Transglutaminase 2, a novel regulator of eicosanoid production in asthma revealed by genome-wide expression profiling of distinct asthma phenotypes

Abstract

Background: A frequent manifestation of asthma, exercise-induced bronchoconstriction (EIB), occurs in 30-50% of asthmatics and is characterized by increased release of inflammatory eicosanoids. The objective of this study was to identify genes differentially expressed in EIB and to understand the function of these genes in the biology of asthma.

Methodology/principal findings: Genome-wide expression profiling of airway leukocytes and epithelial cells obtained by induced sputum was conducted in two groups of subjects with asthma with and without EIB (n = 7 per group), at baseline and following exercise challenge. Based on the results of the gene expression study, additional comparisons were made with a normal control group (n = 10). Localization studies were conducted on epithelial brushings and biopsies from an additional group of asthmatics with EIB (n = 3). Genes related to epithelial repair and mast cell infiltration including beta-tryptase and carboxypeptidase A3 were upregulated by exercise challenge in the asthma group with EIB. A gene novel to asthma pathogenesis, transglutaminase 2 (TGM2), was the most differentially expressed at baseline between the groups. In vivo studies confirmed the increased expression of TGM2 in airway cells and airway lining fluid, and demonstrate that TGM2 is avidly expressed in the asthmatic airway epithelium. In vitro studies using recombinant human enzymes reveal that TGM2 augments the enzymatic activity of secreted phospholipase A(2) (PLA(2)) group X (sPLA(2)-X), an enzyme recently implicated in asthma pathogenesis.

Conclusions/significance: This study found that TGM2, a mediator that is novel to asthma pathogenesis, is overexpressed in asthmatic airways and functions to increase sPLA(2)-X enzymatic activity. Since PLA(2) serves as the first rate-limiting step leading to eicosanoid formation, these results suggest that TGM2 may be a key initiator of the airway inflammatory cascade in asthma.

Figure 1. Comparison of lung function and gene expression between asthmatics with EIB and an asthmatic control group without EIB.

The severity of EIB was markedly greater in the EIB⁺ group (A). Genes that were increased in the EIB⁺ group relative to the EIB⁻ group post-exercise were consistently differentially expressed in the initial and replication cohorts as shown by the strong association between differences in gene expression in each cohort (B). A heatmap of these genes shows the Log₂FC relative to the average expression for each gene in each individual of the two groups post-exercise (C). The fold difference in gene expression between the groups post-exercise identified by the microarray platform was similarly demonstrated by qPCR (D). Within the EIB⁺ group, genes that increased in response to exercise challenge were consistently increased in the initial and replication cohorts (E). A heatmap of these genes shows the Log₂FC relative to the average expression for each gene in each individual of the EIB⁺ cohort (F). The fold increase in gene expression in response to exercise challenge by the microarray platform was similarly demonstrated by qPCR with the exception of CLCA1 and KLK11 (G). To visualize the differences in the pattern of gene expression, differences in gene expression between the groups at baseline are plotted against the change in gene expression with exercise challenge in the EIB⁺ group (H).

Figure 2. Comparison of TGM2 levels in the airways and in vitro function of TGM2 in the activation of secreted PLA₂ activity.

The gene expression of TGM2 by qPCR in airway cells was increased in both asthma groups relative to non-asthmatic controls, and the expression was higher in the EIB⁺ group (A). TGM2 was not detected in induced sputum supernatant from non-asthmatic controls, but was elevated in asthmatics, especially in the EIB⁺ group (B). The bands from one of two Western blots used to quantify the levels of TGM2 in induced sputum supernatant are shown in the panel, with lanes 1–5 representing EIB⁺ asthmatics, lanes 6–10 representing EIB⁻ asthmatics, and lanes 11–15 normal controls (C). A Western blot of epithelial brushings from asthmatics demonstrates higher levels in the epithelium from asthmatics relative to non-asthmatic controls (D). In the blot, the first lane is recombinant human TGM2 (rhTGM2), lanes 3 and 4 are epithelial lysates from 2 different EIB⁺ asthmatics, and lanes 5 and 6 are epithelial lysates from 2 different normal controls. Asthmatic epithelial cells from an EIB⁺ asthmatic in primary culture shown in lanes 8 and 9 also strongly express TGM2. Immunostaining for TGM2 in airway biopsies from an EIB⁺ asthmatic demonstrates immunostaining in the airway epithelium for TGM2 (40×, scale bar is 50 µm) (E). Pre-incubation of recombinant human sPLA₂-X with purified TGM2 from guinea pig liver (F) or with recombinant human TGM2 (G) causes an increase in the PLA₂ activity of the sPLA₂-X enzyme. Denaturing the TGM2 with heat (boiled) or inhibiting the activity of the enzyme by saturating the enzyme with N-carbobenzoxy-Gln-Gly (Inh) demonstrate that the in vitro findings are due to the enzymatic activity of TGM2.