Differential effects of P2Y₁ and P2Y₁₂ nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3‐kinase signalling and cell proliferation in serum‐deprived and nonstarved glioma C6 cells

Abstract

1 We have previously shown that, in glioma C6 cells, two nucleotide ADP-sensitive receptors coexist: P2Y 1 , coupled to PLC and responsible for Ca 2 þ release, and P2Y 12 , negatively coupled to adenylate cyclase. In the present study, we examined the effects of the stimulation of these two receptors on ERK1/2 and PI3-K activation, and cell proliferation in either serum-deprived or nonstarved C6 cells. 2 In response to ADP and its analogues, in serum-starved cells, both p44 ERK1 and p42 ERK2 were activated in a time-dependent manner, as monitored by Western blot analysis using an antiphosphop42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca 2 þ and partially or almost completely by MRS2179 or AR-C69931MX, specific antagonists of the P2Y 1 and P2Y 12 receptors, respectively. The inhibitory effect of antagonists was additive. These data indicate the involvement of both receptors, P2Y 1 and P2Y 12 , in the ERK1/2 activation, but the P2Y 12 receptor contribution predominates. 3 ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. 4 In nonstarved cells, ADP markedly decreased the PI3-K activity. In contrast, in serum-starved cells, ADP evoked an increase in the PI3-K activity. Blocking of the P2Y 1 receptor by MRS2179 additionally increased this ADP response. These results suggest that the P2Y 1 receptor has an inhibitory and the P2Y 12 receptor a stimulatory effect on PI3-K signalling pathway. 5 RT-PCR analysis revealed different mRNA expression of both receptors in starved and nonstarved cells. In nonstarved cells, the P2Y 1 receptor mRNA predominates, whereas in serumdeprived cells the expression of P2Y 12 mRNA becomes more pronounced.

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

“…The P2Y 12 receptor was not expressed in control astrocytoma cells. As a positive control experiment, we also detected the P2Y 12 receptor in rat C6 glioma cells, as reported [15]. Similar positive results were shown for detection of the hP2Y 1 receptor in astrocytoma cells stably transfected with this subtype (data not shown).…”

P2Y1 Gene Deficiency Protects from Renal Disease Progression and Capillary Rarefaction during Passive Crescentic Glomerulonephritis

“…Although no clear-cut studies are available regarding P2Y1-mediated proliferation and apoptosis in endothelial cells in vitro, studies in glioma C6 (43) and astrocytoma (44) cells suggest P2Y1 receptor-mediated effects that are consistent with our findings in vivo. Nucleotidemediated cell proliferation in the glioma cell line C6 (43) could be enhanced via P2Y1 blockade, whereas apoptosis of an astrocytoma cell line was stimulated after de novo introduction of the P2Y1 receptor (44).…”

Effects of P2Y1 receptor on glial fibrillary acidic protein and glial cell line-derived neurotrophic factor production of astrocytes under ischemic condition and the related signaling pathways

“…ATP released by injuried astrocytes activates ERK through P2X 2 and P2Y 1 [25] . ADP can activate ERK1/ERK2 through P2Y 1 and P2Y 12 which leads to cell proliferation of cultured glioma C6 cells [26] . Activation of the JAK/STAT pathway following transient focal cerebral ischemia is mediated through JAK1 and STAT3 in astrocytes, and this signaling pathway involves in the astroglial response to focal cerebral is-chemia [27] .…”