The A3 adenosine receptor induces cytoskeleton rearrangement in human astrocytoma cells via a specific action on Rho proteins

Abstract

In previous studies, we have demonstrated that exposure of astroglial cells to A3 adenosine receptor agonists results in dual actions on cell survival, with "trophic" and antiapoptotic effects at nanomolar concentrations and induction of cell death at micromolar agonist concentrations. The protective actions of A3 agonists have been associated with a reinforcement of the actin cytoskeleton, which likely results in increased resistance of cells to cytotoxic stimuli. The molecular mechanisms at the basis of this effect and the signalling pathway(s) linking the A3 receptor to the actin cytoskeleton have never been elucidated. Based on previous literature data suggesting that the actin cytoskeleton is controlled by small GTP-binding proteins of the Rho family, in the study reported here we investigated the involvement of these proteins in the effects induced by A3 agonists on human astrocytoma ADF cells. The presence of the A3 adenosine receptor in these cells has been confirmed by immunoblotting analysis. As expected, exposure of human astrocytoma ADF cells to nanomolar concentrations of the selective A3 agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (CI-IB-MECA) resulted in formation of thick actin positive stress fibers. Preexposure of cells to the C3B toxin that inactivates Rho-proteins completely prevented the actin changes induced by CI-IB-MECA. Exposure to the A3 agonist also resulted in significant reduction of Rho-GDI, an inhibitory protein known to maintain Rho proteins in their inactive state, suggesting a potentiation of Rho-mediated effects. This effect was fully counteracted by the concomitant exposure to the selective A3 receptor antagonist MRS1191. These results suggest that the reinforcement of the actin cytoskeleton induced by A3 receptor agonists is mediated by an interference with the activation/inactivation cycle of Rho proteins, which may, therefore, represent a biological target for the identification of novel neuroprotective strategies.

FIGURE 1

Schematic representation of the putative relationship between the adenosine A₃ receptor and Rho proteins. Under basal, unstimulated conditions, Rho proteins are maintained in their GDP bound inactive state by Rho-GDI. Stimulation of GDP exchange with GTP via guanine nucleotide exchange factors (GEF) results in release of Rho-GDI, binding of Rho to Rho-GDS and migration of the activated Rho protein to the membrane. Here, Rho can interact with its targets (mainly kinases) that in turn activate a number of proteins, including integrins, CD44, and proteins involved in regulation of the actin cytoskeleton, such as ERM (Ezrin/Radixin/Moesin). Interaction with actin promotes its polymerization and the formation of F-actin stress fibers (as an example, see FIG. 3 b). Return of Rho protein to its inactive state is favoured by proteins that promote the hydrolysis of GTP to GDP (Rho-GAPs) and binding to Rho-GDI. These results suggest that stimulation of the A₃ receptor, likely through PLC, can influence the activation/inactivation cycle of Rho proteins. This could be achieved by either a direct stimulation of GEFs and/or by modulation of Rho-GDI. Data show that exposure to A₃ agonists reduces Rho-GDI availability, by either reducing its expression or by promoting conformational changes that decrease its ability to bind to Rho. This would lead to a potentiation of Rho-mediated effects (thick arrows). See text for further details.

FIGURE 2

Detection of the A₃ adenosine receptor (A₃AR) by immunoblotting analysis in human astrocytoma ADF cells. After cell lysis, the A₃ receptor was detected by immunoprecipitation with a specific antibody and Protein A-sepharose, followed by protein resolution on 11% SDS polyacrilamide gels as described in Materials and Methods. CHO cells transfected with the human A₃ receptor cDNA were used as a positive control. Under such conditions, the A₃ receptor can be detected in both ADF and CHO cells as a specific immunoreactive protein band with an apparent molecular weight of 36 kDa (arrow).

FIGURE 3

Inhibition of Rho prevented Cl-IB-MECA-induced reinforcement of the actin cytoskeleton. In comparison to control cells (a), exposure of ADF cells to 100 nM Cl-IB-MECA for 48 h (b) resulted in marked formation of thick actin stress fibers. Exposure of ADF cells to C3B toxin (c) provoked the retraction of the cell body and the break-down of the actin cytoskeleton. Pretreatment of cells with C3B toxin for three hours before exposure to Cl-IB-MECA (d) fully prevented the actin changes induced by the A₃ agonist.

FIGURE 4

Reduction of Rho-GDI in cultures exposed to Cl-IB-MECA. Cells were exposed to 100 nM Cl-IB-MECA for 48 h in the absence or presence of MRS1191, as indicated. For the Rho-GDI immunoblotting analysis shown in A, cells were lysed, homogenized, proteins separated by SDS-PAGE, and Rho-GDI identified by immunoblot analysis as described in Materials and Methods. For the Rho-GDI immunoprecipitation experiments shown in B, cell homogenates were incubated with the anti-Rho-GDI antibody overnight, and immune complexes separated by centrifugation as described in Materials and Methods. Thus, SDS-PAGE and immunoblot analysis were performed on both supernatants and precipitates. *p < 0.05 with respect to control, **p < 0.05 with respect to control, and Cl-IB-MECA alone, one way ANOVA (Fisher test).