Shue-Fen Luo

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.

Overexpression of HO-1 Protects against TNF-α-Mediated Airway Inflammation by Down-Regulation of TNFR1-Dependent Oxidative Stress

Oxidative stresses are believed to play an important role in the induction of both cell adhesion molecules and pro-inflammatory cytokines, a key event in a variety of inflammatory processes. The enzyme heme oxygenase-1 (HO-1) functions as an antioxidant and serves to protect against tissue injury. In this study, we report that HO-1 was induced in cultured human tracheal smooth muscle cells after either treatment with a potent inducer of HO-1 activity, cobalt protoporphyrin IX, or infection with a recombinant adenovirus that carries the human HO-1 gene. Overexpression of HO-1 protected against tumor necrosis factor (TNF)-alpha-mediated airway inflammation via the down-regulation of oxidative stress, adhesion molecules, and interleukin-6 in both cultured human tracheal smooth muscle cells and the airways of mice. In addition, HO-1 overexpression inhibited TNF-alpha-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, adherence of THP-1 cells, generation of interleukin-6, p47(phox) translocation, and nuclear factor-kappaB activation. HO-1 overexpression also attenuated TNF-alpha-induced oxidative stress, which was abrogated in the presence of both the HO-1 inhibitor, zinc protoporphyrin IX, as well as a carbon monoxide scavenger. In addition, HO-1 overexpression reduced the formation of a TNFR1/c-Src/p47(phox) complex. These results suggest that HO-1 functions as a suppressor of TNF-alpha signaling, not only by inhibiting the expression of adhesion molecules and generation of interleukin-6, but also by diminishing intracellular reactive oxygen species production and nuclear factor-kappaB activation in both cultured human tracheal smooth muscle cells and the airways of mice.

Lipoteichoic Acid Induces HO-1 Expression via the TLR2/MyD88/c-Src/NADPH Oxidase Pathway and Nrf2 in Human Tracheal Smooth Muscle Cells

Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. Increasing reports have indicated that lipoteichoic acid (LTA) exerts as LPS as an immune system-stimulating agent and plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. We report that LTA is an inducer of HO-1 expression mediated through the signaling pathways in human tracheal smooth muscle cells (HTSMCs). LTA-induced HO-1 protein levels, mRNA expression, and promoter activity were attenuated by transfection with dominant negative mutants of TLR2 and MyD88, by pretreatment with the inhibitors of c-Src (PP1), NADPH oxidase (diphenylene iodonium chloride (DPI) and apocynin (APO)), and reactive oxygen species (ROS) scavenger (N-acetyl-l-cysteine) or by transfection with small interfering RNAs of Src and NF-E2-related factor 2 (Nrf2). LTA-stimulated translocation of p47phox and Nrf2 or ROS production was attenuated by transfection with dominant negative mutants of TLR2, MyD88, and c-Src and by pretreatment with DPI or APO. Furthermore, LTA-induced TLR2, MyD88, TNFR-associated factor (TRAF)6, c-Src, and p47phox complex formation was revealed by immunoprecipitation using an anti-TLR2 or anti-c-Src Ab followed by Western blot analysis against an anti-TLR2, anti-MyD88, anti-TRAF6, anti-c-Src, or anti-p47phox Ab. These results demonstrated that LTA-induced ROS generation was mediated through the TLR2/MyD88/TRAF6/c-Src/NADPH oxidase pathway, in turn initiates the activation of Nrf2, and ultimately induces HO-1 expression in HTSMCs.

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.

P2Y2 receptor‐mediated proliferation of C6 glioma cells via activation of Ras/Raf/MEK/MAPK pathway

Extracellular purine and pyrimidine nucleotides have been implicated in the regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the P2Y receptor on C6 glioma cells responsible for stimulating cell proliferation associated with mitogen‐activated protein kinase (MAPK) activation. UTP and ATP produced a similar effect on [3H]‐thymidine incorporation in a time‐ and concentration‐dependent manner, suggesting the involvement of P2Y2 receptor in mediating proliferation of C6 glioma cells. In response to UTP, both p42 and p44 MAPK were activated in a time‐ and concentration‐dependent manner using Western blot analysis with an anti‐phospho‐p42/p44 MAPK antibody. The phosphorylation reached maximal levels after 5u2003min and declining by 30u2003min. Pretreatment with pertussis toxin (PTX) did not change these responses to UTP. Both DNA synthesis and phosphorylation of MAPK in response to UTP were attenuated by tyrosine kinase inhibitors, genistein and herbimycin A, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and removal of Ca2+ by addition of BAPTA/AM plus EGTA. UTP‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2). Furthermore, we showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf‐301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by ATP and UTP. These results conclude that the mitogenic effect of UTP is mediated through a P2Y2 receptor that involves the activation of Ras/Raf/MEK/MAPK pathway. UTP‐mediated MAPK activation was modulated by Ca2+, PKC, and tyrosine kinase associated with cell proliferation in cultured C6 glioma cells.

Cigarette smoke extract induces COX-2 expression via a PKCα/c-Src/EGFR, PDGFR/PI3K/Akt/NF-κB pathway and p300 in tracheal smooth muscle cells

Exposure to cigarette smoke extract (CSE) leads to airway or lung inflammation, which may be mediated through cyclooxygenase-2 (COX-2) expression and its product prostaglandin E2 (PGE2) synthesis. The aim of this study was to investigate the molecular mechanisms underlying CSE-induced COX-2 expression in human tracheal smooth muscle cells (HTSMCs). Here, we describe that COX-2 induction is dependent on PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB signaling in HTSMCs. CSE stimulated the phosphorylation of c-Src, EGFR, PDGFR, and Akt, which were inhibited by pretreatment with the inhibitor of PKCalpha (Gö6976 or Gö6983), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), or PI3K (LY294002). Moreover, CSE induced a significant increase in COX-2 expression, which was reduced by pretreatment with these inhibitors or transfection with siRNA of PKCalpha, Src, or Akt. Furthermore, CSE-stimulated NF-kappaB p65 phosphorylation and translocation were also attenuated by pretreatment with Gö6976, PP1, AG1478, AG1296, or LY294002. CSE-induced COX-2 expression was also mediated through the recruitment of p300 associated with NF-kappaB in HTSMCs, revealed by coimmunoprecipitation and Western blot analysis. In addition, pretreatment with the inhibitors of NF-kappaB (helenalin) and p300 (garcinol) or transfection with p65 siRNA and p300 siRNA markedly inhibited CSE-regulated COX-2 expression. However, CSE-induced PGE2 generation was reduced by pretreatment with the inhibitor of COX-2 (NS-398). These results demonstrated that in HTSMCs, CSE-induced COX-2-dependent PGE2 generation was mediated through PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt leading to the recruitment of p300 with NF-kappaB complex.

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.

Interleukin-1β induces ICAM-1 expression enhancing leukocyte adhesion in human rheumatoid arthritis synovial fibroblasts: Involvement of ERK, JNK, AP-1, and NF-κB

Interleukin‐1β (IL‐1β) has been shown to induce the expression of adhesion molecules on various cell types and contributes to inflammatory responses. However, the molecular mechanisms by which IL‐1β induced intercellular adhesion molecule (ICAM)‐1 expression remain unclear in human rheumatoid arthritis synovial fibroblasts (RASFs). Here, we demonstrated that IL‐1β induces ICAM‐1 gene expression via the de novo protein synthesis through transcription and translation, which is attenuated by pretreatment with actinomycin D and cycloheximide, respectively. IL‐1β‐induced ICAM‐1 expression, extracellular signal‐regulated kinase (ERK) and c‐Jun‐N‐terminal kinase (JNK) phosphorylation, AP‐1 activation, and nuclear factor‐κB (NF‐κB) p65 translocation were attenuated by the inhibitors of MEK1/2 (U0126), JNK (SP600125), AP‐1 (tanshinone IIA), and NF‐κB (helenalin) or transfection with respective short hairpin RNA plasmids. Moreover, IL‐1β‐stimulated NF‐κB p65 translocation was blocked by helenalin, but not by U0126 or SP600125, revealing that MAPKs and NF‐κB pathways were independent on these responses. IL‐1β‐stimulated AP‐1 activation was blocked by U0126 or SP600125, revealing that ERK and JNK linked to AP‐1 on these responses. IL‐1β‐stimulated ICAM‐1 gene expression was attenuated by pretreatment with U0126, SP600125, tanshinone IIA, or helenalin, revealed by ICAM‐1 promoter assay and real‐time RT‐PCR analysis. Finally, up‐regulation of ICAM‐1 enhanced the adhesion of leukocytes to RASFs exposed to IL‐1β. These results suggest that in human RASFs, activation of ERK, JNK, AP‐1, and NF‐κB are essential for IL‐1β‐induced ICAM‐1 expression and leukocyte adhesion. J. Cell. Physiol. 224: 516–526, 2010.

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.

Tumour necrosis factor‐α‐ and interleukin‐1β‐stimulated cell proliferation through activation of mitogen‐activated protein kinase in canine tracheal smooth muscle cells

The elevated levels of inflammatory cytokines such as tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF‐α and IL‐1β on cell proliferation and activation of p42/p44 mitogen‐activated protein kinase (MAPK) in these cells. TNF‐α and IL‐1β induced [3H]‐thymidine incorporation in a time‐ and concentration‐dependent manner. The maximal stimulation of [3H]‐thymidine incorporation induced by TNF‐α and IL‐1β was seen 12u2003h after incubation with these cytokines. In response to TNF‐α and IL‐1β, p42/p44 MAPK was activated with a concentration‐dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF‐α and IL‐1β. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)‐phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)‐PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca2+ by addition of BAPTA/AM plus EGTA. TNF‐α‐ and IL‐1β‐induced [3H]‐thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. These results suggest that the mitogenic effects of TNF‐α and IL‐1β were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF‐α‐ and IL‐1β‐mediated responses were modulated by PLC, Ca2+, PKC, and tyrosine kinase associated with cell proliferation in TSMCs.