Nerve growth factor-induced differentiation of PC12 pheochromocytoma cells has served as a paradigm to study the action of NGF and --by inference-- of neurotrophic factors in general. Several """"""""primary response"""""""" genes --NGFIA, NGFIB and PC4-- induced by NFG in PC12 cells have been cloned and characterized. However, growth factors and tumor promoters induce NGFIA, NGFIB and PC4 expression in a variety of cells. How, then, do neurons express cell-specific responses to neuromodulatory agents? We showed that the expression of individual primary response genes is often """"""""extinguished"""""""" in a cell-type specific manner, e.g., NGFIB cannot be expressed in myeloid cells. We have now cloned a cDNA for a """"""""neuron-restricted"""""""" transcript, PCR-1. PCR-1 is inducible by a number of ligands in PC12 cells, but cannot be induced in a variety of other cell lines. We will isolate cDNAs for additional """"""""neuron-restricted"""""""" primary response genes. NGFIA, NGFIB and PC4 expression can be induced in PC12 cells by EGF, FGF, depolarization and TPA, in addition to NGF. How, then does NGF induce a ligand-specific response? We (and others) showed that distinct ligands induce different quantitative combinations of NGFIA and NGFIB gene expression in PC12 cells. We propose that there exist primary response genes whose expression in PC12 cells be quantitatively much greater in response to NGF than in response to other ligands. We will isolate cDNAs for these """"""""NGF-restricted"""""""" primary response genes. Examination of the expression of PCR-1 and other """"""""neuron-restricted"""""""" primary response genes in other cell lines, in various neuronal populations, and in response to a variety of ligands in PC12 cells will suggest whether such genes play causal roles in neuronal responses to neuromodulatory agents. Examination of induction of """"""""NGF-restricted"""""""" primary response genes in other cell types, in various NGF-responsive neurons, and in neurons responsive to other neurotrophic factors will suggest whether such genes play neuron-specific, unique, causal roles in neurotrophic factor-driven neuronal survival and differentiation. Sequencing both """"""""neuronal-restricted"""""""" cDNAs such as PCR-1 and """"""""NGF- restricted"""""""" cDNAs will suggest potential functions. Both antisense oligonucleotides and antibodies to fusion proteins of the proteins of the products of these genes will be used to determine which of these gene products are necessary for ligand-induced neuronal responses. Sequencing genomic regulatory regions, along with somatic cell genetic analyses, gene fusions and transfections, genomic footprinting, gel-shifts and DNase I chromatin studies will determine the molecular bases for their ligand and cell-type restricted primary response gene expression.
Showing the most recent 10 out of 24 publications
