Expression of the transcription factor REST at different stages of neuronal differentiation: contribution of PKC and WNT/-catenin signaling

A subset of genes implicated in genetic and acquired neurological disorders encode proteins essential to neural patterning and neurogenesis. The neuronal silencing transcription factor REST plays a critical role in elaboration of the neuronal phenotype. In neural progenitor and non-neural cells, REST actively represses a large array of neural-specific genes important to synaptic plasticity and synaptic remodeling. As neural progenitors differentiate, REST downregulation is essential for induction and maintenance of the neural phenotype [1]. In differentiated neurons, derepression of REST is likely to be an important mechanism of insult-induced neuronal death. In this study we provide evidence that, during early stages of neuronal differentiation induced by PKC activation, REST may repress the expression of several genes not yet required by the differentiation program whereas it declines later. Therefore, the decline of REST is a comparatively later event during maturation of neuroroblasts in vitro and requires specific regulatory pathways. A precocious REST down-regulation, obtained using an antisense strategy, causes a more extended neurite network without preventing overt neurogenesis [2]. Nuclear REST expression is increased in SH-SY5Y cells exposed to the phorbol ester PMA (16nM) for 3-days whereas it is down-regulated at later stages. Cell exposure to PKC inhibitors (GF10923X and Gö6976) reverts the effects elicited by PMA alone. REST down-regulation is related to a progressive activity of proteasome: neuroblasts concomitantly exposed to PMA and to the proteasome inhibitor MG132 showed a blockade of neuronal differentiation and REST up-regulation. REST decline is clearly related to morphological differentiation and to the expression of growth cone-associated protein 43 (GAP-43), synapsin I and bIII tubulin, three proteins involved in the early stage of neuronal development. Starting from evidences that phorbol esters, activation of PKC, increase the transcription-activational roles of β-catenin [3,4], we investigated the contribution of WNT/β-catenin pathway on REST down-regulation. In SH-SY5Y cells treated with PMA for 4 days we silenced β-catenin expression using a specific siRNA. In these conditions REST down-regulation was prevented. Therefore, when β-catenin is present, it enters the nucleus to interact with TCF/LEF and mediate transcription of β-TRCP, among others targets. β-TRCP, an E3 ubiquitin ligase, binds and ubiquitinates REST, targeting it to proteasome-dependent degradation. β-TRCP is required for proper neural differentiation only in the presence of REST, confirming that β-TRCP facilitates this process through degradation of REST. We propose that differentiating SH-SY5Y cells, REST is regulated by PKC in a time-dependent manner; it is elevated during early stages of neuronal induction and then declines later. The precocious down-regulation of this transcription factor may increase neurite elongation and accelerate cell differentiation. Therefore, REST is elevated during early steps of neural induction by PKC activator PMA to decline later for the acquisition of neural phenotypes through a pathway activated by PKC and involving the WNT/β-catenin signalling. In differentiated neurons REST expression may be increased by an ischemic insult and PKC activation, inducing REST down-regulation, may protect neurons exposed to ischemia. [1] Ballas et al., 2001, Neuron 31:353-365. [2] Di Toro et al., 2005, Eur J Neurosci 21:46-58. [3] Sun et al., 2001, J Neuroendocrinol 13:249-260. [4] Cook et al., 1996, EMBO J 15(17): 4526-4536.