CNTF is almost exclusively produced in the nervous system and can rescue various types of adult CNS neurons in disease models [45C47]. JNK, and ERK1/2 abolished the effect of tryptase on TNF-production, suggesting that different signaling pathways are involved. Moreover, tryptase-induced activation of MAPKs and AKT was eliminated by FS, implicating that PAR-2 is responsible for transmitting tryptase biosignals to MAPKs and AKT. Tryptase provoked also expression of TGF-and CNTF in astrocytes. The present findings suggest for the first time that tryptase can regulate the release of cytokines from astrocytes via PAR-2-MAPKs or PAR-2-PI3K/AKT signaling pathways, which discloses PAR-2 as a new target actively participating in the 1-Azakenpaullone regulation of astrocytic functions. 1. Introduction As a unique family of G protein-coupled receptors, newfound protease-activated receptors (PARs) are widely expressed around the cells in central nervous system (CNS), including neurons and glial cells [1], regulating cell responses to extracellular serine proteases as cell surface sensors and contributing extensively to the regulation of homeostasis as well as to the dysfunctional responses of these cells required for progression of cerebral diseases [2]. Among the four PARs identified to date, PAR-2 is a unique one activated by trypsin and mast cell tryptase while others (PAR-1, -3, and -4) activated by thrombin [3]. The role of PAR-2, which is usually distributed extensively throughout the nervous system (including CNS and peripheral nervous system), has been principally investigated in peripheral nervous system, where it is known to play major functions in injury, inflammation, neuronal signaling, and nociception [4, 5]. And the physiological role of PAR-2 in CNS remains unclear but its activation has been shown to increase intracellular Ca2+ levels in both neurons and astrocytes [6, H3/h 7] as well as trigger the release of gliotransmitters such as GRO/CINC-1 [8C10] and nitric oxide [11]. Recent group of evidence have revealed that PAR-2 contributes to neuroprotection and/or neurodegeneration in the brain under pathological conditions [12C15]. Therefore, PAR-2 has been suggested to be a novel therapeutic target for the treatment of brain disorders. Tryptase, the major secretory protein of mast cells, is the natural agonist of PAR-2 and can stimulate peripheral mononuclear cells to secrete tumor necrosis factor-alpha (TNF-(TGF-Immunoassay Kit were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). LIVE green reactive oxygen species detection kit was purchased from Molecular Probes Invitrogen (Carlsbad, CA, USA). Specific glial fibrillary acid protein (GFAP) antibody (a marker for astrocytes) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Specific monoclonal antibodies against p38, phospho-p38, SAPK/JNK (c-JUN N-terminal kinase), phospho-SAPK/JNK, p44/42 MAPK (extracellular regulated protein kinases, ERK), phospho-p44/42 MAPK (phospho-ERK) and AKT, and phospho-AKT were obtained from Cell Signaling (Beverly, MA, USA). Specific polyclonal antibodies against TGF-and CNTF were 1-Azakenpaullone purchased from Abcam (Cambridge, MA, UK). 2.2. Primary Astrocyte Cultures Confluent primary astrocyte cultures were prepared from Sprague-Dawley rats as previously described with slight modification. All animal procedures were performed according to the NIH Guide for Animal Care and approved by the institutional animal care 1-Azakenpaullone and use committee. Briefly, postnatal (P1-P2) rats were killed by rapid decapitation, cerebral cortices were triturated and cells were plated on poly-D-lysine precoated culture flasks in DMEM, containing 10%?FBS, 100?U/mL penicillin, and 100?mg/mL streptomycin. Cultures were maintained at 37C in a humidified atmosphere 1-Azakenpaullone of 5% CO2/95% air. Culture medium was replaced 24?h later and then changed every 2-3 days. After reaching a confluent monolayer of cells (10C14 days), microglia were eliminated from astrocytes by shaking off for 5?h at 100?r.p.m. and astrocytes were replated in poly-D-lysine coated culture dishes, 96-well or 6-well plates. The enriched astrocytes were 98% pure as determined by astrocytic marker GFAP. 2.3. Cell Proliferation Assay Cell viability was measured by conversion of Dojindo’s highly water-soluble tetrazolium salt WST-8 to a yellow-colored water-soluble formazan (CCK8 assay). The amount of formazan dye generated by the activity of mitochondrial dehydrogenases in cells is directly proportional to the number of living cells. CCK8 is more sensitive than the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay [26]. Cells were collected and seeded in 96-well plates.