The neurosecretory process, by which neurons and neurosecretory cells store their neurotransmitters and/or neurohormones within unique organelles and discharge them by regulated exocytosis, is the central event of neurosecretion, acquired by the cells in the course of their progression from pluripotent stem cells to restricted lineages. Extensive studies carried out during the last decade (reviewed by Wu and Xie 2006) have shown that this progression depends on a complex signaling network orchestrated by the repressor element 1-silencing transcription factor (REST, also referred to as NRSF), operating also through the involvement of additional factors, active at the transcriptional and post-transcriptional level (Conaco et al. 2006;Ma 2006;Wu and Xie 2006). Binding of REST to a specific DNA sequence distributed in its numerous target genes, the repressor element 1 (RE-1), entails the assembly, at the N and C terminal domains of the repressor, of two protein complexes, including histone and DNA modifying enzymes. The activity of these complexes consists in the repression of transcription. The differential development of Received December 12, 2007; revised manuscript received January 7, 2008; accepted January 8, 2008.Address correspondence and reprint requests to Jacopo Meldolesi, Vita-Salute San Raffaele University, DIBIT, via Olgettina 58, 20132 Milan, Italy. E-mail: meldolesi.jacopo@hsr.itAbbreviations used: DBD, DNA-binding domain; DBD/REST, dominant negative construct of REST; DCV, dense-core vesicle; FITC, fluorescein isothiocyanate; GC, Golgi complex; hChgA and ChgA, human and rat chromogranin A; hChgB and ChgB, human and rat chromogranin B; HDAC, histone deacetylase; ICA512, Islet cell antigen 512; Q-SNARE and R-SNARE, target and vesicle SNAREs; RE-1, responsive element 1; REST, RE-1 silencing transcription (factor); Scg2, secretogranin 2; SLMV, synaptic-like microvesicles; Stx1a, syntaxin 1a; SynI, synapsin I; Syp1, synaptophysin 1; Syt1, synaptotagmin 1; TH, tyrosine hydroxylase; TRITC, tetramethyl-rhodamine isothiocyanate; TSA, trichostatin A; VAMP, vesicle associated protein; wt, wild-type. AbstractThe neurosecretory process is acquired during differentiation and can be lost en block by differentiated cells. To investigate the role of REST/NRSF, a transcription repressor, in the maintenance of the process we studied two PC12 clones, one wt and one defective, expressing low and high levels of endogenous RE-1 silencing transcription (factor) (REST), respectively. Stable transfection of constructs demonstrated that REST represses 10 genes coding for proteins of neurosecretory vesicles and their exocytosis, eight including and two lacking the REST-binding sequence, RE-1. Of these genes, those of chromogranins were strongly repressed by fewfold increases of REST, those of VAMP2 and syntaxin1a required much higher levels. Moreover, in wt cells transfected with an active construct the dense-core vesicles, still competent for regulated exocytosis, were much smaller, with lighter cores; in defective cells, the...

SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although the first SOX14 gene in vertebrates was cloned and characterized more than a decade ago and its expression profile during development was revealed in various animal model systems, the role of this gene during neural development is largely unknown. In the present study we analyzed the expression of SOX14 in human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells. We demonstrated that it is expressed in both cell lines and upregulated during retinoic acid induced neural differentiation. We showed that SOX14 was expressed in both neuronal and non-neuronal differentiated derivatives, as revealed by immunocytochemistry. Since it was previously proposed that increased SOXB2 proteins level interfere with the activity of SOXB1 counteracting partners, we compared expression patterns of SOXB members during retinoic acid induction of embryonal carcinoma cells. We revealed that upregulation of SOX14 expression is accompanied by alterations in the expression patterns of SOXB1 members. In order to analyze the potential cross-talk between them, we generated SOX14 expression construct. The ectopic expression of SOX14 was demonstrated at the mRNA level in NT2/D1, P19 and HeLa cells, while an increased level of SOX14 protein was detected in HeLa cells only. By transient transfection experiments in HeLa cells we showed for the first time that ectopic expression of SOX14 repressed SOX1 expression, whereas no significant effect on SOX2, SOX3 and SOX21 was observed. Data presented here provide an insight into SOX14 expression during in vitro neural differentiation of embryonal carcinoma cells and demonstrate the effect of its ectopic expression on protein levels of SOXB members in HeLa cells. Obtained results contribute to better understanding the role of one of the most conserved SOX proteins.

SummaryThe RE-1-specific silencing transcription factor (REST or NRSF) is a transcription repressor that orchestrates differentiation and also operates in differentiated neurons and neurosecretory cells (neural cells). Its role in proliferation has been investigated so far only in rapidly growing tumors, with conflicting results: suppression in non-neural tumors, stimulation in medulloblastomas. Working with two clones of chromaffin-neuronal PC12 cells, which express different levels of REST, and using genetic complementation and knockdown approaches, we show that REST also promotes proliferation in differentiated neural cells. Mechanistically, this occurs by a signaling pathway involving REST, the GTPase-activating protein tuberin (TSC2) and the transcription co-factor b-catenin. In PC12 cells, raised expression of REST correlates with reduced TSC2 levels, nuclear accumulation and co-transcriptional activation of b-catenin, and increased expression of its target oncogenes Myc and Ccnd1, which might account for the proliferation advantage and the distinct morphology. Rest transcription is also increased, unveiling the existence of a self-sustaining, feed-forward REST-TSC2-b-catenin signaling loop that is also operative in another neural cell model, NT2/D1 cells. Transfection of REST, knockdown of TSC2 or forced expression of active b-catenin recapitulated the biochemical, functional and morphological properties of the high-expressing REST clone in wild-type PC12 cells. Upregulation of REST promoted proliferation and phenotypic changes, thus hindering neurosecretion. The new REST-TSC2-b-catenin signaling paradigm might have an important role in various aspects of neural cell physiology and pathology, including the regulation of proliferation and neurosecretion.

Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4-to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance.

The mechanism by which neurons and neurosecretory cells govern the expression and the exocytic discharge of their clear and dense-core vesicles had remained unclear until recently when studies in the neurosecretory cell model PC12 revealed these processes to be orchestrated by the transcriptional repressor neuron restrictive silencer factor (NRSF)/repressor element-1 silencing transcription factor (REST). In wild-type PC12 fully competent for neurosecretion, NRSF/REST is low. The genes of the proteins involved in neurosecretion [from the secretory to vesicle membrane and plasma membrane proteins, including the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of exocytosis] were all repressed by increases of NRSF/REST expression to various extents when the increase was only a fewfold but were completely or almost completely repressed when the increase was large, as in spontaneously defective PC12 clones. In the first case the dense-core vesicles were still competent for exocytosis but were smaller and less dense than in wild-type cells; in the second they were no longer visible but did reappear when the repression was attenuated by transfection of a dominant-negative construct of NRSF/REST combined with a secretory chromogranin or strengthened by treatment with a blocker of NRSF/REST-associated enzymes, the histone deacetylases.