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8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and α-smooth muscle actin polymerization.

Free Radic Biol Med. 2014 Aug;73:430-8.

 

Luo J1, Hosoki K2, Bacsi A1, Radak Z1, Hegde ML3, Sur S4, Hazra TK4, Brasier AR5, Ba X1, Boldogh I6.
  • 1Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
  • 2Department of Internal Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
  • 3Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
  • 4Department of Internal Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA; Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
  • 5Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA; Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
  • 6Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA; Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA. Electronic address: sboldogh@utmb.edu.

 

Abstract

Reactive oxygen species (ROS) are activators of cell signaling and modify cellular molecules, including DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the prominent lesions in oxidatively damaged DNA, whose accumulation is causally linked to various diseases and aging processes, whereas its etiological relevance is unclear. 8-OxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair (BER) pathway. OGG1 binds free 8-oxoG and this complex functions as an activator of Ras family GTPases. Here we examined whether OGG1-initiated BER is associated with the activation of Rho GTPase and mediates changes in the cytoskeleton. To test this possibility, we induced OGG1-initiated BER in cultured cells and mouse lungs and used molecular approaches such as active Rho pull-down assays, siRNA ablation of gene expression, immune blotting, and microscopic imaging. We found that OGG1 physically interacts with Rho GTPase and, in the presence of 8-oxoG base, increases Rho-GTP levels in cultured cells and lungs, which mediates α-smooth muscle actin (α-SMA) polymerization into stress fibers and increases the level of α-SMA in insoluble cellular/tissue fractions. These changes were absent in cells lacking OGG1. These unexpected data and those showing that 8-oxoG repair is a lifetime process suggest that, via Rho GTPase, OGG1 could be involved in the cytoskeletal changes and organ remodeling observed in various chronic diseases. Copyright © 2014 Elsevier Inc.

KEYWORDS: Base excision repair; Cytoskeleton; Free radicals; OGG1; ROS; Rho–GTP

PMID: 24681335

 

SUPPLEMENT

Chronic inflammatory disorders, including asthma and chronic obstructive pulmonary lung diseases, are characterized by airflow obstruction and airway hyper-responsiveness and differ in clinical harshness, response to therapy, and long-term outcome. A large percentage of asthmatics suffer a permanent decline in respiratory function. Decreased airway function often results from changes in epithelial structure and increased smooth muscle mass leading to sub-epithelial fibrosis and collagen overproduction. Experimental and clinical observations implicate inflammatory cell (eosinophils, macrophages and neutrophils) -derived soluble mediators, including transforming growth factor beta, interleukin (IL) IL4, IL5, and IL13 in airway remodeling. However, the molecular mechanisms driving expression of these cytokines, ILs, and cellular histological changes are not fully understood.

A research group at The University of Texas Medical Branch has made fundamental discoveries showing that repair of oxidatively damaged DNA base lesions (generated by reactive oxygen species; e.g., during allergic inflammatory processes) is associated with gene expression leading to structural changes to the airways. To protect DNA integrity and prevent mutagenenesis by various oxidized DNA base lesions and their biological consequences, genes encoding the oxidized base-specific glycosylases NEIL1, NEIL2, NEIL3, NTH1, and OGG1 are expressed in mammalian cells. These DNA repair enzymes differ in their repair mechanisms and the structure of their catalytic active sites [1], but the overall steps are similar, which include removal of modified base from duplex DNA and the subsequent steps of base excision repair (BER). Together with apurinic/apyrimidinic endonuclease1 or polynucleotide kinase, DNA polymerases and ligases, these enzymes re-establish DNA integrity [2, 3].

Recent studies documented that DNA glycosylases, 8-oxoguanine DNA glycosylase1 (OGG1), which removes the most abundant base lesion [8-oxo-7,8-dihydroguanine (8-oxoG)] during BER is associated with activation of rat viral sarcoma oncogene homolog family small GTPases (RAS) [4-8]. The repair product (the free 8-oxoG base) forms a complex with cytosolic OGG1, and the resulting conformational changes allow its interaction with RAS, RHO and RAC1 GTPases, for which it can function as a guanine nucleotide exchange factor, catalyzing the exchange of GDP for GTP. In a paper published in Free Radical Biology and Medicine (73:430-438) [9], the authors show that upon exposure to oxidative stress only OGG1-expressing cells exhibit increased levels of RHO- and RAC1-GTP, suggesting an important role for OGG1-initiated BER and/or OGG1 in oxidative stress-induced Rho activation. RHO (RHOA) and RAC (RAC1 and RAC2) family members are small G proteins known to mediate the polymerization of tubulin and smooth muscle actin filaments (see Figures A,B), and migration of muscle and non-muscle cells in addition to gene expression mediating cell proliferation and/or differentiation (for details see [10]).

To further these novel observations, a series of studies examined whether 8-oxoG base continuously or intermittently released by OGG1-BER, and the consequent activation of small GTPases (RAS, RHOA and RAC1), induce a distinct set of gene expression changes that result in functional and structural changes resembling those observed in airway remodeling. In these studies, mouse lungs were repeatedly challenged with physiologically relevant doses of 8-oxoG base (the OGG1-BER product), and RNA was isolated. RNA sequencing data analyzed by the PANTHER classification system showed expression of distinct sets of genes (Figures C,D) encoding proteins involved in modulation of the cadherins, actin family cytoskeleton, extracellular matrix, cell adhesion, and cell junction proteins, affecting such biological processes as tissue development, cell-to-cell adhesion, and cell communication and induced histological changes in lungs, including epithelial alterations, increased smooth muscle mass and collagen deposits consistent with airway remodeling [11].

Fig 1. A proposed model for OGG1-BER-driven gene expression for airway remodeling. Environmental and/or inflammatory ROS oxidize guanine to 8-oxoG due to guanine’s lowest oxidation potential among DNA bases. 8-oxoG is continuously removed from DNA by OGG1-BER and transported to the cytoplasm, where it is bound by a cytosolic OGG1. The resulting complex functions as a guanine nucleotide exchange factor and activates (GDP→GTP exchange) small GTPases, RHOA and RAC1 (A). Activated RHOA and RAC1 are directly involved in cytoskeletal network rearrangement (B) and also alter gene expression involved in cell proliferation and differentiation (C). To test the latter hypothesis, we repeatedly added 8-oxoG base to mouse lungs to mimic ongoing OGG1-BER; RNA was isolated 0, 30 and 60 min after the last challenge and analyzed by RNA Sequencing. mRNAs (defined by PANTHER) were clustered according to their expression patterns, and heat maps were generated using GENE-E. The control (time 0 min) was taken as 1-fold (green). The intensity of the colors in the heat map shows the degree of upregulation (red) and dowregulation (green, black). The 207 upregulated (≤5-fold genes were associated with cellular histological changes. These genes were uploaded to GeneMANIA to generate the network shown in panel D.

 

 

 

Xueqing Ba PhD and Istvan Boldogh, DM&B, PhD, DHC

Department of Microbiology and Immunology, Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA

 

References

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[9] Luo, J.; Hosoki, K.; Bacsi, A.; Radak, Z.; Hegde, M. L.; Sur, S.; Hazra, T. K.; Brasier, A. R.; Ba, X.; Boldogh, I. 8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and alpha-smooth muscle actin polymerization. Free Radic Biol Med 73:430-438; 2014.

[10] Gerthoffer, W. T. Migration of airway smooth muscle cells. Proc Am Thorac Soc 5:97-105; 2008.

[11] Aguilera-Aguirre, L., Hosoki, K. Bacsi, A Radák Zs., Sur, S., Hegde, ML., Tian, B., Saavedra-Molina A., Brasier, AR., Ba, X., Boldogh, I. Whole transcriptome analysis reveals the implications of OGG1-intitiated DNA repair signaling in airway remodeling. Free Rad. Biol. Med. In Press; 2015.