Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination

Abstract

The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.

Keywords: DNA endonuclease; DNA transcription; apurinic/apyrimidinic endonuclease 1 (APE1); base excision repair (BER); class switch recombination; immunoglobulin A (IgA); immunology; redox-inhibitor.

Figure 1.

Different APE1 functions in mammalian cells involve independent subdomains.

Figure 2.

APE1 inhibitors APX3330 and compound 3 reduced class switch recombination in CH12F3-treated cells. A–C, representative dot plots (A), percentages of CD19/IgA double-positive cells (B), and amounts of released IgA (C) of CH12F3 under CIT(−) or CIT(+) conditions in absence or in presence of 50 μm APX3330, 0.2 μm compound 3, or both. The data are representative of three independent experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.001; ns, not significant. D, live (trypan negative) and dead (trypan positive) cells evaluated by trypan blue exclusion after 72 h of culture under CIT(−) or CIT(+) condition in the presence of indicated inhibitors. E, percentages of viable, early apoptotic, late apoptotic, and necrotic CH12F3 cells measured by annexin/PI staining. The data are representative of two independent experiments performed in triplicate.

Figure 3.

Re-expression of APE1^WT but not APE1^C65S or APE1^E96A mutant forms restores the ability of APE1-null CH12F3 cells to undergo isotype switching. A–C, representative dot plots (A), percentages of CD19/IgA double-positive cells (B), and amounts of released IgA (C) by switching CH12F3 APE1^+/+/Δ and CH12F3 APE1^Δ/Δ/Δ cells under CIT(−) and CIT(+) conditions. D–F, representative dot plots (D), percentages of GFP/IgA double-positive cells (E), and amounts of released IgA (F) by CH12F3 APE1^+/+/Δ transfected with empty vector or with GFP-APE1^WT, APE1^C65S, or APE1^E96A mutant forms and induced toward IgA switching. G–I, representative dot plots (G), percentages of GFP/IgA double-positive cells (H), and amounts of released IgA (I) by CH12F3 APE1^Δ/Δ/Δ transfected with empty vector, GFP-APE1^WT, APE1^C65S, or APE1^E96A mutant forms and induced toward IgA switching. The data are representative of four independent experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.001; ns, not significant.

Figure 4.

APX3330 impairs the production of IL-6 that is crucial for the optimal CSR. A, levels of secreted IL-6 in the supernatants of CH12F3 cells under CIT(−) or CIT(+) conditions in presence or absence of APE1 inhibitors. B, IL-6 detection in supernatants of not transfected or CH12F3 cells transfected with empty vector or with GFP-APE1^WT, APE1^C65S, or APE1^E96A undergoing CSR. The data in A and B are representative of three independent experiments. C and D, percentages of CD19/IgA double-positive cells (C) and ng/ml of released IgA (D) by switching CH12F3 cells untreated, pretreated with APX3330, or pretreated with APX3330 and induced to CSR in presence of increasing amounts of exogenous recombinant IL-6 (rIL-6) ranging from 0.1 to 10,000 pg/ml. The data are representative of three independent experiments, and comparisons were performed between cells treated with APX3330 versus cells treated with APX330 in presence of IL-6. *, p < 0.05; **, p < 0.005; ***, p < 0.001.

Figure 5.

In vivo effect of APX3330 on T-independent B-cell response to TNP–Ficoll immunization. APX3330-treated mice (50 mg/Kg) and control mice were immunized with TNP–Ficoll, and the levels of specific anti-TNP IgM, IgG₃, and IgA were evaluated in the peripheral blood 1 week after immunization. *, p < 0.05; **, p < 0.005; ns, not significant.