Yin Chen, Ph.D. - US grants

DESCRIPTION: MUC19, a novel gel-forming mucin in salivary glands. Salivary mucins are well recognized to determine the viscoelastic nature of saliva and regulate its physiological activity. The molecular nature of mucin species in salivary secretions is not entirely clear. By using Hidden Markov ModeI(HMM) based genome-wide search method, we have recently identified a novel gel-forming mucin gene, MUC19 that is specifically expressed in various glandular tissues, including the salivary glands. Comparing with the known major salivary gland gel-forming mucin-MUC5B, MUC19 appears to be much larger and more abundantly expressed in salivary glands. Thus, we hypothesize that MUC19 is one of the major contributors to the viscoelastic and protective properties of salivary mucus. Changes in salivary mucus rheology may affect the phyological properties of saliva that is associated with various diseases. Because of the large gene size, the sequence of MUC19 has not been completed. And several essential molecular tools (like MUC19 specific antibodies) are still unavailable. To advance the knowledge and test the functional role of MUC 19 in salivary secretion, we propose in this pilot study to complete the genetic structure of MUC19 and to develop monoclonal antibodies specific to the gene product. In the Aim 1, we will use RT-PCR, RACE, and the molecular cloning approaches to complete the sequence and elucidate the whole genetic structure of MUC19 gene. In Aim 2, we will characterize the expression of MUC 19 at both mRNA level and protein levels using both tissue and saliva samples. Quantitative RT-PCR as well as in situ hybridization will be used to measure the mRNA level of MUC19. MUC19 specific monoclonal antibodies will be developed and used to examine the MUC19 protein in both salivary gland tissues and salivary secretions. The success of this project will have a great impact on the field by providing immediate answer to several long-standing questions like "how many mucin genes are present in the salivary secretion of health and disease?"; "how will different mucin species affect the physiology property of saliva?" etc. It will also generate valuable knowledge for the further exploration of the regulation of MUC 19 in various diseases. Those tools and information will be also essential for our future submission of an RO 1 grant application.

DESCRIPTION (provided by applicant): Airway mucus overproduction and epithelial mucous cell hyperplasia/metaplasia are clinical hallmarks associated with various airway diseases, such as COPD, cystic fibrosis and asthma. Despite the pathological significance of the excess mucus in these diseased airways, the cause and the mechanism of the aberrant mucus production is largely unknown. We have shown a "metaplastic" change of surface airway epithelial cells from the sole expression of the "surface" type of gel-forming mucin -MUC5AC to the co-expression with the submucosal gland-specific MUC5B and MUC19, a novel gel-forming we newly discovered, in airway diseases. Most recently, Muc19 locus has been shown to be genetically associated with salivary gland mucous cell development. A mouse having a single mutation (sld) in Muc19 locus shows mucous cell deficiency in their salivary gland. We have recently found that the mouse containing sld mutation also has much less submucosal gland mucous cells and less Muc19 expression. Thus, Muc19 locus appears to control glandular mucous cell development. Importantly, different from MUC5AC, which is elevated by IL-13 after long term treatment, this newly found gel-forming mucin gene-MUC19, can be stimulated by Th2 cytokines (IL-4 and IL-13) in a short-term (24h). Thus, we hypothesize that the induction of MUC19/Muc19 by Th2 cytokine may represent a key early event, seemingly to promote glandular phenotype, in the development of mucous cell metaplasia in asthmatic airway. To test the hypothesis, four aims are proposed: 1) To test the hypothesis that MUC19 production is significantly elevated in asthmatic airway. 2) To test the hypothesis that Muc19 expression is essential for mucous cell development. 2) To test the hypothesis that Th2 cytokine transcriptionally activates MUC19 gene expression in vitro. 3) To test the hypothesis that Th2 cytokine induced Muc19 expression and mucous cell metaplasia are regulated by the interaction of Stat1 and Stat6 in vivo. The success of this project will significantly advance our understanding of Th2 cytokine effect on development of epithelial mucus overproduction and mucous cell metaplasia, which will accelerate the development of specific epithelium-targeting therapeutic agents to treat asthma.

DESCRIPTION (provided by applicant): TLR3, TICAM1 and human rhinovirus infection Human rhinovirus (RV) infection is associated with the common cold and asthma development and/or exacerbation. To date, we and others have reported a complex of RV-induced epithelial proinflammatory and antiviral pathways, which involve TLR3, RIG1, MDA5 and PKR. Recently, we have shown that TLR3 activation appears to be upstream of all other pathways, and TLR3 desensitization via TICAM1 degradation is critical to antiviral defense by airway epithelium. To further elucidate the underlying mechanism, we propose to first determine the impact of TLR3 desensitization on epithelial anti-viral defense in vitro. In this study, we will determine the kinetics of TICAM1 protein turnover and its downstream cellular antiviral responses induced by dsRNA or by RV. We will also determine the cellular antiviral status during this process. Lastly, we will overexpress TICAM1 to test if this could restore the cellular antiviral response. To evaluate the potential alteration of TLR3 desensitization in asthma, we will determine whether or not Th2-cytokine- treated epithelial cells have altered desensitization, and whether or not human asthmatic epithelial cells are defective in this process as compared to the normal cells. Then, we propose to determine the mechanistic basis of TICAM1 degradation. In this aim, we will test a novel hypothesis that TICAM1 is degraded via chaperone mediated autophagy. This is a high-risk and high-impact project that is well suited for R21 mechanism. If we succeed, we will be able to gain novel insight into the TLR3-TICAM1 mediated airway antiviral defense and to identify a risk factor for asthma exacerbation.

Abstract Arsenic is a ubiquitous environmental toxicant, found in high concentrations in water worldwide; more than 150 million people live in areas with the arsenic content significantly higher than the WHO and USEPA recommended guidelines (10 ppb).The lung is a target organ for arsenic toxicity. Reports from human studies in Chile, Bangladesh, Inner Mongolia and the West Bengal region of India show that chronic exposure to arsenic via drinking water is correlated with increased incidence of chronic cough, chronic bronchitis, shortness of breath, decreased lung function, and obstructive or restrictive lung disease. Evidence from our laboratory and others have contributed to a growing concern that even at 10 ppb, arsenic can alter lung structure and function. In addition, there is also growing evidence that in utero and early postnatal exposures to arsenic can lead to alterations in lung structure and function that contribute to the development of chronic lung disease later in life. In rural areas of United States such as in the southwestern region, a significant percentage of the population receives their water from private, unregulated wells where concentrations of arsenic can far exceed the 10 ppb level. In addition, dusts in the arid Southwestern United States can contain high levels of arsenic and other contaminants. Inhalation of these dusts can increase lung exposures to arsenic that mimic arsenic ingestion induced lung disease. Little data exist concerning the risk from exposure to arsenic containing dusts and the potential interactions between arsenic ingestion in water and dust exposures. Despite the accepted fact that the lung is a major target organ for arsenic toxicity, studies on biomarkers or mechanisms of non-malignant lung diseases following early life arsenic exposures are limited. One exception is Club (formerly Clara) cell secretory protein (hereafter CC16) that is dramatically reduced in blood and lungs of current smokers and is associated with decreased subsequent incidence of airflow limitation, accelerated decline of lung function, and mortality. Studies strongly support CC16 as a biomarker for lung function deterioration and a direct role for CC16 in protecting against chronic lung disease. However, specific function for CC16 in arsenic-induced lung abnormality remains ill-defined. Our overall objective is to determine the mechanism by which arsenic alters CC16 production and the role of reduced CC16 in altering lung structure and function following early life exposure to arsenic. Two Aims will be addressed. Aim.1 will determine the role of CC16 in arsenic-induced lung structural and functional alterations following early life exposures and Aim2 will determine the effect of arsenic exposure on CC16 production and its mode of action in the lung. Results from our study will establish the validity of CC16 as a biomarker for early life arsenic exposure and for the assessment of arsenic toxicity on long-term lung health. The elucidated mechanism will also help to establish therapeutic interventions in the treatment of arsenic-induced lung disease.

PROJECT SUMMARY Human rhinovirus (RV) is the major pathogen of common cold, asthma, and asthma exacerbation. Airway epithelial cells have been the focus for RV research as it is believed to be the primary site for infection and replication. On the other hand, inflammatory cells including monocytes and macrophages are abundant in diseased airways. Although both epithelial cells and monocytes/macrophages play important roles in RV- induced pathogenesis, their interactions are rarely studied. It is a long-held belief that RV can enter monocytes/macrophages and induce proinflammatory cytokine expression in the absence of viral replication. In our recent publication, we demonstrated for the first time that a major-group RV entered, replicated and induced significant cytokine production in monocytic cell line (THP-1) in the presence of airway epithelial cells or their conditioned media (CM). In our preliminary data, we further showed that primary macrophages from bronchoalveolar lavage (BAL) were also able to support RV replication in the presence of CM. These data suggest a significant role of epithelial cells in regulating permissiveness of monocytes/macrophages for RV infection. Interestingly, the only two previously documented events of monocytic RV replication occurred in THP-1 that was differentiated to macrophage by a PMA protocol, but they failed in finding any underlying mechanism. In our study, increased ICAM1 was found to be both necessary and sufficient to drive RV16 replication in THP-1 treated by CM or by the same PMA protocol, suggesting the involvement of a shared pathway between the two treatments. But, our CM model is obviously more physiologically relevant than the PMA model. To identify the potential epithelial factor(s) that drive this change, we performed size fractionation, in-solution digestion, liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis. 4 protein candidates were discovered. Thus, protein(s) in CM likely drive the change of permissiveness of monocytic cells. Therefore, we hypothesize that epithelial factor(s) renders monocytes/macrophages permissive for RV replication via an ICAM1 mediated mechanism, thereby exacerbating inflammation and potentially leading to RV-associated airway illnesses. We will address this hypothesis in two aims. Specific Aim1 will determine the molecular basis of induced permissiveness of monocytes/macrophages for RV infection by testing two related working hypotheses: 1) increased ICAM1 enhances viral entry and cellular viral burden, thereby circumventing antiviral defense; and/or 2) increased ICAM1 weakens cellular antiviral defense. Specific Aim2 will identify and characterize soluble factor(s) in airway epithelium CM that induces the change of permissiveness for RV infection. The success of this project will have significant impact on the development of therapeutics for treating RV-induced airway illnesses by elucidating novel targets (i.e. permissiveness-inducing factor and replicating-RV-bearing monocytes/macrophages).

Abstract: Vanadium is a ductile and malleable transition metal and has a wide application in a range of industries such as automotive, aerospace, chemistry and agriculture industries. A great amount of the environmental vanadium comes from human activity, particularly burning of crude oil and coal, as well as metallurgic and mining activities. The major risk for human exposure to vanadium is through inhalation of particulate matter (PM) generated by fuel combustion. Occupational exposure to vanadium pentoxide (V2O5) in humans were reported to link to asthma and chronic bronchitis. Furthermore, increased vanadium concentration in ambient PM2.5 was found to associate with asthma and asthma exacerbation in young children. The prominent features of asthma and chronic bronchitis include inflammation and mucus hypersecretion. We reported that respiratory exposure to V2O5 caused inflammation and airway mucous cell metaplasia (MCM), a significant increase of mucous cell number causing mucus hypersecretion, in a mouse model of Vanadium Induced Pulmonary Toxicity (VIPT), supporting the observational finding from human exposure. In the present study, we have identified a quantitative trait locus on mouse chromosome 11 containing Ppp3r1, a regulatory component of calcineurin (CN), which was significantly associated with vanadium induced MCM. We further demonstrate that CN inhibition by Cyclosporine A rendered the susceptible A/J mice resistant to VIPT. Thus, we plan to test a novel hypothesis that CN may be an important determinant of the susceptibility to VIPT that particularly affects airway epithelium, a critical barrier and innate immune regulator in almost all lung illnesses. Thus, we propose to first determine the mechanistic role of CN in mediating susceptibility to VIPT using both gain-of-function CN model and CN inhibition model (Aim1). We will also extend our preliminary study that was performed mainly in a high-dose acute V2O5 model to a novel long-term inhalational model that is close to real-life environmental vanadium exposure. We will determine the role of CN in mediating long-term VIPT (Aim2). Successful completion of this proposal will advance our understanding of how vanadium interacts with pulmonary system and elicits toxicity. Additionally, it will be also highly translatable as our novel findings and novel models will inform new therapeutic target for the chronic lung diseases characterized by mucous hypersecretion such as asthma, chronic bronchitis etc. which are also the major illnesses caused by vanadium exposure.

Abstract: Environmental mold (fungal) exposure has long been recognized as a critical risk factor for asthma and asthma exacerbation. The prevalence of fungal sensitization can be up to 48% in asthmatics and fungal asthma is oftentimes poorly managed with frequent exacerbations and hospitalizations. Efforts to reduce indoor fungal exposure by cleaning have been proven impossible in the recent HEAL study. However, an average person exposing to a large number of fungal spores each day, up to 50,000 spores per cubic meter of air during the fungal season, has no detectable respiratory abnormality and not all individuals with fungal sensitization develop asthma. Thus, it is significant to understand the mechanistic basis of this resilience to maintain airway homeostasis despite the impact of abundant asthmagenic substances produced by fungi. Interestingly, IFN signatures were discovered in the fungal asthma model using live Alternaria spores. Epithelial cells were found to sense fungal spores by triggering IFN-I/III production as well as their downstream signaling cascades. IFN-I receptor blockade or deficiency augmented asthmatic phenotypes, suggesting a protective role of IFN-I against asthma. Thus, our overall hypothesis is that fungal spore sensing activates protective IFN-I/III pathway and the impairment of this protection leads to asthma. To test this hypothesis, we will elucidate the protective function of IFN-I/III in the fungal asthma model using a set of knockout mice. Then, we will determine the mechanistic basis of fungal detection and innate defense. Finally yet importantly, we will examine IFN signatures in clinical samples from human asthma with or without fungal sensitization by utilizing datasets and samples from a NIH- supported Asthma Research Program. The completion of this proposal will advance our knowledge about the pathogenesis of fungal asthma, and establish a foundation for the further therapeutic development to treat this type of asthma.

PROJECT ABSTRACT Environmental Health: Transformative Research Undergraduate Experiences (E-H-TRUE) research education program at the University of Arizona (UA) seeks to recruit, educate and retain 5 undergraduate students every year who are underrepresented in environmental health sciences over five-year period. UA has a long record of accomplishment to train and retain under-representative students: more than 40% new freshman students from under-representative groups, raking #1 in PhDs earned by Naïve American students and #8 in the number of PhDs earned by Hispanic/Latino students. UA has received status as an American Indian/Alaska Native and a Hispanic Serving Institution. In E-H-TRUE, we bring together two preeminent programs: the UA Center for Toxicology and the UA Undergraduate Research Program (UBRP) to provide the top-notch research training in environmental health sciences. On the research side, the UA Center for Toxicology, one of leading toxicology research and training centers in the world, is home to the NIEHS-funded Southwest Environmental Health Sciences Center (SWEHSC) and the UA Superfund Research Program. On the undergraduate education side, UBRP has served as a model for high-quality undergraduate research training for more than 30 years. Through established networks by UBRP, we will initiate early contact with freshman/sophomore undergraduates to identify talented undergraduate candidates from under-representative groups who are interested in environmental health science research. The well-designed educational component of E-H-TRUE will include a specifically designed course work in environmental health sciences to expose students to toxicology, immunology, epidemiology and government regulation. Program participants will receive hand-on training in research laboratories. Students will work with their mentors and laboratory collaborators to learn how to review literature, generate hypothesis, design step-by-step experimentation to test hypothesis, document results, and deliver presentable data by due dates. The program will emphasize collaboration and near- peer mentoring by capitalizing on the recently renewed NIEHS T32 training program (continuously funded for 40 years). The E-H-TRUE program will also facilitate community outreach activities in environmental health science through the established outreach core in SWEHSC. Finally, we have implemented a rigorous evaluation plan to measure the success and impact of the program. The main goal of the program is to provide students with research experiences and encourage them pursuing Ph.D. or professional degrees to be active in environmental science research.