Okadaic acid induces down-modulation and shedding of tumor necrosis factor receptors: Comparison with another tumor promoter, phorbol ester

Protein kinase C modulates the receptor for tumor necrosis factor (TNF) in wide variety of different cell types. However, there is no information about the role of phosphatases in the regulation of the TNF receptor. In this report, we investigated the effect of okadaic acid, an inhibitor of serine/threonine phosphatases, on TNF receptors in U-937 cells, a human histiocytic lymphoma cell line. In our study okadaic acid induced a dose- and time-dependent down-modulation of TNF receptors. On exposure of cells to 0.5 μM okadaic acid for 60 min at 37°C, a complete down-regulation of the receptors was observed, but no modulation occurred at 4°C. Scatchard analysis of the binding data on U-937 cells revealed that okadaic acid caused a decrease in the high affinity cell surface receptor number without a significant change in the affinity constant. The down-regulation of the TNF receptor by okadaic acid was not specific to U-937 cells, as it was also observed with several other cell types. Okadaic acid had no significant effect on the internalization of the receptor, but it did induce a shedding of the TNF receptor from its cell surface. On exposure of cells to okadaic acid, a dose-and time-dependent increase in the 40-kDa polypeptide was detected in the medium with anti-p80 antibodies by Western blot analysis. The secreted product was also found to bind TNF. The mechanism by which okadaic acid down-modulates the TNF receptor appears to be quite different from that of phorbol ester. First, okadaic acid synergistically potentiated the effect of phorbol ester. Second, the phorbol ester-mediated down-modulation could be blocked by H-7 and staurosporine, well known protein kinase C inhibitors, but these inhibitors had no effect on okadaic acid-mediated response. Third, phorbol ester-mediated down-modulation of the TNF receptor was reversible after removal of the agent, but the response mediated through okadaic acid was irreversible. Fourth, the TNF receptor was down-regulated by okadaic acid but not by phorbol ester in cells depleted of protein kinase C. Thus, overall, our results demonstrate that serine/threonine phosphatases can down-modulate and induce shedding of TNF receptors by a mechanism distinct from that of protein kinase C.