Nerve growth factor induces the genes for two proteins related to a family of calcium-binding proteins in PC 12 cells

Differential hybridization of a cDNA library from rat pheochromocytoma PC12 cells with cDNA probes from naive PC12 cells and from PC12 cells exposed to nerve growth factor for 7 days identified cDNA sequences of two genes induced by NGF. The mRNA species detected by these cDNA sequences, designated 42A and 42C, reached maximal levels after 24 hr of treatment with NGF and were still significantly higher than control levels after 7 days. Epidermal growth factor transiently induced both mRNAs but at much lower levels. The mRNAs code for 95(42C) and 101(42A) amino acid residue peptides whose sequences are homologous to those of a family of calcium-binding proteins including the S-100 protein. The conservation of primary and secondary structure between 42A, 42C, and the other proteins suggests a possible role for them in the regulation of cell growth and differentiation. In spite of the structural and functional complexity of the nervous system, significant progress has been made in understanding the molecular mechanisms involved in neuronal development. Among the critical steps in this process are the discovery (1) and subsequent work (2, 3) on nerve growth factor (NGF). NGF is one of the best characterized polypeptide growth factors, whose functions in vivo are relatively well understood. It is now known that NGF is responsible for the selective survival of populations of sympathetic and some sensory neurons at the stage in development characterized by neuronal cell death (4) and for the maintenance of the differentiated state of these neurons thereafter (3). Underlying these functions, as well as its probable role in determining axonal growth during nerve regeneration (5), is a retrograde flow of NGF from the target tissues of these neurons to their cell bodies (3). Interest now centers around the molecular mechanisms invoked by this flow of NGF. An important step in the study of the mechanism of NGF action was the establishment of the rat adrenal pheochromocytoma-derived cell line PC12 (6). In the absence of NGF, these cells show characteristics of adrenal chromaffin cells. In response to NGF, they acquire properties of sympathetic neurons, the most dramatic of which is the outgrowth of neurites up to a millimeter long. This process is assumed to approximate the NGF-induced differentiation of embryonic neuroblasts. While many different cellular responses to the factor have been identified (see, e.g., refs. 7 and 8), the exact molecular mechanism of NGF action in PC12 cells remains unclear. The recent cloning of the cDNA for the low-affinity NGF receptor from PC12 cells (9) should permit an analysis, at the molecular level, of the beginning of the intracellular pathway involved in mediating the functions of NGF. To extend this analysis to the final steps of this pathway we have attempted to identify NGF-regulated genes whose products may play a role in mediating cellular responses to NGF. Our strategy has been to clone, by differential hybridization, cDNA copies of messages whose levels are elevated after long exposure to NGF and then select those for which this effect occurs relatively early and specifically. Using this procedure we identified two such sequences coding for peptides with strong sequence homology to the S-100-related family of proteins, which are thought to play various regulatory functions in cells (10-13). In this paper, we present the cloning and sequencing data* and discuss the possibility that this conservation of primary and secondary structure may reflect an important role played by these proteins in cellular differentiation. MATERIALS AND METHODS Cell Culture and RNA Preparation. PC12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% calf serum (HyClone, Logan, UT), 5% horse serum (Irvine Scientific), and antibiotics in a 12% C02/88% air atmosphere. NGF, when present, was at 100 ng/ml and epidermal growth factor (EGF) (Collaborative Research, Waltham, MA) was at 10 ng/ml. The medium was changed every 2 days. The concentration of NGF used produced a dense neurite outgrowth after 7 days in culture. Cytoplasmic RNA was prepared by cell lysis with 0.5% Nonidet-P40, followed by removal of the nuclear pellet by centrifugation and phenol/chloroform extraction of the supernatant (14). The yield was 7 pg of RNA per cell. DNA Cloning and RNA Blot Hybridization Analysis. A cDNA library in plasmid pUC9 was constructed by standard procedures (15), except that the oligo(dT)-cellulose chromatography of RNA and the rigorous size selection of cDNA were omitted. Double-stranded oligo(dC)-tailed cDNA was inserted into the Pst I site of oligo(dG)-tailed pUC9 (Pharmacia). This material was used to transfect competent Escherichia coli HB101 cells (Bethesda Research Laboratories), following the supplier's protocol. A cDNA library in the Okayama-Berg eukaryotic expression vector (16) was constructed using an oligo(dT)-tailed pcDV1 primer and an oligo(dG)-tailed pL1 linker from Pharmacia and following the procedures described in Pharmacia's cDNA cloning manual. For differential hybridization, bacterial colonies were transferred to individual wells of %-well microtiter plates, incubated at 37°C, and inoculated on ampicillin (100 jg/ml) plates with a 96-prong device. The resulting colonies were transferred to Whatman 540 filter paper, fixed, and screened Abbreviations: NGF, nerve growth factor; EGF, epidermal growth factor; CaBP, calcium-binding protein; CFa, cystic fibrosis antigen. *These sequences are being deposited in the EMBL/GenBank data base (Bolt, Beranek, and Newman Laboratories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) [accession nos. J03627 (clone 42C) and J03628 (clone 42A/eX15)]. 1277 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1278 Neurobiology: Masiakowski and Shooter with radioactive cDNA probes (ref. 14; P.M., unpublished results). For screening of libraries with purified inserts, the high-colony-density technique (17) was used. Cytoplasmic RNA samples were separated on 1.5% agarose gels in the presence of formaldehyde (15) and transferred to Hybond-N nylon filters (Amersham) using the manufacturer's protocol. The purified cDNA inserts used as probes were radiolabeled using random oligonucleotides as primers (18). The sizes of RNA bands were estimated using an RNA ladder [0.3-9.5 kilobases (kb)] from Bethesda Research Laboratories as a size marker. Some autoradiograms of RNA blots were analyzed quantitatively using a Microscan 1000 densitometer (Technology Resources, Nashville, TN). DNA Sequencing and Data Processing. DNA sequences of clones 42A and 42C were obtained by both the dideoxynucleotide chain-termination (19) and the base-specific chemical-cleavage (20) methods. Subcloning into phage M13mpl8 and -mpl9 was done following the New England BioLabs manual. Clone 42A was sequenced from both ends and from the internal EcoRI site. Clone 42C was sequenced from both ends and from the internal HincII and Kpn I sites. Clones eX9 and eX15 were sequenced by the chemical-cleavage method using the internal HincIl and EcoRI sites and the flanking BamHI sites of the vector. Sequence data acquisition and initial processing were done using Staden programs (21, 22). For subsequent sequence analysis we used the University of Wisconsin Genetics Computer Group set of programs (23). The FASTP program (24) was used to search the National Biomedical Research Foundation Protein Sequence Database (25, t) and to compare individual protein