Reduction of Nitric Oxide Synthase 2 Expression by Distamycin A Improves Survival from Endotoxemia

Abstract

NO synthase 2 (NOS2) plays an important role in endotoxemia through overproduction of NO. Distamycin A (Dist A) belongs to a class of drugs termed minor-groove DNA binders, which can inhibit transcription factor binding to AT-rich regions of DNA. We and others have previously shown that AT-rich regions of DNA surrounding transcription factor binding sites in the NOS2 promoter are critical for NOS2 induction by inflammatory stimuli in vitro. Therefore, we hypothesized that Dist A would attenuate NOS2 up-regulation in vivo during endotoxemia and improve animal survival. C57BL/6 wild-type (WT) mice treated with Dist A and LPS (endotoxin) showed significantly improved survival compared with animals treated with LPS alone. In contrast, LPS-treated C57BL/6 NOS2-deficient (NOS2−/−) mice did not benefit from the protective effect of Dist A on mortality from endotoxemia. Treatment with Dist A resulted in protection from hypotension in LPS-treated WT mice, but not in NOS2−/− mice. Furthermore, LPS-induced NOS2 expression was attenuated in vivo (WT murine tissues) and in vitro (primary peritoneal and RAW 264.7 murine macrophages) with addition of Dist A. Dist A selectively decreased IFN regulatory factor-1 DNA binding in the enhancer region of the NOS2 promoter, and this IFN regulatory factor-1 site is critical for the effect of Dist A in attenuating LPS induction of NOS2. Our data point to a novel approach in modulating NOS2 expression in vivo during endotoxemia and suggest the potential for alternative treatment approaches for critical illness.

Comprehensive genetic assessment of a functional TLR9 promoter polymorphism: no replicable association with asthma or asthma-related phenotypes

“…Cells were then lysed in 100 ul reporter lysis buffer (Promega, Madison, WI) and the luciferase activity in 50 ul of aliquots of cell lysates was measured using Luciferase Assay System (Promega, Madison, WI) according to the manufacturer's protocol. To monitor the transfection efficiency, a beta-galactosidase assay was conducted using the same lysate according to the published protocol[34]. All constructs were sequenced before transfection and each experiment was independently repeated four times with triplicate wells for each transfection reaction.…”

Nicotinamide Cooperates with Retinoic Acid and 1,25-Dihydroxyvitamin D₃ to Regulate Cell Differentiation and Cell Cycle Arrest of Human Myeloblastic Leukemia Cells

“…The functions of nicotinamide in numerous enzymatic reactions and its relatively low toxicity in vivo have suggested that it might act as a pharmacological agent with potential effects on different pathologies, such as cancer, diabetes and infectious disease [33, 34]. Nicotinamide has been thought to be a potential therapeutic agent in the fields of medicine and nutrition; however, whether nicotinamide works together with other vitamins, such as RA or D3, and its function in cellular processes such as ERK activation involved in cell differentiation remain unclear.…”

The Transcription Factor Nuclear Factor of Activated T Cells c3 Modulates the Function of Macrophages in Sepsis

“…Macrophages express high levels of iNOS and are important sources of iNOS-derived nitric-oxide (NO), which together with the respiratory burst of reactive oxygen species (ROS), leads to effective bactericidal activity [2,6]. NO has a paradoxical role in severe sepsis, where it has a harmful effect by contributing to lung injury in animals and humans [7,8], and a beneficial effect on immune system and in lungs through antimicrobial activity [9–11]. Recently it has been reported that monocyte/macrophage-derived iNOS plays a pivotal role in mediating resolution of lung injury by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair [12].…”