Early Biochemical Steps in Colony Stimulating Factor (CSF) Generation are Induced by Synergy between Phorbol Esters and Calcium Ionophores

Abstract

In many secretory systems receptor triggering by agonists is followed by inositol phospholipid breakdown to diacylglycerol (DAG) and inositol triphosphate (InsP3). DAG activates protein kinase C (PK-C) and InsP3 mobilizes intracellular calcium. Both tumor promoting phorbol esters (TPA) which activate PK-C directly and calcium ionophores which mobilize intracellular calcium bypass inositol phospholipid breakdown. We recently reported that the interaction of TPA and bacterial lipopolysaccharide (LPS) with murine bone marrow cells (BM) is followed by generation of CSF. Optimal generation occurs when TPA and LPS are added simultaneously. To determine whether generation of CSF requires activation of PK-C and calcium mobilization we tested the ability of A23187, a calcium ionophore, to replace LPS. BM and spleen cells were stimulated with TPA and A23187 and the supernatants were assayed for CSF by measuring ³H-thymidine incorporation into a CSF dependent basophil/mast cell line, PT-18. TPA and A23187 acted cooperatively to stimulate generation of CSF similar to the action of TPA and LPS. In addition, trimethoxybenzoate (TMB-8), an inhibitor of calcium mobilization, inhibited CSF production either by TPA and LPS or by TPA and A23187. Synthetic DAG was able to replace TPA in stimulating spleen cells together with A23187 to generate CSF. Generation of CSF by spleen cells can be inhibited by TMB-8 only when added to the cells up to 10 min after stimulation with TPA and A23187. Later addition of TMB-8 had no effect. The results reported suggest that calcium mobilization and activation of PK-C are early biochemical events in the sequence leading to the generation of CSF.