To help elucidate the mechanisms by which nerve growth factor (NGF) regulates gene expression, we have identified and studied four genes (a-2, d-2, d4, and d-S) that are positively regulated by NGF in PC12 cells, including one (d-2) which has previously been identified as a putative transcription factor (NGF I-A). Three of these genes, including d-2, were induced very rapidly at the transcriptional level, but the relative time courses of transcription and mRNA accumulation of each of these three genes were distinct. The fourth gene (d4) displayed no apparent increase in transcription that corresponded to the increase in its mRNA, suggesting that NGF may regulate its expression at a posttranscriptional level. Thus, NGF positively regulates gene expression by more than one mechanism. These genes could also be distinguished on the basis of their response to cyclic AMP. The expression of d-2 and a-2 was increased by cholera toxin and further augmented by NGF; however, cholera toxin not only failed to increase the levels of d-5 and d4 mRNA but also actually inhibited the NGF-dependent increase. The expression of each of these genes, including d-2 (NGF I-A), was also increased by fibroblast growth factor, epidermal growth factor (EGF), phorbol myristate acetate, and in some cases insulin, showing that the regulation of these genes is not unique to NGF. Because each of these genes was expressed in response to phorbol myristate acetate and EGF, their expression may be necessary but is certainly not sufficient for neurite formation. The protein kinase inhibitor K-252a prevented the NGF-associated, but not the acidic FGF-associated, induction of d-2 and d-5 gene expression, suggesting that these two growth factors may regulate gene expression via different cellular pathways. The study of the regulation of the expression of these and other NGF-inducible genes should provide valuable new information concerning how NGF and other growth factors cause neural differentiation.Nerve growth factor (NGF) is a peptide hormone that is essential for the development and the survival of sympathetic nerves, sensory nerves, and certain populations of nerves in the central nervous system (32,52). It also influences many aspects of cell metabolism, including RNA synthesis, protein synthesis, protein phosphorylation, and ion flux (1,11,22,46,54). However, studies of NGF action on neural differentiation have been hampered because NGF influences both cell survival and cell differentiation.PC12 cells, a cloned line derived from a rat pheochromocytoma, has been a useful model system for studying NGFdependent differentiation (18). Although NGF is not required as a survival factor for these cells, it retains the ability to stimulate differentiation and process formation. The heparinbinding peptide growth factors, acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF), also stimulate neurite outgrowth and differentiation in PC12 cells in a pattern that is not distinguishable from that of NGF (47, 51). In the presence of NG...

We have shown previously that the transcription of the gene designated dS is induced by nerve growth factor (NGF) in rat adrenal pheochromocytoma PC-12 cells and that this NGF induction is repressed by cAMP. In this paper we demonstrate that d5 is a member of a gene family that contains several hundred members, which is closely related to retroviruses and retrotransposons, as demonstrated by the following observations: (i) the original dS cDNA hybridized to numerous restriction fragments in genomic DNA;-(i) d5 cDNA hybridized to genomic clones with various intensities, and genomic clones can be isolated with a frequency suggesting that this family includes several hundred members; and (iii) there were minor sequence variations in four independently isolated cDNA clones that were homologous to d5 cDNA. Primer extension studies show that initiation of the 5.7-kilobase d5 mRNA(s) occurs at a unique site relative to a synthetic primer. The 5' end of the cDNA sequence was homologous to Rasheed rat sarcoma virus; and a genomic clone contained several elements that are typical of a long terminal repeat (LTR), including a CCAAT box, a TATA box, a primer binding site, a poly(A) addition signal, and a poly(A) addition site. Furthermore, there is a LTR at the 3' end of at least one of the genes in this family, and there appeared to be a four-base duplication at the probable site of integration into host DNA. Since several members ofthis family retain responses to NGF and cAMP, we conclude that the regulatory elements present in the LTR have been conserved in many members of this family. We have named this family of genes the NICER elements because they are a family of NGF-inducible cAMP-extinguishable retrovirus-like elements.contain regulatory elements that respond to glucocorticoids; and many retroviruses, including human immunodeficiency virus type 1 (HIV-1), formerly called HTLV-III, and avian reticuloendotheliosis virus type 2, contain sequences homologous to the glucocorticoid-responsive elements (9-11). It is reasonable to suggest that retroviruses have evolved their regulatory elements to respond appropriately to various cellular signals during their life cycle.The d5 cDNA clone that originally was isolated as an NGF-inducible gene in rat adrenal pheochromocytoma PC-12 cells is induced not only by NGF but also by epidermal growth factor, fibroblast growth factor, and phorbol 12-myristate 13-acetate (12). The transcription of dS is observed as early as 5 min but returns to basal levels by 3 hr; and its induction by NGF is repressed by cAMP (12). In this paper we show that the d5 gene defines a family of endogenous rat retrovirus-like elements on the basis of several lines of evidence.t First, the 5' end of the d5 cDNA clone has extensive homology to the LTR of Rasheed rat sarcoma virus (13). Second, this 565-base-pair (bp) LTR is repeated at both the 5' and the 3' end of at least one of the d5 genomic clones. Third, this LTR contains several features of a eukaryotic promoter, including a TATA box, a CCAAT bo...

To help elucidate the mechanisms by which nerve growth factor (NGF) regulates gene expression, we have identified and studied four genes (a-2, d-2, d-4, and d-5) that are positively regulated by NGF in PC12 cells, including one (d-2) which has previously been identified as a putative transcription factor (NGF I-A). Three of these genes, including d-2, were induced very rapidly at the transcriptional level, but the relative time courses of transcription and mRNA accumulation of each of these three genes were distinct. The fourth gene (d-4) displayed no apparent increase in transcription that corresponded to the increase in its mRNA, suggesting that NGF may regulate its expression at a posttranscriptional level. Thus, NGF positively regulates gene expression by more than one mechanism. These genes could also be distinguished on the basis of their response to cyclic AMP. The expression of d-2 and a-2 was increased by cholera toxin and further augmented by NGF; however, cholera toxin not only failed to increase the levels of d-5 and d-4 mRNA but also actually inhibited the NGF-dependent increase. The expression of each of these genes, including d-2 (NGF I-A), was also increased by fibroblast growth factor, epidermal growth factor (EGF), phorbol myristate acetate, and in some cases insulin, showing that the regulation of these genes is not unique to NGF. Because each of these genes was expressed in response to phorbol myristate acetate and EGF, their expression may be necessary but is certainly not sufficient for neurite formation. The protein kinase inhibitor K-252a prevented the NGF-associated, but not the acidic FGF-associated, induction of d-2 and d-5 gene expression, suggesting that these two growth factors may regulate gene expression via different cellular pathways. The study of the regulation of the expression of these and other NGF-inducible genes should valuable new information concerning how NGF and other growth factors cause neural differentiation.