Neurotropic viruses can cause massive neuronal dysfunction and destruction that leads to neurological diseases . Based on neural cell composition and the barrier between the peripheral tissues and CNS, astrocytes might play a role in the transmission of virus from peripheral blood flow into the CNS. Recent studies have demonstrated a relationship between elevated levels of MMP-9 and severity of several pathological states in the CNS [5, 6]. MMP-9 has been shown to degrade components of the basal lamina, leading to disruption of the BBB, and to contribute to neuroinflammatory responses in many neurological diseases . Several lines of evidence have shown that reduction of MMP activity by pharmacological inhibitors or gene knock-out strategies protects the brain from BBB disruption, cell death, and advanced neuroinflammation [5, 39]. Previous studies have indicated that several signaling cascades are involved in MMP-9 expression by virus infection [13, 14]. We have previously demonstrated that JEV infection induces MMP-9 expression via NF-κB in RBA-1 cells . Moreover, AP-1 is also known to play an important role in MMP-9 expression in various cell types . However, little is known about the molecular mechanisms of JEV-induced AP-1 activation leading to MMP-9 expression in RBA-1 cells. In this study, the mechanisms underlying JEV-induced MMP-9 expression were investigated using selective pharmacological inhibitors or transfection with siRNAs. The requirement of transcription factors for the regulation of JEV-induced MMP-9 gene expression was determined by reporter gene assays. These results demonstrate that JEV induces MMP-9 expression via a ROS, c-Src, PDGFR, PI3K/Akt, p42/p44 MAPK, p38 MAPK, and JNK1/2-dependent pathway following activation of transcription factor AP-1 (c-Jun and c-Fos) in RBA-1 cells.
Previous studies have reported that the promoter of MMP-9 possesses a series of functional activator element-binding sites, including NF-κB and AP-1 [6, 9]. In addition, AP-1 activity is enhanced by various factors including growth factors, cytokines, physical and chemical stresses, and bacterial and viral infections [21, 28]. However, AP-1 participation in MMP-9 expression is poorly understood in JEV-infected RBA-1 cells. First, we therefore determined the requirement for AP-1 in JEV-induced MMP-9 expression. Our results reveal that JEV infection stimulates expression of MMP-9, which was significantly inhibited by pretreatment with tanshinone and transfection with c-Jun siRNA and c-Fos siRNA. In addition, JEV-induced MMP-9 mRNA expression and promoter activity were attenuated by pretreatment with tanshinone or transfection with a point-mutated AP-1 MMP-9 promoter, indicating that AP-1 participates in MMP-9 expression by JEV infection in RBA-1 cells. Moreover, we demonstrated that JEV-induced AP-1 activation occurs through changes in c-Jun and c-Fos gene transcription and mRNA turnover. These results are consistent with previous studies demonstrating that enhanced expression of MMP-9 in Epstein-Barr virus (EBV)-infected or HBV-infected cells is mediated through activation of AP-1 transcriptional activity [40, 41].
Several factors enhance AP-1 activity through activation of many signaling pathways, such as PDGF-induced activation of AP-1 through p42/p44 MAPK and JNK1/2 in NIH 3T3 mouse fibroblasts [22, 23]. In addition, our previous study reported that EV71 induces AP-1 activation via a c-Src/PDGFR/PI3K/Akt cascade in RBA-1 cells . However, activation of c-Src, PDGFR, and PI3K/Akt by JEV is poorly understood in RBA-1 cells. Therefore, our results from this present study reveal that JEV infection induces expression of c-Jun and c-Fos, and that these expressions are significantly inhibited by pretreatment with AG1296, PP1, or LY294002. In accord with our recent findings of COX-2 expression with EV71 infection in RBA-1 cells , these data suggest that AP-1 activation by JEV infection is mediated through a c-Src, PDGFR, and PI3K/Akt pathway.
Next, we investigated the roles of c-Src, PDGFR, and PI3K/Akt in MMP-9 expression in RBA-1 cells. Our results show that JEV infection stimulates phosphorylation of PDGFR, which is attenuated by pretreatment with AG1296 and PP1. In addition, co-immunoprecipitation assays were performed to ensure that protein levels of p-PDGFR and p-c-Src time-dependently increase in a c-Src-immunoprecipitated complex stimulated by JEV infection, which was inhibited by pretreatment with AG1296 or PP1. Moreover, several studies have reported that Akt is activated following stimulation of receptor tyrosine kinase by different stimuli [30–32]. In addition, in rat brain astrocyte cells or neural cells, PI3K/Akt activation has been shown to be mediated through PDGFR transactivation [42–45]. In this study, pretreatment of RBA-1 cells with AG1296 or PP1 inhibited JEV-stimulated Akt phosphorylation, indicating that activation of PDGFR and c-Src are required for this response. Apart from these, pretreatment with AG1296, PP1, or LY294002; or transfection with siRNA of PDGFR or Akt significantly inhibited JEV-induced MMP-9 protein expression and mRNA accumulation. These data indicate that PI3K/Akt activation is mediated through c-Src-dependent transactivation of PDGFR, which promotes AP-1 activation and eventually leads to MMP-9 expression with JEV infection of RBA-1 cells. This result is consistent with recent studies reporting that MMP-9 expression induced by IL-1β is mediated via activation of c-Src/PDGFR/PI3K/Akt in various cell types [45, 46].
Previous studies have shown that AP-1 activation is also mediated through MAPKs signaling pathways by various factors in various cell types . In addition, our previous study has shown that JEV infection-induced MMP-9 expression is mediated via ROS-p42/p44 MAPK, p38 MAPK, and JNK1/2 in RBA-1 cells . Thus, we also investigated the roles of MAPKs in JEV-induced AP-1 activation. Our results reveal that JEV infection induces expression of c-Jun and c-Fos, which are significantly inhibited by pretreatment with U0126, SP600125, or SB203580. These data indicate that JEV-induced AP-1 activation is dependent on MAPKs in RBA-1 cells. Moreover, the MAPKs signaling cascade can be activated by growth factors such as PDGF . Therefore, we examined whether MAPKs activation by JEV infection is mediated through a c-Src/PDGFR/PI3K/Akt pathway. In this study, pretreatment with AG1296, PP1, or LY294002 inhibited JEV-stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, indicating that activation of c-Src/PDGFR/PI3K/Akt pathway by JEV infection regulates MAPKs activation in RBA-1 cells. These results suggest that expression of MMP-9 with JEV infection is mediated through a c-Src/PDGFR/PI3K/Akt/MAPKs pathway, associated with activation of transcription factor AP-1 in RBA-1 cells. Next, we investigated the role of ROS in activation of a c-Src/PDGFR/PI3K/Akt pathway by JEV infection in RBA-1 cells. Our data reveal that JEV infection-stimulated phosphorylation of PDGFR, c-Src, and Akt are attenuated by pretreatment with APO, DPI, or NAC. These data suggest that ROS plays an important role in JEV-stimulated activation of the c-Src/PDGFR/PI3K/Akt pathway in RBA-1 cells. Although MMP-9 induction is mediated by various stimuli and signaling pathways, such as ROS/ERK1/2, JNK1/2/NF-κB, PKCd/ERK1/2/Elk-1, and Ras/Raf/MEK/ER1/2/NF-κB [27, 47, 48], our results are the first to show a novel role for a ROS-dependent c-Src/PDGFR/PI3K/Akt/MAPKs/AP-1 signaling pathway in JEV-induced MMP-9 expression in RBA-1 cells. In the future, we will investigate the detailed mechanisms underlying JEV-induced MMP-9 expression in RBA-1 cells.