| Summary: | 博士 === 長庚大學 === 生物醫學研究所 === 99 === The enterovirus 71 (EV71) causes severe neurological diseases that are mediated through cyclooxygenase-2 (COX-2) expression in brain. However, the mechanisms underlying EV71-initiated intracellular signaling pathways leading to COX-2 expression remain unknown in neurons. Here, we report that exposure of SK-N-SH cells to EV71 increased COX-2 expression and PGE2 generation in a time- and virus titer-dependent manner, revealed by Western blot, real-time PCR, and PGE2 analyses. These EV71-induced responses were mediated through activation of c-Src/EGFR, p42/p44 MAPK, p38 MAPK, JNK, NF-κB, AP-1, and CREB revealed by using selective pharmacological inhibitors or transfection with respective siRNAs. Consistently, EV71-stimulated translocation of NF-κB into the nucleus and degradation of IκBα in the cytosol was blocked by pretreatment with the selective inhibitors of MEK1/2 (U0126) and NF-κB (Bay11-7085), respectively, suggesting that MEK1/2-p42/p44 MAPK cascade linking to NF-κB was involved in COX-2 expression. EV71-induced AP-1 subunits (c-jun and c-fos mRNA) expression was attenuated by pretreatment with a selective JNK inhibitor SP600125, suggesting that JNK linking to AP-1 was involved in COX-2 expression induced by EV71. In addition, EV71 activates the c-Src/EGFR/p42/p44 MAPK pathway, which induces the association between p300 and CREB, revealed by Western blot and Chromatin immnoprecipitation analyses coupling with pharmacological inhibitors and transfection with siRNAs. Furthermore, EV71 replication was inhibited by pretreatment of SK-N-SH cells with a selective inhibitor of COX-2 activity (celecoxib) or transfection with COX-2 siRNA. However, treatment with PGE2 or forskolin enhanced the replication of EV71, which was significantly inhibited by treatment with H89 (an inhibitor of PKA). Moreover, pretreatment with either AH6809 (an inhibitor of EP2 receptor) or GW627368x (an inhibitor of EP4 receptor) inhibited PGE2-induced viral replication. These findings suggested that EV71-induced COX-2 expression is mediated through c-Src/EGFR, p42/p44 MAPK, p38 MAPK, JNK, NF-κB, AP-1, and CREB. Phosphorylated CREB in turn interacts with p300 and the resulting complex binds to the promoter region of COX-2. The expression of COX-2 and the concomitant release of PGE2 could subsequently enhance viral replication via EP2 and EP4 receptors-dependent cAMP signaling.
Oxidative stress has emerged as a key player in the development and the progression of many pathological conditions, including virus-induced encephalitis. Heme oxygenase-1 (HO-1) plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. Therefore, we investigated the induction of HO-1 level of host cells, which may exert a beneficial effect to minimize viral replication in SK-N-SH cells. In this study, we found that Enterovirus 71 (EV71)-induced the generation of reactive oxygen species (ROS) was measured by using a fluorescent probe CM-H2DCFDA. EV71-induced ROS generation was mediated through activation of integrin β1, EGFR, Rac-1, and NADPH oxidase, revealed by using selective pharmacological inhibitors or transfection with respective siRNAs. In addition, the reduction of viral load was observed by using NADPH oxidase inhibitors (apocynin and diphenyleneiodonium chloride) and ROS scavenger (N-acetylcysteine), or transfection with p47phox siRNA, revealed by Western blot and real-time PCR analyses. Consistently, overexpression of HO-1 attenuated EV71-induced NADPH oxidase/ROS generation and EV71 replication, which were abrogated by pretreatment with an HO-1 inhibitor, zinc protoporphyrin IX (ZnPP IX). Moreover, metabolite of HO-1, carbon monoxide (CO), also diminished ROS formation and EV71 replication, which were reversed by pretreatment with a CO scavenger (hemoglobin). These findings suggest that in SK-N-SH cells, up-regulation of HO-1 serves as a host cellular defense mechanism against EV71 infection.
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