Whole transcriptome analysis reveals a role for OGG1-initiated DNA repair signaling in airway remodeling

Abstract

Reactive oxygen species (ROS) generated by environmental exposures, and endogenously as by-products of respiration, oxidatively modify biomolecules including DNA. Accumulation of ROS-induced DNA damage has been implicated in various diseases that involve inflammatory processes, and efficient DNA repair is considered critical in preventing such diseases. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base-excision repair (OGG1-BER) pathway. Recent studies have shown that the OGG1-BER by-product 8-oxoG base forms a complex with cytosolic OGG1, activating small GTPases and downstream cell signaling in cultured cells and lungs. This implies that persistent OGG1-BER could result in signaling leading to histological changes in airways. To test this, we mimicked OGG1-BER by repeatedly challenging airways with its repair product 8-oxoG base. Gene expression was analyzed by RNA sequencing (RNA-Seq) and qRT-PCR, and datasets were evaluated by gene ontology and statistical tools. RNA-Seq analysis identified 3252 differentially expressed transcripts (2435 up- and 817 downregulated, ≥ 3-fold change). Among the upregulated transcripts, 2080 mRNAs were identified whose encoded protein products were involved in modulation of the actin family cytoskeleton, extracellular matrix, cell adhesion, cadherin, and cell junctions, affecting biological processes such as tissue development, cell-to-cell adhesion, cell communication, and the immune system. These data are supported by histological observations showing epithelial alterations, subepithelial fibrosis, and collagen deposits in the lungs. These data imply that continuous challenge by the environment and consequent OGG1-BER-driven signaling trigger gene expression consistent with airway remodeling.

Keywords: 8-Oxoguanine; Airway remodeling; OGG1-BER.

Fig. 1

Whole transcriptome analysis of gene expression induced by OGG1-BER. Lungs were repeatedly challenged with the product of OGG1-BER (8-oxoG base) and total RNAs were isolated and subjected to RNA-Seq analysis as described under Materials and methods. (A) Hierarchical clustering showing changes at the whole-transcriptome level. Transcript levels (RPKM) were normalized to control (time 0 min). Heat maps were generated using GENE-E. Rows represent identified transcripts and columns the time points (30, 60, and 120 min) after 8-oxoG challenge. The intensity of the red and green colors in the heat map shows the degree of upregulation or downregulation, respectively. (B) Significantly up- and downregulated transcripts in clusters 1 and 2. The total numbers of upregulated (≥3-fold) transcripts are represented by red bars and the downregulated (≤3-fold) ones by green bars. (C) Number of unique and shared transcripts altered by 8-oxoG challenge. Venn diagrams were constructed using the Venny online software. (D) Kinetic changes in number of transcripts induced by 8-oxoG challenge as a function of time.

Fig. 2

Overrepresentation of gene ontology categories. Lungs were challenged with the OGG1-BER product 8-oxoG and RNA-Seq analysis was carried out as described under Materials and methods. An overrepresentation test of the upregulated genes (≥ 3-fold, 60 min) was performed using PANTHER classification system (overrepresentation levels are expressed as − log(P value) and were confirmed by using GSEA (ES, q, and p values are included). (A) Biological process, (B) protein class, (C) cellular component, and (D) molecular function. *ECM, extracellular matrix; Ig, immunoglobulin; NS¹, nervous system; ES, enrichment score; FDR, false discovery rate.

Fig. 3

Signaling pathways induced by the OGG1-BER product 8-oxoG. Lungs were repeatedly challenged with 8-oxoG and RNA was analyzed (Materials and methods). PANTHER analyses were carried out using the list of upregulated genes (≥ 3-fold, 60 min) after multiple challenges with 8-oxoG. (A) Percentage of genes activated in individual signaling pathways. (B) Overrepresentation of signaling pathways. Bars represent−log(P value).

Fig. 4

Validation of gene expression from RNA-Seq data by qRT-PCR. Briefly, 1 μg of pooled RNA was subjected to RNA-Seq or to reverse transcription (cDNA synthesis) for qRT-PCR analysis of selected genes related to tissue remodeling and evaluated by the ΔΔC_t method as described under Materials and methods. Black bars, RNA-Seq; gray bars, qRT-PCR.

Fig. 5

Tissue remodeling-associated gene expression induced by multiple challenges. (A) Experimental design: mice were challenged i.n. with the OGG1-BER product 8-oxoG at days 0, 2, and 4. On days 0 and 4, RNA was isolated after 0, 30, 60, and 120 min for whole transcriptome analysis by RNA-Seq and gene ontology. (B) Differences in RPKM levels between SC and MC at time 0. (C) Fold changes in transcript levels after MC compared to SC at time 0. (D) Fold changes in transcript levels induced by multiple challenges at 60 min compared to time 0. Expression values at 0 min were taken as onefold (D, left).

Fig. 6

Relevance of 8-oxoG-induced gene expression to human tissue eremodeling. Gene lists were generated based on the Gene Cards database and searched against our RNA-Seq datasets to visualize expression patterns of genes linked to human tissue remodeling. (A) Cytoskeleton, (B) cell adhesion, (C) surfactant, (D) extracellular matrix, and (E) cell junction. SC, single challenge; MC, multiple challenges.

Fig. 7

Histological changes in mouse airways after challenge with 8-oxoG released from DNA by OGG1-BER. Mice were challenged with 8-oxoG on days 0, 2, and 4. On day 22, lungs were sectioned, stained, and examined microscopically. (A) Hematoxylin and eosin staining of vehicle-treated (top) and 8-oxoG-challenged (bottom) lungs. (B) Masson’s trichrome-stained lung sections showing collagen deposition (blue, bottom). Original magnifications in (A) and (B) were 96 × (left) and 192 × (right).

Fig. 8

A proposed model for OGG1-BER-driven tissue remodeling. The repair product of OGG1-BER, 8-oxoG base, is bound by cytosolic OGG1 and the complex increases the levels of activated (GTP-bound) small GTPases. Downstream signaling activates transcription factors (TFs) and the expression of mediators, responsible for changes in both the mucosal epithelium (e.g., epithelial–mesenchymal transition) and the subepithelial tissues (e.g., smooth muscle proliferation and increased ECM).