title: Thrombin mediates migration of rat brain astrocytes via PLC, Ca²⁺, CaMKII, PKCα, and AP-1-dependent matrix metalloproteinase-9 expression.
abstract: Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain unclear. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 and migration of RBA-1 cells, which were inhibited by pretreatment with the inhibitor of Gq-coupled receptor (GPAnt2A), Gi/o-coupled receptor (GPAnt2), PC-PLC (D609), PI-PLC (U73122), Ca(2+)-ATPase (thapsigargin, TG), calmodulin (CaMI), CaMKII (KN62), PKC (Gö6976 or GF109203X), MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) or the intracellular calcium chelator (BAPTA/AM) and transfection with siRNA of PKCα, Erk2, JNK1, p38 MAPK, c-Jun, or c-Fos. In addition, thrombin-induced elevation of intracellular Ca(2+) concentration was attenuated by PPACK (a thrombin inhibitor). Thrombin further induced CaMKII phosphorylation and PKCα translocation, which were inhibited by U73122, D609, KN62, TG, or BAPTA/AM. Thrombin also induced PKCα-dependent p42/p44 MAPK and JNK1/2, but not p38 MAPK activation. Finally, we showed that thrombin enhanced c-Fos expression and c-Jun phosphorylation. c-Fos mRNA levels induced by thrombin were reduced by PD98059, SP600125, and Gö6976, but not SB202190. Thrombin stimulated in vivo binding of c-Fos to the MMP-9 promoter, which was reduced by pretreatment with SP600125 or PD98059, but not SB202190. These results concluded that thrombin activated a PLC/Ca(2+)/CaMKII/PKCα/p42/p44 MAPK and JNK1/2 pathway, which in turn triggered AP-1 activation and ultimately induced MMP-9 expression in RBA-1 cells.
authors.affiliation: Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
authors.forename: Chih-Chung
authors.initials: CC
authors.lastname: Lin
authors.forename: I-Ta
authors.initials: IT
authors.lastname: Lee
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Wu
authors.forename: Chiung-Ju
authors.initials: CJ
authors.lastname: Liu
authors.forename: Hsi-Lung
authors.initials: HL
authors.lastname: Hsieh
authors.forename: Li-Der
authors.initials: LD
authors.lastname: Hsiao
authors.forename: Chien-Chung
authors.initials: CC
authors.lastname: Yang
authors.forename: Chuen-Mao
authors.initials: CM
authors.lastname: Yang
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D001921:Brain / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D004792:Enzyme Precursors; D043562:Receptors, G-Protein-Coupled; D018808:Transcription Factor AP-1; D051571:Protein Kinase C-alpha; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; D013917:Thrombin; C120487:pro-matrix metalloproteinase 9; D020780:Matrix Metalloproteinase 9; D002118:Calcium