Hsi-Lung Hsieh

Involvement of p42/p44 MAPK, p38 MAPK, JNK and nuclear factor-kappa B in interleukin-1beta-induced matrix metalloproteinase-9 expression in rat brain astrocytes.

Matrix metalloproteinase (MMP)‐9 expression induced by interleukin‐1β (IL‐1β) was investigated in rat brain astrocyte‐1 (RBA‐1). Here we report that the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐kappa B (NF‐κB) pathways participate in the induction of MMP‐9 expression by IL‐1β. Zymographic, western blotting, and RT‐PCR analyses showed that IL‐1β increased expression of MMP‐9 mRNA and protein, which were inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and JNK (SP600125). In accordance with these findings, IL‐1β stimulated phosphorylation of p42/p44 MAPK, p38, and c‐Jun N‐terminal kinase (JNK), which was attenuated by U0126, SB202190, or SP600125, respectively. Furthermore, this up‐regulation of MMP‐9 mRNA and protein was blocked by a specific NF‐κB inhibitor helenalin. Consistently, IL‐1β‐stimulated translocation of NF‐κB into the nucleus and degradation of inhibitory kappa B‐α (IκB‐α) was revealed by western blotting and immunofluorescence staining, which was blocked by helenalin, but not by U0126, SB202190, or SP600125. Taken together, these results suggest that in RBA‐1 cells, activation of p42/p44 MAPK, p38, JNK and NF‐κB pathways is essential for IL‐1β‐induced MMP‐9 gene expression via transcription and translation processes. An increased understanding of the signal transduction pathways involved in IL‐1β‐induced MMP‐9 expression on RBA‐1 may be of potential therapeutic value in the treatment of inflammatory disease.

Bradykinin induces matrix metalloproteinase-9 expression and cell migration through a PKC-δ-dependent ERK/Elk-1 pathway in astrocytes

Many reports have shown that matrix metalloproteinase (MMP)‐9 plays an important role in brain inflammation and diseases. In our previous study, bradykinin (BK) has been shown to induce proMMP‐9 expression via MAPKs and NF‐κB in rat brain astrocytes (RBA‐1). However, the molecular mechanisms and physiological roles underlying BK‐induced MMP‐9 expression in RBA‐1 remain unclear. Here we reported that BK induced proMMP‐9 expression and promoted RBA‐1 cell migration, via a B2 BK receptor‐activated protein kinase C‐δ (PKC‐δ)‐dependent signaling pathway. Activation of PKC‐δ led to phosphorylation and translocation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and then activated a transcription factor Elk‐1. Phospho‐Elk‐1 bound to MMP‐9 promoter and thereby induced transcription of MMP‐9. The rat MMP‐9 promoter containing an Elk‐1 cis‐binding site (Ets domain), that located at nucleotides −511 to −506 was identified as a crucial domain linking to BK action. Moreover, BK induced recruitment of p300 (as a transcriptional co‐activator) to the MMP‐9 promoter, leading to the acetylation of histone H4 in chromatin and facilitating MMP‐9 gene transcription. Taken together, these results suggested that in RBA‐1 cells, activation of ERK1/2 by a PKC‐δ‐dependent event mediated through Elk‐1 pathway is essential for MMP‐9 gene up‐regulation and cell migration induced by BK.

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.

IL-1β promotes A549 cell migration via MAPKs/AP-1- and NF-κB-dependent matrix metalloproteinase-9 expression

Matrix metalloproteinases (MMPs), in particular MMP-9, is induced by cytokines including IL-1 beta and contributes to airway injury and remodeling. However, the mechanisms underlying IL-1 beta-induced MMP-9 expression and cell migration in human A549 cells remain unclear. Here, we report that the IL-1 beta-induced MMP-9 gene expression was mediated through the activation of p42/p44 MAPK, p38 MAPK, and JNK1/2 in A549 cells, determined by zymographic, RT-PCR, and Western blotting. The involvement of MAPKs in the IL-1beta-induced responses was further ensured by transfection with siRNA of MEK1, p42, p38, or JNK2. Moreover, the IL-1 beta-induced MMP-9 gene expression was also mediated through the translocation of NF-kappaB (p65) into the nucleus and the degradation of I kappaB alpha. In addition, the IL-1 beta-induced c-Jun phosphorylation was reduced by pretreatment with U0126 or SP600125. IL-1 beta stimulated the transcriptional activity of wild-type MMP-9 promoter in A549 cells, which was inhibited by U0126, SB203580, SP600125, and helenalin. In contrast, IL-1 beta had no effect on the cells transfected with a NF-kappaB-mutated MMP-9 promoter construct, suggesting that NF-kappaB is required for this response. Finally, the IL-1 beta-induced MMP-9 expression led to cell migration which was attenuated by pretreatment with U0126, SB203580, SP600125, helenalin, or MMP-2/9 inhibitor. These results suggested that in A549 cells, the activation of p42/p44 MAPK, p38 MAPK, JNK1/2, NF-kappaB, and AP-1 are essential for the IL-1 beta-induced MMP-9 gene expression and cell migration.

IL‐1β induces MMP‐9 expression via a Ca2+‐dependent CaMKII/JNK/c‐JUN cascade in rat brain astrocytes

Interleukin (IL)‐1β has been shown to induce matrix metalloproteinase (MMP)‐9 expression through mitogen‐activated protein kinases, including JNK, in rat brain astrocyte‐1 (RBA‐1) cells. However, little is known about whether JNK activated by Ca2+‐dependent CaMKII is associated with MMP‐9 expression induced by IL‐1β. Here, we report that the Ca2+/CaMKII/JNK/c‐Jun participates in the MMP‐9 expression induced by IL‐1β. Zymographic, Western blotting, and RT‐PCR analyses showed that IL‐1β‐induced expression of MMP‐9 mRNA and protein was attenuated by Ca2+ chelator (BAPTA), and the inhibitors of ER Ca2+‐ATPase (thapsigargin), CaMKII (KN‐62), and JNK1/2 (SP600125). IL‐1β also stimulated phosphorylation of CaMKII and JNK1/2, and increase in intracellular Ca2+ ([Ca2+]i), which were inhibited by pretreatment with BAPTA, thapsigargin (TG), KN‐62, or SP600125. Furthermore, the upregulation of MMP‐9 protein was blocked by transfection with c‐Jun or CaMKII short hairpin RNA (shRNA). We further confirmed that IL‐1β stimulated c‐Jun associated with AP‐1‐binding sites within MMP‐9 promoter (−87 to −80 bp and −511 to −497 bp) by immunoprecipitation and chromatin immunoprecipitation (ChIP)‐PCR assays. The activation and recruitment of c‐Jun to MMP‐9 promoter were inhibited by pretreatment with BAPTA, TG, KN‐62, or SP600125. Moreover, IL‐1β‐induced MMP‐9 gene transcription by AP‐1 was confirmed by transfection with a MMP‐9 promoter‐luciferase reporter plasmid with a distal AP‐1‐binding site (−511 to −497 bp) adjacent to an Ets‐binding site‐mutation (mt‐AP1/Ets‐MMP‐9). These results demonstrated that in RBA‐1 cells, JNK/c‐Jun activation was mediated through a Ca2+‐dependent CaMKII pathway that promoted transcription factor c‐Jun/AP‐1 recruitment and eventually led to increase in MMP‐9 expression by IL‐1β.

PKC-δ/c-Src-mediated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE2 production in rat vascular smooth muscle cells

The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE(2) release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-delta, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-kappaB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-kappaB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-kappaB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(delta)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-kappaB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-kappaB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE(2) release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.

Oxidized low-density lipoprotein induces matrix metalloproteinase-9 expression via a p42/p44 and JNK-dependent AP-1 pathway in brain astrocytes

Upregulation of matrix metalloproteinases (MMPs), especially MMP‐9, by oxidized low‐density lipoprotein (oxLDL) is implicated in many inflammatory diseases including brain injury. However, the signaling mechanisms underlying oxLDL‐induced MMP‐9 expression in astrocytes largely remain unknown. Here we report that oxLDL induces expression of proMMP‐9 via a MAPK‐dependent AP‐1 activation in rat brain astrocyte (RBA)‐1 cells. Results revealed by gelatin zymography, RT‐PCR, and Western blotting analyses showed that oxLDL‐induced proMMP‐9 gene expression was mediated through Akt, JNK1/2, and p42/p44 MAPK phosphorylation in RBA‐1 cells. These responses were attenuated by inhibitors of PI3K (LY294002), JNK (SP600125), and p42/p44 MAPK (PD98059), or transfection with dominant negative mutants and short hairpin RNA. Moreover, we demonstrated that AP‐1 (i.e., c‐Fos/c‐Jun) is crucial for oxLDL‐induced proMMP‐9 expression which was attenuated by pretreatment with AP‐1 inhibitor (curcumin). The regulation of MMP‐9 gene transcription by AP‐1 was confirmed by oxLDL‐stimulated MMP‐9 luciferase activity which was totally lost in cells transfected with the AP‐1 binding site‐mutated MMP‐9 promoter construct (mt‐AP1‐MMP‐9). These results suggested that oxLDL‐induced proMMP‐9 expression is mediated through PI3K/Akt, JNK1/2, and p42/p44 MAPK leading to AP‐1 activation. Understanding the regulatory mechanisms underlying oxLDL‐induced MMP‐9 expression in astrocytes might provide a new therapeutic strategy of brain injuries and diseases.

Tumor necrosis factor-α induces MMP-9 expression via p42/p44 MAPK, JNK, and nuclear factor-κB in A549 cells

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-alpha (TNF-alpha) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-alpha in human A549 cells remain unclear. Here, we showed that TNF-alpha induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-kappaB (helenalin), and transfection with dominant negative mutants of ERK2 (DeltaERK) and JNK (DeltaJNK), and siRNAs for MEK1, p42 and JNK2. TNF-alpha-stimulated phosphorylation of p42/p44 MAPK and JNK were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of DeltaERK and DeltaJNK. Furthermore, the involvement of NF-kappaB in TNF-alpha-induced MMP-9 production was consistent with that TNF-alpha-stimulated degradation of IkappaB-alpha and translocation of NF-kappaB into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-kappaB was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-alpha in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-alpha-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-kappaB MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-alpha-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-kappaB are essential for TNF-alpha-induced MMP-9 gene expression.

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.

Bk-induced cytosolic phospholipase A2 expression via sequential PKC-δ, p42/p44 MAPK, and NF-κB activation in rat brain astrocytes

Bradykinin (BK), an inflammatory mediator, has been shown to induce cytosolic phospholipase A2 (cPLA2) expression implicating in inflammatory responses in various cell types. However, the detailed mechanisms underlying BK‐induced cPLA2 expression in astrocytes remain unclear. RT‐PCR and Western blotting analysis showed that BK induced the expression of cPLA2 mRNA and protein, which was inhibited by Hoe140, suggesting the involvement of B2 BK receptors, confirmed by immunofluorescence staining using anti‐B2 BK receptor antibody. BK‐induced cPLA2 expression and phosphorylation of p42/p44 MAPK was attenuated by PD98059, indicating the involvement of MEK1/2‐p42/p44 MAPK in these responses. BK‐induced cPLA2 expression might be due to the translocation of NF‐κB into nucleus which was inhibited by Hoe140, helenalin, and PD98059, implying the involvement of NF‐κB. Moreover, BK‐induced cPLA2 expression was attenuated by rottlerin, suggesting that PKC‐δ might be involved in these responses. This hypothesis was supported by the transfection with a dominant negative plasmid of PKC‐δ significantly attenuated BK‐induced response. In addition, BK‐stimulated translocation of PKC‐δ from cytosol to membrane fraction was inhibited by rottlerin but not by PD98059, indicating that PKC‐δ might be an upstream component of p42/p44 MAPK. Accordingly, BK‐induced phosphorylation of p42/p44 MAPK was attenuated by rottlerin but not by helenalin. These results suggest that in RBA‐1 cells, BK‐induced cPLA2 expression was sequentially mediated through activation of PKC‐δ, p42/p44 MAPK, and NF‐κB. Understanding the regulation of cPLA2 expression induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.