Wei-Ning Lin

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.

Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: Involvement of Src/EGFR/PI3-K/Akt pathway

In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-kappaB in human tracheal smooth muscle cells (HTSMCs). In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3K) have been shown to be implicated in the expression of several inflammatory target proteins. Here, we reported that LPS-induced up-regulation of VCAM-1 enhanced the adhesion of neutrophils onto HTSMC monolayer, which was inhibited by LY294002 and wortmannin. LPS stimulated phosphorylation of protein tyrosine kinases including Src, PYK2, and EGFR, which were further confirmed using specific anti-phospho-Src, PYK2, or EGFR Ab, respectively, revealed by Western blotting. LPS-stimulated Src, PYK2, EGFR, and Akt phosphorylation and VCAM-1 expression were attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), respectively, or transfection with siRNAs of Src or Akt and shRNA of p110. LPS-induced VCAM-1 expression was also blocked by pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA. LPS-stimulated Akt activation translocated into nucleus and associated with p300 and VCAM-1 promoter region was further confirmed by immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation assays. This association of Akt and p300 to VCAM-1 promoter was inhibited by pretreatment with PP1, AG1478, wortmannin, and SH-5. LPS-induced p300 activation enhanced VCAM-1 promoter activity and VCAM-1 mRNA expression. These results suggested that in HTSMCs, Akt phosphorylation mediated through transactivation of Src/PYK2/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS.

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.

Activation of ROS/NF-κB and Ca2+/CaM kinase II are necessary for VCAM-1 induction in IL-1β-treated human tracheal smooth muscle cells

Histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays a critical role in the expression of inflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1). Oxidative processes have been shown to induce VCAM-1 expression. Here, we investigated the mechanisms underlying IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells (HTSMCs). Our results showed that IL-1beta enhanced HTSMCs-monocyte adhesion through up-regulation of VCAM-1, which was inhibited by pretreatment with selective inhibitors of PKCalpha (Gö6976), c-Src (PP1), NADPH oxidase [diphenylene iodonium (DPI) and apocynin (APO)], intracellular calcium chelator (BAPTA/AM), PI-PLC (U73122), CaM (calmidazolium chloride), CaM kinase II (KN62), p300 (garcinol), NF-kappaB (Bay11-7082), HDAC (trichostatin A), and ROS scavenger [N-acetyl-L-cysteine (NAC)] or transfection with siRNAs of MyD88, PKCalpha, Src, p47(phox), p300, and HDAC4. Moreover, IL-1beta stimulated NF-kappaB and CaMKII phosphorylation through MyD88-dependent PI-PLC/PKCalpha/c-Src/ROS and PI-PLC/Ca2+/CaM pathways, respectively. Activation of NF-kappaB and CaMKII may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylases. These findings suggested that IL-1beta induced VCAM-1 expression via these multiple signaling pathways in HTSMCs. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.

Differential involvement of PKC-dependent MAPKs activation in lipopolysaccharide-induced AP-1 expression in human tracheal smooth muscle cells

Lipopolysaccharide (LPS) has been shown to up-regulate the expression of vascular cell adhesion molecule (VCAM)-1 which contributes to the occurrence of airway inflammatory diseases. Genetic analysis reveals the existence of activator protein-1 (AP-1) binding site on VCAM-1 promoter region. However, the role of AP-1 in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells (HTSMCs) is not known. Here, we show that LPS increased VCAM-1 expression and adhesiveness of HTSMCs through AP-1, since pretreatment with an AP-1 inhibitor tanshinone attenuated LPS-induced VCAM-1 expression and leukocytes adhesion. The implication of AP-1 in LPS-induced VCAM-1 expression was confirmed by animal studies showing that pretreatment of mice with tanshinone attenuated LPS-induced VCAM-1 mRNA expression in airway tissues and accumulation of leukocytes in bronchoalveolar lavage. By using the pharmacological inhibitors and transfection with siRNA of PKC, p42, p38, or JNK2, LPS-induced expression of c-Fos was mediated through protein kinase C (PKC), p42/p44 MAPK and p38 MAPK. While, c-Jun expression was mediated through PKC and mitogen-activated protein kinases (MAPKs, p42/p44 MAPK, p38 MAPK and JNK) in HTSMCs. Pretreatment with the inhibitors of PKCs or MAPKs attenuated LPS-stimulated nuclear translocation and VCAM-1 promoter binding abilities of AP-1, which attenuated promoter activity and gene expression of VCAM-1 and the adhesiveness between HTSMCs and leukocytes. These results indicated that differential regulation of AP-1 through PKCs-dependent MAPKs activation plays central roles in LPS-induced VCAM-1 expression. The altered modulation of this axis with inhibitors or siRNAs may contribute to the improvement of airway inflammatory diseases.

Activation and induction of cytosolic phospholipase A2 by IL-1β in human tracheal smooth muscle cells: Role of MAPKs/p300 and NF-κB

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist‐induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by IL‐1β. However, the mechanisms underlying IL‐1β‐induced cPLA2 expression and PGE2 synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. IL‐1β‐induced cPLA2 protein and mRNA expression, PGE2 production, or phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs of MEK1, p42, p38, and JNK2. IL‐1β‐induced cPLA2 expression was also inhibited by pretreatment with a NF‐κB inhibitor, helenalin or transfection with siRNA of NIK, IKKα, or IKKβ. IL‐β‐induced NF‐κB translocation was blocked by pretreatment with helenalin, but not U0126, SB202190, and SP600125. In addition, transfection with p300 siRNA blocked cPLA2 expression induced by IL‐1β. Moreover, p300 was associated with the cPLA2 promoter, which was dynamically linked to histone H4 acetylation stimulated by IL‐1β. These results suggest that in HTSMCs, activation of MAPKs, NF‐κB, and p300 are essential for IL‐1β‐induced cPLA2 expression and PGE2 secretion. J. Cell. Biochem. 109: 1045–1056, 2010.

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.