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.
Baskey
Jacqueline C
JC
Department of Physiology, University of Western Ontario, and The John R Robarts Research Institute, London, Canada.
Kalisch
Bettina E
BE
Davis
Wanda L
WL
Meakin
Susan O
SO
Rylett
R Jane
RJ
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002795:Choline O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism; D016716:PC12 Cells; D010641:Phenotype; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D020917:Receptor, trkA / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D009569:Nitric Oxide; D020932:Nerve Growth Factor; D002795:Choline O-Acetyltransferase; D020917:Receptor, trkA; D000109:Acetylcholine