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Interleukin-1β (IL-1β) plays a critical mediator in the pathogenesis of eye diseases. The implication of IL-1β in inflammatory responses has been shown to be mediated through up-regulation of inflammatory genes, including matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of IL-1β-induced MMP-9 expression in Statens Seruminstitut Rabbit Corneal Cells (SIRCs) are largely unclear. Here, we demonstrated that in SIRCs, IL-1β induced MMP-9 promoter activity and mRNA expression associated with an increase in the secretion of pro-MMP-9. IL-1β-induced pro-MMP-9 expression and MMP-9 mRNA levels were attenuated by pretreatment with the inhibitor of MEK1/2 (U0126), JNK1/2 (SP600125), NF-κB (Bay11-7082), or AP-1 (Tanshinone IIA) and transfection with siRNA of p42 or JNK2. Moreover, IL-1β markedly stimulated p42/p44 MAPK and JNK1/2 phosphorylation in SIRCs. In addition, IL-1β also enhanced p42/p44 MAPK translocation from the cytosol into the nucleus. On the other hand, IL-1β induced c-Jun and c-Fos mRNA expression, c-Jun phosphorylation, and AP-1 promoter activity. NF-κB translocation, IκBα degradation, and NF-κB promoter activity were also enhanced by IL-1β. Pretreatment with U0126 or SP600125 inhibited IL-1β-induced AP-1 and NF-κB promoter activity, but not NF-κB translocation from the cytosol into the nucleus. Finally, we established that IL-1β could stimulate SIRCs migration via p42/p44 MAPK-, JNK1/2-, AP-1-, and NF-κB-dependent MMP-9 induction. These results suggested that NF-κB and AP-1 activated by JNK1/2 and p42/p44 MAPK cascade are involved in IL-1β-induced MMP-9 expression in SIRCs.

BackgroundIn the brain, the inducible form of heme oxygenase (HO-1) has been recently demonstrated to exacerbate early brain injury produced by intracerebral hemorrhagic stroke which incident rate has been correlated with cigarette smoking previously. Interestingly, cigarette smoke (CS) or chemicals present in CS have been shown to induce HO-1 expression in various cell types, including cerebral endothelial cells. However, the mechanisms underlying CS modulating HO-1 protein expression are not completely understood in the brain vessels.ObjectiveThe aim of the present study was to investigate the mechanisms underlying CS modulating HO-1 protein expression in cerebral endothelial cells.MethodsCultured cerebral endothelial cells (bEnd.3) were used to investigate whether a particulate phase of cigarette smoke extract (PPCSE) regulates HO-1 expression and to investigate the molecular mechanisms involved in HO-1 expression in bEnd.3 cells.ResultsWe demonstrated that PPCSE (30 μg/ml) significantly induced HO-1 protein expression and its enzymatic activity in bEnd.3 cells determined by western blotting and bilirubin formation, respectively. PPCSE-induced HO-1 expression was mediated through phosphatidylcholine phospholipase C (PC-PLC), PKCδ, and PI3K/Akt which were observed by pretreatment with their respective pharmacological inhibitors or transfection with dominant negative mutants of PKCδ and Akt. ROS scavenger (N-acetyl-L-cysteine, NAC) blocked the PPCSE-induced ROS generation and HO-1 expression. Pretreatment with selective inhibitors of PKCδ (rottlerin) and NADPH oxidase [diphenyleneiodonium chloride (DPI) and apocynin (APO)] attenuated the PPCSE-induced NADPH oxidase activity, ROS generation, and HO-1 expression. In addition, we found that PPCSE induced PI3K/Akt activation via NADPH oxidase/ROS-dependent PDGFR phosphorylation.ConclusionsTaken together, these results suggested that PPCSE-induced HO-1 expression is mediated by a PC-PLC/PKCδ/NADPH oxidase-dependent PDGFR/PI3K/Akt pathway in bEnd.3 cells.

BackgroundUp-regulation of cyclooxygenase (COX)-2 and its metabolite prostaglandin E2 (PGE2) are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE2 release remain unclear.Principal FindingsHere, we showed that ATPγS induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin), PKC (Gö6983, Gö6976, Ro318220, and Rottlerin), ROS (Edaravone), NADPH oxidase [diphenyleneiodonium chloride (DPI) and apocynin], Jak2 (AG490), and STAT3 [cucurbitacin E (CBE)] and transfection with siRNAs of PKCα, PKCι, PKCμ, p47phox, Jak2, STAT3, and cPLA2 markedly reduced ATPγS-induced COX-2 expression and PGE2 production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPγS. Moreover, ATPγS-induced ROS generation and p47phox translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPγS stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, Gö6983, Gö6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells.SignificanceTaken together, these results showed that ATPγS induced COX-2 expression and PGE2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA2 signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.