Wei-Hsuan Tung

Cooperation of TLR2 with MyD88, PI3K, and Rac1 in Lipoteichoic Acid–Induced cPLA2/COX-2–Dependent Airway Inflammatory Responses

Lipoteichoic acid (LTA) plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. Cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin (IL)-6 have been demonstrated to engage in airway inflammation. In this study, LTA-induced cPLA(2) and COX-2 expression and PGE(2) or IL-6 synthesis were attenuated by transfection with siRNAs of TLR2, MyD88, Akt, p42, p38, JNK2, and p65 or pretreatment with the inhibitors of PI3K (LY294002), p38 (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-kappaB (helenalin) in human tracheal smooth muscle cells (HTSMCs). LTA also induced cPLA(2) and COX-2 expression and leukocyte count in bronchoalveolar lavage fluid in mice. LTA-regulated PGE(2) or IL-6 production was inhibited by pretreatment with the inhibitors of cPLA(2) (AACOCF(3)) and COX-2 (NS-398) or transfection with cPLA(2) siRNA or COX-2 siRNA, respectively. LTA-stimulated NF-kappaB translocation or cPLA(2) phosphorylation was attenuated by pretreatment with LY294002, SB202190, U0126, or SP600125. Furthermore, LTA could stimulate TLR2, MyD88, PI3K, and Rac1 complex formation. We also demonstrated that Staphylococcus aureus could trigger these responses through a similar signaling cascade in HTSMCs. It was found that PGE(2) could directly stimulate IL-6 production in HTSMCs or leukocyte count in bronchoalveolar lavage fluid in mice. These results demonstrate that LTA-induced MAPKs activation is mediated through the TLR2/MyD88/PI3K/Rac1/Akt pathway, which in turn initiates the activation of NF-kappaB, and ultimately induces cPLA(2)/COX-2-dependent PGE(2) and IL-6 generation.

Enterovirus 71 induces COX-2 expression via MAPKs, NF-κB, and AP-1 in SK–N–SH cells: Role of PGE2 in viral replication

The enterovirus 71 (EV71) causes severe neurological diseases that were 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 PGE(2) generation in a time- and virus titer-dependent manner, revealed by Western blot, real-time PCR, and PGE(2) analyses. These EV71-induced responses were mediated through activation of p42/p44 MAPK, p38 MAPK, JNK, NF-kappaB, and AP-1, revealed by using selective pharmacological inhibitors or transfection with respective siRNAs. Consistently, EV71-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaBalpha in the cytosol was blocked by pretreatment with the selective inhibitors of MEK1/2 (U0126) and NF-kappaB (Bay11-7085), respectively, suggesting that MEK1/2-p42/p44 MAPK cascade linking to NF-kappaB was involved in COX-2 expression. In addition, EV71-induced AP-1 subunits (c-jun and c-fos mRNA) expression was also attenuated by pretreatment with a selective JNK inhibitor SP600125, suggesting that JNK cascade linking to AP-1 was involved in COX-2 expression induced by EV71. These findings suggested that up-regulation of COX-2 associated with the release of PGE(2) from EV71-infected SK-N-SH cells which was mediated through activation of p38 MAPK, JNK, p42/p44 MAPK, NF-kappaB, and AP-1 pathways.

Sphingosine 1‐phosphate induces EGFR expression via Akt/NF‐κB and ERK/AP‐1 pathways in rat vascular smooth muscle cells

Sphingosine 1‐phosphate (S1P) has been shown to regulate expression of several genes in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating epidermal growth factor receptor (EGFR) expression by S1P in aortic VSMCs remain unclear. Western blotting and RT‐PCR analyses showed that S1P induced EGFR mRNA and protein expression in a time‐ and concentration‐dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and phosphatidylinositide 3‐kinase (PI3K; wortmannin), and transfection with dominant negative mutants of ERK and Akt, respectively. These results suggested that S1P‐induced EGFR expression was mediated through p42/p44 MAPK and PI3K/Akt pathways in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt which was attenuated by U0126 and wortmannin, respectively. Furthermore, S1P‐induced EGFR upregulation was blocked by a selective NF‐κB inhibitor helenalin. Immunofluorescent staining and reporter gene assay revealed that S1P‐induced activation of NF‐κB was blocked by wortmannin, but not by U0126, suggesting that activation of NF‐κB was mediated through PI3K/Akt. Moreover, S1P‐induced EGFR expression was inhibited by an AP‐1 inhibitor curcumin and tanshinone IIA. S1P‐stimulated AP‐1 subunits (c‐Jun and c‐Fos mRNA) expression was attenuated by U0126 and wortmannin, suggesting that MEK and PI3K/ERK cascade linking to AP‐1 was involved in EGFR expression. Upregulation of EGFR by S1P may exert a phenotype modulation of VSMCs. This hypothesis was supported by pretreatment with AG1478 or transfection with shRNA of EGFR that attenuated EGF‐stimulated proliferation of VSMCs pretreated with S1P, determined by XTT assay. These results demonstrated that in VSMCs, activation of Akt/NF‐κB and ERK/AP‐1 pathways independently regulated S1P‐induced EGFR expression in VSMCs. Understanding the mechanisms involved in S1P‐induced EGFR expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis. J. Cell. Biochem. 103: 1732–1746, 2008.

Thrombin Induces EGF Receptor Expression and Cell Proliferation via a PKC(δ)/c-Src-Dependent Pathway in Vascular Smooth Muscle Cells

Objection—Thrombin upregulates expression of several proteins in vascular smooth muscle cells (VSMCs) which may contribute to atherosclerosis. Here, we investigated the mechanisms underlying thrombin-induced EGF receptor (EGFR) expression and its effect on VSMCs. Methods and Results—Normal rat VSMCs were used. First, Western blotting and RT-PCR analyses showed that thrombin induces the expression of EGFR at transcription and translation levels in VSMCs. Second, pharmacological inhibitors, dominant negative mutants, and short hairpin RNA interference (shRNA) technology enabled us to demonstrate that thrombin-induced EGFR expression is mediated through PKC(&dgr;)/c-Src–dependent transactivation of EGFR linking to PI3K/Akt and ERK1/2. We further investigated whether the transcription factors AP-1 and NF-&kgr;B are involved in this response by a promoter assay. Finally, data obtained by using EGFR shRNA technology and XTT assay demonstrated that thrombin-enhanced VSMC proliferation was mediated through upregulation of EGFR. Conclusions—Our results demonstrate that thrombin-enhanced VSMC proliferation was mediated through upregulation of EGFR via a PKC(&dgr;)/c-Src–dependent transactivation of EGFR, PI3K-Akt, and ERK, and AP-1/NF-&kgr;B pathway.

Enterovirus 71 induces integrin β1/EGFR-Rac1-dependent oxidative stress in SK-N-SH cells: role of HO-1/CO in viral replication.

Oxidative stress became emerged as a key player in the development and progression of many pathological conditions including virus‐induced encephalitis. Heme oxygenase‐1 (HO‐1) plays a crucial role in defending the body against oxidant‐induced injury during inflammatory processes. Therefore, we investigated the induction of HO‐1 level in 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) and activation of NADPH oxidase. EV71‐induced ROS generation was mediated through activation of integrin β1, an epidermal growth factor receptor (EGFR), Rac1 and NADPH oxidase which revealed by using selective pharmacological inhibitors or transfection with respective siRNAs. In addition, the reduction of viral load was observed with NADPH oxidase inhibitors (apocynin and diphenyleneiodonium chloride), ROS scavenger (N‐acetylcysteine), and transfection with p47phox siRNA in 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) and a cyclic GMP‐dependent protein kinase (PKG) inhibitor (KT5823). These findings suggest that up‐regulation of HO‐1 exerts as a host cellular defense mechanism against EV71 infection in SK‐N‐SH cells. J. Cell. Physiol. 226: 3316–3329, 2011.

Japanese encephalitis virus induces matrix metalloproteinase‐9 in rat brain astrocytes via NF‐κB signalling dependent on MAPKs and reactive oxygen species

BACKGROUND AND PURPOSE Japanese encephalitis virus (JEV) is a member of the family Flaviviridae and JEV infection is a major cause of acute encephalopathy in children, which destroys cells in the CNS, including astrocytes and neurons. However, the detailed mechanisms underlying the inflammatory action of JEV are largely unclear.

EV71 induces COX-2 expression via c-Src/PDGFR/PI3K/Akt/p42/p44 MAPK/AP-1 and NF-κB in rat brain astrocytes†

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 E2 (PGE2) 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 PGE2 production were attenuated by pretreatment with the inhibitors of c‐Src (PP1), PDGFR (AG1296), PI3K (Wortmannin), MEK1/2 (PD98059), NF‐κB (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‐κB translocation from the cytoplasm to the nucleus, IκBα degradation and NF‐κB 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 PGE2 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.

Enterovirus 71 modulates a COX‐2/PGE2/cAMP‐dependent viral replication in human neuroblastoma cells: Role of the c‐Src/EGFR/p42/p44 MAPK/CREB signaling pathway

Enterovirus 71 (EV71) has been shown to induce cyclooxygenase‐2 (COX‐2) expression in human neuroblastoma SK‐N‐SH cells through the action of MAPKs, NF‐κB, and AP‐1. On the other hand, the transcription factor CREB has also been implicated in the expression of COX‐2 in other cell lines. Here, we report that EV71‐induced COX‐2 expression and PGE2 production were both inhibited by pretreatment with the PKA inhibitor H89 or by transfection with CREB siRNA. In addition, EV71‐induced COX‐2 expression and c‐Src/EGFR phosphorylation were both attenuated by transfection with c‐Src siRNA or pretreatment with the inhibitors of c‐Src (PP1) or EGF receptor (EGFR) (AG1478 and EGFR‐neutralizing antibody). We also observed that EV71‐induced p42/p44 MAPK phosphorylation was decreased following pretreatment with AG1478. Moreover, EV71‐induced COX‐2 expression was blocked by pretreatment with the p300 inhibitor GR343 or by transfection with p300 siRNA. Using immunoprecipitation and chromatin immunoprecipitation assays, we observed that EV71 stimulated the association of CREB and p300 with the COX‐2 promoter region. Notably, we also demonstrated that EV71‐induced COX‐2 expression and PGE2 production promoted viral replication via cAMP signaling. In summary, this study demonstrates that EV71 activates the c‐Src/EGFR/p42/p44 MAPK pathway in human SK‐N‐SH cell, which leads to the activation of CREB/p300, and stimulates COX‐2 expression and PGE2 release. J. Cell. Biochem. 112: 559–570, 2011.

Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells: role of ERK1/2, p38 MAPK, and NF-κB

AIMS Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in inflammatory diseases such as atherosclerosis and angiogenesis. As extracellular nucleotides such as ATP have been shown to act via activation of P(2) purinoceptors implicated in various inflammatory diseases, we hypothesized that extracellular nucleotides contribute to vascular diseases via upregulated expression of inflammatory proteins, such as cyclooxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) in VSMCs. METHODS AND RESULTS Western blotting, promoter assay, RT-PCR, and PGE2 immunoassay revealed that ATPgammaS induced expression of COX-2 and prostaglandin (PGE2) synthesis through the activation of p42/p44 MAPK (mitogen-activated protein kinase), p38 MAPK, and nuclear factor-kappaB (NF-kappaB). These responses were attenuated by inhibitors of MAPK/ERK kinase (MEK1/2; U0126), p38 MAPK (SB202190), and NF-kappaB (helenalin), or by tranfection with dominant negative mutants of p42, p38, IkappaB kinase (IKK)alpha, and IKKbeta. Furthermore, the ATPgammaS-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaBalpha was blocked by U0126 and helenalin. In addition, the ATPgammaS-stimulated cPLA2 expression was inhibited by U0126, SB202190, helenalin, celecoxib (a selective COX-2 inhibitor), and PGE2 receptor antagonists (AH6809, GW627368X, and SC-19220). However, the inhibitory effect of celecoxib on cPLA2 expression was reversed by addition of exogenous PGE2. CONCLUSION Our results suggest that in VSMCs, activation of p42/p44 MAPK, p38 MAPK, and NF-kappaB is essential for ATPgammaS-induced COX-2 expression and PGE2 synthesis. Newly synthesized PGE2 was observed to act as an autocrine signal contributing to cPLA2 expression, which may be implicated in inflammatory responses. Collectively, our findings provide insights into the correlation between COX-2 and cPLA2 expression in ATPgammaS-stimulated VSMCs with similar molecular mechanisms and functional coupling to amplify the occurrence of vessel disease-related vascular inflammation.

Lipoteichoic Acid Induces Matrix Metalloproteinase-9 Expression via Transactivation of PDGF Receptors and NF-κB Activation in Rat Brain Astrocytes

Bacterial infections have been shown to be involved in several inflammatory diseases such as brain inflammation. A major factor for these findings is due to the secretion of pro-inflammatory mediators by host cells triggered by the components released from the bacteria. Among these components, lipoteichoic acid (LTA), a component of Gram-positive bacterial cell wall, has been found to be elevated in cerebrospinal fluid of patients suffering from meningitis. Moreover, increased plasma levels of matrix metalloproteinases (MMPs), in particular MMP-9, have been observed in patients with brain inflammatory diseases and may contribute to disease pathology. However, the molecular mechanisms underlying LTA-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain poorly defined. Here, the data with zymographic, Western blotting, RT-PCR, and immunofluorescent staining analyses showed that LTA induced MMP-9 expression and activation via a TLR2-activated c-Src-dependent transactivation of PDGFR pathway. Transactivation of PDGFR led to activation of PI3K/Akt and p42/p44 MAPK and then activated the IKK/NF-κB cascade. The activated-NF-κB translocated into nucleus which bound to κB-binding site of MMP-9 promoter, and thereby turned on transcription of MMP-9. Eventually, upregulation of MMP-9 by LTA enhanced cell migration of astrocytes. Taken together, these results suggested that in RBA-1 cells, activation of NF-κB by a c-Src-dependent PI3K/Akt-p42/p44 MAPK activation mediated through transactivation of PDGFR is essential for MMP-9 gene upregulation induced by LTA. Understanding the regulation of MMP-9 expression and functional changes by LTA/TLR system on astrocytes may provide potential therapeutic targets of Gram-positive bacterial infection in brain disorders.