Inhibitors of diacylglycerol metabolism suppress CCR2 receptor signalling in human monocytes

Priscilla Day, Lisa Burrows, David Richards, Samuel J. Fountain

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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 are used; Fura‐2 loaded cells used to measure intracellular Ca2+ responses; transwell migration; siRNA‐mediated gene knockdown. Key results We observed that CCL2 evokes intracellular Ca2+ signals and stimulates migration in THP‐1 monocytic cells and human CD14+ monocytes in a CCR2‐dependent fashion. Attenuation of diacylglycerol (DAG) catabolism in monocytes by inhibiting DAG kinase (R59949) or DAG lipase (RHC80267) activity suppresses CCL2‐evoked Ca2+ signalling and transwell migration in monocytes. These effects were not due to a reduction in the number of cell surface CCR2 receptors. The effect of DAG kinase or DAG lipase inhibition could be mimicked by the addition of the DAG analogue 1‐oleoyl‐2‐acetyl‐glycerol (OAG) but was not rescued by application of exogenous phosphatidylinositol 4,5‐bisphosphate. Suppressive effects of R59949, RHC80267 and OAG could be partially or fully reversed by the Gö6983 (pan PKC isoenzyme inhibitor) but not by Gö6976 (PKCα and PKCβ inhibitor). RNAi‐mediated knock‐down of DAG kinase α isoenzyme modulated CCL2‐evoked Ca2+ responses in THP‐1 cells. Conclusions & 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 isoenzymes(s) that are sensitive to Gö6983 but not Gö6976.
Original languageEnglish
Pages (from-to)2736-2749
JournalBritish Journal of Pharmacology
Issue number15
Early online date29 Apr 2019
Publication statusPublished - Aug 2019

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