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Nitric oxide regulates mitochondrial oxidative stress protection via the transcriptional coactivator PGC-1

Sara Borniquel^*,,, Inmaculada Valle^*,,, Susana Cadenas, Santiago Lamas^*,, and María Monsalve^*,,,1

^* Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas). Madrid, Spain;

Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; and

Instituto "Reina Sofía de Investigaciones Nefrológicas," Consejo Superior de Investigaciones Cientificas, Madrid, Spain

¹Correspondence: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, Madrid 28029, Spain. E-mail: mmonsalve{at}cnic.es

SPECIFIC AIMS

Nitric oxide (NO) plays a dual role in atherosclerosis and other pathologies associated with mitochondrial reactive oxygen species (ROS). It is generally acknowledged that low NO concentrations, produced mainly by eNOS, play an important antioxidative role that protects cells from oxidative stress. However, when produced in excess in chronic inflammatory processes, NO is an important prooxidant and toxic factor. The molecular mechanisms underlying this dual action are still unclear.

It was recently proposed that long-term exposure of brown adipose and skeletal muscle cell lines to NO donors induces mitochondrial biogenesis via PKG-mediated transcriptional induction of peroxisome proliferators-activated receptor- coactivator 1- (PGC-1). Taking into account our finding that PGC-1 is a key regulator of the mitochondrial ROS protection system in endothelial cells, we investigated whether NO regulates PGC-1 and/or the mitochondrial detoxification system in the vascular endothelium.

In primary vascular endothelial cells, we found that NO donors induce an early down-regulation of PGC-1 expression mediated by protein kinase G (PKG), followed by long-term up-regulation. We conclude that the primary effect of NO is the suppression of PGC-1 expression and that the induction phase must be a secondary process. Changes in PGC-1 levels lead to corresponding variations in the expression of oxidative stress protection genes, suggesting that NO can mediate both prooxidant and antioxidant effects through the regulation of PGC-1 expression in the endothelium.

PRINCIPAL FINDINGS

1. NO modulates PGC-1 expression both positively and negatively
We treated bovine aortic endothelial cells (BAEC) (Fig. 1 ), human umbilical cord vein endothelial cells (HUVEC), and mouse aortic endothelial cells (MAEC) with increasing doses of the slow NO donor, DETA-NO, for up to 48 h. Activation of soluble guanylate cyclase (sGC) and PKG by NO occurs within minutes, and direct transcriptional effects should be apparent after treatment for 6 h. Within the dose range if 31 µM to 500 µM, a short-term down-regulation in PGC-1 mRNA levels preceded a later up-regulation (Fig. 1) . At the highest dose of DETA-NO (1 mM), up-regulation of PGC-1 mRNA expression was evident after 6 h, whereas at the lowest dose (31 µM) up-regulation could not be detected until 48 h. To test the physiological relevance of this regulation, we measured the mRNA levels of two PGC-1 transcriptional targets: cytochrome c (Cyt c) and Mn superoxide dismutase (MnSOD). These genes are hallmarks of two important PGC-1-regulated activities: up-regulation of oxidative phosphorylation (OXPHOS) and protection against mitochondrial oxidative stress. The dose and time response of these genes to DETA-NO paralleled that observed for PGC-1 (Fig. 1) .

View larger version (21K): [in this window] [in a new window]   Figure 1. Dual effect of NO treatment on PGC-1 mRNA expression in BAEC. BAEC were treated with the NO donor DETA-NO (31 µM–1 mM) for 6, 12, 24, and 48 h. The mRNA expression levels of PGC-1 and its target genes Cyt c and MnSOD were determined by qRT-polymerase chain reaction (PCR). Values are relative to those obtained for 18S rRNA. Expression in untreated samples (0 h) has been assigned the value of 1. *P < 0.05, #P < 0.001.

2. Down-regulation of PGC-1 levels by NO is mediated by PKG
To investigate whether the negative effect of NO on PGC-1 expression is mediated by the sGC/PKG pathway, we tested the effect of the sGC inhibitor ODQ (1 µM) in BAEC (Fig. 2 A) and HUVEC treated with DETA-NO (62 µM) for up to 24 h. As before, DETA-NO treatment resulted in a down-regulation of PGC-1 mRNA expression (Fig. 2A , upper panel), but preincubation with the sGC inhibitor ODQ blocked this negative effect.

View larger version (28K): [in this window] [in a new window]   Figure 2. NO down-regulates PGC-1 mRNA through a PKG-dependent mechanism. BAEC were treated as follows. A) DETA-NO (62 µM) ± the soluble guanylate cyclase inhibitor ODQ (1 µM), B) DETA-NO (62 µM) ± the protein kinase G inhibitor KT5823 (1 µM). A, B) Upper panels show PGC-1 mRNA expression at 6, 12, and 24 h of treatment. Lower panels show mRNA expression of PGC-1 target genes after 12 h of treatment. Expression in untreated samples has been assigned the value of 1. *P < 0.05, #P < 0.001.

To determine whether cGMP-dependent protein kinase G (PKG) was mediating the effect of NO on PGC-1, we pretreated cells with the PKG inhibitor KT5823 (1 µM) before exposing them to DETA-NO (62 µM). KT5823 treatment blocked the NO-mediated down-regulation of PGC-1 expression in BAEC (Fig. 2B , upper panel) and HUVEC.

In another test, the cell-permeable cGMP analog 8-Br-cGMP (100 µM) mimicked the effect of NO on PGC-1 expression, with reduced mRNA levels after 6–24 h of treatment (Fig. 2C ). The cAMP analog 8-Br-cAMP had no effect.

3. NO regulates the expression of the mitochondrial oxidative stress protection system
To determine whether NO-mediated PKG activation also regulates genes of the mitochondrial oxidative stress protective system, we examined the mRNA expression levels of MnSOD, peroxiredoxin III (Prx3), peroxiredoxin V (Prx5), thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2), uncoupling protein 2 (uncoupling protein (UCP)-2), and catalase. Treatment of BAEC with 62 µM DETA-NO for 12 h reduced mRNA expression of all of these genes (Fig. 2A , lower panel). Pretreatment with ODQ or KT5823 blocked this effect (Fig. 2B , lower panel) whereas 8-Br-cGMP mimicked it (Fig. 2C ). Similar results were obtained after treatment for 6 and 24 h and in HUVEC and MAEC (data not shown).

4. NO regulation of the mitochondrial oxidative stress protection system is mediated directly by PGC-1
To determine whether the effect of NO on the mitochondrial oxidative stress protection system is mediated by PGC-1, we decoupled PGC-1 from NO regulation by overexpressing PGC-1 from an alternative promoter (cytomeglovirus, or CMV). BAEC infected with the recombinant PGC-1 adenovirus or with a control adenovirus were incubated with DETA-NO for 12 h. DETA-NO treatment of noninfected and control adenovirus-infected cells reduced expression of PGC-1, Cyt c, and MnSOD mRNA by 3-fold. But when cells infected with the PGC-1 adenovirus were treated with DETA-NO, PGC-1 mRNA expression was reduced by only 20%. Most important, mRNA levels of Cyt c, MnSOD, Trx2 were reduced by only 20%, and those of Prx3, Prx5, TrxR2, UCP-2, and catalase did not change. This suggests that NO regulates the mitochondrial oxidative stress protection system through PGC-1.

5. Various tissues from eNOS^–/– mice show reduced mRNA expression of PGC-1 and genes of the mitochondrial oxidative stress protection system
To evaluate the physiological relevance of eNOS expression to the mitochondrial ROS protection system, we examined mRNA and protein expression of PGC-1 and its target genes in heart, spleen, kidney, and aorta from eNOS^–/– and eNOS^+/+ mice. PGC-1 mRNA and protein expression levels were reduced in all tissues from eNOS-deficient mice. Messenger RNA and protein expression levels of the mitochondrial oxidative stress protection system were also reduced in eNOS^–/– mice compared with eNOS^+/+ mice; the differences were greatest in heart, followed by aorta, kidney, and spleen. These results support the notion that long-term exposure to NO is an important positive regulator of the expression of PGC-1 and members of the mitochondrial ROS detoxifying system, and is necessary to maintain physiological PGC-1 levels in a wide variety of tissues, including the heart and the vasculature.

6. PGC- induces the expression of eNOS
The observation that NO modulates PGC-1 expression in vascular endothelial cells prompted us to investigate whether PGC-1 could, in turn, regulate eNOS expression. We therefore overexpressed PGC-1 in BAEC using a recombinant adenovirus. eNOS mRNA and protein levels, as well as production of NO, were evaluated 48 h postinfection. BAEC infected with PGC-1 adenovirus expressed higher levels of eNOS mRNA than noninfected cells or cells infected with a control virus. This transcriptional up-regulation resulted in elevated eNOS protein expression and an elevated production of NO, revealed by the increased accumulation of nitrite in the culture media.

CONCLUSIONS AND SIGNIFICANCE

Identification of the molecular mechanisms that mediate the prooxidative and antioxidative properties of NO has been a challenge for many years. In this study, we have described how NO-mediated regulation of PGC-1 expression, via PKG, modulates the mitochondrial oxidative stress protective system in endothelial cells. We found that NO plays a dual role in the regulation of PGC-1 expression: short-term exposure of murine, bovine, and human primary endothelial cells to NO donors down-regulates PGC-1 and its target genes, while long-term treatments produce an up-regulation.

These results have important physiological implications not only because they can explain how NO action can lead to cellular protection against oxidative stress, but also because they show that at least some of the prooxidant activities of NO are mediated by changes in gene expression and are not just the direct consequence of NO chemistry.

An intriguing question that arises from these studies is why NO down-regulates PGC-1 if it is ultimately going to up-regulate it. We think that the dual action of NO may be related to the phenomenon known as preconditioning. It has been clearly established in several systems that exposure to low concentrations of ROS induces protective mechanisms that prevent oxidative damage under oxidative stress conditions. NO-mediated down-regulation of PGC-1 expression together with NO direct inhibition of mitochondrial respiration would provide an early shutdown of the mitochondrial system that might prevent activation of the mitochondrial apoptotic cascade while also providing a limited ROS burst signal necessary for preconditioning. In fact, it seems feasible that H₂O₂ signaling could play a significant role. We can therefore hypothesize that the initial shutdown of PGC-1 expression is required for the secondary, long-term induction. This notion is supported by the observation that induction of PGC-1 expression by long-term treatment with NO donors is also dependent on PKG activation, which indicates that events that take place in the initial down-regulation phase are required for the long-term induction. However, additional studies will be necessary to elucidate this point.

We believe that the results presented here reveal a novel mechanism through which NO elicits its prooxidant and antioxidant effects. This, in turn, has important implications for how NO can protect the vascular endothelium from oxidative stress, particularly in relation to metabolic dysfunctions such as diabetes, hypercholesterolemia, and hyperlipidemia. In these situations, the role played by PGC-1 could be expected to be particularly important since it is a master regulator of lipid catabolism and mitochondrial function.

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic diagram. NO plays a dual regulatory role in PGC-1 expression. Short-term increases of NO concentration down-regulate of PGC-1 via PKG and, sustained, elevated, NO concentrations up-regulates PGC-1. Reduced PGC-1 expression decreases the mitochondrial detoxification capacity, and therefore increases the mitochondrial production of ROS. Elevated ROS could play a role in the induction of PGC-1 and the mitochondrial oxidative stress protection system that results from sustained, elevated, NO production.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5189fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5189fjev1 20/11/1889    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Borniquel, S. Articles by Monsalve, M. Search for Related Content PubMed PubMed Citation Articles by Borniquel, S. Articles by Monsalve, M.