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

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.

Class III β‐tubulin in human development and cancer

“…The pRb collaborates with HLHs to achieve cooperative induction of terminal mitosis and expression of a repertoire of genes associated with neuronal maturation [Toma et al, 2000]. As indicated above, bHLH transcription factors stimulate the expression of ␤III-tubulin in a neuronal differentiation-dependent manner [Uittenbogaard and Chiamarello, 2002]. Failure to coordinate induction of terminal mitosis due to disrupted cooperation between the mutated or deregulated pRb on one hand and/or bHLH(s) on the other, may explain the expression of ␤III in actively cycling cells in both neuronal and non-neuronal tumors.…”

“…It contains (1) a number of presumptive binding sites for the transcription factors SP-1 and AP-2, (2) a central nervous system enhancer (CNE) characterized by a CAAAAT consensus sequence in the proximal promoter region, and (3) E-box sites that are important in neuronal differentiation [Dennis et al, 2002]. To this end, the basic helix-loop-helix (bHLH) differentiation factor, Nex1/MATH-2, targets and stimulates the expression of ␤III-tubulin along with other genes, such as GAP-43 and NeuroD, which are associated with neuronal differentiation [Uittenbogaard and Chiamarello, 2002]. Interestingly, overexpression of a Xenopus bHLH gene, Xath2, which is the homologue of the murine MATH-2/NEX-1 gene in neurula embryos, induces the expression of the neuron-associated class II ␤-tubulin [Taelman et al, 2001].…”

A high throughput embryonic stem cell screen identifies Oct-2 as a bifunctional regulator of neuronal differentiation

“…Several factors, including Mash1, NeuroD2, and Math1, had previously been observed to induce some level of differentiation when overexpressed in embryonic carcinoma cells (Farah et al 2000). For NeuroD6, an ability to differentiate had previously been demonstrated in rat pheochromocytoma cells (PC12) but not in ES cells (Uittenbogaard and Chiaramello 2002). Two stable lines were chosen and analyzed for each factor and examined 96 h post-induction.…”

Gene regulatory factors in pancreatic development

“…Mitotic silencing is possibly linked to the activation of CDK-inhibitors such as p16, p21, p27, and p57. For instance, Nex, a close homologue of NeuroD, activates p21 in PC12 cells (Uittenbogaard and Chiaramello, 2002), leading to mitotic arrest.…”