyzed 5 hr after replating. A: PC12–Nex1-M(B) cells display substantial neurite regeneration in the absence of NGF compared to PC12 cells. B: Ectopic expression of Nex1 enhances the propensity to initiate neurite outgrowth even in the absence or presence of NGF. The percentage of neurite-bearing cells was scored on at least 300 cells from three distinct experiments. Cells cultured in the absence of NGF after trituration are indicated by open bars, whereas cells grown in the presence of NGF after trituration are represented by hatched bars. This is a representative graph from a triplicate experiment that was repeated four times. The error is the standard deviation of the mean.

Fig. 6
Fig. 6

Overexpression of Nex1 induces expression of several genes associated with neuronal differentiation in the absence of NGF. Western blot analyses comparing the expression of neuronal marker protein using equal amounts of total protein from PC12 cells and the three PC12–Nex1-M(A), PC12–Nex1-M(B), and PC12–Nex1-M(C) clones. The specific antibodies used for this study are described in Materials and Methods. The antigen–antibody complexes were detected by chemiluminescence. Overexpression of Nex1 in untreated cells stimulates the expression of the GAP-43 protein (A), the neuronal specific βIII-tubulin protein (B), the bHLH differentiation factor NeuroD (C), and the mitotic inhibitor p21WAF1 protein (D). As expected, PC12 cells do not express GAP-43 and NeuroD proteins. The p21WAF1 protein and the neuronal-specific βIII-tubulin protein are expressed at much lower levels in PC12 cells.

Fig. 7
Fig. 7

Constitutive expression of mutated Nex1-mut1 blocks NGF-induced differentiation in PC12 cells. PC12 cells were stably transfected with either the eukaryotic expression vector pcDNA6/Nex1-mut1 or the control expression vector pcDNA6/OOF. PC12, control PC12-OOF, and PC12–Nex1-mut1 cells were grown on collagen I-coated plates and differentiated with NGF (25 ng/ml) for 6 days.

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