Overall Lack of Regulated Secretion in a PC12 Variant Cell Clone

doi:10.1074/jbc.271.43.27116

Overall Lack of Regulated Secretion in a PC12 Variant Cell Clone*

From the ^‡ Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology Center, Department of Pharmacology, University of Milan, I-20129 Milan, Italy,
the ^¶ Department of Pharmacology, Faculty of Pharmacy, University of Reggio Calabria, I-88100 Catanzaro, Italy,
the ^∥ Institute for Neurobiology, University of Heidelberg, D-6900 Heidelberg, Germany, and
the ^§ B. Ceccarelli Neurobiology Center and Department of Biological and Technological Research, San Raffaele Institute, I-20132 Milan, Italy

^″ To whom correspondence should be addressed:
CNR Cellular and Molecular Pharmacology Center, I-20129 Milan, Italy
. Tel.: 39-2-70146260; Fax: 39-2-7490574.

Abstract

A stable clone of PC12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretory proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C., and Meldolesi, J. (1992) Eur. J. Neurosci. 4, 944-953) was characterized in detail to clarify the nature of its phenotype and the mechanisms of its establishment. The neuroendocrine nature of the PC12-27 phenotype was documented by specific markers: synapsins, neurofilament subunit H, neuronal kinesin, and α-latrotoxin receptor. Moreover, various intracellular membrane systems of PC12-27, including the endoplasmic reticulum and the Golgi complex, appeared similar to control PC12 in both morphology and marker expression. In contrast, all the investigated markers located either in dense granules (dopamine-β-hydroxylase), in synaptic-like microvesicles (the acetylcholine transporter) or in both these regulated secretory organelles (VAMP2/synaptobrevin-2, synaptotagmin) were missing in PC12-27 cells, and the same was true also for the cytosolic and plasmalemma proteins involved in regulated exocytosis (Rab3, SNAP25, syntaxin). Pulse labeling and in vitro translation experiments revealed the defect to consist in a protein synthesis blockade that mRNA studies (reverse transcription-polymerase chain reaction, Northern blotting, and actinomycin D experiments) revealed to take place primarily at the transcriptional level. The secretion defect of PC12-27 cells was modified neither by various types of long term stimulation nor by nerve growth factor treatment. Moreover, when one of the missing regulated secretory proteins, chromogranin B, was expressed by cDNA transfection, it was secreted, however via the constitutive pathway. Our results demonstrate that PC12-27 cells are fully incompetent for both branches of regulated secretion, those of dense granules and synaptic-like microvesicles, possibly because of the impairment of a general expression control system that appears to operate independently of neuroendocrine cell differentiation.

Footnotes

↵* These studies were supported in part by grants from Consiglio Nazionale delle Ricerche (to P. R. and J. M.), from the EC programs Cooperation in Science and Technology with Central and Eastern European Countries, Biotech (to J. M. and W. B. H.) and Training and Mobility of Researchers, Research Network ERB4061PL95-0350 (to P. R. and W. B. H.), and from Human Frontiers (to J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TGN

trans-Golgi network

ER

endoplasmic reticulum

CgB

chromogranin B

hCgB

human CgB

SgII

secretogranin II

SNAP25

synaptosomal-associated proteins of 25 kDa

PC

proprotein convertase

TPA

12-O-tetradecanoylphorbol-13-acetate

PAGE

polyacrylamide gel electrophoresis

bp

base pair(s)

RT-PCR

reverse transcription-polymerase chain reaction.
↵² M. Passafaro, P. Rosa, F. Clementi, and E. Sher, unpublished data.
↵³ R. H. Kehlenbach and W. B. Huttner, unpublished data.
- Received December 26, 1995.
- Revision received August 1, 1996.