title: IL-1beta induces MMP-9 expression via a Ca2+-dependent CaMKII/JNK/c-JUN cascade in rat brain astrocytes.
abstract: Interleukin (IL)-1beta has been shown to induce matrix metalloproteinase (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 Ca(2+)-dependent CaMKII is associated with MMP-9 expression induced by IL-1beta. Here, we report that the Ca(2+)/CaMKII/JNK/c-Jun participates in the MMP-9 expression induced by IL-1beta. Zymographic, Western blotting, and RT-PCR analyses showed that IL-1beta-induced expression of MMP-9 mRNA and protein was attenuated by Ca(2+) chelator (BAPTA), and the inhibitors of ER Ca(2+)-ATPase (thapsigargin), CaMKII (KN-62), and JNK1/2 (SP600125). IL-1beta also stimulated phosphorylation of CaMKII and JNK1/2, and increase in intracellular Ca(2+) ([Ca(2+)](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-1beta stimulated c-Jun associated with AP-1-binding sites within MMP-9 promoter (-87 to -80 bp 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-1beta-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 Ca(2+)-dependent CaMKII pathway that promoted transcription factor c-Jun/AP-1 recruitment and eventually led to increase in MMP-9 expression by IL-1beta.
authors.affiliation: Department of Physiology and Pharmacology, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
authors.forename: Cheng-Ying
authors.initials: CY
authors.lastname: Wu
authors.forename: Hsi-Lung
authors.initials: HL
authors.lastname: Hsieh
authors.forename: Chi-Chin
authors.initials: CC
authors.lastname: Sun
authors.forename: Chuen-Mao
authors.initials: CM
authors.lastname: Yang
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D015180:Electrophoresis, Gel, Two-Dimensional; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053583:Interleukin-1beta / Q000378:metabolism / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D015854:Up-Regulation
chemical_list: D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048031:JNK Mitogen-Activated Protein Kinases; D020780:Matrix Metalloproteinase 9; D002118:Calcium