Ikuko Takeshita

N‐acetylcysteine attenuates TNF‐α‐induced p38 MAP kinase activation and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells

We have previously shown that tumour necrosis factor‐α (TNF‐α) activates p38 mitogen‐activated protein (MAP) kinase to produce interleukin‐8 (IL‐8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF‐α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti‐oxidants is important for the regulation of cytokine production. However, the effect of N‐acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF‐α‐induced activation of p38 MAP kinase pathway and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF‐α‐induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase‐mediated IL‐8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF‐α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL‐8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC‐treated cells. NAC attenuated TNF‐α‐induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase‐mediated IL‐8 production by TNF‐α‐stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF‐α‐induced activation of p38 MAP kinase pathway and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells, and we emphasize that anti‐oxidant therapy is an important strategy for the treatment of acute lung injury.

ASK1 regulates influenza virus infection-induced apoptotic cell death.

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.

The Rac1/JNK pathway is critical for EGFR-dependent barrier formation in human airway epithelial cells

The airway epithelial barrier provides defenses against inhaled antigens and pathogens, and alterations of epithelial barrier function have been proposed to play a significant role in the pathogenesis of chronic airway diseases. Although the epidermal growth factor receptor (EGFR) plays roles in various physiological and pathological processes on the airway epithelium, the role of EGFR on barrier function in the airway remains largely unknown. In the present study, we assessed the effects of EGFR activation on paracellular permeability in airway epithelial cells (AECs). EGFR activation induced by the addition of EGF increased transepithelial electrical resistance (TER) in AECs. An EGFR-blocking antibody eradicated the development of TER, paracellular influx of dextran, and spatial organization of tight junction. Moreover, the effects of EGFR activation on paracellular permeability were eradicated by knockdown of occludin. To identify the EGFR signaling pathway that regulates permeability barrier development, we investigated the effects of several MAP kinase inhibitors on permeability barrier function. Pretreatment with a JNK-specific inhibitor, but not an ERK- or p38-specific inhibitor, attenuated the development of TER induced by EGFR activation. Rac1 is one of the upstream activators for JNK in EGFR signaling. Rac1 knockdown attenuated the phosphorylation of JNK activation and EGFR-mediated TER development. These results suggest that EGFR positively regulates permeability barrier development through the Rac1/JNK-dependent pathway.

Glucocorticoids enhance airway epithelial barrier integrity.

Asthma is a chronic inflammatory disorder of the airways, but its pathogenesis is incompletely understood. While asthma is a complex disease caused by multiple factors, epithelial barrier damage is a cardinal feature. Glucocorticoids (GCs) are the most effective anti-inflammatory drugs in the treatment of asthma. However, the effects of GCs on the airway epithelial barrier have not been evaluated. Epithelial barrier functions were evaluated in cultured human airway epithelial cell monolayers, Calu-3 and 16HBE. Then, the cells were treated with dexamethasone (Dex), fulticasone propionate (FP), or budesonide (BD) for 5 days. Permeability measured by transepithelial electrical resistance was increased by treatment with Dex, FP, and BD in a dose-dependent manner. Permeability to fluorescein isothiocyanate-labeled dextran was markedly reduced by these treatments. Immunocytostaining revealed that Dex treatment potentiated tight junction formation in these polarized epithelial cells. Knockdown of epidermal growth factor receptor (EGFR) by small interference RNA blunted the effects of Dex on barrier integrity. Although EGFR expression was not affected by Dex treatment, EGFR phosphorylation was enhanced in Dex-treated cells. This is suggesting that EGFR are important for this phenomenon. These findings suggest that GC inhalation therapy can improve epithelial barrier integrity and might contribute to the therapeutic effects of GCs for treating asthma.

Mitogen-activated protein kinase involves neutrophil elastase-induced morphological changes in human bronchial epithelial cells.

Neutrophil elastase (NE) promotes the detachment of airway epithelial cells; however, changes in overall morphology of NE-stimulated bronchial epithelial cell (BEC) monolayer are different from trypsin stimulation. Ras/Raf-initiated-mitogen activated protein kinase (MAPK) also known as extracellular signal-regulated kinase, pathway regulates integrin functions which participate in regulating attachment and detachment of cell and cellular morphology. However, little is known about the role of MAPK in NE-induced changes in overall morphology of BEC. In the present study, we examined the role of MAPK in NE-induced changes in overall morphology of BEC monolayer. To this end, we examined changes in cellular morphology and MAPK activation in NE-stimulated BEC monolayer, and the effect of PD 98059 as the specific inhibitor for MAPK kinase-1 (MEK-1, the upstream regulator of MAPK) on NE-induced changes in cellular morphology and MAPK activation. The results showed that in stimulation of NE, BECs detached and gaps developed, and MAPK activation was observed. PD 98059 attenuated NE-induced changes in cellular morphology as well as MAPK activation. These results indicated that in addition to proteolytic activity of NE on extracellular matrix (ECM), NE-activated MAPK pathway, at least in part, is involved in NE-induced changes in overall morphology and the detachment of BEC monolayer.

Intracellular glutathione regulates tumour necrosis factor-α-induced p38 MAP kinase activation and RANTES production by human bronchial epithelial cells

RANTES plays an important role in the production of allergic inflammation of the airway through its chemotactic activity for eosinophils. The cellular reduction and oxidation (redox) changes are involved in the activation of p38 mitogen‐activated protein (MAP) kinase and the induction of cytokine expression. It has previously been shown that tumour necrosis factor (TNF)‐MA activates p38 mitogen‐activated protein (MAP) kinase to produce cytokine, including RANTES, that N‐acetylcysteine (NAC) attenuates cytokine production by human bronchial epithelial cells (BECs), and that sensitivity to TNFα is inversely correlated with cellular redox state. However, a role of cellular redox regulated by intracellular glutathione (GSH) in TNFα‐induced p38 MAP kinase activation and p38 MAP kinase‐mediated RANTES production by human BECs has not been determined.

N‐acetyl‐L‐cysteine inhibits bleomycin‐induced interleukin‐8 secretion by bronchial epithelial cells

Bleomycin (BLM) has proven effective for the treatment of cancers, but the most serious dose‐limiting side‐effect is the development of pulmonary toxicity. Although the precise mechanism in the pathogenesis of BLM‐induced lung injury has not been determined, oxygen radicals and neutrophils are indicated to play a key role in it. Interleukin‐8 (IL‐8) is thought to be an important mediator of the pathogenesis of acute lung injury.

Regulation by intracellular glutathione of TNF‐α‐induced p38 MAP kinase activation and RANTES production by human pulmonary vascular endothelial cells

Conclusions: These results indicated that TNF‐α‐induced p38 MAP kinase activation and p38 MAP kinase‐mediated RANTES production by human pulmonary vascular endothelial cells are inversely regulated by intracellular GSH levels.

Heregulin activation of ErbB2/ErbB3 signaling potentiates the integrity of airway epithelial barrier

BACKGROUND Members of the ErbB family of the receptor protein tyrosine kinase superfamily mediate heregulin (HRG)-induced cell responses. Here we investigated HRG activation of ErbB receptors, and the role of this activation in the development of the permeability barrier in airway epithelial cells (AECs). METHODS Two airway epithelial-like cell lines, Calu-3 and 16HBE were exposed to HRG or no stimulus and were evaluated with respect to their paracellular permeability as determined by transepithelial electric resistance (TER) and fluorescein isothiocyanate (FITC)-dextran flux. Tight junctions (TJs) were assessed by immunocytochemical localization of occludin and zonula occludens-1. RESULTS HRG promoted the development of the permeability barrier and TJ formation by monolayers of Calu-3 and 16HBE cells. Calu-3 cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4, on their surface. ErbB3 knockdown by small interference RNA (siRNA) blunted the effects of HRG on the permeability barrier. ErbB3 is known as a kinase-dead receptor and relies on other members of the family for its phosphorylation. To identify its heterodimerization partner, we knocked down the expression of other ErbB family receptors. We found that HRGs effect on the permeability barrier could be significantly attenuated by transfecting cells with ErbB2 siRNA but not with EGFR siRNA. CONCLUSION These results indicate that HRG activation of ErbB2/ErbB3 heterodimers is essential for regulation of the permeability barrier in AECs.

p38 MAP kinase regulates RANTES production byTNF‐α‐stimulated human pulmonary vascular endothelial cells

Background: RANTES plays an important role in the production of allergic inflammation of the airway through its chemotactic activity for eosinophils. However, the intracellular signal regulating RANTES expression in human pulmonary vascular endothelial cells has not been determined. In the present study, therefore, we examined the role of p38 mitogen‐activated protein (MAP) kinase in RANTES production by tumor necrosis factor (TNF)‐α‐stimulated pulmonary vascular endothelial cells in order to clarify the signal transduction pathway regulating RANTES production by pulmonary vascular endothelial cells.