Ching-Yi Cheng

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 urokinse-plasminogen activator expression and cell migration through PKCα, JNK1/2, and NF-κB in A549 cells

Breakdown of the extracellular matrix (ECM) is accomplished by the concerted action of several proteases, including the urokinase plasminogen‐activator (uPA) system and matrix metalloproteinases (MMPs), which is crucial for cancer invasion and metastasis. Several reports have shown that the levels of IL‐1β and MMPs in plasma of the patients with lung cancer are significantly elevated and link to the invasion of tumor cells. Therefore, we investigated whether IL‐1β‐induced expression of uPA participated in lung cancer progression. In this study, IL‐1β significantly induced uPA expression and activity via PKCα‐dependent JNK1/2 and NIK cascades, linking to IKKα/β activation, p65 translocation and transcription activity, using pharmacological inhibitors and transfection with dominant negative mutants and siRNAs. IL‐1β‐induced uPA protein and mRNA expression in a time‐ and concentration‐dependent manner, which was inhibited by pretreatment with the inhibitors of JNK1/2 (SP600125), PKC (Ro31‐8220, Gö6976), or NF‐κB (helenalin), and transfection with dominant negative mutants of PKCα, NIK, and IKKβ, and siRNAs of JNK1/2 and p65. IL‐1β stimulated PKCα translocation to plasma membrane leading to phosphorylation of JNK1/2, which was attenuated by PKC inhibitors and transfection with shRNAs of JNK1/2, but not by helenalin. In addition, IL‐1β stimulated p65 phosphorylation and translocation into nucleus concomitant with IκBα phosphorylation and IκBα degradation, which was mediated via activation of PKCα‐dependent JNK1/2–NIK/IKKβ cascade. These results demonstrated that in A549 cells, activation of p50/p65 heterodimer through sequential activation of PKCα–JNK–NIK–IKKβ–NF‐κB was required for IL‐1β‐induced uPA expression associated with migration of tumor cells. J. Cell. Physiol. 219: 183–193, 2009.

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.

IL-1β induces expression of matrix metalloproteinase-9 and cell migration via a c-Src-dependent, growth factor receptor transactivation in A549 cells

BACKGROUND AND PURPOSE Interleukin (IL)‐1β‐induced matrix metalloproteinase (MMP‐9) expression is regulated by mitogen activated protein kinases (MAPKs) and NF‐κB. IL‐1β also stimulates transactivation of growth factor receptors and phosphatidylinositol 3‐kinase (PI3K)/Akt., leading to the expression of inflammatory proteins. Here, we investigated whether these transactivation mechanisms participated in IL‐1β‐induced MMP‐9 expression in A549 cells.

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.

Interleukin-1 receptor antagonist (IL-1RA) prevents apoptosis in ex vivo expansion of human limbal epithelial cells cultivated on human amniotic membrane.

Stem cells of the corneal epithelium have been found to be located exclusively at the anatomical junction between the cornea and the conjunctiva, the limbus. Ex vivo expanded limbal epithelial cells on amniotic membrane (AM) are capable of restoring the corneal surface with limbal stem cell deficiency. Recent studies indicate that intact AM preserves the limbal epithelial phenotype and that distinct epithelial morphology is noted among various culture matrix. However, the factors in response to the interaction between limbal epithelial cells and AM were not well understood. Using Annexin V‐fluorescein isothiocyanate staining, we found that human limbal epithelial cells expanded on intact human AM demonstrated fewer apoptotic cells as compared with those on plastic dishes. To identify the anti‐apoptotic factors, we performed cDNA microarray analysis and showed that interleukin‐1 receptor antagonist (IL‐1RA) was overexpressed in cultures on intact AM, which was confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR), real‐time quantitative PCR (Q‐PCR) and enzyme‐linked immunosorbent assay. In addition, we also noted that the phenomenon of apoptosis detected in cultures on plastic dishes could be reversed by adding recombinant IL‐1RA protein into the media, whereas apoptosis of limbal epithelial cells cultivated on intact AM could be induced by exogenous neutralizing IL‐1RA neutralizing antibody. These results demonstrated that intact human AM may prevent cultured human limbal epithelial cells from undergoing apoptosis. IL‐1RA might be a candidate mediator to exert as an anti‐apoptotic molecule during the interaction between human limbal epithelial cells and intact human AM.

Novel laminin 5 γ2-chain fragments potentiating the limbal epithelial cell outgrowth on amniotic membrane.

PURPOSE Matrix metalloproteases (MMPs)-mediated extracellular matrix (ECM) degradation potentially releases cryptic motility factors involved in somatic stem cell migration and epithelial outgrowth. The authors previously demonstrated that MMP-9 is upregulated in limbal epithelial cells cultivated on amniotic membrane (AM). Here, the authors further investigated whether plasminogen activator (PA)/plasmin regulates MMP-9 activity in this model implicated in the processing of laminin 5 (Ln5), a component of amniotic basement membrane. METHODS Limbal epithelial cells migrated from limbal explants were expanded on intact AM. The activities and proteins of uPA and MMP-9 in limbal epithelial cells were determined by fibrin and gelatin zymography, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Specific pharmacological inhibitors including MMPs inhibitor GM6001, MMP-2/-9 inhibitor, and uPA inhibitor B428 were used to determine whether the PA/plasmin/MMP-9 axis induces cell growth via Ln5 in this model. RESULTS These data showed that MMP-9 activity was attenuated by a selective uPA inhibitor, B428. Furthermore, MMP-9 activity was enhanced by exogenous addition or pre-incubation with plasmin. These results demonstrated that PA/plasmin regulates MMP-9 expression. An interesting proteolytic fragment of Ln5 gamma 2-chain was suppressed by pretreatment with GM6001, B428, or neutralizing antibodies of MMP-9 and uPA, indicating that Ln5 gamma 2-chain is processed by uPA/MMP-9. Moreover, the extent of limbal outgrowth was also retarded by B428. CONCLUSIONS This study suggested that MMP-9 activity was upregulated by PA/plasmin, which in turn processed Ln5 gamma 2-chain to facilitate limbal outgrowth on intact AM.