Mechanisms of thrombin-induced expression of matrix metalloproteinase-9 in rat brain astrocytes

• Main Authors: Chiung Ju Liu, 劉瓊如
• Other Authors: C. M. Yang
• Format: Others
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/39086371835272691447

碩士 === 長庚大學 === 中醫學系天然藥物 === 99 === Thrombin is an important coagulation factor and implicates in the pathogenesis of brain inflammatory diseases such as ischemic stroke, intracerebral hemorrhage, Alzheimer's disease, and Parkinson’s disease. This process may be due to the induction of matrix metalloproteinases-9 (MMP-9) by thrombin in brain. However, the molecular mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA) cells which contributes to inflammatory responses are largely unknown. Western blotting, real-time PCR, and promoter analyses showed that thrombin-induced MMP-9 expression was attenuated by pretreatment with the inhibitors of thrombin proteolytic activity (PPACK), Gi/o protein (GPAnt2), Gq protein (GPAnt2A), MEK1/2 (PD98059), p38 (SB202190), JNK1/2 (SP600125), NF-kB (Bay117082), PI-PLC (U73122), PC-PLC (D609), intracellular/extracellular Ca2+ chelators (BAPTA-AM/EDTA), CaM (CaMI), CaMKII (KN-62), non-selective PKC (GF109203X), Ca2+-dependent PKC (Gö6976), PKC-δ (rottlerin), c-Src (PP1), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), AP-1 (Tanshinone IIA), Jak2 (AG490), and transfection with siRNAs of c-Jun, c-Fos and p65. Moreover, thrombin stimulated phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 which was attenuated by the inhibitors of MEK1/2 (PD98059), p38 MAPK (SB202190), and JNK1/2 (SP600125). Thrombin stimulated both IkBa degradation and NF-kB (p65) nuclear translocation in these cells. Pretreatment with the MAPK inhibitors decreased NF-kB (p65) nuclear translocation stimulated by thrombin. Moreover, pretreatment with PPACK, GPAnt2, and GPAnt2A attenuated thrombin-induced phosphorylation of these kinases, indicating the involvements of Gi/o and Gq proteins, and cross linkage between MAPKs, Ca2+/CaM/CaMKII, RTK transactivation, and Jak2 in these responses. Taken together, these results suggested that in RBA cells, thrombin might activate a GPCR (i.e. protease-activated receptors) coupling to protein Gi/o or Gq protein, PLCs, Ca2+/CaM/CaMKII, PKCs, RTKs, Jak2, and MAPK pathways, leading to NF-kB (p65) nuclear translocation and AP-1 activity to induce MMP-9 expression. Understanding the mechanisms underlying MMP-9 gene regulation may contribute to the therapeutic strategies of brain inflammation.