Neurosecretion Competence A COMPREHENSIVE GENE EXPRESSION PROGRAM IDENTIFIED IN PC12 CELLS*□ S Received for publication, April 18, 2002, and in revised form, June 5, 2002 Published, JBC Papers in Press, June 17, 2002, DOI 10.1074/jbc.M203777200 Christophe Grundschober‡§, Maria Luisa Malosio§¶, Laura Astolfi§, Tiziana Giordano§, Patrick Nef‡, and Jacopo Meldolesi§¶** From the ‡Central Nervous System, F. Hoffmann-La Roche Ltd., Grenzacherstrasse, Basel 4070, Switzerland and the ¶Vita-Salute San Raffaele University and Department of Neuroscience, §San Raffaele Scientific Institute, Department of Biological and Technological Research, Via Olgettina, 58, Milano 20132, Italy The phenotype of neurosecretory cells is character- ized by clear vesicles and dense granules, both dis- charged by regulated exocytosis. However, these organelles are lacking completely in a few neurosecre- tion-incompetent clones of the pheochromocytoma PC12 line, in which other specific features are main- tained (incompetent clones). In view of the heterogene- ity of PC12 cells, a differential characterization of the incompetent phenotype based on the comparison of a single incompetent and a single wild-type clone would have been inconclusive. Therefore, we have compared two pairs of PC12 clones, studying in parallel the tran- script levels of 4,200 genes and 19,000 express sequence tags (ESTs) by high density oligonucleotide arrays. After accurate data processing for quality control and filtra- tion, a total of 755 transcripts, corresponding to 448 genes and 307 ESTs, was found consistently changed, with 46% up-regulated and 54% down-regulated in in- competent versus wild-type clones. Many but not all neu- rosecretion genes were profoundly down-regulated in incompetent cells. Expression of endocytosis genes was normal, whereas that of many nuclear and transcription factors, including some previously shown to play key roles in neurogenesis, was profoundly changed. Addi- tional differences appeared in genes involved in signal- ing and metabolism. Taken together these results demonstrate for the first time that expression of neuro- secretory vesicles and granules is part of a complex gene expression program that includes many other features that so far have not been recognized. Expression of two classes of secretory organelles, small translucent vesicles (clear vesicles) and dense content granules of larger size (DGs), 1 is the typical trait of neurosecretory cells. These organelles resemble in many respects the synaptic or- ganelles of neurons and share with them the property to be discharged by regulated exocytosis. Clear vesicles, DGs, and their synaptic counterparts have attracted uninterrupted at- tention for many years and are, therefore, among the best known organelles not only in terms of composition and struc- ture but also in relation to the processes they are involved in such as assembly, loading of neurotransmitters, exocytotic membrane fusion, and recycling (for reviews see Refs. 1 and 2). In contrast, only a few studies have been devoted to the mech- anisms whereby cells acquire neurosecretion competence. Knowledge in the latter field is therefore limited (see Refs. 3–5). In previous studies, expression of other specific functions was identified as an independent process in the course of cell differentiation. In both neuronal and neurosecretory systems, however, neurosecretion seems to appear concomitantly with the expression of other traits such as neurite outgrowth and synaptogenesis (3, 4, 6, 7). This suggested neurosecretion com- petence is not independent but coordinate with other functions in a wider differentiation program. This interpretation appears open to question, because the isolation, from the well-known neurosecretion competent pheochromocytoma cell line, PC12, of at least three independent clones totally lacking neurosecre- tion (neurosecretion incompetent (NI) clones) (8 –10). These clones express numerous molecular traits (tyrosine hydroxy- lase, neurofilament H, neuronal kinesin) and functional prop- erties (-latrotoxin response) typical of wild-type (WT) PC12 (8, 9), including neurite sprouting following exposure to nerve growth factor. Their incompetence relies on the lack of specific proteins directly involved in regulated exocytosis, which in the WT PC12 are localized not only in the SV and DG membranes and lumina but also in the plasmalemma and cytosol (see Ref. 11). The defective phenotype of NI clones is stable. In contrast to another reported cell model (12), it cannot be reverted by stimulatory treatments or by changes in growth conditions. However, the NI phenotype defect can be rescued by fusion of the NI cells with WT cells (regardless of the species or pheno- type) and following transfection with a WT PC12 cDNA library (11, 13). These data strongly suggest neurosecretion incompe- tence to reflect the lack of expression of a specific program. * This work, supported by grants from the European Union (Grow- beta), the Italian Ministry of University and Research (COFIN and FIRB programs), Telethon (project 1118), and the Armenise-Harvard Foundation, was carried out in the Center of Excellence in Physiopa- thology in Cell Differentiation, awarded to the Vita-Salute San Raffaele University by the Italian Ministry of University and Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains Fig. S1 and Tables I–III. § Both authors contributed equally to this work.  To whom correspondence may be addressed. Tel.: 39-02-2643-2913; Fax: 39-02-2643-2914; E-mail: malosio.marialuisa@hsr.it. ** To whom correspondence may be addressed. Tel.: 39-02-2643- 2770; Fax: 39-02-2643-4813; E-mail: meldolesi.jacopo@hsr.it. 1 The abbreviations used are: DG, dense content granule; NI, neuro- secretion incompetent; WT, wild-type; EST, expressed sequence tag; AD, average difference; CV, coefficient of variation; MFC, minimum detectable -fold change; CG, chromogranin; SG, secretogranin; IP 3 , inositol 1,4,5-trisphosphate; CGA and CGB, chromogranins A and B; H + pump, vacuolar H + pump, vATPase; ICA, islet cell antigen 512; Rab3A and C, small G protein; STX1, syntaxin; SNAP25 and SNAP23, soluble NSF attachment proteins of 25 and 23 kDa; SV2 A–C, synaptic vesicle protein 2 A–C; VAMP 1 and 2, vesicle-associated membrane proteins 1 and 2 (synaptobrevin). THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 39, Issue of September 27, pp. 36715–36724, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. 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