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EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 12, 2005 as doi:10.1096/fj.05-4028fje. |
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B and AP-1 differentially contribute to the induction of Mn-SOD and eNOS during the development of oxidant tolerance
Vascular Cell Biology Laboratory, Centre for Critical Illness Research, Lawson Health Research Institute and Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada
1 Correspondence: E-mail: pkvietys{at}uwo.ca
SPECIFIC AIMS
Exposure of cardiac myocytes to anoxia/reoxygenation (A/R) converts the myocytes to a proinflammatory phenotype, an event prevented by a previous challenge with A/R or H2O2 (oxidant tolerance). Oxidant tolerance is dependent on the induction of Mn-SOD and eNOS. The aim of the present study was to assess the relative roles of the nuclear transcription factors, NF
B and AP-1 in the induction of eNOS and Mn-SOD during development of oxidant tolerance.
PRINCIPAL FINDINGS
1. NFB and AP-1 are involved in the development of oxidant tolerance
As the basic experimental model, cardiomyocytes were exposed to a 30 min period of anoxia, then reoxygenated (A/R). Cardiomyocytes exposed to 30 min of normoxia and reoxygenated (N/R) served as controls. An A/R challenge to cardiomyocytes induced an increase in intracellular oxidant stress (Fig. 1
A) and converted the myocytes to a proinflammatory phenotype, i.e., the myocytes promoted PMN transendothelial migration (Fig. 1B
). If the cardiomyocytes were pretreated with an initial A/R challenge 24 h earlier, the second A/R challenge no longer resulted in 1) an increase in oxidant stress or 2) the conversion of the myocytes to a proinflammatory phenotype. These observations indicate that cardiomyocytes develop oxidant tolerance.
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To determine whether the initial A/R challenge was associated with nuclear translocation of NFB and/or AP-1, nuclear extracts were obtained at different times after A/R and processed for EMSA. Exposure of cardiomyocytes to an A/R challenge resulted in an increase in nuclear binding of NFB within 3–4 h. The initial A/R challenge also induced a rapid and transient increase in nuclear binding of AP-1, peaking 30 min after A/R. Since AP-1 appeared in the nucleus earlier than NFB, we assessed whether NFB activation and translocation to the nucleus was dependent on AP-1. A "decoy" double-stranded oligonucleotide containing the consensus sequence for the AP-1 binding site did not affect the increase in nuclear levels of NFB induced by A/R. These observations indicate that both NFB and AP-1 are translocated to the cardiomyocyte nucleus after the initial A/R challenge. Although AP-1 translocation to the nucleus precedes that of NFB, these two events are not causally linked.
To evaluate the role of NFB in the development of oxidant tolerance in cardiac myocytes, a peptide (SN50) containing the NFB nuclear localization sequence was used. SN50 was added to the myocytes before the first A/R challenge. As shown in Fig. 1
, SN50 prevented the development of oxidant tolerance: the A/R-induced myocyte oxidant stress and their conversion to a proinflammatory phenotype after the second A/R challenge was the same as that noted during the initial A/R challenge. The mutated form of SN50 did not prevent development of the adaptive response. To address the role of AP-1 in the development of oxidant tolerance, the myocytes were treated with the "decoy" oligonucleotide before the initial A/R challenge. The "decoy" prevented the development of oxidant tolerance with respect to both myocyte oxidant stress and their conversion to a proinflammatory phenotype.
Collectively, these observations indicate that nuclear translocation of both NFB and AP-1 is a prerequisite for the development of oxidant tolerance with respect to cardiomyocyte oxidant stress and their conversion to a proinflammatory phenotype.
2. Nuclear translocation of NFB and AP-1 are required for the induction of Mn-SOD during the development of oxidant tolerance
We previously showed that induction of Mn-SOD is required for the development of oxidant tolerance. Thus, in this series of experiments, we assessed the role of NFB and AP-1 in the induction of Mn-SOD during the development of oxidant tolerance. As shown in Fig. 2
A, an initial A/R challenge was not associated with any changes in Mn-SOD protein. However, 24 h later (at the time of the second A/R challenge) Mn-SOD protein was increased. Mn-SOD activity paralleled changes in protein levels. Blockade of NFB activation (MG132) or its translocation to the nucleus (SN50) prevented the A/R-induced increase in myocyte Mn-SOD protein and activity. Blockade of AP-1 translocation to the nucleus using a "decoy" oligonucleotide also prevented the increase in Mn-SOD protein (Fig. 2A
). These findings indicate that nuclear translocation of both NFB and AP-1 are required for the induction of Mn-SOD.
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3. Nuclear translocation of AP-1, but not NFB, is required for the induction of eNOS during the development of oxidant tolerance
As shown in Fig. 2B, an
initial A/R challenge was not associated with any changes in eNOS protein. However, 24 h later (at the time of the second A/R challenge) eNOS protein was increased. Cardiomyocyte NO production (an index of eNOS activity) paralleled changes in eNOS protein levels. A "decoy" oligonucleotide targeting AP-1 prevented the A/R-induced increase in myocyte eNOS protein and NO production. Blockade of NFB activation (MG132) or its translocation to the nucleus (SN50) did not affect the A/R-induced increase in myocyte eNOS protein (Fig. 2B
).
CONCLUSIONS AND SIGNIFICANCE
Herein, using isolated cardiomyocytes, we provide the following novel insights into the role of nuclear transcription factors in the development of oxidant tolerance. First, we provide evidence that, in addition to NFB, AP-1 plays an important role in the development of this adaptive response with respect to 1) myocyte oxidant stress and 2) their conversion to a proinflammatory phenotype. Second, nuclear translocation of both NFB and AP-1 are required for induction of Mn-SOD. Third, nuclear translocation of AP-1, but not NFB, is a prerequisite for the induction of eNOS.
The Mn-SOD and eNOS genes have putative binding sites for both NFB and AP-1 in their promoter regions. Most studies addressing the regulation of Mn-SOD or eNOS genes have focused on either NFB or AP-1. Here we addressed the potential roles of both NFB and AP-1 in the induction of Mn-SOD and eNOS in the same experimental model. Our findings indicate that NFB and AP-1 are both involved in the induction of Mn-SOD by the initial A/R challenge. To our knowledge, this is the first demonstration that nuclear translocation of both NFB and AP-1 is a prerequisite for the induction of Mn-SOD. In addition, we provide evidence that nuclear translocation of AP-1 is a requirement for the induction of eNOS during the development of the A/R-induced adaptive response. However, unlike the case for Mn-SOD, nuclear translocation of NFB was not required for eNOS induction.
Results of the present study may have some bearing on the phenomenon of delayed preconditioning. Delayed preconditioning refers to the adaptive response induced in the heart by an I/R challenge, which renders it resistant to the deleterious effects of a subsequent I/R challenge imposed 24 h or more later. This adaptive response within the myocardium is genetically-mediated (transcription-dependent) and requires protein synthesis. Both SOD and NOS enzyme systems have been implicated. Results of both in vivo (in situ hearts) and in vitro (isolated cardiomyocytes) studies indicate that the activity of the Mn-SOD isoform is increased after the initial I/R challenge, and antisense approaches targeting Mn-SOD can prevent the development of delayed preconditioning. In vivo studies using iNOS deficient mice have implicated iNOS as the effector system. By contrast, in vitro studies using myocytes or endothelial cells isolated from eNOS- or iNOS-deficient mice have implicated eNOS. In terms of the initiating factors involved, NFB has been implicated in the development of delayed preconditioning in both in vivo and in vitro models. In general, the results of in vivo and in vitro studies agree with respect to some of the potential cellular pathways involved in delayed preconditioning. However, further studies are warranted to 1) confirm a role for AP-1 in vivo and 2) address various nuances noted between the in vivo and in vitro studies, e.g., role for eNOS vs. iNOS.
Figure 3
schematically depicts our working hypothesis on the mechanisms involved in the development of oxidant tolerance in cardiomyocytes. We propose that after an A/R challenge the cardiomyocytes incur an oxidant stress that converts the myocytes to a proinflammatory phenotype, i.e., the A/R-conditioned myocytes can promote PMN transendothelial migration. In addition, the oxidant stress induced by the initial A/R challenge results in nuclear translocation of the redox-sensitive transcription factors NFB and AP-1. NFB and AP-1 are involved in transactivating the gene encoding for Mn-SOD, resulting in an increase in Mn-SOD protein and activity during the second A/R challenge. AP-1, but not NFB, is involved in transactivating the gene encoding for eNOS, resulting in an increase in eNOS protein and NO production during the second A/R challenge. The increase in Mn-SOD activity enhances the scavenging of superoxide, thereby reducing the oxidant stress during the second A/R challenge. NO is an anti-inflammatory molecule; thus, the increase in NO production during the second A/R challenge may directly prevent the conversion of cardiomyocytes to a proinflammatory phenotype. Alternatively, NO can interact with superoxide, reducing the oxidant stress. In either case, the concerted interaction of both Mn-SOD and eNOS serves to prevent the conversion of cardiomyocytes to a proinflammatory phenotype. Further studies are warranted to 1) identify other intracellular pathways (additional nuclear transcription factors and/or cell signaling mechanisms upstream) and 2) delineate the specific mechanisms by which an increase in NO and a decrease in oxidant stress prevent the A/R-induced conversion of cardiomyocytes to a proinflammatory phenotype.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4028fje;
This article has been cited by other articles:
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  M. Yang, J. Wu, C. M. Martin, P. R. Kvietys, and T. Rui Important role of p38 MAP kinase/NF-{kappa}B signaling pathway in the sepsis-induced conversion of cardiac myocytes to a proinflammatory phenotype Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H994 - H1001. [Abstract] [Full Text] [PDF]
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