Differential expression of RNA-binding proteins in bronchial epithelium of stable COPD patients

Abstract

PurposeInflammatory gene expression is modulated by posttranscriptional regulation via RNA-binding proteins (RBPs), which regulate mRNA turnover and translation by binding to conserved mRNA sequences. Their role in COPD is only partially defined. This study evaluated RBPs tristetraprolin (TTP), human antigen R (HuR), and AU-rich element-binding factor 1 (AUF-1) expression using lung tissue from COPD patients and control subjects and probed their function in epithelial responses in vitro.Patients and methodsRBPs were detected by immunohistochemistry in bronchial and peripheral lung samples from mild-to-moderate stable COPD patients and age/smoking history-matched controls; RBPs and RBP-regulated genes were evaluated by Western blot, ELISA, protein array, and real-time PCR in human airway epithelial BEAS-2B cell line stimulated with hydrogen peroxide, cytokine combination (cytomix), cigarette smoke extract (CSE), and following siRNA-mediated silencing. Results were verified in a microarray database from bronchial brushings of COPD patients and controls. RBP transcripts were measured in peripheral blood mononuclear cell samples from additional stable COPD patients and controls.ResultsSpecific, primarily nuclear immunostaining for the RBPs was detected in structural and inflammatory cells in bronchial and lung tissues. Immunostaining for AUF-1, but not TTP or HuR, was significantly decreased in bronchial epithelium of COPD samples vs controls. In BEAS-2B cells, cytomix and CSE stimulation reproduced the RBP pattern while increasing expression of AUF-1-regulated genes, interleukin-6, CCL2, CXCL1, and CXCL8. Silencing expression of AUF-1 reproduced, but not enhanced, target upregulation induced by cytomix compared to controls. Analysis of bronchial brushing-derived transcriptomic confirmed the selective decrease of AUF-1 in COPD vs controls and revealed significant changes in AUF-1-regulated genes by genome ontology.ConclusionDownregulated AUF-1 may be pathogenic in stable COPD by altering posttranscriptional control of epithelial gene expression.

Posttranscriptional Gene Regulatory Networks in Chronic Airway Inflammatory Diseases: In silico Mapping of RNA-Binding Protein Expression in Airway Epithelium

“…These changes were associated with the occurrence of several clusters of co-regulated mRBPs, and they impacted relevant pathways deemed pathogenic for COPD. We recently identified in a GEO database of airway epithelial cells from stable COPD patients a significant loss of the RBP AUF-1 compared to smoker controls, matching the loss we identified at protein level in airway samples of patients with stable COPD compared to normal smokers (16). The present study was undertaken on this basis as a broader investigation of epithelial RBP patterns comparing two lung diseases characterized by chronic inflammation and oxidative stress -COPD and SAsince preclinical studies clearly identified the role of this class of posttranscriptional regulators in these pathological processes (14,81,82), yet they are scarcely described in human disease driven by them.…”

“…Canonical pathway analysis indicated that RBP genes in clusters commonly impacted upon RAN signaling and Telomere Extension by Telomerase, along with more cluster-restricted influence on other pathways, such as inhibition of ARE-mediated mRNA degradation and IL-15 expression ( Table 3). In particular, cluster 3 included 40 genes, among which was included HNRNPD, coding for the RBP AUF-1 that we previously identified as repressed in small airway epithelium in airway biopsies of an independent subject cohort of COPD patients compared to smoker controls (16). For cluster 3, IPA analysis identified enrichment of genes involved in RAN signaling, telomere extension, IL-15 expression ( Figure 7B).…”

“…These evidence suggest that shorter leukocyte telomere lengths could be evaluated as a biomarker for clinical outcomes in COPD. Furthermore, these two RBPs were coexpressed in cluster 3 (Figure 7), along with another known TERT-regulating factor, AUF-1 (98), which we initially documented as downregulated in COPD patients vs. controls by immunohistochemistry (16). AUF-1 positively regulates TERT1 at promoter level, but its loss may impact cellular senescence also by concomitant lack of its destabilization function for cell-cycle checkpoint mRNAs (98).…”

“…Involvement of RBPs in airway epithelial responses to inflammatory stimuli and glucocorticoid treatment has been well-characterized in vitro (12)(13)(14)(15) but awaits evidence from patient-based studies. To this end, in a previous characterization of ARE-binding proteins expression in COPD (16) we identified a selective loss of the RBP AU-binding Factor (AUF)−1 in bronchial biopsies in stable COPD patients vs. smoker controls. Besides replicating this finding in vitro upon challenge of the epithelial cell line BEAS-2B with inflammatory cytokines and cigarette smoke extract, we confirmed this loss in primary epithelium, by interrogating a transcriptome database generated from bronchial brushings of COPD patients vs. normal smokers and non-smokers controls (17).…”

“…We therefore set out to evaluate the expression profile of a curated gene list of mRBPs in selected public Gene Expression Omnibus (GEO) transcriptomic databases derived from airway epithelium isolated from bronchial brushings of phenotyped patients affected by COPD, SA and relative control populations, enrolled in relevant translational studies on disease pathophysiology. In particular, we employed for COPD the same database (17) in which we found loss of AUF-1 in COPD samples vs. controls, as observed ex vivo in COPD airway biopsies (16) and a GEO database from SA and healthy controls (HC) enrolled in the Severe Asthma Research Program (SARP) study (19). The list of mRBPs we utilized for this study has been created by annotation of proteins as RBPs generated by domain search, considering known RNA binding domains [among 800 domains extracted from the protein family (Pfam) database], or including proteins known as validated partners of RNP complex (20).…”