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

Abstract

Luca Ricciardi,1,* Jessica Dal Col,1,* Paolo Casolari,2 Domenico Memoli,1 Valeria Conti,1 Alessandro Vatrella,1 Becky M Vonakis,3 Alberto Papi,2 Gaetano Caramori,4 Cristiana Stellato1,3 1Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy; 2Interdipartimental Study Center for Inflammatory and Smoke-related Airway Diseases (CEMICEF), Cardiorespiratory and Internal Medicine Section, University of Ferrara, Ferrara, Italy; 3Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Department of Biomedical Sciences, Dentistry and Morphological and Functional Imaging (BIOMORF), University of Messina, Messina, Italy *These authors contributed equally to this work Purpose: Inflammatory 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 methods: RBPs 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. Results: Specific, 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. Conclusion: Downregulated AUF-1 may be pathogenic in stable COPD by altering posttranscriptional control of epithelial gene expression. Keywords: airway epithelium, AUF-1, COPD, inflammation, posttranscriptional gene regulation

Keywords

• airway epithelium
• AUF-1
• COPD
• inflammation
• posttranscriptional gene regulation