Chiang-Wen Lee

Interleukin-1β induces MMP-9 expression via p42/p44 MAPK, p38 MAPK, JNK, and nuclear factor-κB signaling pathways in human tracheal smooth muscle cells

Matrix metalloproteinases (MMPs) are responsible for degradation of extracellular matrix and play important roles in cell migration, proliferation, and tissue remodeling related to airway inflammation. Interleukin‐1β (IL‐1β) has been shown to induce MMP‐9 production in many cell types and contribute to airway inflammatory responses. However, the mechanisms underlying MMP‐9 expression induced by IL‐1β in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here, we investigated the roles of p42/p44 MAPK, p38 MAPK, JNK, and NF‐κB pathways for IL‐1β‐induced MMP‐9 production in HTSMCs. IL‐1β induced production of MMP‐9 protein and mRNA in a time‐ and concentration‐dependent manner determined by zymographic, Western blotting, and RT‐PCR analyses, which was attenuated by inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), JNK (SP600125), and NF‐κB (helenalin), and transfection with dominant negative mutants of MEK1/2, p38 and JNK, respectively. IL‐1β‐stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK was attenuated by pretreatment with U0126, SB202190, SP600125, or transfection with these dominant negative mutants of MEK, ERK, p38 and JNK, respectively. Furthermore, IL‐1β‐stimulated translocation of NF‐κB into the nucleus and degradation of IκB‐α was blocked by helenalin. Finally, the reporter gene assay revealed that MAPKs and NF‐κB are required for IL‐1β‐induced MMP‐9 luciferase activity in HTSMCs. MMP‐9 promoter activity was enhanced by IL‐1β in HTSMCs transfected with MMP‐9‐Luc, which was inhibited by helenalin, U0126, SB202190, and SP600125. Taken together, the transcription factor NF‐κB, p42/p44 MAPK, p38 MAPK, and JNK that are involved in MMP‐9 expression in HTSMCs exposed to IL‐1β have now been identified. J. Cell. Physiol. 211: 759–770, 2007.

Transcriptional regulation of VCAM-1 expression by tumor necrosis factor-α in human tracheal smooth muscle cells: Involvement of MAPKs, NF-κB, p300, and histone acetylation

Tumor necrosis factor‐α (TNF‐α) has been shown to induce the expression of adhesion molecules in airway resident cells and contribute to inflammatory responses. Here, the roles of mitogen‐activated protein kinases (MAPKs) and NF‐κB in TNF‐α‐induced expression of vascular cell adhesion molecule (VCAM)‐1 were investigated in human tracheal smooth muscle cells (HTSMCs). TNF‐α‐enhanced expression of VCAM‐1 protein and mRNA as well as phosphorylation of p42/p44 MAPK, p38, and JNK were significantly attenuated by inhibitors of MEK1/2 (U0126), p38 (SB202190), and JNK (SP600125). Transfection with dominant negative mutants of MEK1/2, ERK1, ERK2, p38, and JNK attenuated TNF‐α‐induced VCAM‐1 expression. Furthermore, TNF‐α‐induced VCAM‐1 expression was significantly blocked by a selective NF‐κB inhibitor helenalin. TNF‐α‐stimulated translocation of NF‐κB into the nucleus and degradation of IκB‐α was blocked by helenalin, but not by U0126, SB202190, or SP600125. VCAM‐1 promoter activity was enhanced by TNF‐α in HTSMCs transfected with VCAM‐1‐Luc, which was inhibited by helenalin, U0126, SB202190, and SP600125. Most surprisingly, VCAM‐1 expression was also significantly blocked by a selective inhibitor of p300, curcumin. NF‐κB transcription factor and p300 were associated with the VCAM‐1 promoter, which was dynamically linked to histone H3 acetylation stimulated by TNF‐α, as determined by chromatin immunoprecipitation assay. Moreover, the resultant enhancement of VCAM‐1 expression increased the adhesion of polymorphonuclear cells (PMNs) to monolayer of HTSMCs, which was blocked by helenalin, U0126, SB202190, or SP600125. These results suggest that in HTSMCs, activation of MAPK pathways, NF‐κB, and p300 is essential for TNF‐α‐induced VCAM‐1 expression. J. Cell. Physiol. 207: 174–186, 2006.

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.

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.

Transactivation of Src, PDGF receptor, and Akt is involved in IL-1β-induced ICAM-1 expression in A549 cells

In previous study, interleukin‐1β (IL‐1β) has been shown to induce ICAM‐1 expression through MAPKs and NF‐κB in A549 cells. In addition to these pathways, transactivation of non‐receptor tyrosine kinase (Src), PDGF receptors (PDGFRs), and phosphatidylinositol 3‐kinase (PI3K)/Akt has been implicated in the expression of inflammatory genes. Here, we further investigated whether these different mechanisms participating in IL‐1β‐induced ICAM‐1 expression in A549 cells. We initially observed that IL‐1β‐induced ICAM‐1 promoter activity was attenuated by the inhibitors of Src (PP1), PDGFR (AG1296), PI3‐K (LY294002 and wortmannin), and Akt (SH‐5), revealed by reporter gene assay, Western blotting, and RT‐PCR analyses. The involvement of Src and PI3‐K/Akt in IL‐1β‐induced ICAM‐1 expression was significantly attenuated by transfection of A549 cells with dominant negative plasmids of Src, p85 and Akt, respectively. Src, PDGFR, and PI3K/Akt mediated the effects of IL‐1β because pretreatment with PP1, AG1296, and wortmannin also abrogated IL‐1β‐stimulated Src, PDGFR, and Akt phosphorylation, respectively. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked ICAM‐1 expression. We further confirmed that p300 was associated with ICAM‐1 promoter which was dynamically linked to histone H4 acetylation stimulated by IL‐1β, determined by chromatin immunoprecipitation assay. Association of p300 and histone‐H4 to ICAM‐1 promoter was inhibited by LY294002. Up‐regulation of ICAM‐1 enhanced the adhesion of neutrophils onto A549 cell monolayer exposed to IL‐1β, which was inhibited by PP1, AG1296, LY294002, wortmannin, and helenalin. These results suggested that Akt phosphorylation mediated through transactivation of Src/PDGFR promotes the transcriptional p300 activity and eventually leads to ICAM‐1 expression induced by IL‐1β. J. Cell. Physiol. 211: 771–780, 2007.

Tumor Necrosis Factor-α Enhances Neutrophil Adhesiveness: Induction of Vascular Cell Adhesion Molecule-1 via Activation of Akt and CaM Kinase II and Modifications of Histone Acetyltransferase and Histone Deacetylase 4 in Human Tracheal Smooth Muscle Cells

Up-regulation of vascular cell adhesion molecule-1 (VCAM-1) involves adhesions between both circulating and resident leukocytes and the human tracheal smooth muscle cells (HTSMCs) during airway inflammatory reaction. We have demonstrated previously that tumor necrosis factor (TNF)-α-induced VCAM-1 expression is regulated by mitogen-activated protein kinases, nuclear factor-κB, and p300 activation in HTSMCs. In addition to this pathway, phosphorylation of Akt and CaM kinase II has been implicated in histone acetyltransferase and histone deacetylase 4 (HDAC4) activation. Here, we investigated whether these different mechanisms participated in TNF-α-induced VCAM-1 expression and enhanced neutrophil adhesion. TNF-α significantly increased HTSMC-neutrophil adhesions, and this effect was associated with increased expression of VCAM-1 on the HTSMCs and was blocked by the selective inhibitors of Src [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], epidermal growth factor receptor [EGFR; 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline, (AG1478)], phosphatidylinositol 3-kinase (PI3K) [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride(LY294002) and wortmannin],calcium[1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; BAPTA-AM], phosphatidylinositol-phospholipase C (PLC) [1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], protein kinase C (PKC) [12-(2-cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976), rottlerin, and 3-1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro 31-8220)], CaM (calmidazolium chloride), CaM kinase II [(8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62)], p300 (curcumin), and HDAC (trichostatin A) or transfection with short interfering RNAs for Src, Akt, PKCα, PKCμ, and HDAC4. At gene regulation level, reverse-transcriptase polymerase chain reaction and promoter assays revealed that expression of VCAM-1 was also attenuated by these signaling molecule inhibitors. Moreover, TNF-α induced Akt and CaM kinase II phosphorylation via cascades through Src/EGFR/PI3K and PLC/calcium/CaM, respectively. Finally, activation of Akt and CaM kinase II may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylase. These findings revealed that TNF-α induced VCAM-1 expression via multiple signaling pathways. Blockade of these pathways may be selectively targeted to reduce neutrophil adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.

Induction of cyclooxygenase-2 expression in human tracheal smooth muscle cells by interleukin-1β: Involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-κB

Interleukin-1beta (IL-1beta) has been recognized as a potent stimulus for the synthesis of prostaglandin (PG), which has been implicated in inflammatory responses of the airways. However, the mechanisms underlying IL-1beta-induced cyclooxygenase (COX) expression and PGE(2) synthesis via activation of p42/p44 and p38 mitogen-activated protein kinases (MAPKs) in human tracheal smooth muscle cells (HTSMCs) are not completely understood. We found that IL-1beta increased COX-2 expression and PGE(2) synthesis in time- and concentration-dependent manners. Both specific phosphatidylcholine-phospholipase C inhibitor (D609) and protein kinase C inhibitor (GF109203X) attenuated IL-1beta-induced responses in HTSMCs. IL-1beta-induced COX-2 expression and PGE(2) synthesis were also inhibited by an inhibitor of MEK1/2 (PD98059) and inhibitors of p38 MAPK (SB203580 and SB202190), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by the transient activation of p42/p44 and p38 MAPKs induced by IL-1beta. Furthermore, IL-1beta-induced activation of nuclear factor-kappaB (NF-kappaB) was inversely correlated with the degradation of IkappaB-alpha in HTSMCs. IL-1beta-induced COX-2 expression and PGE(2) synthesis were inhibited by the NF-kappaB inhibitor pyrrolidinedithiocarbamate. These findings suggest that the expression of COX-2 is correlated with the release of PGE(2) from IL-1beta-challenged HTSMCs, which is mediated, at least in part, through p42/p44 and p38 MAPKs and NF-kappaB signaling pathways in HTSMCs.

Involvement of MAPKs, NF-κB and p300 co-activator in IL-1β-induced cytosolic phospholipase A2 expression in canine tracheal smooth muscle cells

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during stimulation with interleukin-1beta (IL-1beta). However, the mechanisms underlying IL-1beta-induced cPLA2 expression and PGE2 synthesis by canine tracheal smooth muscle cells (CTSMCs) have not been defined. IL-1beta induced cPLA2 protein and mRNA expression, PGE2 production, and phosphorylation of p42/p44 MAPK, p38 MAPK (ATF2), and JNK (c-Jun) in a time- and concentration-dependent manner, determined by Western blotting, RT-PCR, and ELISA, which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), or transfection with dominant negative mutants of MEK1/2, p38, and JNK, respectively. Furthermore, IL-1beta-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) or transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta. Consistently, IL-1beta stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. NF-kappaB translocation was blocked by helenalin, but not by U0126, SB202190, and SP600125. MAPKs together with NF-kappaB-activated p300 recruited to cPLA2 promoter thus facilitating the binding of NF-kappaB to cPLA2 promoter region and expression of cPLA2 mRNA. IL-1beta-induced cPLA2 expression and PGE2 production was inhibited by actinomycin D and cycloheximide, indicating the involvement of transcriptional and translational events in these responses. These results suggest that in CTSMCs, IL-1beta-induced cPLA2 expression and PGE2 synthesis was independently mediated through activation of MAPKs and NF-kappaB pathways and was connected to p300 recruitment and activation.