TRPV1-mediated transcriptomic effects of cigarette smoke in A549 alveolar epithelial cells

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Abstract

BACKGROUND: TRPV1 cation channels are sensitive to cigarette smoke constituents, particulate matter, and oxidative stress and are considered potential pharmacological targets for chronic obstructive pulmonary disease therapy.

AIM: To delineate TRPV1mediated transcriptomic effects of cigarette smoke extract on A549 epithelial cells.

METHODS: A549 cells were exposed in vitro for 24 hours to the following conditions: 5% cigarette smoke extract; TRPV1 agonist capsaicin (50 µM); TRPV1 antagonist AMG9810 (10 µM); and 5% cigarette smoke extract added 1 hour after AMG9810 (10 µM). Control wells received 0.01% dimethyl sulfoxide. Sequencing was performed on the MGISEQ200 platform in SE50 mode. To isolate the TRPV1dependent component of cigarette smoke extract effects, we used a twostep approach: 1) selection of genes whose cigarette smoke extractinduced expression changes were abolished by AMG9810; 2) crossvalidation of these genes with genes whose expression was altered by capsaicin (considering the direction of the effect). Data processing and analysis included read quality control, read mapping to the transcriptome and quantification, differential gene expression analysis and assessment of the interaction between cigarette smoke extract and AMG9810 effects, enrichment analysis of Gene Ontology, the Kyoto Encyclopedia of Genes and Genomes, and Reactome pathway categories, proteinprotein interaction network construction, and hub gene identification. The significance of gene list overlap was assessed using the hypergeometric test.

RESULTS: Interaction analysis of “cigarette smoke extract:AMG9810” identified 91 genes whose cigarette smoke extractinduced expression changes were significantly modulated by the TRPV1 antagonist. Of these, 18 genes showed increased expression, and 73 showed decreased expression. Crossvalidation with the transcriptional response to capsaicin confirmed the TRPV1dependent nature of these changes for 14 of the 18 upregulated genes (group 1) and for 60 of the 73 downregulated genes (group 2). Group 1 genes did not form functionally enriched categories or interaction networks; the genes of group 2 were functionally unified by their involvement in positive regulation of proliferative responses (e.g., MYC, JUN, FOS, JUNB, FOSL2, EGR1, ATF3) and inhibition of apoptosis (MCL1, TNFAIP3). Hub genes identified from group 2 accounted for more than half of all hub genes identified from the overall set of genes downregulated by cigarette smoke extract.

CONCLUSION: These results suggest that under acute cigarette smoke extract exposure in A549 cells, TRPV1 may mediate delayed, poorly coordinated repair, which is consistent with the processes observed in chronic obstructive pulmonary disease that accompany emphysema development.

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About the authors

Denis E. Naumov

Far Eastern Scientific Center of Physiology and Pathology of Respiration

Author for correspondence.
Email: denn1985@bk.ru
ORCID iD: 0000-0003-3921-8755
SPIN-code: 9818-3790
Scopus Author ID: 55489704800
ResearcherId: F-7421-2017

MD, Cand. Sci. (Medicine), Head, Lab. of Molecular and Translational Research

Russian Federation, Blagoveshchensk

Dina A. Gassan

Far Eastern Scientific Center of Physiology and Pathology of Respiration

Email: dani-shi@mail.ru
ORCID iD: 0000-0003-3718-9962
SPIN-code: 8616-1673
Scopus Author ID: 57201847044
ResearcherId: ААО-1275-2020

MD, Cand. Sci. (Medicine), Head, Lab. of Virus-Associated Developmental Pathologies

Russian Federation, Blagoveshchensk

Olesya O. Nekrasova

Far Eastern Scientific Center of Physiology and Pathology of Respiration

Email: foxy_voxy_on@mail.ru
ORCID iD: 0000-0002-1984-2596
SPIN-code: 7892-4969
Scopus Author ID: 57222023108
ResearcherId: ААО-1284-2020

MD, Cand. Sci. (Medicine), senior research associate, Lab. of Virus-Associated Developmental Pathologies

Russian Federation, Blagoveshchensk

Ivana Yu. Sugaylo

Far Eastern Scientific Center of Physiology and Pathology of Respiration

Email: ivanka_888@mail.ru
ORCID iD: 0000-0002-9170-1245
SPIN-code: 4808-8924
Scopus Author ID: 57222021421
ResearcherId: AAC-8166-2022

MD, Cand. Sci. (Medicine), research associate, Lab. of Molecular and Translational Research

Russian Federation, Blagoveshchensk

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Supplementary files

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1. JATS XML
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2. Supplement 1. Key differential expression parameters for genes whose activity is significantly altered by cigarette smoke extract and modulated by the TRPV1 antagonist AMG9810
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3. Supplement 2. Gene ontology, Kyoto Encyclopedia of Genes and Genomes, and Reactome categories enriched with TRPV1dependent genes whose expression was significantly downregulated in response to cigarette smoke extract
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4. Fig. 1. Principal component analysis: distribution of control samples (ctrl), cells exposed to cigarette smoke extract (cse), capsaicin (caps), TRPV1 antagonist AMG9810 (amg), and cigarette smoke extract in the presence of AMG9810 (csea) in the coordinates of two principal components, PC1 and PC2.

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5. Fig. 2. Volcano plot for the CSE:AMG interaction factor in A549 cells, showing how TRPV1 blockade (AMG9810) modifies the transcriptomic response to cigarette smoke extract.

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6. Fig. 3. Venn diagram showing the overlap of gene lists for upregulated and downregulated genes in response to capsaicin or cigarette smoke extract that are corrected by TRPV1 blockade.

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