Ssion via a c-Jun/AP-1 signal pathway.LTA-induced proMMP-9 expression requires
Ssion via a c-Jun/AP-1 signal pathway.LTA-induced proMMP-9 expression requires JNK1/2 activationData were estimated using a GraphPad Prism Program (GraphPad, San Diego, CA, USA). Quantitative data were analyzed using ANOVA followed by Tukey’s honestly Chloroquine (diphosphate)MedChemExpress Chloroquine (diphosphate) significant difference tests between individual groups. Data were expressed as mean ?SEM. A value of P < 0.05 was considered significant.ResultsLTA-induced proMMP-9 expression is mediated through c-Jun/AP-Recently, we have demonstrated that LTA induces proMMP-9 up-regulation in astrocytes [30]. Moreover, MMP-9 promoter contains AP-1 binding sites that are essential for induction of several inflammatory genes such as MMP-9 [22,34]. Therefore, we first determined whether AP-1 was involved in LTA-induced proMMP-9 expression in RBA-1 cells, an AP-1 inhibitor tanshinone IIA (TSIIA) was used. The concentration of LTA at 50 g/ml was used throughout this study according to our previous report (Hsieh et al., 2010) [30]. The conditioned media were collected and analyzed for de novo synthesis and activity of MMPs by gelatin zymography. As shown in Figure 1A, pretreatment with TSIIA (0.110 M) significantly attenuated LTA-induced proMMP9 expression and activity. Moreover, pretreatment with TSIIA (10 M) also markedly inhibited LTA (50 g/ml, 16 h)-induced MMP-9 mRNA expression, determined by RT-PCR (Figure 1B), suggesting that AP-1 is an important factor in LTA-induced proMMP-9 expression. To further determine whether an AP-1 subunit c-Jun was essential for LTA-induced proMMP-9 expression, cells were incubated with 50 g/ml LTA for the indicated time intervals. As shown in Figure PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26577270 1C (upper panel), LTA stimulated phosphorylation of c-Jun in a time-dependent manner. There was a significant increase within 60 min and reached a maximal response by 90 min. Pretreatment with TSIIA (10 M) attenuated LTA-stimulated c-Jun phosphorylation (Figure 1C, lower panel). To confirm the crucial role of c-Jun in these responses, as shown in Figure 1D, transfection with cJun shRNA for 24 h down-regulated endogenous c-Jun protein expression (upper panel), and significantly attenuated LTA-induced proMMP-9 expression in RBA-1 cells (lower panel). These results suggested that LTASeveral studies have demonstrated that JNK, a member of MAPK family, mediates up-regulation of MMP-9 in RBA-1 cells [20,25]. Thus, to investigate whether JNK1/ 2 also involved in LTA-induced proMMP-9 expression, a pharmacological inhibitor of JNK1/2, SP600125 was used. As shown in Figure 2A, pretreatment with SP600125 (1 M) significantly inhibited the LTAinduced proMMP-9 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26437915 expression during the period of observation. Moreover, LTA-induced MMP-9 mRNA expression was also significantly blocked by pretreatment with SP600125, determined by RT-PCR (Figure 2B). To further determine whether LTA-induced proMMP-9 expression was mediated through JNK1/2 phosphorylation, the kinetics of JNK1/2 phosphorylation stimulated by LTA was assessed by western blot using an anti-phospho-JNK1/2 antibody. As shown in Figure 2C, LTA stimulated JNK1/2 phosphorylation in a time-dependent manner with a maximal response within 60-90 min, which was significantly inhibited by pretreatment with SP600125 (1 mM) during the period of observation. Pretreatment with SP600125 (1 M) also almost completely inhibited LTA-stimulated c-Jun phosphorylation (Figure 2C), suggesting that JNK was an upstream signal molecule of c-Jun/AP-1 cascade. Thus, to further ensure that JNK was involved in LTA-induc.