Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics. Here, we define a signaling pathway from chloride channel calcium-activated 1 (CLCA1) to MAPK13 that is responsible for IL-13-driven mucus production in human airway epithelial cells. The same pathway was also highly activated in the lungs of humans with excess mucus production due to COPD. We further validated the pathway by using structure-based drug design to develop a series of novel MAPK13 inhibitors with nanomolar potency that effectively reduced mucus production in human airway epithelial cells. These results uncover and validate a new pathway for regulating mucus production as well as a corresponding therapeutic approach to mucus overproduction in inflammatory airway diseases.

Epithelial hyperplasia and metaplasia are common features of inflammatory and neoplastic disease, but the basis for the altered epithelial phenotype is often uncertain. Here we show that long-term ciliated cell hyperplasia coincides with mucous (goblet) cell metaplasia after respiratory viral clearance in mouse airways. This chronic switch in epithelial behavior exhibits genetic susceptibility and depends on persistent activation of EGFR signaling to PI3K that prevents apoptosis of ciliated cells and on IL-13 signaling that promotes transdifferentiation of ciliated to goblet cells. Thus, EGFR blockade (using an irreversible EGFR kinase inhibitor designated EKB-569) prevents virus-induced increases in ciliated and goblet cells whereas IL-13 blockade (using s-IL-13Rα2-Fc) exacerbates ciliated cell hyperplasia but still inhibits goblet cell metaplasia. The distinct effects of EGFR and IL-13 inhibitors after viral reprogramming suggest that these combined therapeutic strategies may also correct epithelial architecture in the setting of airway inflammatory disorders characterized by a similar pattern of chronic EGFR activation, IL-13 expression, and ciliated-to-goblet cell metaplasia. IntroductionEpithelial cell hyperplasia and metaplasia are common consequences of inflammation and may be associated with protective as well as pathogenic outcomes. In the lung, airway epithelial remodeling can be life threatening, since mucous cell metaplasia is the foundation for hypersecretion that can obstruct the airway lumen. Despite the critical nature of this process, little is known about how mucous cell metaplasia develops in the setting of acute or chronic inflammatory disease. Particularly, little is known about the mechanism for what is likely the most common cause of mucous cell metaplasia in the lung, i.e., respiratory viral infection, since previous work has focused on bacterial, allergic, and carcinogenic stimuli. Perhaps because of the paucity of mechanistic information, no effective and specific pharmacologic treatment is currently available to treat epithelial cell metaplasia in general or mucous cell metaplasia in particular.In this context, recent work on mucous cell metaplasia has often focused on signaling pathways initiated by activation of the IL-13 receptor (IL-13R) and EGFR (also designated ErbB1 and HER1). The experimental role of IL-13R was established when a decoy receptor for IL-13 (soluble IL-13Rα2-Fc) was found to inhibit allergen-induced mucous (goblet) cell formation in mice (1, 2). These reports have been followed by evidence that IL-13 can directly drive mucin gene expression in airway epithelial cells cultured under physiologic conditions and in vivo (3-6). Moreover, IL-13 is often overexpressed in the setting of mucous cell metaplasia in asthma

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Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

STAT (signal transducer and activator of transcription) proteins combine with cytokine receptors and receptor-associated kinases in distinct protein/protein interactions that are critical for STAT-dependent signal transduction events, but the nature of any subsequent STAT interactions with DNA-binding proteins in the nucleus is less certain. Based on assays of DNA/protein binding and activity of transfected reporter plasmids, we determined that occupation of contiguous DNA-binding sites for Stat1 (the first member of the STAT family) and the transcriptional activator Sp1 are both required for full activation of the intercellular adhesion molecule-1 gene by interferon-␥. Thus, Stat1 binding to DNA cannot by itself be equated with biologic actions of Stat1. In co-immunoprecipitation experiments, we also obtained evidence of direct and selective Stat1/Sp1 interaction (in primary culture cells without overexpression), further indicating that Stat1/Sp1 synergy confers an element of specificity in the pathway leading to cytokine-activated transcription and cytokine-dependent immunity and inflammation. STAT1 proteins act as critical intermediates in cytokine-dependent gene activation based on their dual capacities for signal transduction (at the cell surface) and activation of transcription (in the nucleus) (1). Signal transduction depends on programmed assembly of cytokine receptors, receptor-associated JAK kinases, and in some cases serine kinases, that recruit and activate specific STAT proteins (2-4). Phosphorylated/activated STATs then dimerize, translocate to the nucleus, and direct transcription of specific target genes. For example, the first member of the STAT family (designated Stat1␣) undergoes tyrosine 701 and serine 727 phosphorylation in response to IFN-␥ (5). This activation step is triggered by IFN-␥-dependent oligomerization of the IFN-␥ receptor and consequent cross-phosphorylation of receptor-associated Jak1 and Jak2 kinases and the receptor ␣-chain (6). Receptor phosphorylation enables ␣-chain recruitment of Stat1 via its SH2 domain. Stat1 then undergoes phosphorylation and release from the receptor as a homodimer that can translocate to the nucleus and bind to a specific DNA element (7,8). Thus, distinct protein/protein interactions are critical for Stat1-dependent signal transduction events at the IFN-␥ receptor, but the nature of Stat1 interactions with other proteins (especially other transcription factors) in the nucleus is less certain. In the present report, we take advantage of a primary cell culture model with selective IFN-␥ responsiveness of the intercellular adhesion molecule-1 (ICAM-1) gene (9, 10) in order to study the basis for Stat1-dependent transcription. The results offer the first evidence that Stat1-mediated transactivation depends on synergistic interaction with another transcriptional activator (Sp1). EXPERIMENTAL PROCEDURESMaterials-Recombinant human IFN-␥ was from Genentech (San Francisco, CA); unlabeled dATP and dGTP were from Boehringer Mannheim; [␣-32 P]dCTP was from DuPo...

The action of adenoviral E1A oncoprotein on host immune-response genes has been attributed to interaction with p300/CBP-type transcriptional coactivators in competition with endogenous transcription factors such as signal transducer and activator of transcription (STAT) proteins. However, we show that mutant forms of E1A that no longer bind p300/CBP can still interact directly with Stat1 (via E1A N-terminal and Stat1 C-terminal residues) and block IFNgamma-driven, Stat1-dependent gene activation and consequent function during early-phase infection in the natural host cell. The results provide a distinct and more specific mechanism for E1A-mediated immune suppression and an alternative model of IFNgamma-driven enhanceosome formation that may allow for other adaptors (in addition to p300/CBP) to link Stat1 to the basal transcription complex.

Ciliated airway epithelial cells are critical for mucosal barrier function, including host defense against pathogens. This cell population is often the primary target and thereby the first line of defense against many common respiratory viruses. It is also the precursor for mucous cells and thereby promotes mucociliary clearance of infectious and other noxious agents. Cells with motile cilia in other organs (e.g., brain and reproductive organs) may also have roles in development and reproduction. However, definitive proof of ciliated cell function is hampered by the lack of strategies to specifically target this cell population for loss of function in vivo. To this end, cell type-specific gene promoters have been combined with the Cre/LoxP system to disrupt genes in airway and alveolar epithelial cell populations expressing surfactant protein C (SP-C) or Clara cell secretory protein (CCSP). By contrast, an analogous system to disrupt gene function in ciliated airway epithelial cells was still needed. Here we report the generation and analysis of mouse lines with a FOXJ1 promoter driving the Cre recombinase and show that this system mediates genomic recombination specifically in ciliated cells. The pattern of recombination recapitulates endogenous FOXJ1 promoter function, being restricted to ciliated cells present in pulmonary airways as well as choroid plexus, ependyma, oviduct, and testis. This transgenic mouse system thereby offers a new strategy for specific knockouts of genes in ciliated cells. It should prove extremely useful for defining ciliated cell function in airway mucosal immunity as well as development and reproduction.

Background: CLCA proteins activate CaCCs; CLCAs have roles in cancer and inflammatory lung diseases, but their mechanism of action is unknown. Results: CLCA proteins must undergo self-cleavage via their own novel metalloprotease domain in the N terminus to activate CaCCs. Conclusion: Self-cleavage unmasks the N-terminal fragment, which alone activates CaCCs. Significance: This work identifies a unique ion channel activation mechanism defining framework to understand CLCA functions in diseases.