PC12nnr5 cells expressing TrkA receptors undergo morphological but not cholinergic phenotypic differentiation in response to nerve growth factor.

We investigated mechanisms underlying nerve growth factor-mediated morphological differentiation and expression of cholinergic neuronal phenotype. In PC12, but not PC12nnr5 cells, nerve growth factor induces neurite-like outgrowths and enhances cholinergic phenotype; stable expression of TrkA receptors in nnr5 cells (called B5P cells) restores morphological differentiation but not expression of choline acetyltransferase. Transfection with an AP-1 luciferase reporter gene revealed that PC12 but not B5P cells expressed nerve growth factor-induced functional AP-1 activity. RT-PCR analysis of nerve growth factor-mediated changes in AP-1 transcription factors showed rapid increases in c-fos and junB mRNA in PC12 and B5P cells, while increases in c-jun were small. Using DNA-protein gel shift assays we determined that nerve growth factor stimulates AP-1 binding in both PC12 and B5P cells, and identified c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD in AP-1 complexes. In Fos/Jun functional luciferase reporter assays, nerve growth factor stimulated phosphorylation of c-Fos in both PC12 and B5P cells, but phosphorylation of c-Jun only in PC12, and not in B5P cells. These data indicate that mechanisms relating to AP-1 transcription factor complexes underlying nerve growth factor-mediated enhancement of cholinergic gene expression may differ from those required for morphological differentiation.