Figure 3

LTA-induced Ca²⁺ release from internal TG-sensitive Ca²⁺ store plays a role in LTA-induced MMP-9 expression. (A, B) Cells were pretreated with BAPTA/AM or TG for 1 h and then incubated with 50 μg/ml LTA for 24 h. Conditioned media were collected and analyzed by gelatin zymography. The cell lysates were assayed by western blot using an anti-GAPDH antibody as a control. (C-E) For Ca²⁺ mobilization, confluent cultures of RBA-1 cells on glass coverslips were loaded with Fura-2/AM and fluorescent measurement of [Ca²⁺]_i was carried out in a dual excitation wavelength spectrophotometer, with excitation at 340 nm and 380 nm. (C) Cells were incubated in Ca²⁺-containing normal buffer or (D) Ca²⁺-free buffer and then exposed to LTA at 100 s. (E) In a Ca²⁺-free buffer, cells were pretreated with 1 μM TG for 3 min, exposed to LTA at 100 s, and then 2 mM Ca²⁺ was added to the cells. The figure represents one of at least five similar experiments. (F) Effects of calcium inhibitors on LTA-induced phosphorylation of JNK and c-Jun, RBA-1 cells were pretreated with BAPTA or TG for 1 h and then incubated with 50 mg/ml LTA for the indicated time intervals. Phosphorylation of JNK and c-Jun was determined by western blot using an anti-phospho-JNK or phospho-c-Jun antibody. Data are expressed as mean ± SEM (A, B) or mean (F) of three independent experiments (n = 3). *P < 0.05; ^#P < 0.01, as compared with the cells exposed to LTA alone. The figure represents one of at least three individual experiments.