Mechanisms Targeting the Unfolded Protein Response in Asthma.

Lung cells are constantly exposed to various internal and external stressors that disrupt protein homeostasis. To cope with these stimuli, cells evoke a highly conserved adaptive mechanism called the unfolded protein response (UPR). UPR stressors can impose greater protein secretory demands on the endoplasmic reticulum (ER), resulting in the development, differentiation, and survival of these cell types to meet these increasing functional needs. Dysregulation of the UPR leads to the development of the disease. The UPR and ER stress are involved in several human conditions, such as chronic inflammation, neurodegeneration, metabolic syndrome, and cancer. Furthermore, potent and specific compounds that target the UPR pathway are under development as future therapies. The focus of this review is to thoroughly describe the effects of both internal and external stressors on the ER in asthma. Furthermore, we discuss how the UPR signaling pathway is activated in the lungs to overcome cellular damage. We also present an overview of the pathogenic mechanisms, with a brief focus on potential strategies for pharmacological interventions.

Modulation of blood inflammatory markers by benralizumab in patients with eosinophilic airway diseases.

BACKGROUND: Benralizumab, a humanized, afucosylated, monoclonal antibody that targets interleukin-5 receptor α, depletes eosinophils and basophils by enhanced antibody-dependent cell-mediated cytotoxicity. It demonstrated efficacy for patients with moderate to severe asthma and, in a Phase IIa trial, for chronic obstructive pulmonary disease (COPD) with eosinophilic inflammation. We investigated effects of benralizumab 100 mg every 8 weeks (first three doses every 4 weeks) subcutaneous on blood inflammatory markers through proteomic and gene-expression analyses collected during two Phase II studies of patients with eosinophilic asthma and eosinophilic COPD. METHODS: Serum samples for proteomic analysis and whole blood for gene expression analysis were collected at baseline and 52 weeks (asthma study) or 32 weeks (COPD study) post-treatment. Proteomic analyses were conducted on a custom set of 90 and 147 Rules-Based Medicine analytes for asthma and COPD, respectively. Gene expression was profiled by Affymetrix Human Genome U133 plus 2 arrays (~ 54 K probes). Gene set variation analysis (GSVA) was used to determine transcriptomic activity of immune signatures. Treatment-related differences between analytes, genes, and gene signatures were analyzed for the overall population and for patient subgroups stratified by baseline blood eosinophil count (eosinophil-high [≥300 cells/µL] and eosinophil-low [< 300 cells/µL]) via t-test and repeated measures analysis of variance. RESULTS: Eosinophil chemokines eotaxin-1 and eotaxin-2 were significantly upregulated (false discovery rate [FDR] < 0.05) by approximately 2.1- and 1.4-fold in the asthma study and by 2.3- and 1.7-fold in the COPD study following benralizumab treatment. Magnitude of upregulation of these two chemokines was greater for eosinophil-high patients than eosinophil-low patients in both studies. Benralizumab was associated with significant reductions (FDR < 0.05) in expression of genes associated with eosinophils and basophils, such as CLC, IL-5Rα, and PRSS33; immune-signaling complex genes (FCER1A); G-protein-coupled receptor genes (HRH4, ADORA3, P2RY14); and further immune-related genes (ALOX15 and OLIG2). The magnitude of downregulation of gene expression was greater for eosinophil-high than eosinophil-low patients. GSVA on immune signatures indicated significant treatment reductions (FDR < 0.05) in eosinophil-associated signatures. CONCLUSIONS: Benralizumab is highly selective, modulating blood proteins or genes associated with eosinophils or basophils. Modulated protein and gene expression patterns are most prominently altered in eosinophil-high vs. eosinophil-low patients. TRIAL REGISTRATION: NCT01227278 and NCT01238861 .

IL-18 associated with lung lymphoid aggregates drives IFNγ production in severe COPD.

BACKGROUND: Increased interferon gamma (IFNγ) release occurs in Chronic Obstructive Pulmonary Disease (COPD) lungs. IFNγ supports optimal viral clearance, but if dysregulated could increase lung tissue destruction. METHODS: The present study investigates which mediators most closely correlate with IFNγ in sputum in stable and exacerbating disease, and seeks to shed light on the spatial requirements for innate production of IFNγ, as reported in mouse lymph nodes, to observe whether such microenvironmental cellular organisation is relevant to IFNγ production in COPD lung. RESULTS: We show tertiary follicle formation in severe disease alters the dominant mechanistic drivers of IFNγ production, because cells producing interleukin-18, a key regulator of IFNγ, are highly associated with such structures. Interleukin-1 family cytokines correlated with IFNγ in COPD sputum. We observed that the primary source of IL-18 in COPD lungs was myeloid cells within lymphoid aggregates and IL-18 was increased in severe disease. IL-18 released from infected epithelium or from activated myeloid cells, was more dominant in driving IFNγ when IL-18-producing and responder cells were in close proximity. CONCLUSIONS: Unlike tight regulation to control infection spread in lymphoid organs, this local interface between IL-18-expressing and responder cell is increasingly supported in lung as disease progresses, increasing its potential to increase tissue damage via IFNγ.

A Sputum Proteomic Signature That Associates with Increased IL-1ß Levels and Bacterial Exacerbations of COPD.

PURPOSE: Activation of the interleukin-1ß (IL-1ß) signaling pathway has been implicated in COPD, but the proportion of COPD subjects whose disease is principally driven by activation of this pathway is poorly understood. In this study, we sought to differentiate an IL-1ß-associated sputum signature from other inflammation-associated COPD phenotypes. METHODS: Luminex-multiplex assays were used to study IL-1ß-mediated signature proteins within airway epithelium, smooth muscle, and vascular endothelial cell cultures. The IL-1ß-mediated signature was tested in a longitudinal study comprising of 35 paired stable-COPD and acute exacerbation (AECOPD) sputum samples. The presence of respiratory pathogens (H. influenzae, M. catarrhalis, S. pneumoniae, and P. aeruginosa) was evaluated by sputum cultures. RESULTS: Five proteins namely TNF-α, GCSF, IL-6, CD-40L, and MIP-1ß were found to be IL-1ß-regulated across all donors and cell types. All five of these IL-1ß-mediated proteins were significantly increased (p < 0.05) in sputum corresponding to AECOPD events showing at least a twofold increase in IL-1ß (IL-1ß(+) events, 18 of 35 total events), relative to preceding stable-COPD state. Sputum IL-1ß levels showed no significant association (p > 0.05, spearman) with known markers of other major COPD inflammation phenotypes. In addition, there was a significant association with bacterial presence in sputum culture with an odds ratio of 9 (95 % CI 1.56, 51.9) in IL-1ß(+) events versus IL-1ß(-) events. CONCLUSION: Our findings provide insights into potential markers of IL-1ß-associated AECOPD, and reaffirm association between IL-1ß pathway activation and airway bacterial infection in COPD. Taken together, our findings could help identify COPD patient subsets who may benefit from therapies targeting IL-1ß pathway.

Irreversible airway obstruction in asthma: what we lose, we lose early.

Asthma, a syndrome manifested by airway inflammation and obstruction, globally contributes significantly to morbidity and mortality. Although current evidence identifies risk factors that evoke asthma, critical questions concerning susceptibility factors that induce severe persistent disease remain unclear. Early onset of asthma decreases lung function that may be unrecognized until later in adulthood when patients experience dyspnea on exertion and attenuated quality of life. This review highlights current evidence in predicting the onset of asthma and identifying those patients at greatest risk for severe persistent disease.

Trichostatin A abrogates airway constriction, but not inflammation, in murine and human asthma models.

Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (R(L)) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSA-treated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca(2+). The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in R(L) and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca(2+) in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca(2+) in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.

Regulator of G-protein signaling-5 inhibits bronchial smooth muscle contraction in severe asthma.

Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein-coupled receptor (GPCR)-induced Ca(2+) mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca(2+) mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling-5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca(2+) to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.

Beta-agonist-associated reduction in RGS5 expression promotes airway smooth muscle hyper-responsiveness.

Although short-acting and long-acting inhaled ß(2)-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent ß(2)-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. Here we provide evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to ß(2)-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not increase surface receptor amounts or expression of G proteins and downstream effectors (phospholipase Cß and myosin light chain). In contrast, ß-agonists decreased expression of regulator of G protein signaling 5 (RGS5), which is an inhibitor of G-protein-coupled receptor (GPCR) activity. RGS5 knockdown in HuASM increased agonist-evoked intracellular calcium flux and myosin light chain (MLC) phosphorylation, which are prerequisites for contraction. PCLS from Rgs5(-/-) mice contracted more to carbachol than those from WT mice, indicating that RGS5 negatively regulates bronchial smooth muscle contraction. Repetitive ß(2)-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction signaling pathways as a result of reduced RGS5 expression.

C-027 inhibits IgE-mediated passive sensitization bronchoconstriction and acts as a histamine and serotonin antagonist in human airways.

Atopic asthma is poorly controlled by current therapies. Newer therapies and novel antihistamines are, therefore, required to treat patients whose atopic asthma is not controlled. For the first time, C-027 is shown to antagonize histamine, IgE-mediated and serotonin-induced contraction in human airways and vessels. Human precision-cut lung slices (PCLS, 250 µm thick), containing an airway or blood vessel, were pretreated with either C-027 (2 hours) or with vehicle alone and were contracted with histamine or serotonin. Known antihistamine was used as a comparator in antihistamine studies. Also, human airways were contracted via IgE passive sensitization in the presence or absence of C-027 or fexofenadine. Affinity of C-027 toward human G-protein coupled receptors was also determined, as well as the drug's biodistribution in murine model. C-027 was shown to have the highest affinity toward human histamine and serotonin receptors. Subsequently, C-027 was shown to antagonize histamine- and serotonin-induced airway and vascular smooth muscle contraction, respectively, and histamine-released bronchocontraction mediated by IgE passive sensitization in human small airways. C-027 also inhibited histamine-mediated single-cell calcium ion release. Low levels of C-027 were found in murine brain tissue. Collectively, these data suggest that C-027 markedly inhibits IgE-induced bronchoconstriction and antagonizes histamine and serotonin-contraction with little biodistribution in the brain. The compound may offer a future therapy for allergen-induced airway hyperresponsiveness in patients with asthma.

Glucocorticoid receptor interacting protein-1 restores glucocorticoid responsiveness in steroid-resistant airway structural cells.

Glucocorticoid (GC) insensitivity represents a profound challenge in managing patients with asthma. The mutual inhibition of transcriptional activity between GC receptor (GR) and other regulators is one of the mechanisms contributing to GC resistance in asthma. We recently reported that interferon regulatory factor (IRF)-1 is a novel transcription factor that promotes GC insensitivity in human airway smooth muscle (ASM) cells by interfering with GR signaling (Tliba et al., Am J Respir Cell Mol Biol 2008;38:463-472). Here, we sought to determine whether the inhibition of GR function by IRF-1 involves its interaction with the transcriptional co-regulator GR-interacting protein 1 (GRIP-1), a known GR transcriptional co-activator. We here found that siRNA-mediated GRIP-1 depletion attenuated IRF-1-dependent transcription of the luciferase reporter construct and the mRNA expression of an IRF-1-dependent gene, CD38. In parallel experiments, GRIP-1 silencing significantly reduced GR-mediated transactivation activities. Co-immunoprecipitation and GST pull-down assays showed that GRIP-1, through its repression domain, physically interacts with IRF-1 identifying GRIP-1 as a bona fide transcriptional co-activator for IRF-1. Interestingly, the previously reported inhibition of GR-mediated transactivation activities by either TNF-alpha and IFN-gamma treatment or IRF-1 overexpression was fully reversed by increasing cellular levels of GRIP-1. Together, these data suggest that the cellular accumulation of IRF-1 may represent a potential molecular mechanism mediating altered cellular response to GC through the depletion of GRIP-1 from the GR transcriptional regulatory complexes.

Deletion of microsomal prostaglandin E synthase-1 does not alter ozone-induced airway hyper-responsiveness.

Nonsteroidal anti-inflammatory drugs ameliorate pain and fever by inhibiting cyclooxygenase (COX) and suppressing prostanoid formation. Microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes formation of PGE(2) from the COX product PGH(2) and has emerged as a therapeutic target. Inhibition of mPGES-1, however, renders the PGH(2) substrate available for diversion to other PG synthases. To address the possibility that substrate diversion augments formation of PGs that might modulate bronchial tone, we assessed the impact of mPGES-1 deletion in a mouse model of ozone-induced airway hyper-responsiveness. Ozone exposure increased total lung resistance to inhaled methacholine in wild-type mice. Deletion of mPGES-1 had little effect on total lung resistance in either naive or ozone-exposed animals. The carbachol-induced narrowing of luminal diameter in intrapulmonary airways of lung slices from acute ozone-exposed mice was also unaltered by mPGES-1 deletion. Likewise, although concentrations of PGE(2) were reduced in bronchoalveolar lavage fluid, whereas 6-keto-PGF(1alpha), PGD(2), and PGF(2alpha), all were increased, deletion of mPGES-1 failed to influence cell trafficking into the airways of either naive or ozone-exposed animals. Despite biochemical evidence of PGH(2) substrate diversion to potential bronchomodulator PGs, deletion of mPGES-1 had little effect on ozone-induced airway inflammation or airway hyper-responsiveness. Pharmacologically targeting mPGES-1 may not predispose patients at risk to airway dysfunction.

Inhibition of myristoylated alanine-rich C kinase substrate (MARCKS) protein inhibits ozone-induced airway neutrophilia and inflammation.

Evidence suggests inhibition of leukocyte trafficking mitigates, in part, ozone-induced inflammation. In the present study, the authors postulated that inhibition of myristoylated alanine-rich C kinase substrate (MARCKS), an 82-kDa protein with multiple biological roles, could inhibit ozone-induced leukocyte trafficking and cytokine secretions. BALB/c mice (n = 5/cohort) were exposed to ozone (100 ppb) or forced air (FA) for 4 hours. MARCKS-inhibiting peptides, MANS, BIO-11000, BIO-11006, or scrambled control peptide RNS, were intratracheally administered prior to ozone exposure. Ozone selectively enhanced bronchoalveolar lavage (BAL) levels of killer cells (KCs; 6 +/- 0.9-fold), interleukin-6 (IL-6; 12.7 +/- 1.9-fold), and tumor necrosis factor (TNF; 2.1 +/- 0.5-fold) as compared to cohorts exposed to FA. Additionally, ozone increased BAL neutrophils by 21% +/- 2% with no significant (P > .05) changes in other cell types. MANS, BIO-11000, and BIO-11006 significantly reduced ozone-induced KC secretion by 66% +/- 14%, 47% +/- 15%, and 71.1% +/- 14%, and IL-6 secretion by 69% +/- 12%, 40% +/- 7%, and 86.1% +/- 11%, respectively. Ozone-mediated increases in BAL neutrophils were reduced by MANS (86% +/- 7%) and BIO-11006 (84% +/- 2.5%), but not BIO-11000. These studies identify for the first time the novel potential of MARCKS protein inhibitors in abrogating ozone-induced increases in neutrophils, cytokines, and chemokines in BAL fluid. BIO-11006 is being developed as a treatment for chronic obstructive pulmonary disorder (COPD) and is currently being evaluated in a phase 2 clinical study.

Signal transducer and activator of transcription 3 is required for abnormal proliferation and survival of TSC2-deficient cells: relevance to pulmonary lymphangioleiomyomatosis.

Tumor suppressor complex TSC1/TSC2 represents a key negative regulator of mammalian target of rapamycin (mTOR)-S6 kinase 1 signaling. Mutational inactivation of TSC1 or TSC2, linked to a rare lung disease, lymphangioleiomyomatosis (LAM), manifests as neoplastic growth of smooth-muscle (SM)-like cells and cystic destruction of the lungs that induces loss of pulmonary function. However, the precise mechanisms of abnormal cell growth in LAM remain uncertain. Here, we demonstrate increased signal transducer and activator of transcription (STAT) 3 expression, phosphorylation, and nuclear localization in SM-like cells in LAM lungs and in TSC2-null xenographic tumors. Treatment of TSC2-null tumors with mTOR inhibitor rapamycin attenuated STAT3 expression and phosphorylation. Increased STAT3 level and activation were also observed in LAM-dissociated (LAMD) cell cultures compared with normal human bronchus fibroblasts (HBFs) from LAM patients. Although interferon (IFN)-gamma inhibited proliferation of HBFs, IFN-gamma treatment had little effect on proliferation of LAMD and TSC2-null cells. Re-expression of TSC2 or treatment with rapamycin inhibited IFN-gamma-induced STAT3 phosphorylation and synergized with IFN-gamma in inhibiting TSC2-null and LAMD cell proliferation. Reduction of STAT3 protein levels or activity using specific small interfering RNA or inhibitory peptide, respectively, decreased proliferation and induced apoptosis in TSC2-null and LAMD cells and sensitized cells to growth-inhibitory and proapoptotic effects of IFN-gamma. Collectively, our data demonstrate that STAT3 activation is required for proliferation and survival of cells with TSC2 dysfunction, that STAT3 impedes growth-inhibitory and proapoptotic effects of IFN-gamma, and that TSC2- and rapamycin-dependent inhibition of STAT3 restores antiproliferative effects of IFN-gamma. Thus, STAT3 may provide a novel therapeutic target for diseases associated with TSC1/TSC2 dysfunction.

Airway smooth muscle as an immunomodulatory cell.

Although pivotal in regulating bronchomotor tone in asthma, airway smooth muscle (ASM) also modulates airway inflammation in asthma. ASM myocytes secrete or express a wide array of immunomodulatory mediators in response to extracellular stimuli, and in chronic severe asthma, increases in ASM mass may also render the airway irreversibly obstructed. Although the mechanisms by which ASM secretes cytokines and chemokines are shared with those regulating immune cells, there exist unique ASM signaling pathways that may provide novel therapeutic targets. This review provides an overview of our current understanding of the proliferative as well as synthetic properties of ASM.

Transcriptional regulation of cytokine function in airway smooth muscle cells.

The immuno-modulatory properties of airway smooth muscle have become of increasing importance in our understanding of the mechanisms underlying chronic inflammation and structural remodeling of the airway wall in asthma and chronic obstructive pulmonary disease (COPD). ASM cells respond to many cytokines, growth factors and lipid mediators to produce a wide array of immuno-modulatory molecules which may in turn orchestrate and perpetuate the disease process in asthma and COPD. Despite numerous studies of the cellular effects of cytokines on cultured ASM, few have identified intracellular signaling pathways by which cytokines modulate or induce these cellular responses. In this review we provide an overview of the transcriptional mechanisms as well as intracellular signaling pathways regulating cytokine functions in ASM cells. The recent discovery of toll-like receptors in ASM cells represents a significant development in our understanding of the immuno-modulatory capabilities of ASM cells. Thus, we also review emerging evidence of the inflammatory response to toll-like receptor activation in ASM cells.

Cytokines induce an early steroid resistance in airway smooth muscle cells: novel role of interferon regulatory factor-1.

We have previously shown that long-term treatment of airway smooth muscle (ASM) cells with a combination of TNF-alpha and IFN-gamma impaired steroid anti-inflammatory action through the up-regulation of glucocorticoid receptor beta isoform (GRbeta) (Mol Pharmacol 2006;69:588-596). We here found that steroid actions could also be suppressed by short-term exposure of ASM cells to TNF-alpha and IFN-gamma (6 h) as shown by the abrogated glucocorticoid responsive element (GRE)-dependent gene transcription; surprisingly, neither GRalpha nuclear translocation nor GRbeta expression was affected by cytokine mixture. The earlier induction of CD38, a molecule recently involved in asthma, seen with TNF-alpha and IFN-gamma combination but not with cytokine alone, was also completely insensitive to steroid pretreatment. Chromatin-immunoprecipitation (IP) and siRNA strategies revealed not only increased binding of interferon regulatory factor 1 (IRF-1) transcription factor to CD38 promoter, but also its implication in regulating CD38 gene transcription. Interestingly, the capacity of fluticasone to completely inhibit TNF-alpha-induced IRF-1 expression, IRF-1 DNA binding, and transactivation activities was completely lost in cells exposed to TNF-alpha and IFN-gamma in combination. This early steroid dysfunction seen with cytokine combination could be reproduced by enhancing IRF-1 cellular levels using constitutively active IRF-1, which dose-dependently inhibited GRE-dependent gene transcription. Consistently, reducing IRF-1 cellular levels using siRNA approach significantly restored steroid transactivation activities. Collectively, our findings demonstrate for the first time that IRF-1 is a novel alternative GRbeta-independent mechanism mediating steroid dysfunction induced by pro-asthmatic cytokines, in part via the suppression of GRalpha activities.

Histamine stimulation of MMP-1(collagenase-1) secretion and gene expression in gastric epithelial cells: role of EGFR transactivation and the MAP kinase pathway.

BACKGROUND AND AIMS: GPCR stimulation by various ligands including histamine has been shown to transactivate the epidermal growth factor receptor (EGFR). This study examines the functional interactions between the H2 receptor and the EGFR in the regulation of matrix metalloproteinase-1 (MMP-1) secretion and gene expressions in cultured gastric epithelial cells. METHODS: AGS cells were incubated for up to 24 h with either histamine or heparin binding-epidermal growth factor (HB-EGF) and MMP-1 release was determined by immunoassay. MMP-1 responses to histamine and HB-EGF were further tested by the use of H2 receptor antagonist, EGFR inhibitor and mitogen activator protein kinase (MAPK) inhibitor. The role of EGFR in MMP-1 release was further tested in cells transfected with specific EGFR siRNA. EGFR and ERK1/2 phosphorylation was determined by Western blot analysis. MMP-1 gene expression was determined by RNase protection assay (RPA). RESULTS: Histamine and HB-EGF caused a dose-dependent release of MMP-1 with maximal responses that were 2.7- and 4.5-fold greater, respectively, than control, P<0.001. Famotidine prevented histamine-mediated MMP-1 release and AG1478 and EGFR siRNA completely inhibited MMP-1 secretion stimulated by both histamine and HB-EGF. Both histamine and HB-EGF stimulation of MMP-1 release was associated with activation of ERK1/2. MAPK inhibition also prevented histamine-and HB-EGF-induced MMP-1 secretion. Results of MMP-1 gene expression, either stimulatory or inhibitory, paralleled responses to MMP-1 secretion. CONCLUSION: Histamine stimulation of the H2 receptor on AGS cells evoked MMP-1 secretion and gene up regulation that was dependent on transactivation of the EGFR and downstream activation of MAPK.

IL-4 induced MUC4 enhancement in respiratory epithelial cells in vitro is mediated through JAK-3 selective signaling.

BACKGROUND: Recent studies have identified MUC4 mucin as a ligand for activation of ErbB2, a receptor tyrosine kinase that modulates epithelial cell proliferation following epithelial damage in airways of asthmatics. In this study, we investigated the potential role of IL-4, one of the Th2 inflammatory cytokines persistent in asthmatic airways, in regulating MUC4 expression using a cell line NCI-H650. METHODS: Real time PCR analysis was performed to determine concentration and time dependent effects of IL-4 upon MUC4 expression. Nuclear run on experiments were carried out to explore potential transcriptional modulation. Western blotting experiments using a monoclonal antibody specific to ASGP-2 domain of MUC4 were performed to analyze MUC4 glycoprotein levels in plasma membrane fractions. To analyze potential signal transduction cascades, IL-4 treated confluent cultures were co-incubated, separately with a pan-JAK inhibitor, a JAK-3 selective inhibitor or a MEK-1, 2 (MAPK) inhibitor at various concentrations before MUC4 transcript analysis. Corresponding transcription factor activation was tested by western blotting using a monoclonal p-STAT-6 antibody. RESULTS: MUC4 levels increased in a concentration and time specific fashion reaching peak expression at 2.5 ng/ml and 8 h. Nuclear run on experiments revealed transcriptional enhancement. Corresponding increases in MUC4 glycoprotein levels were observed in plasma membrane fractions. Pan-JAK inhibitor revealed marked reduction in IL-4 stimulated MUC4 levels and JAK3 selective inhibitor down-regulated MUC4 mRNA expression in a concentration-dependent fashion. In accordance with the above observations, STAT-6 activation was detected within 5 minutes of IL-4 stimulus. No effect in MUC4 levels was observed on using a MAPK inhibitor. CONCLUSION: These observations signify a potential role for IL-4 in MUC4 up-regulation in airway epithelia.

IL-9 modulated MUC4 gene and glycoprotein expression in airway epithelial cells.

Compromised epithelial cell integrity is a common feature associated with chronic lung inflammatory states such as asthma. While epithelial cell damage is largely due to sustained effects of inflammatory mediators localized to airways, the subsequent process of epithelial cell differentiation is attributed to members of the transmembrane receptor tyrosine kinase family called the ErbB's. MUC4, a large molecular weight membrane-bound glycoprotein, has recently been identified as a potential ligand for the ErbB-2 receptor. In this study, we investigated the possible role of interleukin-9 (IL-9), a Th2 cytokine, on MUC4 expression using a lung cancer cell line, NCI-H650. We determined that IL-9 up-regulates MUC4 expression in a time and concentration-dependent fashion. Nuclear run-on assays indicated transcriptional regulation of MUC4 while no post-transcriptional mRNA stabilization was observed by actinomycin D chase experiments. IL-9 also increased MUC4 glycoprotein expression as determined by Western blots using a monoclonal antibody specific for a non-tandem repeat region on ASGP-2 region of MUC4. Furthermore, a JAK3-selective inhibitor 4-(4'-hydroxyphenyl) amino-6, 7-dimethoxyquinazoline (WHI-P131), substantially reduced IL-9-induced MUC4 mRNA expression in a dose-dependent fashion. These results implicate a potential role for IL-9 upon MUC4 expression in human airway epithelial cells.