EV71 Induces COX-2 Expression Via c-Src/PDGFR/PI3K/Akt/p42/ p44 MAPK/AP-1 and NF-kB in Rat Brain Astrocytes y WEI-HSUAN TUNG, 1 I-TA LEE, 1 HSI-LUNG HSIEH, 2 AND CHUEN-MAO YANG 1 * 1 Department of Physiology and Pharmacology, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan 2 Department of Nursing, Division of Basic Medical Sciences, Chang Gung Institute of Technology, Tao-Yuan, Taiwan Enterovirus 71 (EV71) induces the expression of cyclooxgenase (COX)-2 served as a major neurotoxic factor in CNS injury. However, the mechanisms underlying EV71-initiated intracellular signaling pathways leading to COX-2 expression remain unknown. Therefore, we investigated the mechanisms underlying EV71-induced COX-2 expression and prostaglandin E 2 (PGE 2 ) production in rat brain astrocytes (RBA)-1, determined by Western blotting, RT-PCR, and promoter assay. Here, we reported that EV71-indued COX-2 expression and PGE 2 production were attenuated by pretreatment with the inhibitors of c-Src (PP1), PDGFR (AG1296), PI3K (Wortmannin), MEK1/2 (PD98059), NF-kB (helenalin), and AP-1 (Tanshinone) and transfection with shRNA or siRNA of c-Src, PDGFR, p85, c-Jun, c-Fos, ERK1, or ERK2. We further observed that EV71-induced activation of Akt and p42/p44 MAPK were mediated via c-Src and PDGFR. Pretreatment with PP1 attenuated EV71-stimulated phosphorylation of Src, PDGFR, Akt, and p42/p44 MAPK. Inhibition of PI3K by Wortmannin attenuated EV71-induced Akt and p42/p44 MAPK phosphorylation, but had no effect on PDGFR phosphorylation, suggesting that PDGFR is an upstream and p42/p44 MAPK is a downstream component of PI3K/Akt in these responses. EV71-stimulated NF-kB translocation from the cytoplasm to the nucleus, IkBa degradation and NF-kB promoter activity were attenuated by pretreatment with helenalin, but not AG1296, Wortmannin, and PD98059. EV71-induced c-Jun mRNA expression was attenuated by pretreatment with PD98059, AG1296, or Wortmannin. These results demonstrate that in RBA-1 cells, EV71-induced COX-2 expression associated with PGE 2 production is mediated through activation of c-Src/PDGFR/PI3K/Akt/p42/p44 MAPK to initiate the expression of AP-1. J. Cell. Physiol. 224: 376–386, 2010. ß 2010 Wiley-Liss, Inc. Enterovirus (EV) 71 is a non-enveloped RNA virus within Enterovirus B genus of the Picornaviridae family (Palacios and Oberste, 2005). EV71 infection could cause clinical syndromes, including hand-foot and mouth disease (HFMD), pulmonary edema, herpangina, and myocarditis (Hsueh et al., 2000; Lin et al., 2002). EV71 is also a highly neurotropic virus, which has been shown to cause aseptic meningitis, poliomyelitis-like paralysis, or encephalitis, and then lead to death in neonates. Thus, EV71 has been regarded as the most important neurotropic EV after the eradication of the poliovirus (Liu et al., 2007). Furthermore, Coxsackievirus B3 (CVB3), B4 (CVB4), and Theiler’s murine encephalomyelitis virus (TMEV) have been shown to induce the expression of inflammatory mediators in human and mouse astrocytes (Kwon et al., 2004). Astrocytes are neuroectodermally derived cells, which are critical for normal functions of the CNS, including synaptic transmission, maintenance of the blood–brain barrier, support of axons, and ion and neurotransmitter buffering. Moreover, astrocytes may be critical for early recruitment of inflammatory cells in the initiation of virus-induced diseases (Carpentier et al., 2008). However, little is known about the expression of inflammatory mediators in astrocytes following infection with EV71. Cyclooxygenase (COX) is the rate-limiting step in the production of prostaglandins (PGs). Arachidonic acid is the principal substrate for COX (Consilvio et al., 2004). COX-1 is constitutively expressed in most tissues and participated in normal physiological processes. However, COX-2 is initially characterized as an inducible enzyme that is expressed in response to various inflammatory stimuli (O’Banion, 1999). The expression of COX-2 is dramatically increased and plays as major toxic mediator in acute CNS injury, such as encephalitis (Hinson and Tyor, 2001; Chen and Reis, 2002). The induction of neuronal COX-2 expression predominantly occurs in brain areas where macrophages and microglia are infected with virus (Rohrenbeck et al., 1999). In addition, several reports have shown that the induction of COX-2 expression participates in the modulation of inflammatory responses following viral infection (Steer and Corbett, 2003). Moreover, the detail mechanisms of EV71-induced COX-2 expression in astrocytes remain largely unknown. Previous studies have reported that several proinflammatory factors induce the expression of COX-2 via various intracellular signaling pathways. For instance, IL-1b has been shown to induce COX-2 expression and PGE 2 production concomitant via p38 MAPK and JNK1/2 in renal mesangial cells (Guan et al., 1998). Moreover, p38 MAPK and p42/p44 MAPK play the key roles in IL-1b-mediated PGE 2 release, with differences at the level of cytosolic phospholipase A 2 (cPLA 2 )/ COX-2 expression in both neuroglioma and neuroblastoma cells (Moolwaney and Igwe, 2005). The transcriptional factors, Contract grant sponsor: National Science Council, Taiwan; Contract grant numbers: NSC96-2320-B-182-003, NSC98-2320- B-182-004-MY3, NSC97-2321-B-182-007, NSC98-2321-B-182- 004. Contract grant sponsor: Chang Gung Medical Research Foundation, Taiwan; Contract grant numbers: CMRPD150253, CMRPD150313, CMRPD170492. y This article was published online 23 Mar 2010. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 26 April 2010. *Correspondence to: Chuen-Mao Yang, Department of Pharmacology, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan. E-mail: chuenmao@mail.cgu.edu.tw Received 10 November 2009; Accepted 9 February 2010 Published online in Wiley InterScience (www.interscience.wiley.com.), 23 March 2010. DOI: 10.1002/jcp.22133 ORIGINAL ARTICLE 376 Journal of Journal of Cellular Physiology Cellular Physiology ß 2010 WILEY-LISS, INC.