IL-1 beta Induces MMP-9 Expression Via a Ca2+-Dependent CaMKII/JNK/c-Jun Cascade in Rat Brain Astrocytes

Interleukin (IL)-1 beta has been shown to induce matrix metal-loproteinase (MMP)-9 expression through mitogen-activated protein kinases, including JNK, in rat brain astrocyte-1 (RBA-1) cells. However, little is known about whether JNK activated by Ca2+-dependent CaMKII is associated with MMP-9 expression induced by IL-1 beta. Here, we report that the Ca2+/CaMKII/JNK/c-Jun participates in the MMP-9 expression induced by IL-1 beta. Zymographic, Western blotting, and RT-PCR analyses showed that IL-1 beta-induced expression of MMP-9 mRNA and protein was attenuated by Ca2+ chelator (BAPTA), and the inhibitors of ER Ca2+-ATPase (thapsigargin), CaMKII (KN-62), and JNK1/2 (SP600125). IL-1 beta also stimulated phosphorylation of CaMKII and JNK1/2, and increase in intracellular Ca2+([Ca2+](i)), which were inhibited by pretreatment with BAPTA, thapsigargin (TG), KN-62, or SP600125. Furthermore, the upregulation of MMP-9 protein was blocked by transfection with c-Jun or CaMKII short hairpin RNA (shRNA). We further confirmed that IL-1 beta stimulated c-Jun associated with AP-1-binding sites within MMP-9 promoter (-87 to -80 by and -511 to -497 bp) by immunoprecipitation and chromatin immunoprecipitation (ChIP)-PCR assays. The activation and recruitment of c-Jun to MMP-9 promoter were inhibited by pretreatment with BAPTA, TG, KN-62, or SP600125. Moreover, IL-1 beta-induced MMP-9 gene transcription by AP-1 was confirmed by transfection with a MMP-9 promoter-luciferase reporter plasmid with a distal AP-1-binding site (-511 to -497 bp) adjacent to an Ets-binding site-mutation (mt-AP1/Ets-MMP-9). These results demonstrated that in RBA-1 cells, JNK/c-Jun activation was mediated through a Ca2+-dependent CaMKII pathway that promoted transcription factor c-Jun/AP-1 recruitment and eventually led to increase in MMP-9 expression by IL-1 beta. (C) 2009 Wiley-Liss, Inc.