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1.  CaMKII Is Essential for the Proasthmatic Effects of Oxidation 
Science translational medicine  2013;5(195):195ra97.
Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca2+/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl− current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyper-reactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent pro-asthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.
doi:10.1126/scitranslmed.3006135
PMCID: PMC4331168  PMID: 23884469
2.  Foxa3 Induces Goblet Cell Metaplasia and Inhibits Innate Antiviral Immunity 
Rationale: Goblet cell metaplasia accompanies common pulmonary disorders that are prone to recurrent viral infections. Mechanisms regulating both goblet cell metaplasia and susceptibility to viral infection associated with chronic lung diseases are incompletely understood.
Objectives: We sought to identify the role of the transcription factor FOXA3 in regulation of goblet cell metaplasia and pulmonary innate immunity.
Methods: FOXA3 was identified in airways from patients with asthma and chronic obstructive pulmonary disease. We produced transgenic mice conditionally expressing Foxa3 in airway epithelial cells and developed human bronchial epithelial cells expressing Foxa3. Foxa3-regulated genes were identified by immunostaining, Western blotting, and RNA analysis. Direct binding of FOXA3 to target genes was identified by chromatin immunoprecipitation sequencing correlated with RNA sequencing.
Measurements and Main Results: FOXA3 was highly expressed in airway goblet cells from patients with asthma and chronic obstructive pulmonary disease. FOXA3 was induced by either IL-13 or rhinovirus. Foxa3 induced goblet cell metaplasia and enhanced expression of a network of genes mediating mucus production. Paradoxically, FOXA3 inhibited rhinovirus-induced IFN production, IRF-3 phosphorylation, and IKKε expression and inhibited viral clearance and expression of genes required for antiviral defenses, including MDA5, RIG-I, TLR3, IRF7/9, and nuclear factor-κB.
Conclusions: FOXA3 induces goblet cell metaplasia in response to infection or Th2 stimulation. Suppression of IFN signaling by FOXA3 provides a plausible mechanism that may serve to limit ongoing Th1 inflammation during the resolution of acute viral infection; however, inhibition of innate immunity by FOXA3 may contribute to susceptibility to viral infections associated with chronic lung disorders accompanied by chronic goblet cell metaplasia.
doi:10.1164/rccm.201306-1181OC
PMCID: PMC3977731  PMID: 24392884
rhinovirus; IFN; transcription factors; mucus
3.  Rhinovirus-16 Induced Release of IP-10 and IL-8 Is Augmented by Th2 Cytokines in a Pediatric Bronchial Epithelial Cell Model 
PLoS ONE  2014;9(4):e94010.
Background
In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation.
Methods
Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID50. Phosphatidyl inositol 3-kinase (PI3K) and P38 mitogen activated protein kinase (MAPK) inhibitors and dexamethasone were used to investigate differences in signaling pathways.
Results
The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone.
Conclusion
Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.
doi:10.1371/journal.pone.0094010
PMCID: PMC3976391  PMID: 24705919
4.  Regulation of A Disintegrin And Metalloprotease-33 Expression by Transforming Growth Factor-β 
The asthma susceptibility gene, a disintegrin and metalloprotease-33 (ADAM33), is selectively expressed in mesenchymal cells, and the activity of soluble ADAM33 has been linked to angiogenesis and airway remodeling. Transforming growth factor (TGF)-β is a profibrogenic growth factor, the expression of which is increased in asthma, and recent studies show that it enhances shedding of soluble ADAM33. In this study, we hypothesized that TGF-β also affects ADAM33 expression in bronchial fibroblasts in asthma. Primary fibroblasts were grown from bronchial biopsies from donors with and those without asthma, and treated with TGF-β2 to induce myofibroblast differentiation. ADAM33 expression was assessed using quantitative RT-PCR and Western blotting. To examine the mechanisms whereby TGF-β2 affected ADAM33 expression, quantitative methylation-sensitive PCR, chromatin immunoprecipitation, and nuclear accessibility assays were conducted on the ADAM33 promoter. We found that TGF-β2 caused a time- and concentration-dependent reduction in ADAM33 mRNA expression in normal and asthmatic fibroblasts, affecting levels of splice variants similarly. TGF-β2 also induced ADAM33 protein turnover and appearance of a cell-associated C-terminal fragment. TGF-β2 down-regulated ADAM33 mRNA expression by causing chromatin condensation around the ADAM33 promoter with deacetylation of histone H3, demethylation of H3 on lysine-4, and hypermethylation of H3 on lysine-9. However, the methylation status of the ADAM33 promoter did not change. Together, these data suggest that TGF-β2 suppresses expression of ADAM33 mRNA in normal or asthmatic fibroblasts. This occurs by altering chromatin structure, rather than by gene silencing through DNA methylation as in epithelial cells. This may provide a mechanism for fine regulation of levels of ADAM33 expression in fibroblasts, and may self-limit TGF-β2–induced ectodomain shedding of ADAM33.
doi:10.1165/rcmb.2011-0030OC
PMCID: PMC3359905  PMID: 22227561
a disintegrin and metalloprotease-33; myofibroblast; transforming growth factor-β; histone modification
5.  Kruppel-like factor 5 is Required for Formation and Differentiation of the Bladder Urothelium 
Developmental biology  2011;358(1):79-90.
SUMMARY
Kruppel-like transcription factor 5 (Klf5) was detected in the developing and mature murine bladder urothelium. Herein we report a critical role of KLF5 in the formation and terminal differentiation of the urothelium. The ShhGfpCre transgene was used to delete the Klf5floxed alleles from bladder epithelial cells causing prenatal hydronephrosis, hydroureter, and vesicoureteric reflux. The bladder urothelium failed to stratify and did not express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and the uroplakins. The effects of Klf5 deletion were unique to the developing bladder epithelium since maturation of the epithelium comprising the bladder neck and urethra were unaffected by the lack of KLF5. mRNA analysis identified reductions in Pparγ, Grhl3, Elf3, and Ovol1expression in Klf5 deficient fetal bladders supporting their participation in a transcriptional network regulating bladder urothelial differentiation. KLF5 regulated expression of the mGrhl3 promoter in transient transfection assays. The absence of urothelial Klf5 altered epithelial-mesenchymal signaling leading to the formation of an ectopic alpha smooth muscle actin positive layer of cells subjacent to the epithelium and a thinner detrusor muscle that was not attributable to disruption of SHH signaling, a known mediator of detrusor morphogenesis. Deletion of Klf5 from the developing bladder urothelium blocked epithelial cell differentiation, impaired bladder morphogenesis and function causing hydroureter and hydronephrosis at birth.
doi:10.1016/j.ydbio.2011.07.020
PMCID: PMC3180904  PMID: 21803035
bladder; vesicoureteral reflux; KLF5; GRHL3; urothelium; micro-CT
6.  Airway Epithelial Transcription Factor NK2 Homeobox 1 Inhibits Mucous Cell Metaplasia and Th2 Inflammation 
Rationale: Airway mucous cell metaplasia and chronic inflammation are pathophysiological features that influence morbidity and mortality associated with asthma and other chronic pulmonary disorders. Elucidation of the molecular mechanisms regulating mucous metaplasia and hypersecretion provides the scientific basis for diagnostic and therapeutic opportunities to improve the care of chronic pulmonary diseases.
Objectives: To determine the role of the airway epithelial–specific transcription factor NK2 homeobox 1 (NKX2-1, also known as thyroid transcription factor-1 [TTF-1]) in mucous cell metaplasia and lung inflammation.
Methods: Expression of NKX2-1 in airway epithelial cells from patients with asthma was analyzed. NKX2-1+/− gene targeted or transgenic mice expressing NKX2-1 in conducting airway epithelial cells were sensitized to the aeroallergen ovalbumin. In vitro studies were used to identify mechanisms by which NKX2-1 regulates mucous cell metaplasia and inflammation.
Measurements and Main Results: NKX2-1 was suppressed in airway epithelial cells from patients with asthma. Reduced expression of NKX2-1 in heterozygous NKX2-1+/− gene targeted mice increased mucous metaplasia in the small airways after pulmonary sensitization to ovalbumin. Conversely, mucous cell metaplasia induced by aeroallergen was inhibited by expression of NKX2-1 in the respiratory epithelium in vivo. Genome-wide mRNA analysis of lung tissue from ovalbumin-treated mice demonstrated that NKX2-1 inhibited mRNAs associated with mucous metaplasia and Th2-regulated inflammation, including Spdef, Ccl17, and Il13. In vitro, NKX2-1 inhibited SPDEF, a critical regulator of airway mucous cell metaplasia, and the Th2 chemokine CCL26.
Conclusions: The present data demonstrate a novel function for NKX2-1 in a gene network regulating mucous cell metaplasia and allergic inflammation in the respiratory epithelium.
doi:10.1164/rccm.201101-0106OC
PMCID: PMC3175541  PMID: 21562130
asthma; goblet cell; respiratory epithelium; NK2 homeobox 1
7.  Intersections between Pulmonary Development and Disease 
Recent advances in cellular, molecular, and developmental biology have revolutionized our concepts regarding the process of organogenesis that have important implications for our understanding of both lung formation and pulmonary disease pathogenesis. Pulmonary investigators have long debated whether developmental processes are recapitulated during normal repair of the lung or in the setting of chronic pulmonary diseases. Although the cellular events involved in lung morphogenesis and those causing pulmonary disease are likely to include processes that are distinct, there is increasing evidence that the pathogenesis of many lung disorders involves the same genetic machinery that regulates cell growth, specification, and differentiation during normal lung development.
doi:10.1164/rccm.201103-0495PP
PMCID: PMC3175542  PMID: 21642246
lung; morphogenesis; transcription; respiratory

Results 1-7 (7)