Inhibitors of DAG metabolism suppress CCR2 signalling in human monocytes.

ABSTRACT

BACKGROUND AND PURPOSE: CCL2 is an inflammatory chemokine that stimulates the recruitment of monocytes into tissue via activation of the GPCR CCR2. EXPERIMENTAL APPROACH: Freshly isolated human monocytes and THP-1 cells were used. Fura-2 loaded cells were used to measure intracellular Ca2+ responses. Transwell migration to measure chemotaxis. siRNA-mediated gene knock-down was used to support pharmacological approaches. KEY RESULTS: CCL2 evoked intracellular Ca2+ signals and stimulated migration in THP-1 monocytic cells and human CD14+ monocytes in a CCR2-dependent fashion. Attenuation of DAG catabolism in monocytes by inhibiting DAG kinase (R59949) or DAG lipase (RHC80267) activity suppressed CCL2-evoked Ca2+ signalling and transwell migration in monocytes. These effects were not due to a reduction in the number of cell surface CCR2. The effect of inhibiting DAG kinase or DAG lipase could be mimicked by addition of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) but was not rescued by application of exogenous phosphatidylinositol 4,5-bisphosphate. Suppressive effects of R59949, RHC80267, and OAG were partially or fully reversed by Gö6983 (pan PKC isoenzyme inhibitor) but not by Gö6976 (PKCα and PKCß inhibitor). RNAi-mediated knock-down of DAG kinase α isoenzyme modulated CCL2-evoked Ca2+ responses in THP-1 cells. CONCLUSIONS AND IMPLICATIONS Taken together, these data suggest that DAG production resulting from CCR2 activation is metabolised by both DAG kinase and DAG lipase pathways in monocytes and that pharmacological inhibition of DAG catabolism or application suppresses signalling on the CCL2-CCR2 axis via a mechanism dependent upon a PKC isoenzyme that is sensitive to Gö6983 but not Gö6976.