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Background We reported that DNA-dependent protein kinase (DNA-PK) is critical for the expression of nuclear factor κB–dependent genes in TNF-α–treated glioblastoma cells, suggesting an involvement in inflammatory diseases. Objective We sought to investigate the role of DNA-PK in asthma. Methods Cell culture and ovalbumin (OVA)– or house dust mite–based murine asthma models were used in this study. Results DNA-PK was essential for monocyte adhesion to TNF-α–treated endothelial cells. Administration of the DNA-PK inhibitor NU7441 reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness, and OVA-specific IgE production in mice prechallenged with OVA. Such effects correlated with a marked reduction in lung vascular cell adhesion molecule 1 expression and production of several cytokines, including IL-4, IL-5, IL-13, eotaxin, IL-2, and IL-12 and the chemokines monocyte chemoattractant protein 1 and keratinocyte-derived chemokine, with a negligible effect on IL-10/IFN-γ production. DNA-PK inhibition by gene heterozygosity of the 450-kDa catalytic subunit of the kinase (DNA-PKcs +/− ) also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma by using house dust mite, a human allergen. Remarkably, such protection occurred without causing severe combined immunodeficiency. Adoptive transfer of T H 2-skewed OT-II wild-type CD4 + T cells reversed IgE and T H 2 cytokine production but not airway hyperresponsiveness in OVA-challenged DNA-PKcs +/− mice. DNA-PK inhibition reduced IL-4, IL-5, IL-13, eotaxin, IL-8, and monocyte chemoattractant protein 1 production without affecting IL-2, IL-12, IFN-γ, and interferon-inducible protein 10 production in CD3/CD28-stimulated human CD4 + T cells, potentially by blocking expression of Gata3 . These effects occurred without significant reductions in T-cell proliferation. In mouse CD4 + T cells in vitro DNA-PK inhibition severely blocked CD3/CD28-induced Gata3 and T-bet expression in CD4 + T cells and prevented differentiation of T H 1 and T H 2 cells under respective T H 1- and T H 2-skewing conditions. Conclusion Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.

Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca2+-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca2+ chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca2+ independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca2+ chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca2+-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis. Background: The mechanism by which apoptotic internucleosomal DNA fragmentation occurs remains unclear. Results: CAD and DNAS1L3 cooperate to process chromatin degradation during apoptosis. DNAS1L3 achieves such function by translocating from the ER to the nucleus. Conclusion: The results provide new insight on the mechanism by which chromatin degradation takes place during apoptosis. Significance: Our results exemplify the complexity of chromatin degradation during apoptosis.