Abstract
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.
Highlights
Retinoic acid (RA),1 a biologically active metabolite of vitamin A, has been shown to play an essential role in maintaining the differentiated phenotype in a variety of tissues (1, 2)
In light of the 6 – 8-fold increase of PKC␣ protein in RA-treated B16 melanoma cells (10) and a selective enrichment of nuclear-associated PKC␣ (13), we determined whether RA increased AP-1 activity
B16 melanoma cells were transiently transfected with a pGL-2 plasmid containing four tandem AP-1 consensus sequences inserted 5Ј to an SV-40 promoter driving the expression of luciferase
Summary
Retinoic acid (RA),1 a biologically active metabolite of vitamin A, has been shown to play an essential role in maintaining the differentiated phenotype in a variety of tissues (1, 2). In light of the RA-induced increase in PKC␣ protein in B16 cells, we determined whether there was any change in AP-1 activity. Nuclear extracts from control and treated cells (10 g of protein) were incubated with 30,000 dpm of probe in a buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 40 mM NaCl, 12% (v/v) glycerol, and 0.5 g of poly(dI-dC) in a total reaction volume of 25 l.
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