RNA-Seq transcriptome profiling identifies CRISPLD2 as a glucocorticoid responsive gene that modulates cytokine function in airway smooth muscle cells

Abstract

Asthma is a chronic inflammatory respiratory disease that affects over 300 million people worldwide. Glucocorticoids are a mainstay therapy for asthma because they exert anti-inflammatory effects in multiple lung tissues, including the airway smooth muscle (ASM). However, the mechanism by which glucocorticoids suppress inflammation in ASM remains poorly understood. Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone--a potent synthetic glucocorticoid (1 µM for 18 hours). Based on a Benjamini-Hochberg corrected p-value <0.05, we identified 316 differentially expressed genes, including both well known (DUSP1, KLF15, PER1, TSC22D3) and less investigated (C7, CCDC69, CRISPLD2) glucocorticoid-responsive genes. CRISPLD2, which encodes a secreted protein previously implicated in lung development and endotoxin regulation, was found to have SNPs that were moderately associated with inhaled corticosteroid resistance and bronchodilator response among asthma patients in two previously conducted genome-wide association studies. Quantitative RT-PCR and Western blotting showed that dexamethasone treatment significantly increased CRISPLD2 mRNA and protein expression in ASM cells. CRISPLD2 expression was also induced by the inflammatory cytokine IL1β, and small interfering RNA-mediated knockdown of CRISPLD2 further increased IL1β-induced expression of IL6 and IL8. Our findings offer a comprehensive view of the effect of a glucocorticoid on the ASM transcriptome and identify CRISPLD2 as an asthma pharmacogenetics candidate gene that regulates anti-inflammatory effects of glucocorticoids in the ASM.

Figure 1. RNA-Seq profiling of DEX-treated ASM cells.

A) Volcano plot of overall gene-based differential expression results for four cell lines treated with DEX vs. left untreated (each dot corresponds to a gene). The y-axis corresponds to the negative log (base 10) of P-values while the x-axis corresponds to the negative log (base 2) of the fold change for difference in expression when cells were stimulated with DEX. There were 316 differentially expressed genes according to an adjusted p-value <0.05 (blue dots). B) Validation of known GC-responsive genes through qRT-PCR. After ASM cells were treated with 1 µM DEX for 18 h, the mRNA levels of indicated genes were measured by qRT-PCR and the folds of change in mRNA induced by DEX were calculated. Each column bar represents an individual cell line. Experiments for each cell line were performed in triplicate, and the error bars are SE values corresponding to a cell line's replicates. The dotted line indicates a fold change of 1. ** P<0.005, * P<0.05 (t test).

Figure 2. Validation of GC responsive genes.

After cells from three individual ASM lines were treated with 1 µM DEX for 18 h, the mRNA levels of indicated genes were measured by qRT-PCR and the folds of change induced by DEX were calculated. Each column bar represents an individual cell line; experiments for each cell line were performed in triplicate. Error bars are SE values corresponding to a cell line's replicates.

Figure 3. CRISPLD2 is a GC- and IL1β-responsive gene.

ASM cells were treated with 100A) increased CRISPLD2 mRNA expression as measured by qRT-PCR, B) increased CRISPLD2 protein expression as measured by immuno-blotting. ASM cells were treated with 5 ng/mL IL1β for 24 h, resulting in C) increased CRISPLD2 mRNA expression as measured by qRT-PCR, and D) increased CRISPLD2 protein expression as measured by immuno-blotting. CRISPLD2 mRNA levels were measured in triplicate. CRISPLD2 protein levels are shown as normalized blot densitometry values, and the error bars are SE values across three independent experiments. * P<0.05 (t test).

Figure 4. CRISPLD2 regulates the response to inflammatory cytokines.

A) Effect of CRISPLD2-specific siRNA on CRISPLD2 mRNA and protein levels. ASM cells were transfected with CRISPLD2-specific siRNA or non-targeting (NT) siRNA, and 72 h later, CRISPLD2 mRNA and protein levels were determined by qRT-PCR (levels normalized to those in control cells transfected with NT siRNA) and immuno-blotting, respectively. The effect of CRISPLD2 knockdown on IL1β-induced cytokine expression was assessed by transfecting ASM cells with CRISPLD2-specific or NT siRNA, and 72 h later, treating cells for 24 h with B) 5 ng/mL IL1β, C) 100 nM DEX, or D) 5 ng/mL IL1β and 100 nM DEX. IL6 expression was determined by qRT-PCR. Normalized mRNA levels are shown. Experiments were performed in triplicate, and the error bars are SE values for three samples. * P<0.05 (t test).