Mitogenic signaling by ATP/P2Y purinergic receptors in astrocytes: involvement of a calcium-independent protein kinase C, extracellular signal-regulated protein kinase pathway distinct from the phosphatidylinositol-specific phospholipase C/calcium pathway

Abstract

Activation of ATP/P2Y purinergic receptors stimulates proliferation of astrocytes, but the mitogenic signaling pathway linked to these G-protein-coupled receptors is unknown. We have investigated the role of extracellular signal-regulated protein kinase (ERK) in P2Y receptor-stimulated mitogenic signaling as well as the pathway that couples P2Y receptors to ERK. Downregulation of protein kinase C (PKC) in primary cultures of rat cerebral cortical astrocytes greatly reduced the ability of extracellular ATP to stimulate ERK. Because occupancy of P2Y receptors also leads to inositol phosphate formation, calcium mobilization, and PKC activation, we explored the possibility that signaling from P2Y receptors to ERK is mediated by a phosphatidylinositol-specific phospholipase C (PI-PLC)/calcium pathway. However, neither inhibition of PI-PLC nor chelation of calcium significantly reduced ATP-stimulated ERK activity. Moreover, a preferential inhibitor of calcium-dependent PKC isoforms, Gö 6976, was significantly less effective in blocking ATP-stimulated ERK activity than GF102903X, an inhibitor of both calcium-dependent and -independent PKC isoforms. Furthermore, ATP stimulated a rapid translocation of PKCdelta, a calcium-independent PKC isoform, but not PKCgamma, a calcium-dependent PKC isoform. ATP also stimulated a rapid increase in choline, and inhibition of phosphatidylcholine hydrolysis blocked ATP-evoked ERK activation. These results indicate that P2Y receptors in astrocytes are coupled independently to PI-PLC/calcium and ERK pathways and suggest that signaling from P2Y receptors to ERK involves a calcium-independent PKC isoform and hydrolysis of phosphatidylcholine by phospholipase D. In addition, we found that inhibition of ERK activation blocked extracellular ATP-stimulated DNA synthesis, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y receptors.

Fig. 1.

Signaling from P2Y receptors to ERK is dependent on PKC. Primary rat astrocyte cultures were treated with 100 μm ATP for 15 min (●–●), with 100 nm TPA for 24 hr before application of 100 μm ATP for 15 min (○–○), or were untreated (×–×). Homogenates were centrifuged, and supernatants containing equivalent amounts of protein were applied to a 1 ml Hi-Trap Q anion exchange column as described in Materials and Methods. Fractions (1 ml) were collected at a flow rate of 0.5 ml/min, and proteins were eluted with a linear, 60 ml NaCl gradient (0–400 mm). Two peaks of ERK activity eluted at 110 and 175 mm NaCl. Inset, Immunoblot of column fractions from the peak ERK regions and a fraction between the peaks. The indicated column fractions and the cellular extract (EXT) from the ATP-treated, normal cells were subjected to SDS-PAGE, and immunoblots were probed with anti-ERK1/2. The arrows indicate the two ERK isoforms p42 and p44, before and after fractionation by anion exchange chromatography. Similar results were obtained by chromatography on a 1 ml Resource Q (Amersham Pharmacia Biotech) column.

Fig. 2.

Inhibition of PI-specific PLC does not block ATP-stimulated ERK activity. In A, primary rat astrocyte cultures were treated with 10 μm U73122 for 30 min before addition of ATP (100 μm, 15 min). ERK activity data (mean ± SEM) were obtained from three experiments, each conducted with duplicate culture plates. ERK activity in untreated cultures was 43.5 ± 8.2 pmol phosphate transferred per minute per milligram of protein. In B, primary rat astrocyte cultures were treated with 10 μm U73122 for 30 min before addition of ATP (500 μm, 30 min), and inositol phosphates were extracted and measured as described in Materials and Methods.

Fig. 3.

Effects of inhibitors of calcium-independent and -dependent PKC isoforms on signaling from P2Y receptors to ERK. InA, primary rat astrocyte cultures were treated with ATP (100 μm, 5 min) or with Gö 6976 (1–5 μm, 20 min), which preferentially inhibits calcium-dependent PKC isoforms, or GF109203X (1–5 μm, 20 min), an inhibitor of both calcium-dependent and -independent PKC isoforms, before addition of ATP (100 μm, 5 min). ERK activity data were obtained from a minimum of three experiments, each conducted with duplicate culture plates. In B, primary rat astrocyte cultures were treated with ATP (100 μm, 5 min) or with Gö 6976 (2.5 μm) or GF109203X (2.5 μm) for 20 min before addition of ATP (100 μm, 5 min), cells were lysed, and lysates containing equivalent amounts of protein were subjected to SDS-PAGE. Immunoblots were probed with antibodies that recognize phosphorylated ERK1/ERK2 (top panel) or ERK1/ERK2 (bottom panel).

Fig. 4.

Extracellular ATP stimulates translocation of a calcium-independent PKC isoform. In A, primary rat astrocyte cultures were treated with or without (CON) 100 μm ATP for 15 sec, or with 100 nm TPA for 5 min, and particulate fractions were prepared as described in Materials and Methods. Equal amounts of protein (3.9 μg) were subjected to SDS-PAGE and analyzed by immunoblotting with monoclonal antibodies raised against PKCδ or PKCγ. In B, astrocytes were treated with 100 μm ATP for the times indicated, and particulate fractions were obtained. Equal amounts of protein within each experiment (ranging from 4 to 5 μg) were analyzed by immunoblotting with a monoclonal antibody raised against PKCδ. Values given are mean ± SEM; densitometric analysis (Bio-Rad GS-670) was conducted on a minimum of four to six independent experiments, each representing different culture seedings.

Fig. 5.

Extracellular ATP rapidly stimulates choline formation. Primary rat astrocyte cultures were incubated with³H-choline (2.5 μCi/ml) for 48 hr followed by 24 hr in choline-free culture medium before treatment with ATP (100 μm) for the indicated times. Choline formation was measured as described in Materials and Methods. Similar results were obtained in two additional experiments.

Fig. 6.

Inhibition of PC hydrolysis decreases the ability of ATP to stimulate ERK activity. Primary rat astrocytes were treated without or with the indicated concentrations of D609 for 60 min before addition of ATP (100 μm) for 5 min. In A, phosphorylated ERK1/ERK2 (top panel) and ERK1/ERK2 (bottom panel) were detected by immunoblotting as described in Materials and Methods. InB, ERK activity data (mean ± SEM) were obtained from three independent experiments, each representing different seedings and conducted with duplicate culture plates. “0” indicates cultures treated with ATP only. ERK activity in untreated cultures was 43.3 ± 4.9 pmol phosphate transferred per minute per milligram of protein. **p < 0.001; *p < 0.05 for comparisons of ATP versus D609 + ATP.

Fig. 7.

Extracellular ATP-evoked mitogenic signaling is mediated by the ERK/MAPK cascade. In A, primary rat astrocyte cultures were treated with ATP (100 μm, 5 min), or with PD 098059 (50 μm) for 30 min before addition of ATP (100 μm, 5 min), cells were lysed, and lysates containing equivalent amounts of protein were subjected to SDS-PAGE. Immunoblots were probed with antibodies that recognize phosphorylated ERK1/ERK2 (top panel) or ERK1/ERK2 (bottom panel). In B, primary rat astrocyte cultures were treated with ATP (100 μm), or with PD 098059 (50 μm) for 30 min before addition of ATP, and DNA synthesis was determined as described in Materials and Methods. Values are given as the mean ± SE of the mean and were obtained from three independent experiments, each conducted in quadruplicate and with different seedings. ³H-thymidine incorporation in untreated cultures was 1055 ± 358 cpm per culture well. *p < 0.001 for comparison of ATP versus PD 098059 + ATP.