Search

Exposure to diesel exhaust particles (DEPs) is known to cause serious health problems, owing to a steady increase in the number of diesel vehicles worldwide. DEPs comprise approximately 90% particle mass existing in the fine size range (≤2.5 μm) and are mainly absorbed in the respiratory tract. However, limited information is available on the effects of DEP exposure on the respiratory tract in humans. Here, we investigated the effect and signaling pathways of DEPs on the expression of mucin, especially MUC5AC and MUC5B, in human airway epithelial cells by reverse-transcriptase polymerase chain reaction (PCR), real-time PCR, enzyme-linked immunosorbent assay, western blotting, and immunofluorescence staining. The signaling pathways activated following DEP-induced expression of MUC5AC and MUC5B in airway epithelial cells were analyzed by evaluating Toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38), and nuclear factor kappa B (NF-κB) phosphorylation with western blot and small-interfering RNA (siRNA) analyses. DEPs significantly increased MUC5AC and MUC5B expression in human airway epithelial cells that was closely related to TLR4, MAPK (ERK 1/2 and p38), and NF-κB pathway activation. This is the first report to demonstrate the DEP-mediated increase in MUC5AC and MUC5B expression via the TLR4-mediated activation of ERK1/2, p38 MAPK, and NF-κB signaling pathways in human airway epithelial cells.

Adipokines, a group of proteins including leptin, visfatin, resistin, and adiponectin, are produced by adipocytes. Among adipokines, resistin is implicated in insulin resistance and inflammatory response modulation. Mucus hypersecretion has been greatly linked to airway diseases, such as asthma, chronic obstructive pulmonary disease, and rhinosinusitis. Increasing evidence has indicated that adipokines, such as leptin and visfatin, play important regulatory roles in various biological processes involved in mucus secretion. However, the effects of resistin on mucin expression in human airway epithelial cells, as well as the underlying mechanisms, have not been investigated yet. We showed that resistin affected mucin expression in human airway epithelial cells via the mitogen-activated protein kinase/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Resistin increased MUC5AC and MUC5B expression in NCI-H292 and primary human nasal epithelial cells. Additionally, it significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and NF-κB. ERK1/2 and p38 specific inhibitors significantly attenuated resistin-induced MUC5AC/5B expression; however, NF-κB inhibitor reduced resistin-induced MUC5AC, but not MUC5B, expression. Knockdown of ERK1, ERK2, and p38 by ERK1, ERK2, and p38 small interfering RNA (siRNA), respectively, significantly blocked resistin-induced MUC5AC and MUC5B mRNA expression. In addition, NF-κB siRNA attenuated resistin-induced MUC5AC, but not MUC5B, expression. These results suggested that resistin induced MUC5AC and MUC5B expression via activation of different signaling pathways in human airway epithelial cells.

Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine and a potent factor for B- and T-cell growth and differentiation. Recent studies have demonstrated an association of TSLP with allergic and inflammatory airway diseases. However, no study on the effect of TSLP on expression of mucin genes in airway epithelial cells has been reported. Therefore, the effects and brief signaling pathways of TSLP on expression of mucin genes in human airway epithelial cells were investigated in this study. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effect and signaling pathway of TSLP on expression of mucin genes were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). In human NCI-H292 airway epithelial cells, TSLP increased MUC5B expression. TSLP significantly activated phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase (MAPK). U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor) significantly attenuated TSLP-induced MUC5B mRNA expression. Knockdown of ERK1, ERK2, and p38 MAPK by ERK1, ERK2, and p38 MAPK siRNA significantly blocked TSLP-induced MUC5B mRNA expression. In the primary cultures of normal nasal epithelial cells, TSLP significantly increased MUC5B mRNA expression, which was significantly attenuated after pretreatment with U0126 and SB203580. These results suggest that TSLP induces MUC5B expression via the ERK1/2 and p38 MAPK signaling pathway in human airway epithelial cells.

The biologic actions of insulin-like growth factor-1(IGF-1) are associated with cell growth, differentiation, migration, and survival. IGF-1 constitutes the pathogenic factor in formation of nasal polyps and the regulatory factor in expression of mucins. However, the effect of IGF-1 on MUC8 and MUC5B expression has not been reported. Therefore, in this study, the effect and brief signaling pathway of IGF-1 on MUC8 and MUC5B expression were investigated in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal human nasal epithelial cells, the effect and signaling pathway of IGF-1 on MUC8 and MUC5B expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA (siRNA) for mitogen-activated protein kinase (MAPK). IGF-1 induced MUC8 and MUC5B expression, and activated phosphorylation of ERK1/2 and p38 MAPK. U0126 (ERK1/2 inhibitor) and SB203580 (p38 MAPK inhibitor) inhibited IGF-1 induced MUC8 and MUC5B mRNA expression. In addition, the knockdown of ERK1 and p38 MAPK by siRNA significantly blocked IGF-1 induced MUC8 and MUC5B mRNA expression; the knockdown of ERK2 MAPK by siRNA did not. These results demonstrate for the first time that IGF-1 induced MUC8 and MUC5B expression is regulated by activation of the ERK1 and p38 MAPK signaling pathway in human airway epithelial cells.

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.

This study demonstrates for the first time the effects of IL-1beta on the regulation of protein production as well as MUC2 gene transcription in cultured human airway epithelial cells. The effect of IL-1beta on the regulation of MUC2 protein was determined by flow cytometric analysis. The expression level of MUC2 induced by IL-1beta increased in a dose-dependent manner. MUC2 transcripts were detected after 2 h of exposure to IL-1beta and reached maximal level after 8 h. Actinomycin D experiments indicated that the IL-1beta-mediated MUC2 expression was controlled by transcriptional regulation. Both RT-PCR and FACS analysis showed that budesonide concomitantly attenuated IL-1beta mediated MUC2 gene as well as protein production levels. Use of the glucocorticoid receptor antagonist, RU-486, restored the inhibitory effect of budesonide on the IL-1beta-mediated MUC2 protein as well as gene. The data suggest that IL-1beta up-regulates MUC2 gene by transcriptional regulation and that budesonide suppresses the IL-1beta-medicated MUC2 expression via decreased transcriptional activation.