title: Transient receptor potential canonical 3 (TRPC3) mediates thrombin-induced astrocyte activation and upregulates its own expression in cortical astrocytes.
abstract: Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca(2+)-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca(2+) influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca(2+) influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH(2)-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca(2+) signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca(2+) signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca(2+)-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH(2), a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own expression.
authors.affiliation: Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Shirakawa
authors.forename: Shinya
authors.initials: S
authors.lastname: Sakimoto
authors.forename: Kenji
authors.initials: K
authors.lastname: Nakao
authors.forename: Aiko
authors.initials: A
authors.lastname: Sugishita
authors.forename: Masakazu
authors.initials: M
authors.lastname: Konno
authors.forename: Shota
authors.initials: S
authors.lastname: Iida
authors.forename: Ayaka
authors.initials: A
authors.lastname: Kusano
authors.forename: Emina
authors.initials: E
authors.lastname: Hashimoto
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Shuji
authors.initials: S
authors.lastname: Kaneko
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D001896:Boron Compounds / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005911:Gliosis / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D011714:Pyrans / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000096:biosynthesis / Q000235:genetics; D050052:TRPC Cation Channels / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*
chemical_list: D001896:Boron Compounds; C550156:N-(6-(4-chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano(2,3-b)pyridin-4-yl)-5-methyl-1H-pyrazole-3-carboxamide; D009414:Nerve Growth Factors; D009842:Oligopeptides; D011714:Pyrans; D011725:Pyridines; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579159:S100b protein, mouse; C579158:S100b protein, rat; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; C515806:seryl-arginyl-leucyl-leucyl-argininamide; C109986:2-aminoethoxydiphenyl borate; D013917:Thrombin