Unconventional activation of PRKDC by TNF-α: deciphering its crucial role in Th1-mediated inflammation beyond DNA repair as part of the DNA-PK complex

Background: The DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID. Methods: We investigated the central role of PRKDC in inflammation and its potential association with DNA repair. We also elucidated the relationship between inflammatory cytokines (e.g., TNF-α) and PRKDC by analyzing its connections to inflammatory kinases. Human cell lines, primary human endothelial cells, and mouse fibroblasts were used to conduct the in vitro studies. For animal studies, LPS- and oxazolone-induced mouse models of acute lung injury (ALI) and delayed-type hypersensitivity (DHT) were used. Wild-type, PRKDC+/−, or Ku70+/− mice used in this study. Results: A ~ 50% reduction in PRKDC markedly blocked TNF-α-induced expression of inflammatory factors (e.g., ICAM-1/VCAM-1). PRKDC regulates Th1-mediated inflammation, such as DHT and ALI, and its role is highly sensitive to inhibition achieved by gene heterozygosity or pharmacologically. In endothelial or epithelial cells, TNF-α promoted rapid PRKDC phosphorylation in a fashion resembling that induced by, but independent of, DSBs. Ku70 heterozygosity exerted little to no effect on ALI in mice, and whatever effect it had was associated with a specific increase in MCP-1 in the lungs and systemically. While Ku70 knockout blocked VP-16-induced PRKDC phosphorylation, it did not prevent TNF-α − induced phosphorylation of the kinase, suggesting Ku70 dispensability. Immunoprecipitation studies revealed that PRKDC transiently interacts with p38MAPK. Inhibition of p38MAPK blocked TNF-α-induced PRKDC phosphorylation. Direct phosphorylation of PRKDC by p38MAPK was demonstrated using a cell-free system. Conclusions: This study presents compelling evidence that PRKDC functions independently of the DNA-PK complex, emphasizing its central role in Th1-mediated inflammation. The distinct functionality of PRKDC as an individual enzyme, its remarkable sensitivity to inhibition, and its phosphorylation by p38MAPK offer promising therapeutic opportunities to mitigate inflammation while sparing DNA repair processes. These findings expand our understanding of PRKDC biology and open new avenues for targeted anti-inflammatory interventions

Poly (ADP) Ribose Polymerase (PARP)-1 as a novel therapeutic target for alleviating the manifestations of Atopic Dermatitis

American Society for Pharmacology and Experimental Therapeutics Annual Meeting, 2024; May 16 - May 19, 2024; Arlington, V

Common and differential roles of inducible NO synthase and poly (ADP-ribose)polymerase in allergen-induced inflammation and airway hyperresponsiveness: a potential connection to NO levels (HYP5P.320)

Abstract The potential role of iNOS as a viable therapeutic target for the treatment of asthma was severely hampered by the negative clinical trial results showing that a selective iNOS inhibitor did not affect airway hyperreactivity (AHR) or airway inflammation after allergen challenge in human subjects with asthma. Our laboratory has shown that iNOS inhibition is protective against airway inflammation upon acute, but not chronic, exposures to ovalbumin. More importantly, iNOS inhibition protected against lung fibrosis suggesting that iNOS inhibition may be protective against some aspects of asthma. Here, we show that iNOS inhibition, pharmacologically by L-Nil or by gene knockout, provided an excellent protection against AHR upon an acute, but not chronic, exposure to ovalbumin. Our laboratory also established a reciprocal relationship between iNOS and PARP-1. However, PARP inhibition, protected against inflammation and AHR upon acute and chronic exposure to ovalbumin. It is interesting that PARP-1 inhibition does not completely abrogate expression of iNOS leaving the possibility that the protective effect of PARP inhibition against inflammation and AHR may be associated with reduction but not the complete inhibition of iNOS and associated production of moderate levels of NO. Studies are being conducted to verify this hypothesis and clarify the intricate roles of iNOS in asthma pathogenesis allowing the possibility of being a viable target for the treatment of the disease.</jats:p

The catalytic subunit of DNA-PK has a unique function in inflammation independently of Ku70 and DNA repair: a new opportunity to target the enzyme without interfering with DNA repair

Abstract Our laboratory demonstrated a critical role for DNA-dependent protein kinase (DNA-PK) in asthma pathogenesis via modulating the pertinent immune responses. DNA-PK is DNA repair enzyme composed of a catalytic subunit (DNA-PKcs) and two DNA-binding subunits (Ku70 and Ku80). Human cells express high levels of DNA-PK, surprisingly such high levels do not confer increased ability to repair DNA damage. Here we show that the role of DNA-PK in promoting inflammatory responses is independent of its function in the DNA repair. We examined the effect(s) of partial depletion of Ku70 on Ovalbumin (OVA) induced lung inflammation in mouse model of the disease. Of note, depletion of Ku70 by gene heterozygosity causes deficiency in DNA-PK-dependent DNA repair. Unlike the protective effects provided by DNA-PKcs gene heterozygosity, Ku70 heterozygosity did not alter the OVA-induced eosinophilia, mucus hypersecretion, Th2 cytokines production, or OVA-specific IgE upon OVA challenge. Interestingly, Ku70 heterozygosity enhanced methacholine-induced AHR over that of WT mice. Using a cell culture system, we demonstrate that while IL-4 and TNF-α are potent inducers of DNA-PKcs, such activation did not coincide with any detectable DNA damage or repair responses. Remarkably, while Ku70−/− blocked DNA-PKcs autophosphorylation in response to the DNA damage agent, etoposide, it did not affect the kinase in response to TNFα. Our findings suggest that the mechanism by which DNA-PK functions in inflammation is completely unrelated to its role in DNA repair, thus, unraveling a completely novel function for the kinase. More importantly, this provides a window of opportunity to target DNA-PK in inflammatory diseases without interfering with DNA repair processes.</jats:p

DNA-dependent protein kinase plays a role in inflammation independent of DNA-damage repair function and its inhibition blocks allergic inflammation in mice and modulates human CD4+ T cell function without causing SCID (HYP7P.264)

Abstract We reported that DNA- dependent protein kinase (DNA-PK) is critical for the expression of NF-κB-dependent genes in TNF-α -treated glioblastoma cells including VCAM-1. Here we show that DNA-PK protein level and function are critical for VCAM-1 expression in TNF-α-treated human endothelial and mouse lung smooth muscle cells and are required for monocytes adhesion. Interestingly, DNA-PK activation and subsequent VCAM-1 expression in presence of TNF occurred independently of DNA breaks/repair. Administration of the DNA-PK inhibitor, NU7441, and DNA-PK heterozygosity reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness (AHR), and OVA-specific IgE production in asthma model of mice. Such effects correlated with a marked reduction in lung VCAM-1 expression and production of several inflammatory cytokines. Remarkably, such protection occurred without causing SCID. These results were confirmed in a chronic model of asthma using house dust mite. DNA-PK inhibition reduced Th2 cytokines production without affecting Th1 cytokines production and cell proliferation in CD3/CD28-stimulated human CD4+T cells potentially by blocking gata-3 expression. In mouse CD4+T cells, DNA-PK inhibition, in vitro, severely blocked CD3/CD28-induced gata-3 and t-bet expression and prevented differentiation of Th1 and Th2 cells. Thus, our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.</jats:p