Effect of retinoic acid on phorbol ester-stimulated differentiation and protein kinase C-dependent phosphorylation in the U937 human monoblastoid cell.

Phorbol esters stimulate differentiation of certain human leukemic cell lines. Although activation of protein kinase C may mediate certain effects of phorbol esters, controversy exists as to the role of protein kinase C activation in phorbol ester-induced differentiation. Retinoic acid modulates responses to phorbol esters in several cell types. Retinoic acid has also been found to alter protein kinase C-dependent phosphorylation in leukemic cells. We correlated the effects of retinoic acid on protein kinase C-dependent phosphorylation and differentiation stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester, in the human monoblastoid U937 cell line. At concentrations less than 1 nM, which were 100-fold less than those directly stimulating differentiation, retinoic acid potentiated TPA-induced differentiation of the U937 cell as assessed by enhanced adherence to plastic and acquisition of nonspecific esterase activity. TPA-stimulated decreases in cellular proliferation were not affected by retinoic acid treatment. Without altering the sensitivity to TPA, retinoic acid increased the maximal response to this agent. Retinoic acid enhanced TPA-stimulated phosphorylation of a Mr 48,000 substrate in intact 32P-labeled U937 cells and also increased the protein kinase C-dependent phosphorylation of a similar Mr 48,000 substrate and a Mr 80,000 substrate in cellular extracts. In cellular extracts the retinoic acid-induced enhancement of protein kinase C-dependent phosphorylation was predominantly localized to the cytosolic fraction. Increases in protein kinase C-dependent phosphorylation were evident within a 12-h exposure to 1 nM retinoic acid and were observed at retinoic concentrations of 0.01 to 1 nM. A retinoic acid-induced increase in the protein kinase C-dependent phosphorylation of an exogenous substrate, histone, was observed following diethylaminoethyl extraction of cytosol, but not a solubilized particulate fraction. The conditions of retinoic acid treatment increasing protein kinase C activity and enhancing protein kinase C-dependent phosphorylation of endogenous substrates were similar to those conditions potentiating phorbol ester-induced differentiation. Thus, the retinoic acid-induced amplification of phorbol ester signal transduction at the level of protein kinase C activation could mediate the effects of this vitamin on phorbol ester-induced differentiation.