Rosiglitazone-induced SERCA2b inhibition: implications for monocyte cytoskeletal remodelling and diabetic microangiopathy

Abstract

In type 2 diabetes, exposure of monocytic cells to inflammatory stimuli can lead to aberrant cytoskeletal remodelling and contribute to the development of diabetic microangiopathy. Previous studies within our research group have demonstrated that Rosiglitazone significantly reduces formyl-methionyl-leucyl-proline (f-MLP)-induced actin polymerization in monocytes by increasing cytosolic calcium ion concentrations ([Ca]i) in both resting and f-MLP-stimulated cells. As the timescale of this effect resembled inhibition of SERCA2b, the ‘housekeeping’ Ca2þ pump enzyme that sequesters Ca2þ into the ER compensatory to non-specific leakage into the cytoplasm, the present study sought to test the hypothesis that Rosiglitazone-induced increases in [Ca]i are brought about by inhibition of SERCA2b, and also investigated how such changes are transduced into cytoskeletal remodelling. Coupled-enzyme Ca2þ-ATPase assays revealed a SERCA2b activity of 49+ 5 nmol/mg/min in monocytic microsomes but pre-incubation with Rosiglitazone (20 mM; 30 min) induced statistically significant inhibition of this activity (P, 0.05). Permeabilization of cultured monocytes in external solutions containing differing [Ca]free was used to manipulate [Ca ]i, and F-actin content measured using flow cytometric analysis of FITC-phalloidin fluorescence. At [Ca]i .100 nM, a significant decrease in cells’ F-actin content was observed (P, 0.05). In conditions of minimal [Ca]i, f-MLP exerted a polymerizing effect on the actin cytoskeleton; however, as [Ca]i was increased to .100 nM, this polymerizing effect was significantly reduced. Finally, as a literature search identified the Ca2þ-dependent actin-modulating protein gelsolin as a candidate for transducing changes in [Ca]i into actin cytoskeletal remodelling, western blot experiments using anti-gelsolin antibodies were used to detect 88 kDa immunogenic bands in monocytic protein extracts, thus confirming the expression of gelsolin in monocytes. In conclusion, the current study demonstrates the importance of [Ca]i in modulating actin cytoskeletal remodelling, and suggests that Rosiglitazone, via its ability to affect monocytic Ca2þ signalling processes, may confer a level of protection against the development of diabetic microangiopathy.