Characterization of retinoic acid-induced AP-1 activity in B16 mouse melanoma cells.
Retinoic acid (RA) induces differentiation of B16 mouse melanoma cells, which is accompanied by an increase in protein kinase Calpha (PKCalpha) as well as a selective enrichment of nuclear PKCalpha. We report here that RA also increases AP-1 activity in these cells. Transient transfection of B16 cells with luciferase reporter gene constructs indicated that RA induced a concentration-dependent increase in AP-1 activity. Acute treatment (2 h) of B16 cells with phorbol dibutyrate (PDB) increased AP-1 activity by 10-fold. RA treatment did not change the expression of Jun family members; however, it decreased the expression of c-Fos. In contrast acute PDB treatment induced c-Fos expression, while having little effect on c-Jun. Five DNA-protein complexes were formed with nuclear extracts from B16 cells and an oligonucleotide containing an AP-1 consensus sequence. Several complexes were decreased in cells treated with RA. Conversely, certain complexes were increased in cells acutely treated with PDB. The slowest migrating complexes were shown to contain Fos family members. Down-regulation of PKC inhibited both the acute PDB-induced and the RA-induced increase in AP-1 activity. The selective PKC enzyme inhibitor, bisindolylmaleimide, reduced PDB-stimulated AP-1 activity, but enhanced RA-induced AP-1 activity. These results together with our previous studies suggest the intriguing possibility that PKC protein, but not enzyme activity, may be required for RA-induced AP-1 activity.
Desai
S H
SH
Department of Biochemistry and Molecular Biology, Marshall University School of Medicine, Huntington, West Virginia 25755, USA.
Niles
R M
RM
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D007641:Keratolytic Agents / Q000494:pharmacology*; D008545:Melanoma / Q000378:metabolism*; D051379:Mice; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D014212:Tretinoin / Q000494:pharmacology*
D007527:Isoenzymes; D007641:Keratolytic Agents; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014212:Tretinoin; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha