EFFECTS OF CHRONIC PHORBOL ESTER TREATMENT ON PROTEIN-KINASE-C ACTIVITY, CONTENT, AND GENE-EXPRESSION IN THE HUMAN MONOBLASTOID U937 CELL

Immediate and sustained signal transduction is involved in mediating p horbol ester-induced changes in growth and differentiation. Activation of protein kinase C (PKC) is the initial step in phorbol ester-induce d signal transduction. By virtue of preferential down-regulation of in dividual isoforms and generation of proteolytically derived kinase act ivities, the signal transduced by sustained activation of this pathway may differ substantially from that generated initially upon applicati on of the phorbol ester. To examine the effect of chronic phorbol este r-induced activation of this pathway, the relationship between PKC act ivity/content and AP-1 binding activity and gene expression was studie d in the U937 cell. Phorbol ester-induced differentiation of the U937 cell into a monocyte/macrophage-like cell requires sustained activatio n of the PKC pathway. AP-1 binding activity was enhanced by 12-0-tetra decanoylphorbol-13-acetate (TPA) and in a temporally dependent manner, with conversion of a high to low mobility band shift occurring after a 12-h exposure to TPA. After a 72-h exposure, AP-1 binding activity w as maximally increased by 1 nm TPA and remained elevated to a similar degree even after treatment with 600 nm TPA. Enhanced AP-1 binding act ivity was dependent upon- continuous exposure to TPA and was not secon dary to differentiation. A 72-h treatment with one nm TPA maximally in creased expression of c-jun, krox-24, and jun-B mRNA transcripts. Expo sure to higher TPA concentrations decreased the content of these trans cripts. Maximal expression of collagenase and plasminogen activator re ceptor transcripts required exposure to much higher TPA concentrations (100 nm). Enhanced expression of mRNA transcripts required continuous exposure to phorbol esters and decreased to uninduced levels upon rem oval of this agent. A 72-h exposure to 0.1-1.0 nm TPA increased PKC ac tivity and content. Exposure to high TPA concentrations (>10 nm) decre ased PKC activity to near negligible levels and abolished detection of Ca2+-dependent isoforms. Transient transfection with a collagenase pr omoter-chloramphenicol acetyltransferase reporter construct and a PKC construct constitutively activated in the absence of TPA demonstrated that expression induced from the AP-1-containing collagenase promoter was due to PKC activation rather than down-regulation of endogenous is oforms. These results demonstrate that alterations occurring within th e PKC pathway after chronic exposure to TPA may serve as a mechanism t o enhance the diversity of cellular responses elicited by phorbol este rs.