Abstract
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 microM okadaic acid for 60 min at 37 degrees C, a complete down-regulation of the receptors was observed, but no modulation occurred at 4 degrees 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.
Highlights
Protein kinase C modulates the receptor for tumor Tumor necrosis factor (TNF)’was originally characterized necrosis factor (TNF)in wide variety of different cell as a monokine that induces the necrosis of certain tumors types
The disappearance of TNF cell surface binding was temperature-dependent since exposure of cells to okadaic acid at 4 "C had no effect on the cell surface TNF receptor
U-937 cells were incubated with either PMA (5 nM) or okadaic acid (0.25 PM) for 2 h at 37 "C, washed, and thenincubated for either 0 or 8 h at 37 "C.Thereafter specific binding of TNF was determined as described under"Experimental Procedures." Cell viability of treated and untreatedcells was determined by trypan blue dye exclusion
Summary
Okadaic Acid Down-regulates the TNF Receptor-To analyze the effect of okadaic acid on the TNF receptor, U-937 cells were treated with different concentrations of this phosphataseinhibitor. The decrease in theamount of specific binding of TNF to U-937 cells correlated with the increase in okadaic acid concentration. Incubation of U-937 cells with 0.5 PM okadaic acid at 37 "C for 1h decreased more than 95% of the TNF receptors. Exposure of cells to 0.5 PM okadaic acid at 37 "C for 30 min was sufficient (Fig. 1, lower panel) to decrease the receptors by approximately 50%, and by 60 min essentially all of the TNF binding was eliminated. The disappearance of TNF cell surface binding was temperature-dependent since exposure of cells to okadaic acid at 4 "C had no effect on the cell surface TNF receptor
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