I-Ta Lee

Inflammatory signalings involved in airway and pulmonary diseases.

In respiratory diseases, there is an increased expression of multiple inflammatory proteins in the respiratory tract, including cytokines, chemokines, and adhesion molecules. Chemokines have been shown to regulate inflammation and immune cell differentiation. Moreover, many of the known inflammatory target proteins, such as matrix metalloproteinase-9 (MMP-9), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2), are associated with airway and lung inflammation in response to various stimuli. Injuriously environmental stimuli can access the lung through either the airways or the pulmonary and systemic circulations. The time course and intensity of responses by resident and circulating cells may be regulated by various inflammatory signalings, including Src family kinases (SFKs), protein kinase C (PKC), growth factor tyrosine kinase receptors, nicotinamide adenine dinucleotide phosphate (NADPH)/reactive oxygen species (ROS), PI3K/Akt, MAPKs, nuclear factor-kappa B (NF-κB), activator protein-1 (AP-1), and other signaling molecules. These signaling molecules regulate both key inflammatory signaling transduction pathways and target proteins involved in airway and lung inflammation. Here, we discuss the mechanisms involved in the expression of inflammatory target proteins associated with the respiratory diseases. Knowledge of the mechanisms of inflammation regulation could lead to the pharmacological manipulation of anti-inflammatory drugs in the respiratory diseases.

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.

Induction of COX-2/PGE2/IL-6 is crucial for cigarette smoke extract-induced airway inflammation: Role of TLR4-dependent NADPH oxidase activation

Exposure to cigarette smoke extract (CSE) leads to airway and lung inflammation through an oxidant-antioxidant imbalance. Cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) have been shown to play critical roles in respiratory inflammation. Here, we show that COX-2/PGE(2)/IL-6 induction is dependent on Toll-like receptor 4 (TLR4)/NADPH oxidase signaling in human tracheal smooth muscle cells (HTSMCs). CSE induced COX-2 expression in vitro in HTSMCs and in vivo in the airways of mice. CSE also directly caused an increase in TLR4. Moreover, CSE-regulated COX-2, PGE(2), and IL-6 generation was inhibited by pretreatment with TLR4 Ab; inhibitors of c-Src (PP1), NADPH oxidase (diphenylene iodonium chloride and apocynin), p38 MAPK (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-kappaB (helenalin); a ROS scavenger (N-acetyl-l-cysteine); and transfection with siRNA of TLR4, MyD88, TRAF6, Src, p47(phox), p38, p42, JNK2, or p65. CSE-induced leukocyte numbers in BAL fluid were also reduced by pretreatment with these inhibitors. Furthermore, CSE induced p47(phox) translocation and TLR4/MyD88/TRAF6 and c-Src/p47(phox) complex formation. We found that PGE(2) enhanced IL-6 production in HTSMCs and leukocyte count in BAL fluid. In addition, treatment with nicotine could induce COX-2, PGE(2), and IL-6 generation in in vivo and in vitro studies. These results demonstrate that CSE-induced ROS generation was mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase pathway, in turn initiated the activation of MAPKs and NF-kappaB, and ultimately induced COX-2/PGE(2)/IL-6-dependent airway inflammation.

Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells

BackgroundIn bacteria-induced glomerulonephritis, Toll-like receptor 4 (TLR4) activation by lipopolysaccharide (LPS, a key component of the outer membranes of Gram-negative bacteria) can increase oxidative stress and the expression of vascular cell adhesion molecule-1 (VCAM-1), which recruits leukocytes to the glomerular mesangium. However, the mechanisms underlying VCAM-1 expression induced by LPS are still unclear in human renal mesangial cells (HRMCs).ResultsWe demonstrated that LPS induced VCAM-1 mRNA and protein levels associated with an increase in the promoter activity of VCAM-1, determined by Western blot, RT-PCR, and promoter assay. LPS-induced responses were inhibited by transfection with siRNAs of TLR4, myeloid differentiation factor 88 (MyD88), Nox2, Nox4, p47phox, c-Src, p38 MAPK, activating transcription factor 2 (ATF2), and p300 or pretreatment with the inhibitors of reactive oxygen species (ROS, edaravone), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], c-Src (PP1), p38 MAPK (SB202190), and p300 (GR343). LPS induced NADPH oxidase activation, ROS production, and p47phox translocation from the cytosol to the membrane, which were reduced by PP1 or c-Src siRNA. We observed that LPS induced TLR4, MyD88, c-Src, and p47phox complex formation determined by co-immunoprecipitation and Western blot. We further demonstrated that LPS stimulated ATF2 and p300 phosphorylation and complex formation via a c-Src/NADPH oxidase/ROS/p38 MAPK pathway. Up-regulation of VCAM-1 led to enhancing monocyte adhesion to HRMCs challenged with LPS, which was inhibited by siRNAs of c-Src, p47phox, p38 MAPK, ATF2, and p300 or pretreatment with an anti-VCAM-1 neutralizing antibody.ConclusionsIn HRMCs, LPS-induced VCAM-1 expression was, at least in part, mediated through a TLR4/MyD88/ c-Src/NADPH oxidase/ROS/p38 MAPK-dependent p300 and ATF2 pathway associated with recruitment of monocyte adhesion to kidney. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in renal diseases.

Cooperation of TLR2 with MyD88, PI3K, and Rac1 in Lipoteichoic Acid–Induced cPLA2/COX-2–Dependent Airway Inflammatory Responses

Lipoteichoic acid (LTA) plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. Cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin (IL)-6 have been demonstrated to engage in airway inflammation. In this study, LTA-induced cPLA(2) and COX-2 expression and PGE(2) or IL-6 synthesis were attenuated by transfection with siRNAs of TLR2, MyD88, Akt, p42, p38, JNK2, and p65 or pretreatment with the inhibitors of PI3K (LY294002), p38 (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-kappaB (helenalin) in human tracheal smooth muscle cells (HTSMCs). LTA also induced cPLA(2) and COX-2 expression and leukocyte count in bronchoalveolar lavage fluid in mice. LTA-regulated PGE(2) or IL-6 production was inhibited by pretreatment with the inhibitors of cPLA(2) (AACOCF(3)) and COX-2 (NS-398) or transfection with cPLA(2) siRNA or COX-2 siRNA, respectively. LTA-stimulated NF-kappaB translocation or cPLA(2) phosphorylation was attenuated by pretreatment with LY294002, SB202190, U0126, or SP600125. Furthermore, LTA could stimulate TLR2, MyD88, PI3K, and Rac1 complex formation. We also demonstrated that Staphylococcus aureus could trigger these responses through a similar signaling cascade in HTSMCs. It was found that PGE(2) could directly stimulate IL-6 production in HTSMCs or leukocyte count in bronchoalveolar lavage fluid in mice. These results demonstrate that LTA-induced MAPKs activation is mediated through the TLR2/MyD88/PI3K/Rac1/Akt pathway, which in turn initiates the activation of NF-kappaB, and ultimately induces cPLA(2)/COX-2-dependent PGE(2) and IL-6 generation.

Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells.

Salvianolic acid B (Sal B), a water‐soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo‐E‐deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP‐2 and MMP‐9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo‐E‐deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP‐2 and MMP‐9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP‐2 and barely detectable levels for MMP‐9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS‐induced cell migration through the inactivation of MMP‐2 and MMP‐9 protein synthesis as well as the downregulation of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2) and c‐Jun NH2‐terminal kinase (JNK). These results demonstrate that Sal B has anti‐migration properties on smooth muscle cells and may explain its anti‐atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 100: 372–384, 2007.

Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes

BackgroundJapanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear.MethodsIn this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs.ResultsHere, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs.ConclusionFrom these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases.

Cigarette smoke particle-phase extract induces HO-1 expression in human tracheal smooth muscle cells: role of the c-Src/NADPH oxidase/MAPK/Nrf2 signaling pathway.

Heme oxygenase-1 (HO-1) is known as an oxidative stress protein that is up-regulated by various stimuli. HO-1 has been shown to protect cells against oxidative damage. Cigarette smoke is a potential inflammatory mediator that causes chronic obstructive pulmonary disease and asthma. In this study, we report that cigarette smoke particle-phase extract (CSPE) is an inducer of HO-1 expression mediated through various signaling pathways in human tracheal smooth muscle cells (HTSMCs). CSPE-induced HO-1 protein, mRNA expression, and promoter activity were attenuated by pretreatment with a ROS scavenger (N-acetyl-l-cysteine) and inhibitors of c-Src (PP1), NADPH oxidase [diphenylene iodonium chloride (DPI) and apocynin (APO)], MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs for Src, p47(phox), NOX2, p42, p38, JNK2, or NF-E2-related factor 2 (Nrf2). CSPE-stimulated translocation of p47(phox) and Nrf2, ROS production, and NADPH oxidase activity was attenuated by transfection with siRNAs for Src, p47(phox), and NOX2 or pretreatment with PP1, DPI, or APO. Furthermore, CSPE-induced NOX2, c-Src, and p47(phox) complex formation was revealed by immunoprecipitation using an anti-NOX2, anti-p47(phox), or anti-c-Src Ab followed by Western blot against anti-NOX2, anti-p47(phox), or anti-c-Src Abs. These results demonstrate that CSPE-induced ROS generation is mediated through a c-Src/NADPH oxidase/MAPK pathway and in turn initiates the activation of Nrf2 and ultimately induces HO-1 expression in HTSMCs.

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.