Thomas J. Mariani

Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Three unique and highly compartmentalized mammalian superoxide dismutases have been biochemically and molecularly characterized to date. SOD1, or CuZn-SOD (EC 1.15.1.1), was the first enzyme to be characterized and is a copper and zinc-containing homodimer that is found almost exclusively in intracellular cytoplasmic spaces. SOD2, or Mn-SOD (EC 1.15.1.1), exists as a tetramer and is initially synthesized containing a leader peptide, which targets this manganese-containing enzyme exclusively to the mitochondrial spaces. SOD3, or EC-SOD (EC 1.15.1.1), is the most recently characterized SOD, exists as a copper and zinc-containing tetramer, and is synthesized containing a signal peptide that directs this enzyme exclusively to extracellular spaces. What role(s) these SODs play in both normal and disease states is only slowly beginning to be understood. A molecular understanding of each of these genes has proven useful toward the deciphering of their biological roles. For example, a variety of single amino acid mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis. Knocking out the SOD2 gene in mice results in a lethal cardiomyopathy. A single amino acid mutation in human SOD3 is associated with 10 to 30-fold increases in serum SOD3 levels. As more information is obtained, further insights will be gained.

K-ras activation generates an inflammatory response in lung tumors

Activating mutations in K-ras are one of the most common genetic alterations in human lung cancer. To dissect the role of K-ras activation in bronchial epithelial cells during lung tumorigenesis, we created a model of lung adenocarcinoma by generating a conditional mutant mouse with both Clara cell secretory protein (CC10)-Cre recombinase and the Lox-Stop-Lox K-rasG12D alleles. The activation of K-ras mutant allele in CC10 positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately adenocarcinoma. Surprisingly, K-ras activation in the bronchiolar epithelium is associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response with a median survival of 8 weeks. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that increased significantly with age. Cell lines derived from these tumors directly produced MIP-2, LIX and KC. This model demonstrates that K-ras activation in the lung induces the elaboration of inflammatory chemokines and provides an excellent means to further study the complex interactions between inflammatory cells, chemokines and tumor progression.

Airway lipoxin A4 generation and lipoxin A4 receptor expression are decreased in severe asthma.

RATIONALE Airway inflammation is common in severe asthma despite antiinflammatory therapy with corticosteroids. Lipoxin A(4) (LXA(4)) is an arachidonic acid-derived mediator that serves as an agonist for resolution of inflammation. OBJECTIVES Airway levels of LXA(4), as well as the expression of lipoxin biosynthetic genes and receptors, in severe asthma. METHODS Samples of bronchoalveolar lavage fluid were obtained from subjects with asthma and levels of LXA(4) and related eicosanoids were measured. Expression of lipoxin biosynthetic genes was determined in whole blood, bronchoalveolar lavage cells, and endobronchial biopsies by quantitative polymerase chain reaction, and leukocyte LXA(4) receptors were monitored by flow cytometry. MEASUREMENTS AND MAIN RESULTS Individuals with severe asthma had significantly less LXA(4) in bronchoalveolar lavage fluids (11.2 +/- 2.1 pg/ml) than did subjects with nonsevere asthma (150.1 +/- 38.5 pg/ml; P < 0.05). In contrast, levels of cysteinyl leukotrienes were increased in both asthma cohorts compared with healthy individuals. In severe asthma, 15-lipoxygenase-1 mean expression was decreased fivefold in bronchoalveolar lavage cells. In contrast, 15-lipoxgenase-1 was increased threefold in endobronchial biopsies, but expression of both 5-lipoxygenase and 15-lipoxygenase-2 in these samples was decreased. Cyclooxygenase-2 expression was decreased in all anatomic compartments sampled in severe asthma. Moreover, LXA(4) receptor gene and protein expression were significantly decreased in severe asthma peripheral blood granulocytes. CONCLUSIONS Mechanisms underlying pathological airway responses in severe asthma include lipoxin underproduction with decreased expression of lipoxin biosynthetic enzymes and receptors. Together, these results indicate that severe asthma is characterized, in part, by defective lipoxin counterregulatory signaling circuits.

The SERPINE2 Gene Is Associated with Chronic Obstructive Pulmonary Disease

RATIONALE Chronic obstructive pulmonary disease (COPD) is a complex disease influenced by multiple genes and environmental factors. A region on chromosome 2q has been shown to be linked to COPD. A positional candidate gene from the chromosome 2q region SERPINE2 (Serpin peptidase inhibitor, clade E [nexin, plasminogen activator inhibitor type 1], member 2), was previously evaluated as a susceptibility gene for COPD in two association studies, but the results were contradictory. OBJECTIVES To identify the relationship between SERPINE2 polymorphisms and COPD-related phenotypes using family-based and case-control association studies. METHODS In the present study, we genotyped 25 single nucleotide polymorphisms (SNPs) from SERPINE2 and analyzed qualitative and quantitative COPD phenotypes in 635 pedigrees with 1,910 individuals and an independent case-control population that included 973 COPD cases and 956 control subjects. The family data were analyzed using family-based association tests. The case-control data were analyzed using logistic regression and linear models. MEASUREMENTS AND MAIN RESULTS Six SNPs demonstrated significant associations with COPD phenotypes in the family-based association analysis (0.0016<or=p<or=0.042). Five of these SNPs demonstrated replicated associations in the case-control analysis (0.021<or=p<or=0.031). In addition, the results of haplotype analyses supported the results from single SNP analyses. CONCLUSIONS These data provide further support for SERPINE2 as a COPD susceptibility gene.

Conditional Stabilization of β‐Catenin Expands the Pool of Lung Stem Cells

Maintenance of classic stem cell hierarchies is dependent upon stem cell self‐renewal mediated in part by Wnt/β‐catenin regulation of the cell cycle. This function is critical in rapidly renewing tissues due to the obligate role played by the tissue stem cell. However, the stem cell hierarchy responsible for maintenance of the conducting airway epithelium is distinct from classic stem cell hierarchies. The epithelium of conducting airways is maintained by transit‐amplifying cells in the steady state; rare bronchiolar stem cells are activated to participate in epithelial repair only following depletion of transit‐amplifying cells. Here, we investigate how signaling through β‐catenin affects establishment and maintenance of the stem cell hierarchy within the slowly renewing epithelium of the lung. Conditional potentiation of β‐catenin signaling in the embryonic lung results in amplification of airway stem cells through attenuated differentiation rather than augmented proliferation. Our data demonstrate that the differentiation‐modulating activities of stabilized β‐catenin account for expansion of tissue stem cells.

Integration of genomic and genetic approaches implicates IREB2 as a COPD susceptibility gene.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and is influenced by both genetic determinants and smoking. We identified genomic regions from 56 lung-tissue gene-expression microarrays and used them to select 889 SNPs to be tested for association with COPD. We genotyped SNPs in 389 severe COPD cases from the National Emphysema Treatment Trial and 424 cigarette-smoking controls from the Normative Aging Study. A total of 71 autosomal SNPs demonstrated at least nominal significance with COPD susceptibility (p = 3.4 x 10(-6) to 0.05). These 71 SNPs were evaluated in a family-based study of 127 probands with severe, early-onset COPD and 822 of their family members in the Boston Early-Onset COPD Study. We combined p values from the case-control and family-based analyses, setting p = 5.60 x 10(-5) as a conservative threshold for significance. Three SNPs in the iron regulatory protein 2 (IREB2) gene met this stringent threshold for significance, and four other IREB2 SNPs demonstrated combined p < 0.02. We demonstrated replication of association for these seven IREB2 SNPs (all p values < or = 0.02) in a family-based study of 3117 subjects from the International COPD Genetics Network; combined p values across all cohorts for the main phenotype of interest ranged from 1.6 x 10(-7) to 6.4 x 10(-4). IREB2 protein and mRNA were increased in lung-tissue samples from COPD subjects in comparison to controls. In summary, gene-expression and genetic-association results have implicated IREB2 as a COPD susceptibility gene.

Deletion of Keap1 in the lung attenuates acute cigarette smoke-induced oxidative stress and inflammation.

Exposure to cigarette smoke (CS) is the primary factor associated with the development of chronic obstructive pulmonary disease (COPD). CS increases the level of oxidants in the lungs, resulting in a depletion of antioxidants, which promotes oxidative stress and the destruction of alveolar tissue. In response to CS, pulmonary epithelial cells counteract increased levels of oxidants by activating Nrf2-dependent pathways to augment the expression of detoxification and antioxidant enzymes, thereby protecting the lung from injury. We hypothesize that increasing the pathways activated by Nrf2 will afford protection against CS-induced lung damage. To this end we have developed a novel mouse model in which the cytosolic inhibitor of Nrf2, Keap1, is genetically deleted in Clara cells, which predominate in the upper airways in mice. Deletion of Keap1 in Clara cells resulted in increased expression of Nrf2-dependent genes, such as Nqo1 and Gclm, as determined by microarray analysis and quantitative PCR. Deletion of Keap1 in airway epithelium decreased Keap1 protein levels and significantly increased the total level of glutathione in the lungs. Increased Nrf2 activation protected Clara cells against oxidative stress ex vivo and attenuated oxidative stress and CS-induced inflammation in vivo. Expression of KEAP1 was also decreased in human epithelial cells through siRNA transfection, which increased the expression of Nrf2-dependent genes and attenuated oxidative stress. In conclusion, activating Nrf2 pathways in tissue-specific Keap1 knockout mice represents an important genetic approach against oxidant-induced lung damage.

Molecular Biomarkers for Quantitative and Discrete COPD Phenotypes

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disorder with complex pathological features and largely unknown etiology. The identification of biomarkers for this disease could aid the development of methods to facilitate earlier diagnosis, the classification of disease subtypes, and provide a means to define therapeutic response. To identify gene expression biomarkers, we completed expression profiling of RNA derived from the lung tissue of 56 subjects with varying degrees of airflow obstruction using the Affymetrix U133 Plus 2.0 array. We applied multiple, independent analytical methods to define biomarkers for either discrete or quantitative disease phenotypes. Analysis of differential expression between cases (n = 15) and controls (n = 18) identified a set of 65 discrete biomarkers. Correlation of gene expression with quantitative measures of airflow obstruction (FEV(1)%predicted or FEV(1)/FVC) identified a set of 220 biomarkers. Biomarker genes were enriched in functions related to DNA binding and regulation of transcription. We used this group of biomarkers to predict disease in an unrelated data set, generated from patients with severe emphysema, with 97% accuracy. Our data contribute to the understanding of gene expression changes occurring in the lung tissue of patients with obstructive lung disease and provide additional insight into potential mechanisms involved in the disease process. Furthermore, we present the first gene expression biomarker for COPD validated in an independent data set.

Epithelial cell PPARγ contributes to normal lung maturation

Peroxisome proliferator‐activated receptor (PPAR)‐γ is a member of the nuclear hormone receptor superfamily that can promote cellular differentiation and organ development. PPARγ expression has been reported in a number of pulmonary cell types, including inflammatory, mesenchymal, and epithelial cells. We find that PPARγ is prominently expressed in the airway epithelium in the mouse lung. In an effort to define the physiological role of PPARγ within the lung, we have ablated PPARγ using a novel line of mice capable of specifically targeting the airway epithelium. Airway epithelial cell PPARγ‐targeted mice display enlarged airspaces resulting from insufficient postnatal lung maturation. The increase in airspace size is accompanied by alterations in lung physiology, including increased lung volumes and decreased tissue resistance. Genome‐wide expression profiling reveals a reduction in structural extracellular matrix (ECM) gene expression in conditionally targeted mice, suggesting a disruption in epithelial‐mesenchymal interactions necessary for the establishment of normal lung structure. Expression profiling of airway epithelial cells isolated from conditionally targeted mice indicates PPARγ regulates genes encoding known PPARγ targets, additional lipid metabolism enzymes, and markers of cellular differentiation. These data reveal airway epithelial cell PPARγ is necessary for normal lung structure and function.—Simon, D. M., Arikan, M. C., Srisuma, S., Bhattacharya, S., Tsai, L. W., Ingenito, E. P., Gonzalez, F., Shapiro, S. D., and Mariani, T. J. Epithelial cell PPARγ contributes to normal lung maturation. FASEB J. 20, E710–E720 (2006)

Airway Epithelial STAT3 Is Required for Allergic Inflammation in a Murine Model of Asthma

The STAT3 transcription factor is critical for cytokine signaling and the acute phase response, but its role in allergic asthma is largely undefined. To investigate the role of STAT3 in mediating allergic inflammation, we used chemical and genetic approaches to inactivate STAT3 in the airway epithelium of mice. In a murine model of chronic asthma, we demonstrate that the administration of house dust mite (HDM) leads to robust STAT3 activation in the airway epithelium, smooth muscle, and immune cells in the lungs of C57BL/6 mice. To investigate the role of STAT3 in HDM-induced airway inflammation, a conditional knockout of STAT3 in the airway epithelium was generated, e-STAT3−/−. We determined that e-STAT3−/− mice had a significant decrease in HDM-induced airway eosinophilia, lung Th2 accumulation, and chemokines compared with wild-type animals. Importantly, the e-STAT3−/− mice had a significant decrease in airway hyperresponsiveness to methacholine. The administration of two STAT kinase inhibitors diminished STAT3 activation and markedly abrogated the HDM-induced lung inflammation. These findings suggest that STAT3 acts as a novel epithelial regulator of the allergic response by altering Th2 cell recruitment and effector function, and thus, targeting this molecule may provide the basis for a novel asthma therapy.