Figure 8

LTA induces astrocytic migration through Ca²⁺/CaMKII-dependent c-Jun/AP-1 increasing MMP-9 expression. RBA-1 cells were plated on coverslips, and grown to confluence. The coverslips were transferred to new 10-cm dishes containing serum-free medium for 24 h. The cells were pretreated with TG, KN-62, SP600125, or TSIIA (containing 10 μM hydroxyurea, a cell proliferation inhibitor) for 1 h and then incubated with 50 μg/ml LTA for 48 h. Phase contrast images of RBA-1 cells were taken at 48 h showing the response to LTA (n = 3). The number of LTA-induced cell migrations at 48 h were counted and summarized in (A). (B) LTA-induced MMP-9 expression via Ca²⁺/CaMKII-dependent PDGFR/PI3K/JNK/c-Jun cascade is observed in primary culture rat brain astrocytes. The rat primary culture astrocytes were pretreated with TG, CaMI, KN-62, AG1296, LY294002, SP600125, or TSIIA for 1 h and then incubated with LTA for 24 h. The conditioned media were analyzed by zymographic analysis. Data are expressed as mean ± SEM (A) or mean (B) of three independent experiments (n = 3). *P < 0.05; ^#P < 0.01, as compared with the cells exposed to LTA alone. (C) Schematic representation of the LTA-mediated signaling pathways linked to proMMP-9 expression and cell migration in RBA-1 cells. Stimulation of LTA results in release of intracellular Ca²⁺ from internal stores and activation of CaM/CaMKII-dependent transactivation of PDGFR and PI3K-JNK cascades, leading to activation of c-Jnu/AP-1. Activated c-Jun/AP-1 turns on MMP-9 gene expression and promotes cell motility of RBA-1 cells. This signaling pathway might contribute to sustained expression of MMP-9 which is required for RBA-1 cell migration.