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Zöller, M. ; Mastalerz, M. ; Dick, E. ; Hennen, E. ; Chakraborty, A. ; Merl-Pham, J. ; Marchi, H. ; Le Gleut, R. ; Jeridi, A. ; Prasse, A.* ; Jäger, B.* ; Santofimia‐Castaño, P.* ; Stoleriu, M.-G. ; Hilgendorff, A. ; Yildirim, A.Ö. ; Hauck, S.M. ; Staab-Weijnitz, C.A.*

Quantitative Proteomics Identifies Nuclear Protein 1 as a Regulator of Cell Death in Differentiated Primary Human Bronchial Epithelial Cells.

Am. J. Respir. Crit. Care Med. 211, A6855 - A6855 (2025)
DOI
Rationale: The airway epithelium serves as the first line of defense against cigarette smoke (CS), a major risk factor for chronic lung diseases. To the best of our knowledge, a comprehensive proteomic assessment of CS-induced changes in primary human bronchial epithelial cells (phBECs) differentiated at the air-liquid interface (ALI) has not been reported. Also, the relevance of CS-induced pathways for idiopathic pulmonary fibrosis (IPF) is poorly understood. An improved understanding thereof may unravel novel preventive strategies. Objective: To uncover novel key regulators and molecular pathways induced by CS in phBECs including such that may be relevant to IPF pathogenesis. Methods: Chronically CS-exposed, fully differentiated phBECs were analyzed by label-free tandem mass spectrometry (MS/MS)-based proteomics followed by pathway enrichment analysis using Ingenuity Pathway Analysis (IPA). Selected results were validated by ELISA and immunoblotting. The function of Nuclear protein 1 (NUPR1) was further explored in control and IPF-derived phBECs with and without CS-exposure using the NUPR1-specific inhibitor ZZW-115. Gene expression, cell type composition, barrier integrity, cytotoxicity, and cell death mechanisms were assessed using quantitative PCR, immunofluorescent stainings, transepithelial electrical resistance measurement, lactate dehydrogenase, BODIPY assay, and flow cytometry. Results: Out of 4861 detected proteins, proteomics revealed significantly altered levels for 186 proteins (adj. p<0.05) in response to CS. Subsequent pathway enrichment analysis suggested 11 key regulators. Results included many known CS-responsive genes involved in xenobiotic metabolism and antioxidant defense as well as activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) and Aryl hydrocarbon receptor (AhR) dependent pathways, validating the overall approach. Interestingly, IPA suggested NUPR1 as the top activated key regulator. In cultured phBECs, NUPR1 localized to basal and ciliated cells. NUPR1 inhibition with ZZW-115 in phBECs confirmed regulation of selected NUPR1 target genes suggested by IPA and revealed modulation of ferroptosis-associated genes. Inhibition of NUPR1 dose-dependently induced cell death and loss of epithelial integrity, with the underlying mechanisms varying across donor-derived cells, not consistently tied to a specific cell death type, and surprisingly little affected by prior CS exposure. Interestingly, IPF-derived phBECs showed a higher susceptibility to cytotoxicity after NUPR1 inhibition relative to control cells. Conclusion: Our proteomics analysis identified NUPR1 as a multifaceted regulator of cell death in differentiated phBECs. The results warrant further investigation of the function of NUPR1 in IPF airways.
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Publication type Article: Journal article
Document type Meeting abstract
Corresponding Author
ISSN (print) / ISBN 1073-449X
e-ISSN 1535-4970
Journal American Journal of Respiratory and Critical Care Medicine
Quellenangaben Volume: 211, Issue: Abstracts, Pages: A6855 - A6855 Article Number: , Supplement: ,
Publisher American Thoracic Society
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Lung Health and Immunity (LHI)
CF Metabolomics & Proteomics (CF-MPC)
CF Statistical Consulting (CF-STATCON)