Yin Chen

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• Li, H., Castro, M., Denlinger, L. C., Erzurum, S. C., Fahy, J. V., Gaston, B., Israel, E., Jarjour, N. N., Levy, B. D., Mauger, D. T., Moore, W. C., Wenzel, S. E., Zein, J., Bleecker, E. R., Meyers, D. A., Chen, Y., Li, X., & , N. S. (2023). Investigations of a combination of atopic status and age of asthma onset identify asthma subphenotypes. The Journal of asthma : official journal of the Association for the Care of Asthma, 60(10), 1843-1852.
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  Subphenotypes of asthma may be determined by age onset and atopic status. We sought to characterize early or late onset atopic asthma with fungal or non-fungal sensitization (AAFS or AANFS) and non-atopic asthma (NAA) in children and adults in the Severe Asthma Research Program (SARP). SARP is an ongoing project involving well-phenotyped patients with mild to severe asthma.
• Liang, W., Gu, M., Zhu, L., Yan, Z., Schenten, D., Herrick, S., Li, H., Samrat, S. K., Zhu, J., & Chen, Y. (2023). The main protease of SARS-CoV-2 downregulates innate immunity via a translational repression. Signal transduction and targeted therapy, 8(1), 162.
• Samrat, S. K., Bashir, Q., Zhang, R., Huang, Y., Liu, Y., Wu, X., Brown, T., Wang, W., Zheng, Y. G., Zhang, Q. Y., Chen, Y., Li, Z., & Li, H. (2023). A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases. Emerging microbes & infections, 12(1), 2204164.
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  SARS-CoV-2 has caused a global pandemic with significant humanity and economic loss since 2020. Currently, only limited options are available to treat SARS-CoV-2 infections for vulnerable populations. In this study, we report a universal fluorescence polarization (FP)-based high throughput screening (HTS) assay for SAM-dependent viral methyltransferases (MTases), using a fluorescent SAM-analogue, FL-NAH. We performed the assay against a reference MTase, NSP14, an essential enzyme for SARS-CoV-2 to methylate the N7 position of viral 5'-RNA guanine cap. The assay is universal and suitable for any SAM-dependent viral MTases such as the SARS-CoV-2 NSP16/NSP10 MTase complex and the NS5 MTase of Zika virus (ZIKV). Pilot screening demonstrated that the HTS assay was very robust and identified two candidate inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the NSP14 MTase with low micromolar IC. We used three functional MTase assays to unambiguously verified the inhibitory potency of these molecules for the NSP14 N7-MTase function. Binding studies indicated that these molecules are bound directly to the NSP14 MTase with similar low micromolar affinity. Moreover, we further demonstrated that these molecules significantly inhibited the SARS-CoV-2 replication in cell-based assays at concentrations not causing cytotoxicity. Furthermore, NSC111552 significantly synergized with known SARS-CoV-2 drugs including nirmatrelvir and remdesivir. Finally, docking suggested that these molecules bind specifically to the SAM-binding site on the NSP14 MTase. Overall, these molecules represent novel and promising candidates to further develop broad-spectrum inhibitors for the management of viral infections.
• Lian, Q., Zhang, K., Zhang, Z., Duan, F., Guo, L., Luo, W., Mok, B. W., Thakur, A., Ke, X., Motallebnejad, P., Nicolaescu, V., Chen, J., Ma, C. Y., Zhou, X., Han, S., Han, T., Zhang, W., Tan, A. Y., Zhang, T., , Wang, X., et al. (2022). Differential effects of macrophage subtypes on SARS-CoV-2 infection in a human pluripotent stem cell-derived model. Nature communications, 13(1), 2028.
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  Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19), with macrophages as one of the main cell types involved. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. Here, we establish a lung and macrophage co-culture system derived from human pluripotent stem cells (hPSCs), modeling the host-pathogen interaction in SARS-CoV-2 infection. We find that both classically polarized macrophages (M1) and alternatively polarized macrophages (M2) have inhibitory effects on SARS-CoV-2 infection. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Moreover, M1 macrophages suppress the growth and enhance apoptosis of lung cells. Inhibition of viral entry using an ACE2 blocking antibody substantially enhances the activity of M2 macrophages. Our studies indicate differential immune response patterns in distinct macrophage phenotypes, which could lead to a range of COVID-19 disease severity.
• Zhang, X., Chen, Z., Bhadani, R., Cao, S., Lu, M., Lytal, N., Chen, Y., & An, L. (2022). NISC: Neural Network-Imputation for Single-Cell RNA Sequencing and Cell Type Clustering. Frontiers in genetics, 13, 847112.
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  Single-cell RNA sequencing (scRNA-seq) reveals the transcriptome diversity in heterogeneous cell populations as it allows researchers to study gene expression at single-cell resolution. The latest advances in scRNA-seq technology have made it possible to profile tens of thousands of individual cells simultaneously. However, the technology also increases the number of missing values, i. e, dropouts, from technical constraints, such as amplification failure during the reverse transcription step. The resulting sparsity of scRNA-seq count data can be very high, with greater than 90% of data entries being zeros, which becomes an obstacle for clustering cell types. Current imputation methods are not robust in the case of high sparsity. In this study, we develop a Neural Network-based Imputation for scRNA-seq count data, NISC. It uses autoencoder, coupled with a weighted loss function and regularization, to correct the dropouts in scRNA-seq count data. A systematic evaluation shows that NISC is an effective imputation approach for handling sparse scRNA-seq count data, and its performance surpasses existing imputation methods in cell type identification.
• Zhou, X., Zhu, L., Bondy, C., Wang, J., Luo, Q., & Chen, Y. (2022). AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway. International journal of molecular sciences, 23(10).
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  Current options for preventing or treating influenza are still limited, and new treatments for influenza viral infection are urgently needed. In the present study, we serendipitously found that a small-molecule inhibitor (AG1478), previously used for epidermal growth factor receptor (EGFR) inhibition, demonstrated a potent activity against influenza both in vitro and in vivo. Surprisingly, the antiviral effect of AG1478 was not mediated by its EGFR inhibitory activity, as influenza virus was insensitive to EGFR blockade by other EGFR inhibitors or by siRNA knockdown of EGFR. Its antiviral activity was also interferon independent as demonstrated by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout approach. Instead, AG1478 was found to target the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1)-ADP-ribosylation factor 1 (ARF1) system by reversibly inhibiting GBF1 activity and disrupting its Golgi-cytoplasmic trafficking. Compared to known GBF1 inhibitors, AG1478 demonstrated lower cellular toxicity and better preservation of Golgi structure. Furthermore, GBF1 was found to interact with a specific set of viral proteins including M1, NP, and PA. Additionally, the alternation of GBF1 distribution induced by AG1478 treatment disrupted these interactions. Because targeting host factors, instead of the viral component, imposes a higher barrier for developing resistance, GBF1 modulation may be an effective approach to treat influenza infection.
• Chau, B., Witten, M. L., Cromey, D., Chen, Y., & Lantz, R. C. (2021). Lung developmental is altered after inhalation exposure to various concentrations of calcium arsenate. Toxicology and applied pharmacology, 432, 115754.
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  Exposure to dust from active and abandoned mining operations may be a very significant health hazard, especially to sensitive populations. We have previously reported that inhalation of real-world mine tailing dusts during lung development can alter lung function and structure in adult male mice. These real-world dusts contain a mixture of metal(loid)s, including arsenic. To determine whether arsenic in inhaled dust plays a role in altering lung development, we exposed C57Bl/6 mice to a background dust (0 arsenic) or to the background dust containing either 3% or 10% by mass, calcium arsenate. Total level of exposure was kept at 100 μg/m. Calcium arsenate was selected since arsenate is the predominant species found in mine tailings. We found that inhalation exposure during in utero and postnatal lung development led to significant increases in pulmonary baseline resistance, airway hyper-reactivity, and airway collagen and smooth muscle expression in male C57Bl/6 mice. Responses were dependent on the level of calcium arsenate in the simulated dust. These changes were not associated with increased expression of TGF-β1, a marker of epithelial to mesenchymal transition. However, responses were correlated with decreases in the expression of club cell protein 16 (CC16). Dose-dependent decreases in CC16 expression and increases in collagen around airways was seen for animals exposed in utero only (GD), animals exposed postnatally only (PN) and animals continuously exposed throughout development (GDPN). These data suggest that arsenic inhalation during lung development can decrease CC16 expression leading to functional and structural alterations in the adult lung.
• Daines, M., Pereira, R., Cunningham, A., Pryor, B., Besselsen, D. G., Liu, Y., Luo, Q., & Chen, Y. (2021). Novel Mouse Models of Fungal Asthma. Frontiers in cellular and infection microbiology, 11, 683194.
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  is a ubiquitous fungus and a major allergen associated with the development of asthma. Inhalation of intact spores is the primary cause of human exposure to fungal allergen. However, allergen-rich cultured fungal filtrates are oftentimes used in the current models of fungal sensitization that do not fully reflect real-life exposures. Thus, establishing novel spore exposure models is imperative. In this study, we established novel fungal exposure models of both adult and neonate to live spores. We examined pathophysiological changes in the spore models as compared to the non-exposure controls and also to the conventional filtrate models. While both filtrate- and spore-exposed adult BALB/c mice developed elevated airway hyperresponsiveness (AHR), filtrates induced a greater IgE mediated response and higher broncholavage eosinophils than spores. In contrast, the mice exposed to spores had higher numbers of neutrophils. Both exposures induced comparable levels of lung tissue inflammation and mucous cell metaplasia (MCM). In the neonatal model, exposure to spores resulted in a significant increase of AHR in both adult and neonatal mice. Increased levels of IgE in both neonatal and adult mice exposed to spores was associated with increased eosinophilia in the treatment groups. Adult demonstrated increased numbers of lymphocytes that was paralleled by increased IgG1 production. Both adults and neonates demonstrated similarly increased eosinophilia, IgE, tissue inflammation and MCM.
• Gu, P., Wang, D., Zhang, J., Wang, X., Chen, Z., Gu, L., Liu, M., Meng, F., Yang, J., Cai, H., Xiao, Y., Chen, Y., & Cao, M. (2021). Protective function of interleukin-22 in pulmonary fibrosis. Clinical and translational medicine, 11(8), e509.
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  Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive scarring disease with unknown etiology. The evidence of a pathogenic role for transforming growth factor-beta (TGF-β) in the development and progression of IPF is overwhelming. In the present study, we investigated the role of interleukin-22 (IL-22) in the pathogenesis of IPF by regulating the TGF-β pathway. We measured parameters and tissue samples from a clinical cohort of IPF. IL-22R knock out (IL-22RA1 ) and IL-22 supplementation mouse models were used to determine if IL-22 is protective in vivo. For the mechanistic study, we tested A549, primary mouse type II alveolar epithelial cell, human embryonic lung fibroblast, and primary fibroblast for their responses to IL-22 and/or TGF-β1. In a clinical cohort, the expression level of IL-22 in the peripheral blood and lung tissues of IPF patients was lower than healthy controls, and the lower IL-22 expression was associated with poorer pulmonary function. IL-22R mice demonstrated exacerbated inflammation and fibrosis. Reciprocally, IL-22 augmentation by intranasal instillation of recombinant IL-22 repressed inflammation and fibrotic phenotype. In vitro, IL-22 treatment repressed TGF-β1 induced gene markers representing epithelial-mesenchymal-transition and fibroblast-myofibroblast-transition, likely via the inhibition of TGF-β receptor expression and subsequent Smad2/3 activation. IL-22 appears to be protective against pulmonary fibrosis by inhibiting TGF-β1 signaling, and IL-22 augmentation may be a promising approach to treat IPF.
• Kato, K., Chang, E. H., Chen, Y., Lu, W., Kim, M. M., Niihori, M., Hecker, L., & Kim, K. C. (2020). MUC1 contributes to goblet cell metaplasia and MUC5AC expression in response to cigarette smoke in vivo. American journal of physiology. Lung cellular and molecular physiology, 319(1), L82-L90.
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  Goblet cell metaplasia (GCM) and mucin overproduction are a hallmark of chronic rhinosinusitis (CRS) and chronic obstructive pulmonary disease (COPD). In the airways, cigarette smoke (CS) induces activation of the epidermal growth factor receptor (EGFR) leading to GCM and overexpression of the gel-forming mucin MUC5AC. Although previous studies have demonstrated that a membrane-bound mucin, MUC1, modulates the activation of CS-induced EGFR, the role of MUC1 in CS-induced GCM and mucin overproduction has not been explored. In response to CS exposure, wild-type (WT) rats displayed Muc1 translocation from the apical surface of airway epithelium to the intracellular compartment of hyperplastic intermediate cells, EGFR phosphorylation, GCM, and Muc5ac overproduction. Similarly, human CRS sinonasal tissues demonstrated hyperplasia of intermediate cells enriched with MUC1 in the intracellular compartment, which was accompanied by GCM and increased MUC5AC expression. To further evaluate the role of Muc1 in vivo, a Muc1 knockout (KO) rat (MUC in humans and Muc in animals) was developed. In contrast to WT littermates, Muc1-KO rats exhibited no activation of EGFR, and were protected from GCM and Muc5ac overproduction. Genetic knockdown of MUC1 in human lung or Muc1 knockout in primary rat airway epithelial cells led to significantly diminished EGF-induced MUC5AC production. Together, these findings suggest that MUC1-dependent EGFR activation mediates CS-induced GCM and mucin overproduction. Strategies designed to suppress MUC1-dependent EGFR activation may provide a novel therapeutic approach for treating mucin hypersecretion in CRS and COPD.
• Liu, Y., Liu, F., Liang, W., Zhu, L., Lantz, R. C., Zhu, J., & Chen, Y. (2020). Arsenic represses airway epithelial mucin expression by affecting retinoic acid signaling pathway. Toxicology and applied pharmacology, 394, 114959.
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  Arsenic is a ubiquitous environmental toxicant, found in high concentrations worldwide. Although abundant research has dealt with arsenic-induced cancers, studies on mechanisms of non-malignant lung diseases have not been complete. In addition, decades of research have mostly concentrated on high-dose arsenic exposure, which has very limited use in modeling the biological effects of today's low-dose exposures. Indeed, accumulated evidence has shown that low-dose arsenic exposure (i.e. ≤100 ppb) may also alter lung homeostasis by causing host susceptibility to viral infection. However, the underlying mechanism of this alteration is unknown. In this study, we found that low-dose sodium arsenite (As (III)) repressed major airway mucins-MUC5AC and MUC5B at both mRNA and protein levels. We further demonstrated that this repression was not caused by cellular toxicity or mediated by the reduction of a common mucin-inducing pathway-EGFR. Other established mucin activators- dsRNA, IL1β or IL17 were not able to override As (III)-induced mucin repression. Interestingly, the suppressing effect of As (III) appeared to be partially reversible, and supplementation of all trans retinoic acid (t-RA) doses dependently restored mucin gene expression. Further analyses indicated that As (III) treatment significantly reduced the protein level of retinoic acid receptors (RARα, γ and RXRα) as well as RARE promoter reporter activity. Therefore, our study fills in an important knowledge gap in the field of low-dose arsenic exposure. The interference of RA signaling, and mucin gene expression may be important pathogenic factors in low-dose arsenic induced lung toxicity.
• Wu, X., Lee, B., Zhu, L., Ding, Z., & Chen, Y. (2020). Exposure to mold proteases stimulates mucin production in airway epithelial cells through Ras/Raf1/ERK signal pathway. PloS one, 15(4), e0231990.
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  Environmental mold (fungus) exposure poses a significant threat to public health by causing illnesses ranging from invasive fungal diseases in immune compromised individuals to allergic hypertensive diseases such as asthma and asthma exacerbation in otherwise healthy people. However, the molecular pathogenesis has not been completely understood, and treatment options are limited. Due to its thermo-tolerance to the normal human body temperature, Aspergillus. fumigatus (A.fumigatus) is one of the most important human pathogens to cause different lung fungal diseases including fungal asthma. Airway obstruction and hyperresponsiveness caused by mucus overproduction are the hallmarks of many A.fumigatus induced lung diseases. To understand the underlying molecular mechanism, we have utilized a well-established A.fumigatus extracts (AFE) model to elucidate downstream signal pathways that mediate A.fumigatus induced mucin production in airway epithelial cells. AFE was found to stimulate time- and dose-dependent increase of major airway mucin gene expression (MUC5AC and MUC5B) partly via the elevation of their promoter activities. We also demonstrated that EGFR was required but not sufficient for AFE-induced mucin expression, filling the paradoxical gap from a previous study using the same model. Furthermore, we showed that fungal proteases in AFE were responsible for mucin induction by activating a Ras/Raf1/ERK signaling pathway. Ca2+ signaling, but ROS, both of which were stimulated by fungal proteases, was an indispensable determinant for ERK activation and mucin induction. The discovery of this novel pathway likely contributes to our understanding of the pathogenesis of fungal sensitization in allergic diseases such as fungal asthma.
• Zhou, J. J., Zhai, J., Zhou, H., Chen, Y., Guerra, S., Robey, I., Weinstock, G. M., Weinstock, E., Dong, Q., Knox, K. S., & Twigg, H. L. (2020). Supraglottic Lung Microbiome Taxa Are Associated with Pulmonary Abnormalities in an HIV Longitudinal Cohort. American journal of respiratory and critical care medicine, 202(12), 1727-1731.
• Daines, M., Zhu, L., Pereira, R., Zhou, X., Bondy, C., Pryor, B. M., Zhou, J., & Chen, Y. (2019). Alternaria induces airway epithelial cytokine expression independent of protease-activated receptor. Respirology (Carlton, Vic.).
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  A novel fungal allergen, Alternaria (Alt), has been previously shown to associate with the pathogenesis of allergic rhinitis and bronchial asthma, particularly in arid and semi-arid regions. Airway epithelial cells are among the first to encounter Alt, and epithelial cytokine production and subsequent airway inflammation are early events in the response to Alt exposure. However, the underlying mechanism is unclear. As protease-activated receptor 2 (PAR2) has been implicated in most of the Alt-induced biological events, we investigated the regulation of airway inflammation and epithelial cytokine expression by PAR2.
• Liu, X., Zheng, P., Zheng, S. G., Zhai, Y., Zhao, X., Chen, Y., Cai, C., Wu, Z., Huang, Z., Zou, X., Liao, C., & Sun, B. (2019). Co-sensitization and cross-reactivity of Blomia tropicalis with two Dermatophagoides species in Guangzhou, China. Journal of clinical laboratory analysis, 33(9), e22981.
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  Contradictory results have been reported previously in the analyses of cross-reactivity among Blomia tropicalis (Blo t), Dermatophagoides pteronyssinus (Der p), and Dermatophagoides farinae (Der f). This study aims to investigate the characteristics of co-sensitization and the IgE cross-reactivity among them and attempts to identify whether patients are sensitized to Blo t due to cross-reaction or true sensitization.
• Zhang, J., Hu, Y., Hau, R., Musharrafieh, R., Ma, C., Zhou, X., Chen, Y., & Wang, J. (2019). Identification of NMS-873, an allosteric and specific p97 inhibitor, as a broad antiviral against both influenza A and B viruses. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 133, 86-94.
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  Influenza virus infection causes substantial morbidity and mortality worldwide. The limited efficacy of oseltamivir in delayed treatment, coupled with the increasing incidences of oseltamivir-resistant strains, calls for next-generation of antiviral drugs. In this study, we discovered NMS-873, an allosteric and specific p97 inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. NMS-873 shows potent antiviral activity with low-nanomolar ECs against multiple human influenza A and B viruses, including adamantine-, oseltamivir-, or double resistant strains. Our data further showed that silencing of p97 via siRNA or inhibiting p97 by NMS-873 both inhibited virus replication and retained viral ribonucleoproteins (vRNPs) in the nucleus, confirming p97 is the drug target. Mechanistic studies have shown that the nuclear retention of vRNP with NMS-873 treatment is a combined result of two effects: the reduced viral M1 protein level (indirect effect), and the disruption of p97-NP interactions (direct effect). Taken together, our results suggest that p97 could be a novel antiviral target and its inhibitor, NMS-873, is a promising antiviral drug candidate.
• Zhou, J., Jenkins, T. G., Jung, A. M., Jeong, K. S., Zhai, J., Jacobs, E. T., Griffin, S. C., Dearmon-Moore, D., Littau, S. R., Peate, W. F., Ellis, N. A., Lance, P., Chen, Y., & Burgess, J. L. (2019). DNA methylation among firefighters. PloS one, 14(3), e0214282.
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  Firefighters are exposed to carcinogens and have elevated cancer rates. We hypothesized that occupational exposures in firefighters would lead to DNA methylation changes associated with activation of cancer pathways and increased cancer risk. To address this hypothesis, we collected peripheral blood samples from 45 incumbent and 41 new recruit non-smoking male firefighters and analyzed the samples for DNA methylation using an Illumina Methylation EPIC 850k chip. Adjusting for age and ethnicity, we performed: 1) genome-wide differential methylation analysis; 2) genome-wide prediction for firefighter status (incumbent or new recruit) and years of service; and 3) Ingenuity Pathway Analysis (IPA). Four CpGs, including three in the YIPF6, MPST, and PCED1B genes, demonstrated above 1.5-fold statistically significant differential methylation after Bonferroni correction. Genome-wide methylation predicted with high accuracy incumbent and new recruit status as well as years of service among incumbent firefighters. Using IPA, the top pathways with more than 5 gene members annotated from differentially methylated probes included Sirtuin signaling pathway, p53 signaling, and 5' AMP-activated protein kinase (AMPK) signaling. These DNA methylation findings suggest potential cellular mechanisms associated with increased cancer risk in firefighters.
• Chen, Y., Liao, S., Harper, R., Zhou, X., Bondy, C., Lizzarraga, R., Ran, D., An, L., & Zhu, L. (2018). The Club Cell Marker SCGB1A Downstream of FOXA1 is Reduced in Asthma. American Journal of Respiratory Cell and Molecular Biology. doi:DOI: 10.1165/rcmb.2018-0199OC
• Zhu, L., An, L., Ran, D., Lizarraga, R., Bondy, C., Zhou, X., Harper, R. W., Liao, S. Y., & Chen, Y. (2018). The Club Cell Marker SCGB1A1 Downstream of FOXA2 is Reduced in Asthma. American journal of respiratory cell and molecular biology.
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  Human SCGB1A1 protein has been shown to be significantly reduced in bronchoalveolar lavage, sputum and serum from human asthmatics as compared to healthy individuals. However, the mechanism of this reduction and its functional impact have not been entirely elucidated. By mining online datasets, we found that the message RNA of SCGB1A1 was significantly repressed in brushed human airway epithelial cells from asthmatics and this repression appeared to be associated with the reduced expression of FOXA2. Consistently, both Scgb1A1 and FoxA2 were downregulated in an ovalbumin (OVA)-induced mouse model of asthma. Furthermore, compared to wild-type mice, Scgb1a1 knockout mice had increased airway hyperreactivity and inflammation when the mice were exposed to OVA, confirming the anti-inflammatory role of Scgb1a1 in the protection against asthma phenotypes. To search for potential asthma-related stimuli of SCGB1A1 repression, we tested T-helper 2 (Th2) cytokines. Both IL4 and IL13 repressed epithelial expression of SCGB1A1 and FOXA2. Importantly, infection of epithelial cells with human rhinovirus similarly reduced expression of these two genes, which suggests that FOXA2 may be the common regulator of SCGB1A1. To establish the causal role of reduced FOXA2 in SCGB1A1 repression, we demonstrated that FOXA2 was required for SCGB1A1 expression at base line. FOXA2 overexpression was sufficient to drive the promoter activity and the expression of SCGB1A1, and it was also able to restore the repressed SCGB1A1 expression in IL13-treated or RV infected cells. Taken together, we conclude that low levels of epithelial SCGB1A1 in asthma is caused by reduced FOXA2 expression.
• Chen, Y., Vasquez, M. M., Zhu, L., Lizarraga, R. E., Krutzsch, M., Einspahr, J., Alberts, D. S., Di, P. Y., Martinez, F. D., & Guerra, S. (2017). Effects of Retinoids on Augmentation of Club Cell Secretory Protein. American journal of respiratory and critical care medicine.
• Lv, J., He, X., Wang, H., Wang, Z., Kelly, G. T., Wang, X., Chen, Y., Wang, T., & Qian, Z. (2017). TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages. BMC pulmonary medicine, 17(1), 194.
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  Macrophages stand at the forefront of both innate and adapted immunity through their capacities to recognize, engulf, and eliminate foreign particles, and to stimulate adapted immune cells. They are also involved in controlling pro- and anti-inflammatory pathways. Macrophage activity against Mycobacterium tuberculosis (M. tuberculosis) has been shown to involve Toll-like receptor (TLR) activation and ROS production. Previous studies have shown that lipopolysaccharide (LPS), through TLR4, could activate macrophages, improve their bactericidal ROS production, and facilitate anti-infective immune responses. We sought to better understand the role of the TLR4-NOX2 axis in macrophage activation during M. tuberculosis infection.
• Zhou, X., Lizarraga, R., Zhu, L., & Chen, Y. (2017). Human Airway Epithelial Cells Direct Significant Rhinovirus Replication in Monocytic Cells by Enhancing ICAM1 Expression. American journal of respiratory cell and molecular biology. doi:10.1165/rcmb.2016-0271OC
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  Human rhinovirus (RV) is the major cause of common cold, and it also plays a significant role in asthma and asthma exacerbation. Airway epithelium is the primary site of RV infection and production. In contrast, monocytic cells (e.g. monocytes and macrophages) are believed to be non-permissive for RV replication. Instead, RV has been shown to modulate inflammatory gene expressions in these cells via a replication-independent mechanism. In the present study, RV16 (a major-group RV) replication was found to be significantly enhanced in monocytes when co-cultivated with airway epithelial cells. This effect appeared to be mediated by secretory components from epithelial cells, which stimulated RV16 replication and significantly elevated the expression of a number of proinflammatory cytokines. The lack of such effect on RV1A, a minor-group RV that enters the cell by a different receptor, suggests that ICAM1, the receptor for major-group RVs, may be involved. Indeed, conditioned media from epithelial cells significantly increased ICAM1 expression in monocytes. Consistently, ICAM1 overexpression and ICAM1 knockdown enhanced and blocked RV production, respectively, confirming the role of ICAM1 in this process. Thus, this is the first report demonstrating that airway epithelial cells direct significant RV16 replication in monocytic cells via an ICAM1-dependent mechanism. This finding will open a new venue for the study of RV infection in airway disease and its exacerbation.
• Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H., Acevedo Arozena, A., Adachi, H., Adams, C. M., Adams, P. D., Adeli, K., Adhihetty, P. J., Adler, S. G., Agam, G., Agarwal, R., Aghi, M. K., Agnello, M., Agostinis, P., Aguilar, P. V., Aguirre-Ghiso, J., , Airoldi, E. M., et al. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy, 12(1), 1-222.
• Velichko, S., Zhou, X., Zhu, L., Anderson, J. D., Wu, R., & Chen, Y. (2016). A Novel Nuclear Function for the Interleukin-17 Signaling Adaptor Protein Act1. PloS one, 11(10), e0163323.
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  In the context of the human airway, interleukin-17A (IL-17A) signaling is associated with severe inflammation, as well as protection against pathogenic infection, particularly at mucosal surfaces such as the airway. The intracellular molecule Act1 has been demonstrated to be an essential mediator of IL-17A signaling. In the cytoplasm, it serves as an adaptor protein, binding to both the intracellular domain of the IL-17 receptor as well as members of the canonical nuclear factor kappa B (NF-κB) pathway. It also has enzymatic activity, and serves as an E3 ubiquitin ligase. In the context of airway epithelial cells, we demonstrate for the first time that Act1 is also present in the nucleus, especially after IL-17A stimulation. Ectopic Act1 expression can also increase the nuclear localization of Act1. Act1 can up-regulate the expression and promoter activity of a subset of IL-17A target genes in the absence of IL-17A signaling in a manner that is dependent on its N- and C-terminal domains, but is NF-κB independent. Finally, we show that nuclear Act1 can bind to both distal and proximal promoter regions of DEFB4, one of the IL-17A responsive genes. This transcriptional regulatory activity represents a novel function for Act1. Taken together, this is the first report to describe a non-adaptor function of Act1 by directly binding to the promoter region of IL-17A responsive genes and directly regulate their transcription.
• Wang, T., Shimizu, Y., Kelly, G., Wu, X., Wang, L., Chen, Y., Garcia, J., Xu, X., & Qian, Z. (2016). Particulate Matter Disrupts Human Lung Endothelial Cell Barrier Integrity via Rho-dependent Pathways. Pulmonary Circulation.
• Lee, W., Chen, Y., Wang, W., & Mosser, A. (2015). Growth of human rhinovirus in H1-HeLa cell suspension culture and purification of virions. Methods in molecular biology (Clifton, N.J.), 1221, 49-61.
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  HeLa cell culture is the most widely used system for in vitro studies of the basic biology of human rhinovirus (HRV). It is also useful for making sufficient quantities of virus for experiments that require highly concentrated and purified virus. This chapter describes the protocols for producing a large amount of HeLa cells in suspension culture, using these cells to grow a large quantity of virus of HeLa-adapted HRV-A and -B serotypes, and making highly concentrated virus stock and highly purified virions. These purified HRV virions are free of cellular components and suitable for experiments that are sensitive to cellular contaminations.
• Lee, W., Chen, Y., Wang, W., & Mosser, A. (2015). Infectivity assays of human rhinovirus-A and -B serotypes. Methods in molecular biology (Clifton, N.J.), 1221, 71-81.
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  Infectivity is a fundamental property of viral pathogens such as human rhinoviruses (HRVs). This chapter describes two methods for measuring the infectivity of HRV-A and -B serotypes: end point dilution (TCID50) assay and plaque assay. End point dilution assay is a quantal, not quantitative, assay that determines the dilution of the sample at which 50 % of the aliquots have infectious virus. It can be used for all the HRV-A and -B serotypes and related clinical isolates that grow in cell culture and induce cytopathic effect (CPE), degenerative changes in cells that are visible under a microscope. Plaque assay is a quantitative assay that determines the number of infectious units of a virus in a sample. After an infectious unit of virus infects one cell, the infected cell produces progeny viruses that then infect and kill a circle of adjacent cells. This circle of dead cells detaches from the dish and thus leaves a clear hole in a cell monolayer. Plaque assay works only for HeLa-adapted HRV-A and -B serotypes that can make visible plaques on the cell monolayer. Currently the end point dilution assay and plaque assay have not been developed for the newly discovered HRV-C.
• Zhu, L., Di, P. Y., Wu, R., Pinkerton, K. E., & Chen, Y. (2015). Repression of CC16 by cigarette smoke (CS) exposure. PloS one, 10(1), e0116159.
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  Club (Clara) Cell Secretory Protein (CCSP, or CC16) is produced mainly by non-ciliated airway epithelial cells including bronchiolar club cells and the change of its expression has been shown to associate with the progress and severity of Chronic Obstructive Pulmonary Disease (COPD). In an animal model, the lack of CC16 renders the animal susceptible to the tumorigenic effect of a major CS carcinogen. A recent population-based Tucson Epidemiological Study of Airway Obstructive Diseases (TESAOD) has indicated that the low serum CC16 concentration is closely linked with the smoke-related mortality, particularly that driven by the lung cancer. However, the study of CC16 expression in well-defined smoke exposure models has been lacking, and there is no experimental support for the potential causal link between CC16 and CS-induced pathophysiological changes in the lung. In the present study, we have found that airway CC16 expression was significantly repressed in COPD patients, in monkey CS exposure model, and in CS-induced mouse model of COPD. Additionally, the lack of CC16 exacerbated airway inflammation and alveolar loss in the mouse model. Therefore, CC16 may play an important protective role in CS-related diseases.
• Oslund, K. L., Zhou, X., Lee, B., Zhu, L., Duong, T., Shih, R., Baumgarth, N., Hung, L., Wu, R., & Chen, Y. (2014). Synergistic up-regulation of CXCL10 by virus and IFN γ in human airway epithelial cells. PloS one, 9(7), e100978.
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  Airway epithelial cells are the first line of defense against viral infections and are instrumental in coordinating the inflammatory response. In this study, we demonstrate the synergistic stimulation of CXCL10 mRNA and protein, a key chemokine responsible for the early immune response to viral infection, following treatment of airway epithelial cells with IFN γ and influenza virus. The synergism also occurred when the cells were treated with IFN γ and a viral replication mimicker (dsRNA) both in vitro and in vivo. Despite the requirement of type I interferon (IFNAR) signaling in dsRNA-induced CXCL10, the synergism was independent of the IFNAR pathway since it wasn't affected by the addition of a neutralizing IFNAR antibody or the complete lack of IFNAR expression. Furthermore, the same synergistic effect was also observed when a CXCL10 promoter reporter was examined. Although the responsive promoter region contains both ISRE and NFκB sites, western blot analysis indicated that the combined treatment of IFN γ and dsRNA significantly augmented NFκB but not STAT1 activation as compared to the single treatment. Therefore, we conclude that IFN γ and dsRNA act in concert to potentiate CXCL10 expression in airway epithelial cells via an NFκB-dependent but IFNAR-STAT independent pathway and it is at least partly regulated at the transcriptional level.
• Zhu, L., Lee, B., Zhao, F., Zhou, X., Chin, V., Ling, S. C., & Chen, Y. (2014). Modulation of airway epithelial antiviral immunity by fungal exposure. American journal of respiratory cell and molecular biology, 50(6), 1136-43.
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  Multiple pathogens, such as bacteria, fungi, and viruses, have been frequently found in asthmatic airways and are associated with the pathogenesis and exacerbation of asthma. Among these pathogens, Alternaria alternata (Alt), a universally present fungus, and human rhinovirus have been extensively studied. However, their interactions have not been investigated. In the present study, we tested the effect of Alt exposure on virus-induced airway epithelial immunity using live virus and a synthetic viral mimicker, double-stranded RNA (dsRNA). Alt treatment was found to significantly enhance the production of proinflammatory cytokines (e.g., IL-6 and IL-8) induced by virus infection or dsRNA treatment. In contrast to this synergistic effect, Alt significantly repressed type I and type III IFN production, and this impairment led to elevated viral replication. Mechanistic studies suggested the positive role of NF-κB and mitogen-activated protein kinase pathways in the synergism and the attenuation of the TBK1-IRF3 pathway in the inhibition of IFN production. These opposite effects are caused by separate fungal components. Protease-dependent and -independent mechanisms appear to be involved. Thus, Alt exposure alters the airway epithelial immunity to viral infection by shifting toward more inflammatory but less antiviral responses.
• Zhu, L., Barret, E. C., Yuxue, X. u., Liu, Z., Manoharan, A., & Chen, Y. (2013). Regulation of Cigarette Smoke (CS)-Induced Autophagy by Nrf2. PLoS ONE, 8(4).
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  PMID: 23585825;PMCID: PMC3621864;Abstract: Cigarette smoke (CS) has been reported to induce autophagy in airway epithelial cells. The subsequent autophagic cell death has been proposed to play an important pathogenic role in chronic obstructive pulmonary disease (COPD); however, the underlying molecular mechanism is not entirely clear. Using CS extract (CSE) as a surrogate for CS, we found that it markedly increased the expressions of both LC3B-I and LC3B-II as well as autophagosomes in airway epithelial cells. This is in contrast to the common autophagy inducer (i.e., starvation) that increases LC3B-II but reduces LC3B-I. Further studies indicate that CSE regulated LC3B at transcriptional and post-translational levels. In addition, CSE, but not starvation, activated Nrf2-mediated adaptive response. Increase of cellular Nrf2 by either Nrf2 overexpression or the knockdown of Keap1 (an Nrf2 inhibitor) significantly repressed CSE-induced LC3B-I and II as well as autophagosomes. Supplement of NAC (a GSH precursor) or GSH recapitulated the effect of Nrf2, suggesting the increase of cellular GSH level is responsible for Nrf2 effect on LC3B and autophagosome. Interestingly, neither Nrf2 activation nor GSH supplement could restore the repressed activities of mTOR or its downstream effctor-S6K. Thus, the Nrf2-dependent autophagy-suppression was not due to the re-activation of mTOR-the master repressor of autophagy. To search for the downstream effector of Nrf2 on LC3B and autophagosome, we tested Nrf2-dependent genes (i.e., NQO1 and P62) that are also increased by CSE treatment. We found that P62, but not NQO1, could mimic the effect of Nrf2 activation by repressing LC3B expression. Thus, Nrf2->P62 appears to play an important role in the regulation of CSE-induced LC3B and autophagosome. © 2013 Zhu et al.
• Floyd, A. M., Zhou, X., Evans, C., Rompala, O. J., Zhu, L., Wang, M., & Chen, Y. (2012). Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface. PLoS ONE, 7(12).
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  PMID: 23272068;PMCID: PMC3525643;Abstract: MUC5AC is the most abundant gel-forming mucin in the ocular system. However, the specific function is unknown. In the present study, a Muc5ac knockout (KO) mouse model was subject to various physiological measurements as compared to its wide-type (WT) control. Interestingly, when KO mice were compared to WT mice, the mean tear break up time (TBUT) values were significantly lower and corneal fluorescein staining scores were significantly higher. But the tear volume was not changed. Despite the lack of Muc5ac expression in the conjunctiva of KO mice, Muc5b expression was significantly increased in these mice. Corneal opacification, varying in location and severity, was found in a few KO mice but not in WT mice. The present results suggest a significant difference in the quality, but not the quantity, of tear fluid in the KO mice compared to WT mice. Dry eye disease is multifactorial and therefore further evaluation of the varying components of the tear film, lacrimal unit and corneal structure of these KO mice may help elucidate the role of mucins in dry eye disease. Because Muc5ac knockout mice have clinical features of dry eye, this mouse model will be extremely useful for further studies regarding the pathophysiology of the ocular surface in dry eye in humans. © 2012 Floyd et al.
• Hao, Y., Kuang, Z., Walling, B. E., Bhatia, S., Sivaguru, M., Chen, Y., Gaskins, H. R., & Lau, G. W. (2012). Pseudomonas aeruginosa pyocyanin causes airway goblet cell hyperplasia and metaplasia and mucus hypersecretion by inactivating the transcriptional factor FoxA2. Cellular Microbiology, 14(3), 401-415.
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  PMID: 22103442;Abstract: The redox-active exotoxin pyocyanin (PCN) can be recovered in 100μM concentrations in the sputa of bronchiectasis patients chronically infected with Pseudomonas aeruginosa (PA). However, the importance of PCN within bronchiectatic airways colonized by PA remains unrecognized. Recently, we have shown that PCN is required for chronic PA lung infection in mice, and that chronic instillation of PCN induces goblet cell hyperplasia (GCH), pulmonary fibrosis, emphysema and influx of immune cells in mouse airways. Many of these pathological features are strikingly similar to the mouse airways devoid of functional FoxA2, a transcriptional repressor of GCH and mucus biosynthesis. In this study, we postulate that PCN causes and exacerbates GCH and mucus hypersecretion in bronchiectatic airways chronically infected by PA by inactivating FoxA2. We demonstrate that PCN represses the expression of FoxA2 in mouse airways and in bronchial epithelial cells cultured at an air-liquid interface or conventionally, resulting in GCH, increased MUC5B mucin gene expression and mucus hypersecretion. Immunohistochemical and inhibitor studies indicate that PCN upregulates the expression of Stat6 and EGFR, both of which in turn repress the expression of FoxA2. These studies demonstrate that PCN induces GCH and mucus hypersecretion by inactivating FoxA2. © 2011 Blackwell Publishing Ltd.
• Tao, S., Zhu, L., Lee, P., Lee, W., Knox, K., Chen, J., Di, Y. P., & Chen, Y. (2012). Negative control of TLR3 signaling by TICAM1 down-regulation. American Journal of Respiratory Cell and Molecular Biology, 46(5), 660-667.
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  PMID: 22205631;PMCID: PMC3359907;Abstract: Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM1, also called TRIF) is an important adaptor protein in TLR3 and TLR4 signaling pathways that mediate proinflammatory cytokine and IFN responses. Negative regulation of TICAM1 by exogenous viral protease or by endogenous caspase and proteasome have been reported to shut down TICAM1-mediated signaling. In this study, we discovered that down-regulation of TICAM1, but not other components in this signaling pathway, occurred in a natural process of TLR3 activation induced by double-stranded RNA or human rhinovirus (RV) infection in airway epithelial cells and various other cell types. TICAM1 was essential for IFN expression, and the loss of TICAM1 significantly elevated RV production. The low level of TICAM1 protein expression, caused by the prior double-stranded RNA treatment, led to a lack of IFN production upon additional treatment, suggesting receptor desensitization. In follow-up studies, TICAM1 down-regulation was found to be dependent on TLR3 but not RIG1, MDA5, or PKR and appeared to be regulated post-translationally. Neither proteasome nor caspase inhibitors could prevent TICAM1 down-regulation. Instead, a lysosome-mediated process appeared to be involved, suggesting a novel mechanism that is different from previous reports. In conclusion, TICAM1 down-regulation is an essential step in TLR3 activation, and its function is to stop TLR3-mediated IFN production. Copyright © 2012 by the American Thoracic Society.
• Dongfang, Y. u., Walters, D. M., Zhu, L., Lee, P., & Chen, Y. (2011). Vanadium pentoxide (V₂O₅) induced mucin production by airway epithelium. American Journal of Physiology - Lung Cellular and Molecular Physiology, 301(1), 31-39.
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  PMID: 21531775;PMCID: PMC3129903;Abstract: Exposure to environmental pollutants has been linked to various airway diseases and disease exacerbations. Almost all chronic airway diseases uch as chronic obstructive pulmonary disease and asthma are aused by complicated interactions between gene and environment. ne of the major hallmarks of those diseases is airway mucus verproduction (MO). Excessive mucus causes airway obstruction and significantly increases morbidity and mortality. Metals are major components of environmental particulate matters (PM). Among them, vanadium has been suggested to play an important role in PM-induced mucin production. Vanadium pentoxide (V2O5) is the most common commercial source of vanadium, and it has been associated with occupational chronic bronchitis and asthma, both of which are MO diseases. However, the underlying mechanism is not entirely clear. In this study, we used both in vitro and in vivo models to demonstrate the robust inductions of mucin production by V2O5. Furthermore, the follow-up mechanistic study revealed a novel v-raf-1 murine leukemia viral oncogene homolog 1-IKK-NF-κB pathway that mediated V2O5- induced mucin production. Most interestingly, the reactive oxygen species and the classical mucin-inducing epidermal growth factor receptor (EGFR)-MAPK pathway appeared not to be involved in this process. Thus the V2O5-induced mucin production may represent a novel EGFR-MAPK-independent and environmental toxicant-associated MO model. Complete elucidation of the signaling pathway in this model will not only facilitate the development of the treatment for V2O5-associated occupational diseases but also advance our understanding on the EGFR-independent mucin production in other chronic airway diseases. © 2011 the American Physiological Society.
• Fujisawa, T., Chang, M. M., Velichko, S., Thai, P., Hung, L., Huang, F., Phuong, N., Chen, Y., & Reen, W. u. (2011). NF-κB mediates IL-1β- and IL-17A-induced MUC5B expression in airway epithelial cells. American Journal of Respiratory Cell and Molecular Biology, 45(2), 246-252.
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  PMID: 20935193;PMCID: PMC3175554;Abstract: A major pathological feature of chronic airway diseases is the elevated expression of gel-forming mucins. NF-κB activation in airway epithelial cells has been shown to play a proinflammatory role in chronic airway diseases; however, the specific role of NF-κB in mucin gene expression has not been characterized. In this study, we show that the proinflammatory cytokines, IL-1β and IL-17A, both of which use the NF-κB pathway, are potent inducers of MUC5B mRNA expression in both well differentiated primary normal human bronchial epithelial cells and the human bronchial epithelial cell line, HBE1. MUC5B induction by these cytokines was both time- and dose-dependent, and was attenuated by the small molecule inhibitor, NF-κB inhibitor III, as well as p65 small interfering RNA, suggesting that the regulation of MUC5B expression by these cytokines is via an NF-κB-based transcriptional mechanism. Deletion analysis of the MUC5B promoter demonstrated that IL-1β- and IL-17A-induced promoter activity resides within the -4.17-kb to -2.56-kb region relative to the transcriptional start site. This region contains three putative κB-binding sites (NF-κB-1, -3,786/-3,774; NF-κB-2, -3,173/-3,161; and NF-κB-3, -2,921/-2,909). Chromatin immunoprecipitation analysis confirmed enhanced binding of the p50 NF-κB subunit to the NF-κB-3 site after cytokine stimulation. We conclude that an NF-κB-based transcriptional mechanism is involved in MUC5B regulation by IL-1β and IL-17A in airway epithelium. This is the first demonstration of the participation of NF-κB and its specific binding site in cytokine-mediated airway MUC5B expression.
• Zhu, L., Lee, P., Dongfang, Y. u., Tao, S., & Chen, Y. (2011). Cloning and characterization of human MUC19 gene. American Journal of Respiratory Cell and Molecular Biology, 45(2), 348-358.
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  PMID: 21075863;PMCID: PMC3175562;Abstract: The most recently discovered gel-forming mucin, MUC19, is expressed in both salivary glands and tracheal submucosal glands. We previously cloned the 3′-end partial sequence (AY236870), and here report the complete sequencing of the entire MUC19 cDNA. One highly variable region (HVR) was discovered in the 5′ end of MUC19. A total of 20 different splicing variants were detected in HVR, and 18 variants are able to translate into proteins along with the rest of the MUC19 sequence. The longest variant of MUC19 consists of 182 exons, with a transcript of approximately 25 kb. A central exon of approximately 12 kb contains highly repetitive sequences and has no intron interruption. The deduced MUC19 protein has the bona fide gel-forming mucin structure, VWD-VWD-VWD-"threonine/serine-rich repeats"-VWC-CT. An unusual structural feature of MUC19, which is lacking in other gel-forming mucins, is its long amino terminus upstream of the first VWD domain. The long amino terminus is mostly translated from the sequences in HVR, and contains serine-rich repetitive sequences. To validate the integrity of the MUC19 sequence, primers from both the 3′ and 5′ end were used to demonstrate a similar tissue expression pattern of MUC19 in trachea and salivary glands. In addition, antibodies were developed against either the amino (N) or carboxy (C) terminus of MUC19, and similar antibody staining patterns were observed in both salivary and tracheal submucosal glands. In conclusion, we have cloned and elucidated the entire MUC19 gene, which will facilitate understanding of the function and regulation of this important, yet understudied, mucin gene in airway diseases.
• Zhu, L., Lee, P., Lee, W., Zhao, Y., Dongfang, Y. u., & Chen, Y. (2009). Rhinovirus-induced major airway mucin production involves a novel TLR3-EGFR-dependent pathway. American Journal of Respiratory Cell and Molecular Biology, 40(5), 610-619.
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  PMID: 18978302;PMCID: PMC2677440;Abstract: Mucociliary clearance is a critical innate defense system responsible for clearing up invading pathogens including bacteria and virus. Although the right amount of mucusis good, excessivemucus causes airway obstruction and tends to precipitate disease symptoms. Rhinovirus (RV) is a common cold virus that causes asthma and chronic obstructive pulmonary disease exacerbation. Mucus overproduction has been linked to the pathogenesis of RV-induced diseases and disease exacerbations. However, the molecular mechanism is not clear. In this study, using one of the major airway mucin-MUC5AC as marker, we found that both major and minor groups of RV induced mucin production in primary human epithelial cells and cell line. RV1A (a minor group of RV) could induce mucous cell metaplasia in vivo. Viral replication was needed for RV-induced mucin expression, and this induction was also dependent on TLR3, suggesting the involvement of double-stranded (ds) RNA signaling. Indeed, dsRNA alone could also induce mucin expression. TLR3-mediated mucin induction was negatively regulated by MyD88, and only partially dependent on TRIF, which suggests a departure from well-documented TLR3 signaling paradigm that mediates inflammatory and other innate defense gene inductions. In addition, TLR3 signaling activated epidermal growth factor receptor (EGFR) through inductions of the expression of EGFR ligands (transforming growth factor-α and amphiregulin), which in turn activated EGFR-ERK signaling and mucin expression through an autocrine/paracrine loop. This novel coupling of antiviral defense machinery (i.e., TLR3) and major epithelial proliferation/repair pathway (i.e., EGFR) might play an important role in viral-induced airway remodeling and airway disease exacerbation.
• Zhu, L., Jingbo, P. i., Wachi, S., Andersen, M. E., Reen, W. u., & Chen, Y. (2008). Identification of Nrf2-dependent airway epithelial adaptive response to proinflammatory oxidant-hypochlorous acid challenge by transcription profiling. American Journal of Physiology - Lung Cellular and Molecular Physiology, 294(3), L469-L477.
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  PMID: 18156441;Abstract: In inflammatory diseases of the airway, a high level (estimated to be as high as 8 mM) of HOCl can be generated through a reaction catalyzed by the leukocyte granule enzyme myeloperoxidase (MPO). HOCl, a potent oxidative agent, causes extensive tissue injury through its reaction with various cellular substances, including thiols, nucleotides, and amines. In addition to its physiological source, HOCl can also be generated by chlorine gas inhalation from an accident or a potential terrorist attack. Despite the important role of HOCl-induced airway epithelial injury, the underlying molecular mechanism is largely unknown. In the present study, we found that HOCl induced dose-dependent toxicity in airway epithelial cells. By transcription profiling using GeneChip, we identified a battery of HOCl-inducible antioxidant genes, all of which have been reported previously to be regulated by nuclear factor erythroid-related factor 2 (Nrf2), a transcription factor that is critical to the lung antioxidant response. Consistent with this finding, Nrf2 was found to be activated time and dose dependently by HOCl. Although the epidermal growth factor receptor-MAPK pathway was also highly activated by HOCl, it was not involved in Nrf2 activation and Nrf2-dependent gene expression. Instead, HOCl-induced cellular oxidative stress appeared to lead directly to Nrf2 activation. To further understand the functional significance of Nrf2 activation, small interference RNA was used to knock down Nrf2 level by targeting Nrf2 or enhance nuclear accumulation of Nrf2 by targeting its endogenous inhibitor Keap1. By both methods, we conclude that Nrf2 directly protects airway epithelial cells from HOCl-induced toxicity. Copyright © 2008 the American Physiological Society.
• Y., D., Reen, W. u., Chen, Y., Tarasova, N., & Chang, M. M. (2007). PMA stimulates MUC5B gene expression through an Sp1-based mechanism in airway epithelial cells. American Journal of Respiratory Cell and Molecular Biology, 37(5), 589-597.
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  PMID: 17600309;PMCID: PMC2048678;Abstract: We previously showed that the MUC5B gene expression was elevated by phorbol 12-myristate 13-acetate (PMA) through an epidermal growth factor receptor-independent Ras/MEKK1/JNK and P38 signaling-based transcriptional mechanism. In the current study, we elucidated the molecular basis of this transcriptional regulation using promoter-reporter gene expression and chromatin immunoprecipitation (ChIP) assays with primary human bronchial epithelial cells that are cultured at the air-liquid interface. We have observed that PMA-induced MUC5B promoter activity is blocked by the Sp1-binding inhibitor, mithramycin A, in a dose-dependent manner. Deletion analysis with the MUC5B promoter construct demonstrated that both basal and PMA-induced promoter-reporter activities reside within the -222/-78 bp region relative to the transcriptional start site. NoShift transcriptional factor assays demonstrated that PMA stimulated Sp1 binding, but not STAT1 and c-Myc binding. Immunoprecipitation studies also verified the enhanced phosphorylation of Sp1 after PMA treatment. Site-directed mutagenesis and transfection studies demonstrated the involvement of Sp1-1 (-122/-114) and the Sp1-2 (-197/-186) cis elements in the basal and PMA-induced MUC5B promoter activity. The ChIP assay with anti-RNA polymerase II reconfirmed the PMA-induced MUC5B promoter activity by showing enhanced RNA polymerase II-DNA complex containing putative MUC5B Sp1-1, Sp1-2, or Sp1-3 sites. However, the ChIP assay using anti-Sp1 antibody demonstrated that the PMA-stimulated binding is only at Sp1-2. These results suggested an Sp1-based transcriptional mechanism with Sp1-1 as the regulator of basal MUC5B promoter activity and Sp1-2 as the regulator of PMA-induced MUC5B gene expression in the human airway epithelial cells.
• Chen, Y., Hamati, E., Lee, P., Lee, W., Wachi, S., Schnurr, D., Yagi, S., Dolganov, G., Boushey, H., Avila, P., & Reen, W. u. (2006). Rhinovirus induces airway epithelial gene expression through double-stranded RNA and IFN-dependent pathways. American Journal of Respiratory Cell and Molecular Biology, 34(2), 192-203.
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  PMID: 16210696;PMCID: PMC2644182;Abstract: Rhinovirus (RV) infection is the major cause of common colds and of asthma exacerbations. Because the epithelial cell layer is the primary target of RV infection, we hypothesize that RV-induced airway disease is associated with the perturbation of airway epithelial gene expression. In this study, well differentiated primary human airway epithelial cells were infected with either RV16 (major group) or RV1B (minor group). Transcriptional gene profiles from RV-infected and mock-infected control cells were analyzed by Affymetrix Genechip, and changes of the gene expression were confirmed by real-time RT-PCR analysis. At 24 h after infection, 48 genes induced by both viruses were identified. Most of these genes are related to the IFN pathway, and have been documented to have antiviral functions. Indeed, a significant stimulation of IFN-β secretion was detected after RV16 infection. Neutralizing antibody specific to IFN-β and a specific inhibitor of the Janus kinase pathway both significantly blocked the induction of RV-inducible genes. Further studies demonstrated that 2-aminopurine, a specific inhibitor double-stranded RNA-dependent protein kinase, could block both IFN-β production and RV-induced gene expression. Thus, IFN-β-dependent pathway is a part of the double-stranded RNA-initiated pathway that is responsible for RV-induced gene expression. Consistent with its indispensable role in the induction of antiviral genes, deactivation of this signaling pathway significantly enhanced viral production. Because increase of viral yield is associated with the severity of RV-induced airway illness, the discovery of an epithelial antiviral signaling pathway in this study will contribute to our understanding of the pathogenesis of RV-induced colds and asthma exacerbations.
• Reen, W. u., Harper, R., Kao, C., Thai, P., Daphne, W. u., Chen, Y., & M., M. (2006). New insights into airway mucous cell differentiation. Journal of Organ Dysfunction, 2(1), 30-36.
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  Abstract: Chronic lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis are characterized by persistent airway inflammation and the overproduction of mucus in airways. So-called "goblet (mucous) cell metaplasia/hyperplasia" is the pathologic feature in these diseased airways, in which the normal mucociliary epithelium is replaced by goblet/mucous cells. The nature of the goblet/mucous cell population which arises in these diseased airways is unknown. Our recent studies have shown that trans-differentiation of surface epithelial cells occurs so that they express the submucosal gland-type mucin gene, MUC5B , in addition to a general elevation of all mucin gene products in surface epithelial cells as well as in the submucosal area. In contrast to MUC5B , the surface type of mucin gene, MUC5AC , is restrictedly expressed in the surface epithelium. Using a panel of cytokines - interleukin (IL)-1a, -1ß, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -15, -16, -17, -18, and -19, and tumor necrosis factor-a - we have found to our surprise that only IL-6 and -7 can directly stimulate both MUC5AC and MUC5B expression in well-differentiated and polarized primary human airway epithelial cell cultures. Other cytokines, such as the Th2 type IL-4, -5, -9, and -13, cannot. Inhibitor and signaling transduction studies revealed the presence of an IL-6 paracrine/autocrine loop and the dependence on extracellular signal-regulated kinase signaling activation in IL-17-stimulated mucin gene expression. Further studies are needed to connect cytokine-based mucin gene expression and the trans-differentiation phenomenon in airway diseases. © 2006 Taylor & Francis.
• Harper, R. W., Changhong, X. u., Eiserich, J. P., Chen, Y., Kao, C., Thai, P., Setiadi, H., & Reen, W. u. (2005). Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium. FEBS Letters, 579(21), 4911-4917.
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  PMID: 16111680;Abstract: Partially reduced metabolites of molecular oxygen, superoxide (O 2.-) and hydrogen peroxide (H2O2), are detected in respiratory tract lining fluid, and it is assumed that these are key components of innate immunity. Whether these reactive oxygen species (ROS) are produced specifically by the respiratory epithelium in response to infection, or are a non-specific by-product of oxidant-producing inflammatory cells is not well characterized. Increasing evidence supports the hypothesis that the dual function NAD(P)H oxidases/peroxidases, Duox1 and Duox2, are important sources of regulated H2O2 production in respiratory tract epithelium. However, no studies to date have characterized the regulation of Duox gene expression. Accordingly, we examined Duox1 and Duox2 mRNA expression by real-time PCR in primary respiratory tract epithelial cultures after treatment with multiple cytokines. Herein, we determined that Duox1 expression was increased several-fold by treatment with the Th2 cytokines IL-4 and IL-13, whereas Duox2 expression was highly induced following treatment with the Th1 cytokine IFN-γ. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H2O2 production was similarly increased by the addition of Th1 or Th2 cytokines. These results demonstrate for the first time the regulation of Duox expression by immunomodulatory Th1 and Th2 cytokines, and suggest a mechanism by which ROS production can be regulated in the respiratory tract as part of the host defense response. © 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
• Kao, C., Huang, F., Chen, Y., Thai, P., Wachi, S., Kim, C., Tam, L., & Reen, W. u. (2005). Up-regulation of CC chemokine ligand 20 expression in human airway epithelium by IL-17 through a JAK-independent but MEK/NF-κB-dependent signaling pathway. Journal of Immunology, 175(10), 6676-6685.
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  PMID: 16272323;Abstract: CCL20, like human β-defensin (hBD)-2, is a potent chemoattractant for CCR6-positive immature dendritic cells and T cells in addition to recently found antimicrobial activities. We previously demonstrated that IL-17 is the most potent cytokine to induce an apical secretion and expression of hBD-2 by human airway epithelial cells, and the induction is JAK/NF-κB-dependent. Similar to hBD-2, IL-17 also induced CCL20 expression, but the nature of the induction has not been elucidated. Compared with a panel of cytoldnes (DL-1α, 1β, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 18, 16, 18, IFN-γ, GM-CSF, and TNF-α), IL-17 was as potent as IL-1α, 1β, and TNF-α, with a time- and dose-dependent phenomenon in stimulating CCL20 expression in both well-differentiated primary human and mouse airway epithelial cell culture systems. The stimulation was largely dependent on the treatment of polarized epithelial cultures from the basolateral side with IL-17, achieving an estimated 4- to 10-fold stimulation at both message and protein levels. More than 90% of induced CCL20 secretion was toward the basolateral compartment (23.02 ± 1.11 ng/chamber/day/basolateral vs 1.82 ± 0.82 ng/chamber/day/apical). Actinomycin D experiments revealed that enhanced expression did not occur at mRNA stability. Inhibitor studies showed that enhanced expression was insensitive to inhibitors of JAK/STAT, p38, JNK, and PI3K signaling pathways, but sensitive to inhibitors of MEK1/2 and NF-κB activation, suggesting a MEK/NF-κB-based mechanism. These results suggest that IL-17 can coordinately up-regulate both hBD-2 and CCL20 expressions in airways through differentially JAK-dependent and -independent activations of NF-κB-based transcriptional mechanisms, respectively. Copyright © 2005 by The American Association of Immunologists, Inc.
• Thai, P., Chen, Y., Dolganov, G., & Reen, W. u. (2005). Differential regulation of MUC5AC/Muc5ac and hCLCA-1/mGob 5 expression in airway epithelium. American Journal of Respiratory Cell and Molecular Biology, 33(6), 523-530.
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  PMID: 16151054;PMCID: PMC2715330;Abstract: This study demonstrates that the two biomarkers, MUC5AC/ Muc5ac and hCLCA1/Gob5, which are frequently associated with surface mucous/goblet cells in asthmatic airways, are differentially regulated. Intratracheal instillation of IL-13 (0.5 μg/mouse lung) elicited 8- and 110-fold induction of Muc5ac and Gob5 messages, respectively, within 24 h in wild-type mouse lung, whereas these inductions were abrogated in Stat6 knockout mice. The induction of MUC5AC/Muc5ac message could not be duplicated in vitro with primary tracheobronchial epithelial (TBE) cells derived from wild-type mice or humans, despite significant inductions still seen for hCLCA1/Gob5. Further studies with JAK inhibitors and STAT6 signaling showed active signaling of the JAK/STAT6 pathway in these primary TBE cultures by IL-13 in the regulation of hCLCA1 expression. Dual immunofluorescent staining with antibodies specific to MUC5AC and hCLCA1 revealed a differential nature of the expression of these two biomarkers by distinct cell types of primary TBE cultures. Finally, MUC5AC expression could be elevated by a bacterial product, peptidoglycan, without any induction of hCLCA1. Thus, these results suggest that the two biomakers of the metaplastic airway mucous cell type are differentially regulated by JAK/STAT6-dependent and -independent pathways.
• Chen, Y., Zhao, Y. H., Kalaslavadi, T. B., Hamati, E., Nehrke, K., Le, A. D., Ann, D. K., & Reen, W. u. (2004). Genome-Wide Search and Identification of a Novel Gel-Forming Mucin MUC19/Muc19 in Glandular Tissues. American Journal of Respiratory Cell and Molecular Biology, 30(2), 155-165.
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  PMID: 12882755;Abstract: Gel-forming mucins are major contributors to the viscoelastic properties of mucus secretion. Currently, four gel-forming mucin genes have been identified: MUC2, MUC5AC, MUCSB, and MUC6. All these genes have five major cysteine-rich domains (four von Willebrand factor [vWF] C or D domains and one Cystine-knot [CT] domain) as their distinctive features, in contrast to other non-gel-forming type of mucins. The CT domain is believed to be involved in the initial mucin dimer formation and have very succinct relationship between different gel-forming mucins across different species. Because of gene duplication and evolutional modification, it is very likely that other gel-forming mucin genes exist. To search for new gel-forming mucin candidate genes, a "Hidden Markov Model"(HMM) was built from the common features of the CT domains of those gel-forming mucins. By using this model to screen all protein databases as well as the six-frame translated expression sequence tag and translated human genomic databases, we identified a locus located at the peri-centromere region of human chromosome 12 and the corresponding homologous region of mouse chromosome 15. We cloned the 3′ end of this gene and its mouse homolog. We found one vWF C domain, one CT domain, and various mucin-like threonine/serine-rich repeats. Phylogenetic analysis indicated the close relationship between this gene and the submaxillary mucin from porcine and bovine. A polydispersed signal was observed on the Northern blot, which indicates very large mRNA size. Further analysis of the upstream genomic sequences generated from human and mouse genome projects revealed three additional vWF D domains and many mucin-like threonine/serine- rich repeats. The expression of this gene is restricted to the mucous cells of various glandular tissues, including sublingual gland, submandibular gland, and submucosal gland of the trachea. Based on the chronological convention, we have given the name MUC19 to the human ortholog and Muc19 to the mouse.
• Harper, R., Changhong, X. u., Peter, D. i., Chen, Y., Privalsky, M., & Reen, W. u. (2004). Identification of a novel MAGE D2 antisense RNA transcript in human tissues. Biochemical and Biophysical Research Communications, 324(1), 199-204.
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  PMID: 15465002;Abstract: Using cDNA microarray analysis, we identified a cDNA clone, DD74, from primary human bronchial epithelial cells, which exhibits increased expression in vitro after treatment with all-trans retinoic acid. This clone corresponded to MAGE D2 mRNA, a gene previously identified to be upregulated in several cancer tissues. Surprisingly, in situ hybridization of lung tissue demonstrated positive hybridization signals with sense, but not antisense, MAGE D2-specific cRNA probes. Examination of several cell lines by Northern blot hybridization confirmed significant expression of two RNA bands. With strand-specific riboprobes, we identified a 2.0 kb RNA transcript with the antisense probe as expected and identified a 4.1 kb transcript by the sense probe. Further sequence analysis of the 4.1 kb transcript revealed at least a 509 nucleotide sequence exactly complementary to the 2.0 kb MAGE D2 mRNA sequence. This MAGE D2i sequence contains unique structural features not shared with those of previously described antisense transcripts. Identification of this transcript potentially has important implications for future studies examining MAGE D2 expression patterns in cancer and normal tissues. © 2004 Elsevier Inc. All rights reserved.
• Kao, C., Chen, Y., Thai, P., Wachi, S., Huang, F., Kim, C., Harper, R. W., & Reen, W. u. (2004). IL-17 markedly up-regulates β-defensin-2 expression in human airway epithelium via JAK and NF-κB signaling pathways. Journal of Immunology, 173(5), 3482-3491.
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  PMID: 15322213;Abstract: Using microarray gene expression analysis, we first observed a profound elevation of human β-defensin-2 (hBD-2) message in IL-17-treated primary human airway epithelial cells. Further comparison of this stimulation with a panel of cytokines (IL-1α, 1β, 2-13, and 15-18; IFN-γ; GM-CSF; and TNF-α demonstrated that IL-17 was the most potent cytokine to induce hBD-2 message (>75-fold). IL-17-induced stimulation of hBD-2 was time and dose dependent, and this stimulation also occurred at the protein level. Further studies demonstrated that hBD-2 stimulation was attenuated by IL-17R-specific Ab, but not by IL-1R antagonist or the neutralizing anti-IL-6 Ab. This suggests an IL-17R-mediated signaling pathway rather than an IL-17-induced IL-1αβ and/or IL-6 autocrine/paracrine loop. hBD-2 stimulation was sensitive to the inhibition of the JAK pathway, and to the inhibitors that affect NF-κB translocation and the DNA-binding activity of its p65 NF-κB subunit. Transient transfection of airway epithelial cells with an hBD-2 promoter-luciferase reporter gene expression construct demonstrated that IL-17 stimulated promoter-reporter gene activity, suggesting a transcriptional mechanism for hBD-2 induction. These results support an IL-17R-mediated signaling pathway involving JAK and NF-κB in the transcriptional stimulation of hBD-2 gene expression in airway epithelium. Because IL-17 has been identified in a number of airway diseases, especially diseases related to microbial infection, these findings provide a new insight into how IL-17 may play an important link between innate and adaptive immunity, thereby combating infection locally within the airway epithelium.
• Chen, Y., Thai, P., Zhao, Y., Ho, Y., DeSouza, M. M., & Reen, W. u. (2003). Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop. Journal of Biological Chemistry, 278(19), 17036-17043.
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  PMID: 12624114;Abstract: Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleukin-1α, 1β, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor α). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B and MUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5B expression through the ERK signaling pathway.
• Kao, C. Y., Chen, Y., Zhao, Y. H., & Reen, W. u. (2003). ORFeome-based search of airway epithelial cell-specific novel human β-defensin genes. American Journal of Respiratory Cell and Molecular Biology, 29(1), 71-80.
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  PMID: 12600824;Abstract: β-Defensin is one of the major host defense shields produced by various tissues and organs against microbial infection. To date, four human β-defensins (DEFBs) gene products that share a consensus six-cysteine motif have been discovered. The hidden Markov model (HMM) profile was constructed from the common features of those known β-defensin peptides to search for additional novel DEFB genes. A genome-wide search of the profile against ORFeome-based peptide databases (e.g., Ensembl project) led to the identification of six new DEFB members that also shared the conserved six-cysteine motif. Phylogenetic analysis supported a close relationship of these six new members with existing DEFB genes. Polymerase Chain Reaction studies of human tissue cDNA panels confirmed the expression of all six novel DEFB genes in various tissues. Two of them, DEFB106 and DEFB109, were expressed in the lung. A pilot study with cRNA probes for in situ hybridization and a synthetic propeptide for the functional characterization demonstrated the tissue-/ cell-specific expression and the strong antimicrobial activity of DEFB106. These results support the utility of ORFeome-based HMM search in gene discovery for members of a specific gene family. The novel DEFB genes identified in this study may significantly contribute to overall antimicrobial host defenses.
• Yoneda, K., Chang, M. M., Chmiel, K., Chen, Y., & Reen, W. u. (2003). Application of high-density DNA microarray to study smoke- and hydrogen peroxide-induced injury and repair in human bronchial epithelial cells. Journal of the American Society of Nephrology, 14(SUPPL. 3), S284-S289.
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  PMID: 12874447;Abstract: Recent advances in high-density DNA microarray technique allow the possibility to analyze thousands of genes simultaneously for their differential gene expression patterns in various biologic processes. Through clustering analysis and pattern recognition, the significance of these differentially expressed genes can be recognized and correlated with the biologic events that may take place inside the cell and tissue. High-density DNA microarray nylon membranes were used to explore gene expression and regulation associated with smoke-and hydrogen peroxide-induced injury and repair in differentiated human bronchial epithelial cells in vitro. At least three phases of change in gene expression could be recognized. The first phase seems to be an immediate event in response to oxidant injury. This phase includes the induction of bcl-2 and mdm2 genes that are involved in the regulation of apoptosis, and the mitogen-activated protein kinase phosphatase 1 that functions as a regulator for various mitogen-activated protein kinase activities. The second phase, usually 5 h later, includes the induction of various stress proteins and ubiquitin, which are important in providing the chaperone mechanism and the turnover of damaged macromolecules. The third phase, which is 5 to 10 h later, includes the induction of genes that seem to be involved in reducing oxidative stress by metabolizing the cellular level of reactive oxygen species. In this phase, enzymes associated with tissue and cell remodeling are also elevated. These results demonstrated a complex gene expression array by bronchial epithelial cells in response to a single insult of oxidants that are relevant to environmental pollutants.
• Chen, Y., Zhao, Y. H., & Reen, W. u. (2001). In silico cloning of mouse Muc5b gene and upregulation of its expression in mouse asthma model. American Journal of Respiratory and Critical Care Medicine, 164(6), 1059-1066.
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  PMID: 11587997;Abstract: Using a BLAST-searching approach, we identified a mouse expressed sequence tag (EST) clone (AA038672) showing great similarity to the 3′ end of the human MUC5B gene. The clone was named "3pmmuc5b-1" after complete nucleotide sequencing (Genbank Accession, AF369933). A subsequent search of the mouse genome database with the 3pmmuc5b-1 sequence identified two overlapping genomic clones (AC020817 and AC020794) that contained the sequence of both 3pmmuc5b-1 and the mouse Muc5ac gene. Like their human homologs, the genomic order of the mouse Muc genes is 5′-Muc5ac-Muc5b-3′. These results suggest that the newly identified EST clone, 3pmmuc5b-1, is part of the 3′ portion of the mouse Muc5b gene. In situ hybridization demonstrated that this putative mouse Muc5b message was expressed in a restricted manner in the sublingual gland region of the tongue and the submucosal gland region of the mouse trachea in a normal animal. However, the gene expression was greatly enhanced in airway surface epithelium and the submucosal gland region in ovalbumin-induced asthmatic mice. These results were consistent with previous studies of human airway tissues. We therefore conclude that this newly cloned mouse Muc5b gene could be used as a marker for studying aberrant mucin gene expression in mouse models of various airway diseases.
• Chen, Y., Zhao, Y. H., & Wu, R. (2001). Differential regulation of airway mucin gene expression and mucin secretion by extracellular nucleotide triphosphates. American Journal of Respiratory Cell and Molecular Biology, 25(4), 409-417.
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  PMID: 11694445;Abstract: The effects of extracellular nucleotide triphosphates on the stimulation of mucin production by airway epithelial cells were examined. The order of potency in stimulating mucin secretion in primary cultures of human tracheobronchial epithelial cells is: uridine 5′-triphosphate (UTP) ≈ adenosine 5′-triphosphate (ATP) ≈ ATP-γ-S > uridine 5′-diphosphate ≈ adenosine 5′-diphosphate > α,β-methylene ATP >> adenosine. However, only UTP can increase mucin gene (MUC5AC, MUC5B) expression; ATP and other analogues have no stimulatory effect. The stimulation of MUC5AC and MUC5B expression by UTP is time- and dose-dependent. A similar effect on the elevation of mucous cell population in mouse airway epithelium can be demonstrated in vivo by an intratracheal instillation of UTP-saline solution. The stimulatory effect of UTP or ATP on mucin secretion was inhibited by pertussis toxin, U73122, and Calphostin C, but not by PD98059, suggesting a G-protein/phospholipase (PL) C/protein kinase (PK) C-dependent and mitogen-activated protein kinase (MAPK)-independent signaling pathway. However, the stimulatory effect of UTP on mucin gene expression was sensitive to pertussis toxin and PD98059, but not to Calphostin C and U73122, suggesting a G-protein/MAPK-dependent and PLC/PKC-independent signaling pathway. These findings are the first demonstration that UTP, a pyrimidine nucleotide triphosphate, can enhance both mucin secretion and mucin gene expression through different signaling pathways.
• Chen, Y., Zhao, Y. H., Di, Y. -., & Wu, R. (2001). Characterization of human mucin 5B gene expression in airway epithelium and the genomic clone of the amino-terminal and 5′-flanking region. American Journal of Respiratory Cell and Molecular Biology, 25(5), 542-553.
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  PMID: 11713095;Abstract: Human mucin (MUC) 5B gene expression in human airway epithelium was studied in both tissue sections and cultures of tracheobronchial epithelial (TBE) cells. In situ hybridization demonstrated that MUC5B message was expressed mainly in the mucous cells of submucosal glands of normal human airway tissues. Nevertheless, an elevated MUC5B message level could be seen in surface goblet cells from patients with airway diseases and inflammation. Regardless of the airway tissue sources, MUC5B message was regulated by all-trans-retinoic acid (RA) and culture conditions in both primary and passage-1 cultures of TBE cells. MUC5B message, to a lesser extent, was also found in the immortalized epithelial cell line HBE1, but not in BEAS-2B cells. To elucidate the molecular mechanism of MUC5B gene expression, a genomic clone was obtained and sequenced for the amino terminal and the 5′-flanking region of MUC5B gene. A luciferase reporter construct containing 4,169 base pairs of the 5′-flanking region of MUC5B gene demonstrated a cell type-specific basal promoter activity in transfection studies. Both RA and the air-liquid interface culture condition further enhanced this promoter activity. These results suggest that the 5′-flanking region of MUC5B gene contains cis-elements that are potentially involved in the regulation of MUC5B gene expression.
• Wu, R., & Chen, Y. (2001). Fishing for inflammatory cytokine-inducible genes with an old trick. American Journal of Physiology - Lung Cellular and Molecular Physiology, 280(5 25-5), L839-L840.
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  PMID: 11290505;
• Yoneda, K., Peck, K., Chang, M. M., Chmiel, K., Sher, Y. P., Chen, J., Yang, P. C., Chen, Y., & Wu, R. (2001). Development of high-density DNA microarray membrane for profiling smoke- and hydrogen peroxide-induced genes in a human bronchial epithelial cell line.. American journal of respiratory and critical care medicine, 164(10 Pt 2), S85-89.
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  PMID: 11734474;Abstract: Development of the high-density DNA microarray technique permits the analysis of thousands of genes simultaneously for their differential expression patterns in various biological processes. Through clustering analysis and pattern recognition, the significance of differentially expressed genes can be recognized and correlated with biological events that may take place inside the cell and tissue. With this notion in mind, high-density DNA microarray nylon membrane with colorimetry detection was used to profile the expression of smoke- and hydrogen peroxide-inducible genes in a human bronchial epithelial cell line, HBE1. On the basis of the time course of expression, at least three phases of change in gene expression could be recognized. The first phase is an immediate event in response to oxidant injury. This phase includes induction of the bcl-2 and mdm-2 genes, which are involved in the regulation of apoptosis, and the mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) gene, that functions as a regulator of various mitogen-activated protein kinase activities. The second phase, usually 5 h later, includes the induction of various stress proteins and ubiquitin, which are important in providing the chaperone mechanism and the turnover of damaged macromolecules. The third phase, which is 5-10 h later, includes the induction of genes that are apparently involved in reducing oxidative stress by metabolizing reactive oxygen species. In this phase, enzymes associated with tissue and cell remodeling are also elevated. These results demonstrate a complex gene expression array by bronchial epithelial cells in response to the insult of oxidants that are relevant to environmental pollutants.
• Deng, J., Chen, Y., & Reen, W. u. (2000). Induction of cell cornification and enhanced squamous-cell marker SPRR1 gene expression by phorbol ester are regulated by different signaling pathways in human conducting airway epithelial cells. American Journal of Respiratory Cell and Molecular Biology, 22(5), 597-603.
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  PMID: 10783132;Abstract: Phorbol ester is a strong inducer for both cell cornification and squamous-cell marker SPRR1 gene expression in conducting airway epithelial cells. However, the signaling pathways involved in the regulation of both events have not been completely elucidated. The current study focuses on the common and divergent pathways involved in the induction of these two activities by phorbol-13-myristate-12-acetate (PMA). Using a protein kinase (PK) C inhibitor, bisindolylmaleimide I, PMA-induced cell cornification and SPRR1 gene expression were abolished. Further, a PKC activator, indolactam V, induced cell cornification in the absence of PMA treatment. These results suggest a PKC-dependent signaling pathway for both gene induction and enhanced cell cornification by PMA. However, a mitogen-activated protein kinase-specific inhibitor, PD98059, could only block the gene induction event but failed to prevent cell cornification induced by PMA. These results suggest that diverse signaling pathways after PKC activation by PMA are involved in the regulation of these two events.