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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 23, 2002 as doi:10.1096/fj.01-0664fje. |
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* Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and
Division of Experimental Surgery, National Children’s Medical Research Center, Tokyo, Japan
2Correspondence: Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Ross 1023, 720 Rutland Ave., Baltimore MD 21205, USA. E-mail: kirani{at}jhmi.edu
SPECIFIC AIMS
The primary goals of this study were to 1) determine whether redox factor 1 (ref-1) protects against reperfusion/reoxygenation injury and 2) examine the relationship between the ref-1 and the small GTPase rac1 in regulating oxidative stress in the setting of reperfusion/reoxygenation. Recombinant adenoviruses were used to overexpress ref-1 (using Adref-1) or down-regulate endogenous ref-1 (using AdFer; antisense ref-1) and to activate rac1-dependent signaling (using Adrac1V12) in mouse liver in vivo and isolated hepatocytes. Reperfusion/reoxygenation-induced parameters, including oxidative stress, apoptosis, and activation of nuclear factor kappa B (NF-
B), were measured.
PRINCIPAL FINDINGS
1. Ref-1 inhibits reoxygenation/reperfusion-induced and rac1-regulated oxidative stress
The most interesting and novel findings of this study were that overexpression of ref-1 suppressed reperfusion/reoxygenation-stimulated activation of rac1 and oxidative stress, and inhibited reactive oxygen species (ROS) production induced by expression of an activated allele of rac1 (Fig. 1
). Taken together, these findings imply that ref-1, by modulating the activation of rac1, plays a pivotal role in regulating the redox state of hepatocytes and hepatic tissue.
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2. Ref-1 suppresses NF-B activation, and oxidative tissue injury
Oxidative stress regulated by the rac1 GTPase has been implicated in NF-B activation and tissue injury, particularly in the context of reperfusion/reoxygenation. Consistent with the above findings concerning the role of ref-1 in suppressing activity of rac1, overexpression of ref-1 mitigated reperfusion-stimulated NF-B activation (Fig. 2
a), apoptosis, and tissueinjury. Overexpressing ref-1 did not suppress activation of NF-B by H2O2 in hepatocytes (Fig. 2
b). Moreover, down-regulation of endogenous ref-1 augmented reperfusion-induced NF-B DNA binding activity.
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CONCLUSIONS AND SIGNIFICANCE
Redox factor 1 is a ubiquitously expressed dual function protein. It is an endonuclease in the base excision repair of oxidatively modified DNA and a reducing protein that promotes the DNA binding properties of many transcription factors. The present study demonstrates that ref-1 also participates in the control of cellular oxidative stress by regulating the activity of the rac1 GTPase. This novel property of ref-1 may be an additional means by which it inhibits oxidative cell death and tissue injury, and it opens the door for future studies to characterize the precise mechanism(s) for the regulation of rac1 activity by ref-1.
Suppression of postreperfusion NF-B activity by ref-1 is consistent with its ability to suppress rac1 activation and rac1-regulated oxidative stress, but in contrast to reports indicating that ref-1 promotes NF-B DNA binding in cell-free systems. These discrepant findings should be considered in the context of different and perhaps opposing mechanisms for the regulation of NF-B activity by ref-1 in vivo. Such opposing mechanisms exist for thioredoxin, another redox protein that associates with ref-1 and is involved in regulating NF-B activity.
In summary, this report provides the first evidence that ref-1 has a causal role in protection against in vivo oxidative injury. More important, by demonstrating that ref-1 suppresses rac1 activation and rac1-regulated oxidative stress, it provides a novel mechanistic basis for this protective effect. The data suggest that ref-1 is part of a feedback loop that modulates NF-B activity and ROS production (Fig. 3
). Although the data presented are in hepatic cells and tissue, the functional relationship between ref-1 and rac1 described here may have broad applicability in understanding how these widely expressed and conserved proteins regulate the redox balance of other mammalian cells and tissues.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0664fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.01-0664fje. 
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N). All viruses were used at 2 x 109 PFU/animal. b) Superoxide levels in animals coinfected with Adßgal, Adref-1, and Adrac1V12. PFU/animal: + 2 x 109; ++ 4 x 109; +++ 6 x 109. n = 4 mice in each group. Representative data from two independent experiments are shown. *P < 0.05 compared with Adßgal. c) Rac1 activity in uninfected, Adßgal-, and Adref-1-infected cells under normoxia and after H/R. Hypoxia 4 h; reoxygenation 10 min. Hepatocytes infected at 10 MOI of virus. A representative blot from two independent experiments is shown.
