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.

Gliosis is characterized by hypertrophic and hyperplastic responses of astrocytes to brain injury. To determine whether injury of astrocytes produced by an in vitro model of brain trauma activates extracellular signal-regulated protein kinase (ERK), a key regulator of cellular proliferation and differentiation, astrocytes cultured on deformable SILASTIC membranes were subjected to rapid, reversible strain (stretch)-induced injury. Activation of ERK was observed 1 min after injury, was maximal from 10 to 30 min, and remained elevated for 3 hr. Activation of ERK was dependent on the rate and magnitude of injury; maximum ERK activation was observed after a 20-60 msec, 7.5 mm membrane displacement. ERK activation was blocked by inhibiting MEK, the upstream activator of ERK. Activation of ERK was reduced when calcium influx was diminished. When extracellular ATP was hydrolyzed by apyrase or ATP/P2 receptors were blocked, injury-induced ERK activation was significantly reduced. P2 receptor antagonist studies indicated a role for P2X2 and P2Y1, but not P2X1, P2X3, or P2X7, receptors in injury-induced ERK activation. These findings demonstrate for the first time that ATP released by mechanical injury is one of the signals that triggers ERK activation and suggest a role for extracellular ATP, P2 purinergic receptors, and calcium-dependent ERK signaling in the astrocytic response to brain trauma.

TAM induces typical apoptosis in ER(+) or ER(-) human breast cancer cells. TAM induction of apoptosis in MCF-7 cells involves the estrogen receptor, and requires the synthesis of new protein and mRNA. TAM induction of apoptosis in MDA-231 cells depends primarily on protein synthesis. TAM-induced cytotoxicity and DNA damage appear to be explained in part by the induction of apoptosis.

1 Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P 2Y receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P 2Y receptors expressed in astrocytes have not been de®ned and it is not known whether all P 2Y receptor subtypes are coupled to Erk by identical or distinct signalling pathways. 2 We found that the P 2Y receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the ®rst kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. 3 Reverse transcriptase-polymerase chain reaction (RT ± PCR) studies using primer pairs for cloned rat P 2Y receptors revealed that rat cortical astrocytes express P 2Y 1 , a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P 2Y 2 and P 2Y 4 , subtypes in rats for which ATP and UTP are equipotent. Transcripts for P 2Y 6 , a pyrimidine-preferring receptor, were not detected. 4 ATP did not increase cyclic AMP levels, suggesting that P 2Y 11 , an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. 5 These signal transduction and RT ± PCR experiments reveal dierences in the activation of cRaf-1 by P 2Y receptor agonists that are inconsistent with properties of the P 2Y 1 , P 2Y 2 and P 2Y 4 receptors shown to be expressed in astrocytes, i.e. ATP =UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P 2Y receptors coupled to the Erk cascade dier from the recombinant P 2Y receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.

Abbreviations used: A2m, a-2-macroglobulin; BBB, blood-brain barrier; Cebpb, CCAAT/enhancer-binding protein b; Cdkn1a, cyclindependent kinase inhibitor 1A; Csf2rb, colony-stimulating factor 2 receptor b; FPI, fluid-percussion brain injury; GFAP, glial fibrillary acidic protein; gp130, glycoprotein 130; GCSF, granulocyte colonystimulating factor; GSK-3b, glycogen synthase kinase-3b; Il2rc, interleukin-2 receptor c; Il4ra, interleukin-4 receptor a; Jak, Janus kinase; Mmp3, matrix metalloproteinase 3; Mpl, myeloproliferative leukemia virus oncogene; Nos2, nitric oxide synthase 2; Osm, oncostatin M; qPCR, quantitative RT-PCR; STAT3, signal transducer and activator of transcription-3; Socs3, suppressor of cytokine signaling 3; SFK, Src family kinase; TBI, traumatic brain injury. Department of Neurological Surgery, The Miami Project to Cure Paralysis, University of Miami MillerSchool of Medicine, Miami, Florida, USA Abstract Astrocytes respond to trauma by stimulating inflammatory signaling. In studies of cerebral ischemia and spinal cord injury, astrocytic signaling is mediated by the cytokine receptor glycoprotein 130 (gp130) and Janus kinase (Jak) which phosphorylates the transcription factor signal transducer and activator of transcription-3 (STAT3). To determine if STAT3 is activated after traumatic brain injury (TBI), adult male Sprague-Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery, and then the ipsilateral cortex and hippocampus were analyzed at various posttraumatic time periods for up to 7 days. Western blot analyses indicated that STAT3 phosphorylation significantly increased at 30 min and lasted for 24 h post-TBI. A significant increase in gp130 and Jak2 phosphorylation was also observed. Confocal microscopy revealed that STAT3 was localized primarily within astrocytic nuclei. At 6 and 24 h post-TBI, there was also an increased expression of STAT3 pathway-related genes: suppressor of cytokine signaling 3, nitric oxide synthase 2, colony stimulating factor 2 receptor b, oncostatin M, matrix metalloproteinase 3, cyclin-dependent kinase inhibitor 1A, CCAAT/enhancer-binding protein b, interleukin-2 receptor c, interleukin-4 receptor a, and a-2-macroglobulin. These results clarify some of the signaling pathways operative in astrocytes after TBI and demonstrate that the gp130-Jak2-STAT3 signaling pathway is activated after TBI in astrocytes.