PGC-1α regulates the mitochondrial antioxidant defense system in vascular endothelial cells

Abstract

Objective: Mitochondrial production of oxidants contributes to a variety of pathological conditions including the vascular complications of diabetes, neurodegenerative diseases, and cellular senescence. We postulated that a transcriptional coactivator, peroxisome proliferator activated receptor-γ coactivator 1α (PGC-1α), a major regulator of oxidative metabolism and mitochondrial biogenesis, could be involved in the transcriptional regulation of the mitochondrial antioxidant defense system in vascular endothelial cells.

Methods and results: We show that PGC-1α is present in human, bovine, and mouse endothelial cells and positively modulates the expression of the mitochondrial detoxification system. Endothelial cells that overexpress PGC-1α show reduced accumulation of reactive oxygen species (ROS), increased mitochondrial membrane potential, and reduced apoptotic cell death both in basal and oxidative stress conditions. Downregulation of PGC-1α levels by siRNA reduces the expression of mitochondrial detoxification proteins.

Conclusions: These results unveil a novel regulatory pathway that links mitochondrial activity and mitochondrial oxidative stress protective systems. In addition, they suggest that PGC-1α could play a crucial protective role in vascular complications of diabetes, where the mitochondrial metabolism of glucose has been shown to result in oxidative stress and vascular endothelial cell dysfunction.

PGC-1α, Endothelium, Oxidative stress, Mitochondria, Glucose

1. Introduction

Excessive production of reactive oxygen species (ROS) in the mitochondria plays a major role in the development of several diseases including the vascular and neurological complications of diabetes [29,46], neurodegenerative diseases like Parkinson's disease [13], Alzheimer [1] and epilepsy [9], ageing [11] and cancer [31,32]. In diabetic patients, hyperglycemia causes increased ROS production [29] that eventually leads to endothelial dysfunction. Endothelial dysfunction is a sensitive indicator of cardiovascular disease, predicts its prognosis and is closely associated with the development of atherosclerosis [7].

The Peroxisome Proliferator Activated Receptor (PPAR) γ Coactivator 1-α (PGC-1α) [42] is a transcriptional coactivator identified as an upstream regulator of lipid catabolism, mitochondrial number and function. Consistent with its emerging role as a central regulator of energy metabolism, PGC-1α is abundantly expressed in tissues with high metabolic rates.

Increased mitochondrial activity and electron flow through the ETC (electron transport chain) may be responsible for an increased production of superoxide (O₂⁻) [20], that when dismutated generates the highly toxic hydrogen peroxide (H₂O₂), unless adequate detoxification mechanisms are activated. We postulated the existence of a mechanism for the coordinated regulation of mitochondrial oxidative metabolism and ROS protection systems should exist. As PGC-1α is known to regulate mitochondrial activity, we decided to test whether PGC-1α could play such a role. This idea was supported by recent observations that indicated increased levels of Manganese Superoxide Dismutase (MnSOD) mRNA in C2C12 myotubes that overexpressed PGC-1α [39]. As a model system we chose primary endothelial cells, where excessive production of O₂⁻ and H₂O₂ in the mitochondria has been shown to mediate high-glucose dependent oxidative stress [29].

Until recently, the only well-known antioxidant mitochondrial protein was MnSOD [25]. A complete mitochondrial detoxification system has now been described. It is composed of Peroxiredoxin III [44], Peroxiredoxin V [18], mitochondrial Thioredoxin [37], mitochondrial Thioredoxin Reductase [8,22,26,41], and mitochondrial splicing variants of Glutaredoxin [24] and Glutathione Reductase [16]. The mitochondrial Uncoupling Protein 2 (UCP-2) forms a proton channel that has been proposed to prevent superoxide production [5,21].

We found that PGC-1α expression in primary endothelial cells results in the up-regulation of mitochondrial number and activity and of a full set of oxidative stress protective genes. This regulatory activity helps to prevent increased ROS accumulation, endothelial dysfunction and apoptosis in high glucose conditions.

2. Methods

2.1. Cell culture

Human Umbilical Vein Endothelial Cells (HUVEC) were isolated from umbilical cord veins and cultured as previously described [28]. Umbilical cords were obtained from Ruber International Hospital (Madrid, Spain) with the approval of the donors and the ethics committee of the institution. Bovine Aortic Endothelial Cells (BAEC) from bovine aortas were isolated and cultured as previously described [23]. Aortas were obtained from an authorized slaughterhouse. Mouse Aortic Endothelial Cells (MAEC) were isolated and cultured as previously described [14] from C57B mice provided by our animal facility. The protocols used conform to the Declaration of Helsinki and to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH Publication No. 85-23).

2.2. Adenoviral vectors and infection

Three copies of the HA tag were inserted at the N-terminus of PGC-1α, and subcloned in pShuttle-CMV (Quantum). Adenoviruses were generated and purified according to the manufacturer's instructions. Confluent HUVEC or BAEC cells were exposed to adenoviral vectors at an moi of 50 for 12 h.

2.3. RNA purification and analysis

Total RNA was isolated with Trizol. The expression levels were determined by reverse transcription (RT) of total RNA followed by PCR analysis (RT-PCR). One microgram of total RNA was reverse transcribed by extension of random primers. The PCR program used was as follows: 2 min at 94 °C; 30–40 cycles of 30″ at 92 °C, 30″ at 55 °C and 30″ at 72 °C; and a final extension step of 5′ at 72 °C. A PCR reaction was also performed on total RNA that had not been reverse transcribed to control for the absence of genomic DNA amplification. PCR products were resolved on 1% agarose gels. Relative expression levels were determined by Real-Time PCR (rPCR) in a 7000 Sequence Detection System (Applied Biosystem). Thermal conditions were: 2′ at 50 °C, 10′ at 95 °C; and 45 cycles of 15″ at 95 °C, 30″ at 55–60 °C, 30″ at 72 °C.

2.4. Western blot

Whole-cell extracts (WCE) were prepared as previously described [27]. Protein samples were resolved on SDS-PAGE, transferred onto Immobilon™-P membrane (Millipore), and analyzed by Western blotting with chemi-luminescence detection (ECL, Amersham).

2.5. siRNA

The siRNAs were synthesized using the Silencer siRNA Construction Kit from Ambion, from the following DNA oligos:

• GAPDH siRNA: Sense control DNA and Antisense Control DNA (provided with the kit)

• PGC-1α siRNA: 585/s 5′-aacgctttattgctccatgaacctgtctc-3′, 585/as 5′-aattcatggagcaataaagcgcctgtctc-3.

1 μg of siRNA was transfected into 70% confluent BAEC cells grown on 6 well plates with Lipofectamine 2000 (Invitrogen), following the manufacturer's instructions. Cells were harvested 48 h post-transfection.

2.6. Apoptosis

2.6.1. Flow cytometry

Cells were resuspended in 100 μl of annexin-binding buffer (10 mM HEPES, 140 mM NaCl and 25 mM CaCl₂, pH 7.4), plus 2.5 μl of Alexa fluor 488 annexin V conjugate. Cells were incubated at room temperature (RT) for 15 min prior to the addition of 400 μl of annexin-binding buffer, and propidium iodide (PI) 2 μg/ml final concentration. Following an additional 10-min incubation period, the cells were analyzed on a CYAN MLE (Dako Cytomation).

2.6.2. Caspase 3 and Caspase 9 activity

2.6.2.1. Confocal microscopy

Cells were grown on glass bottom dishes, incubated for 30 min at RT with 0.1% substrates of Caspases 3 (FITC-DEVD-FMK) or 9 (Red-LEHD-FMK), plus 0.1% HOECHST42, and visualized on a Radiance 2100 confocal microscope (BioRad). Control samples were pre-incubated with the caspase inhibitor Z-VAD.

2.6.2.2. Fluorimetric quantification

50 μl of WCE (1 mg/ml) were incubated with fluorescent substrates for Caspase-3 and Caspase-9 at 20 μM final concentration. The fluorescent products were detected in a FluoroscanAscent (Thermo Labsystems).

2.7. Mitochondrial transmembrane potential (ΔΨ_m)

Changes in ΔΨ_m were determined as differences in the tetramethylrhodamine methyl ester (TMRM) versus MitoTracker Green signal ratio. Fluorochromes were added to the cell culture medium at 50 nM. Cells were incubated for 30 min at 37 °C, trypsinized and resuspended in PBS. Fluorescence intensity was measured by flow cytometry.

2.8. Reactive oxygen species

Cells were incubated with 2 μM 5-chloromethyl-2′ 7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) for 30 min (added directly to the media). The cells were then trypsinized and resuspended in PBS. Fluorescence intensity was measured by flow cytometry.

2.9. Chromatin immunoprecipitation (ChIP)

The experimental conditions used have been described previously [35]. PGC-1α was immunoprecipitated with a polyclonal α-PGC-1α antibody. The immunoprecipitated DNA was analyzed by rPCR.

2.10. Statistics

Data are expressed as means ± S.D. values. Statistical evaluation of the data was performed by analysis of variance (ANOVA) or non-parametric tests as appropriate. Values were considered to be statistically significant at P<0.05. n ≥ 3 in all the experiments.

2.11. Materials

DMNQ (2,3-dimetoxi-1-naphthoquinone) was from Calbiochem. Trizol reagent is from Invitrogen. 488 annexin V conjugate, CM-H₂DCFDA, TMRM and MitoTracker Green were from Molecular Probes. The fluorescent substrates for Caspase-3 (Ac-DEVD-AMC) and Caspase-9 (Ac-LEHD-AFC) were from PharMingen and Biochem. Substrates of Caspases (CaspGLOW staining kit) were from Alexis Biochemicals, HOECHST42 was from Sigma.

The following antibodies were used: Mouse monoclonal α-cytochrome c (Clone 7H8.2C12) was purchased from BD PharMingen; mouse monoclonal α-Peroxiredoxin-V (Clone 44) was from BD Biosciences; mouse monoclonal α-GAPDH was from Chemicon and rabbit polyclonal α-Caspase-3 (H-277), α-PGC-1 (H-300) and α-UCP-2 (C-20) antibodies were from Santa Cruz Biotechnology. Rabbit polyclonal α-MnSOD (SOD-110) was from Stressgen; rabbit polyclonal α-catalase was from Chemicon Int.; and rabbit polyclonal α-Peroxiredoxin III, α-Thioredoxin 2 and α-Thioredoxin Reductase 2 were form LabFrontiers. Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (α-goat) and Amersham (α-rabbit and α-mouse).

3. Results

3.1. PGC-1α is present in vascular endothelial cells

PGC-1α is a highly regulated protein and its levels vary greatly among tissues and in changing metabolic situations[33]. It was therefore important to determine if PGC-1α was present in the vascular endothelium. We tested HUVEC and BAEC for the presence of PGC-1α mRNA by RT-PCR. As shown in Fig. 1A, a band corresponding to the PGC-1α fragment was detected in cDNA samples but not in RNA controls. The identity of the band was confirmed by DNA sequencing.

Fig. 1

PGC-1α is present in endothelial cells of human and bovine origin. PGC-1α expression in HUVECs drives the induction of ETC genes. (A) The presence of PGC-1α mRNA was determined by RT-PCR analysis of total RNA. PGC-1α protein was detected by Western blot of WCE. Cells infected with a recombinant PGC-1α adenovirus (Ad-PGC-1α) were used as positive control (lower panel). (B) HUVEC were infected with increasing doses of Ad-PGC-1 (0, 5, 12.5, 25, 50, 100 moi), and/or a complementary dose of Ad, and induction of ETC genes was monitored by RT-PCR analysis with specific oligonucleotides, β-actin was used as a negative control. (C) Mitochondrial mass was determined by flow cytometry of cells labeled with Mitotracker Green. Levels are expressed as percentage relative to the values obtained for non-infected cells. (*) P ≥ 0.05.

We also tested the presence of PGC-1α protein in HUVEC and BAEC, WCE by Western blot. As a positive control we used cells infected with a recombinant adenovirus expressing PGC-1α. We could reproducibly identify PGC-1α in both HUVEC and BAEC cells (Fig. 1A).

We then decided to investigate whether PGC-1α activity in the endothelium would result in the induction of oxidative metabolism genes, as has been described in other cell systems [45]. We constructed a recombinant adenovirus expressing HA-tagged PGC-1-α (Ad-PGC-1) to ensure the ubiquitous expression of PGC-1α in endothelial cells. HUVEC were infected with increasing doses (moi 1–100) of Ad-PGC-1 or the control empty virus (Ad), and induction of the ETC genes, Cytochrome C (Cyt C), Cytochrome Oxidase subunits II and IV (COX_II, COX_IV) and ATP Synthetase β subunit (ATPaseß), was tested 72 h post-infection by RT-PCR. We observed a dose-dependent induction of the mRNA expression levels for all the ETC genes tested (Fig. 1B).

To determine if the induction of ETC gene expression did result in an increased mitochondrial mass, we labeled HUVEC infected with Ad, Ad-PGC-1, or non-infected control with Mitotracker Green. Labeled cells were analyzed by flow cytometry. 72 h post-infection, Ad-PGC-1 cells had 50% higher mitochondrial content than control cells (Fig. 1C, upper panel). Fig. 1C lower panel shows the fluorescence signal distribution in a representative experiment. A clear shift to higher fluorescence values can be observed in Ad-PGC-1 cells.

3.2. PGC-1α induces oxidative stress protective genes

In order to evaluate PGC-1α activity under mitochondrial oxidative stress, we analyzed whether PGC-1α was active and able to regulate ETC and lipid metabolism genes in 30 mM glucose [12]. HUVEC infected with Ad, Ad-PGC-1 or non-infected control, were cultured in low (5 mM) and high (30 mM) glucose for 48 h. The results, obtained by rRT-PCR analysis (Fig. 2A) showed that PGC-1α is able to induce ETC (Cyt C, ATPaseβ, and COX_IV) and lipid catabolism genes, Medium-Chain Acyl-CoA Dehydrogenase (MCAD), Carnitine Palmitoyltransferase I (CPT-I), Long-Chain Acyl Co-A Oxidase (ACO) in oxidative stress conditions (Table 1).

Fig. 2

PGC-1α induces the expression of ETC genes, lipid catabolism genes, and oxidative stress protective genes in endothelial cells exposed to 5 mM and 30 mM glucose. Cells were cultured in low or high glucose media for 48 h, and total RNA was analyzed by rRT-PCR. Values are relative to those obtained for 18S rRNA. FN-A (fibronectin A) is a negative control. Levels obtained for the control, untreated samples have been assigned the arbitrary value of 1. (A) and (B) HUVEC. (A) Expression of ETC (CytC, COX_IV, ATPaseβ), and lipid catabolism genes MCAD, CPT-I, ACO. Inset shows a magnified view of the lower panel. (B) Induction of oxidative stress genes. UCP-2, MnSOD, Catalase, Prx3, Prx5, TRX2, TRXR2 and, Cu/ZnSOD. (C) BAEC. (*) P ≥ 0.05.

Table 1

Primer pairs used in rRT-PCR to quantify changes in gene expression

Target	Forward	Reverse
hACO	5′-ccgagagatcgagaacatgatc-3′	5′-ccaagcctcgaaggtgagttc-3′
hATPasεβ	5′-gttccatcctgtcagggactatg-3′	5′-tgtgctctcacccaaatgctgg-3′
hCOX IV	5′-acgagctcatgaaagtgttgtg-3′	5′-aatgcgatacaactcgactttctc-3′
hMCAD	5′-gatttagttttgagttcaccgaac-3′	5′-tccaagacctccacacagttctc-3′
hMnSOD	5′-aggttagatttagccttattccac-3′	5′-ttactttttgcaagccatgtatctttc-3′
hPrx 3	5′-cctttggatttcacctttgtgtg-3′	5′-caaaccaccattctttcttggtg-3′
hPrx5	5′-ccaatcaaggtgggagatgcc-3′	5′-gcaggtgtgtcttggaacatc-3′
hTRX2	5′-gtccacaccactgtgcgtgg-3′	5′-ttgcagggagatggctcagcg-3′
hCat	5′-gacaatcagggtggtgctcc-3′	5′-gaatcgcattcttaggcttctc-3′
hCPT1	5′-actgtgaacaggtatctacagtc-3′	5′-tacagcagatccatggcataatag-3′
hPGC-1	5′-ggcagtagatcctcttcaagatc-3′	5′-tcacacggcgctcttcaattg-3′
hCytc	5′-cttcggagcgggagtgttcg-3′	5′-cagatgatgcctttgttcttattg-3′
hFNA	5′-cagaaatgactattgaaggcttgc-3′	5′-ccacggatgagctgtcaggag-3′
hUCP-2	5′-tacaaagccggattccggcagc-3′	5′-ctccttggatctgtaaccggac-3′
hCOX II	5′-cattattcctagaaccaggcgac-3′	5′-gaattaattctaggacgatgggc-3′
hCu/ZnSOD	5′-cggaggctttgaaggtgtgg–3′	5′-ctccaacatgcctctcttcatcc-3′
bUCP-2	5′-cggctacagatccaaggagaa-3′	5′-ccgatgccagcatgctcaga-3′
bMnSOD	5′-ggaacaacaggtcttatccccct-3′	5′-ttacttgctgcaagccgtgtatc-3′
bTRX2	5′-cagggagatggctcagcga-3′	5′-gaccacaccattgtgcatgga-3′
hmCytC	5′-gccaataagaacaaaggcatca-3′	5′-gttttgtaataaataaggcagtgg-3′
bGAPDH	5′-agtggggtgatgctggtgctg-3′	5′-cgcctgcttcaccaccttctt-3′
18S rRNA	Pre-developed TaqMan Assay Reagent 18S RNA, Applied Biosystems
TRXR2	Assay-on-Demand 138372 (TaqMan MGB probe), Applied Biosystems

We then analyzed the effect of PGC-1α expression on the mRNA levels of the mitochondrial ROS detoxification machinery: MnSOD, Peroxirredoxins 3 and 5 (Prx3, Prx5) mitochondrial Thioredoxin (TRX2), and mitochondrial Thioredoxin Reductase (TRXR2), UCP-2, and catalase. UCP-2 has been shown to be regulated at the transcriptional level by PGC-1α [45]. Catalase is an enzyme that degrades H₂O₂ and is mainly localized in peroxisomes. We observed that PGC-1α was able to induce the expression of all these genes in both normal (5 mM glucose) and oxidative stress (30 mM glucose) conditions. Importantly, the mRNA levels of a cytoplasmic detoxification enzyme, Cu/Zn Superoxide Dismutase (Cu/ZnSOD) were not modulated by PGC-1α, suggesting that the effect of PGC-1α is specific for the mitochondrial system (Fig. 2B). We have extended our observations to other endothelial cell systems: BAEC (Fig. 2C) and MAEC (our unpublished results).

3.3. PGC-1 associates with the promoters of MnSOD, UCP-2 and Prx5 genes

In order to evaluate whether PGC-1α induction of oxidative stress protective genes was the result of the direct interaction of PGC-1α with their regulatory promoter regions, we carried out ChIP experiments. HUVEC, were cultured in low or high glucose for 48 h. Following chromatin preparation, the DNA fragments were immunoprecipitated and analyzed by rPCR. As shown in Fig. 3, MnSOD, UCP-2 and Prx5 promoter fragments were specifically enriched in the IP (α-PGC-1) fraction of Ad-PGC-1. Importantly, UCP-2 had already been proposed to be a direct transcriptional target of PGC-1α [45]. We conclude that the effects of PGC-1α are likely to be mediated by its direct association with the promoter regions of these genes (Table 2).

Fig. 3

PGC-1α associates with the promoter region of MnSOD, UCP-2 and Prx5 genes in HUVEC cells, as determined by ChIP analysis. HUVEC infected with Ad-PGC-1-were cultured in low or high glucose media for 48 h. Following formaldehyde treatment, PGC-1α was immunoprecipitated from sonicated WCE. In control IP-reactions a non-relevant antibody was used. The co-immunoprecipitated DNA was purified and analyzed by rPCR. β-actin was used as a non-PGC-1α-target negative control. Values refer to the X-fold enrichment of a particular fragment in the IP sample relative to the input. (*) P ≥ 0.05.

Table 2

Primer pairs used in ChIP rPCR analysis

Target	Forward	Reverse
hp1.3MnSOD	5′-gttcctcttcgcctgactgtt-3′	5′-ctgaaccgtttccgttgctt-3′
hp3MnSOD	5′-gacttttgtccttccccttgc-3′	5′-gatgcaggctttctgtcttcaa-3′
hpPrx5	5′-tgcatggtcctcagaaactcct-3′	5′-gagataattttggcctcttgcc-3′
hpUCP2	5′-gcaggcctttgcatctgttct-3′	5′-tagcttttgcgctgagctctg-3′
hgβactin	5′-caccttccagcagatgtgga-3′	5′-agcatttgcggtggacgatgg-3′

The primer set hp1.3MnSOD is located aproximately 1.3 kb upstream of the SOD2 transcription start site.

The primer set hp3MnSOD is located aproximately 3.4 kb upstream of the SOD2 transcription start site.

The primer set hpPrx5 is located aproximately 1 kb upstream of the prxV transcription start site.

The primer set hpUCP2 is located right upstream of the ucp-2 transcription start site.

The primer set hgβactin is located within the βactin CDS.

3.4. PGC-1α increases cellular levels of mitochondrial antioxidant proteins

We then assessed whether cells overexpressing PGC-1α modified the cellular content of antioxidant proteins. The results shown in Fig. 4A indicate that PGC-1α expression increases the levels of MnSOD, Catalase, Prx5, Prx3, UCP-2, TRXR2, and TRX2, in both normal glucose and oxidative stress conditions (30 mM glucose) (Table 1). Similar results were obtained in different endothelial cells systems like BAEC (Fig. 4B) and MAEC (our unpublished results).

Fig. 4

PGC-1α increases the expression of antioxidant proteins in low (5 mM) and high (30 mM) glucose conditions. Cells were cultured in low or high glucose media for 48 h, and WCE were analyzed by Western blot with specific antibodies. (A) HUVEC. (B) BAEC.

3.5. Expression of PGC-1α reduces the accumulation of ROS

To establish whether the observed induction of oxidative stress proteins would increase the cellular detoxification capacity, we incubated HUVEC, with 20 μM DMNQ, a quinone that blocks ETC Complex II [10], and induces a dose-dependent production of superoxide [6]. The level of cellular ROS was estimated by CM-H₂DCFDA labeling. We observed that PGC-1α expression reduced the amount of ROS accumulated in DMNQ treated cells by about 50% (Fig. 5A). Since DMNQ is expected to induce the same ROS production in the absence or presence of PGC-1α, we concluded that PGC-1α expression does increase the cell detoxification capacity.

Fig. 5

PGC-1α expression increases the mitochondrial detoxification capacity. (A) HUVEC were treated with 20 μM DMNQ for 2.5 h and 30 min. (B) HUVEC, (C) BAEC were cultured in low or high glucose media for 48 h. Cells were labeled with CM-H₂DCFA and total intracellular ROS content was estimated by flow cytometry. Levels obtained for control, untreated samples, have been assigned the arbitrary value of 1 or 100%. (*) P ≥ 0.05.

We then investigated whether PGC-1α would reduce the levels of ROS present in cells treated with high glucose. HUVEC accumulate about 30% more ROS in 30 mM than in 5 mM glucose. PGC-1α expression reduced ROS levels by 50% in both low and high glucose conditions (Fig. 5B). Importantly, Ad-PGC-1 cells in 30 mM glucose have about 25% less ROS than control cells in 5 mM glucose, suggesting that PGC-1α can prevent high glucose dependent oxidative stress. A similar result was obtained in BAEC cells (Fig. 5C).

3.6. PGC-1 can prevent mitochondrial dysfunction

High glucose treatment of HUVEC and other cell types causes a drop in mitochondrial potential that can be attributed to the inhibition of the ETC by ROS [34], and precedes the activation of the apoptotic cascade. The reduced accumulation of ROS, in PGC-1α expressing cells, might help to prevent mitochondrial dysfunction. Therefore, we determined ΔΨ_m in HUVEC, cultivated in low or high glucose for 48 h. MitoTracker Green labeling was used to estimate mitochondrial mass. Changes in ΔΨ_m were calculated as differences in the TMRM/MitoTracker Green fluorescence ratio. High glucose treatment reduced mitochondrial potential in control HUVEC by 30%. Mitochondrial ΔΨ_m was about 50% higher in cells expressing PGC-1α. High glucose treatment reduced ΔΨ_m in Ad-PGC-1 cells by only 15%, and therefore was still about 25% higher than in controls in low glucose, supporting the notion that PGC-1α can prevent mitochondrial dysfunction in stress conditions (Fig. 6A).

Fig. 6

PGC-1α expression prevents mitochondrial dysfunction and cell death. (A) HUVECs that express PGC-1α have a higher ΔΨ_m both in low and high glucose. Relative ΔΨ_m values were calculated as the ratio of the TMREM/Mitotracker Green fluorescence, and referred to the levels obtained for the control, untreated samples, which were assigned the arbitrary value of 100%. (B) PGC-1α protects HUVEC from apoptotic cell death induced by oxidative stress. HUVEC were exposed to oxidative stress conditions (250 μmol/L H₂O₂ for 12 h (upper panel) or high glucose media for 24 h (lower panel)), labeled with an annexin V-FITC conjugate and PI, and apoptotic cell death was determined by flow cytometry.

3.7. PGC-1α expression prevents apoptotic cell death

To investigate whether PGC-1α activity, was relevant to cell survival, we monitored apoptotic cell death of HUVEC under oxidative stress. It has been reported that exposure to ROS, such as H₂O₂, or stress inducers that are not themselves ROS, like high glucose, induces apoptosis in HUVEC and other endothelial cell types [3,30,48,12]. HUVEC were exposed to 250 μmol/L H₂O₂, and cell death was monitored by flow cytometry. After 12 h of treatment 90–100% of control cells had undergone apoptosis, but 50% PGC-1α expressing cells were still alive (PI⁻, annexin V⁻) (Fig. 6B). We concluded that the higher detoxification capacity of PGC-1α expressing cells could prevent apoptotic cell death in oxidative stress conditions.

We next studied whether PGC-1α could prevent apoptotic death induced by high glucose in HUVEC. We observed that in low glucose about 10% of the control cells died every 24 h. PGC-1α expressing cells had a slightly lower proportion of apoptotic cells (9%) than controls. In high glucose 20% of the control cells died every 24 h. Only about 12% of PGC-1α-expressing cells died per day in 30 mM glucose (Fig. 6B), supporting the notion that PGC-1α expression protects cells from oxidative stress-induced apoptosis.

3.8. PGC-1α prevents the activation of caspase-9 and caspase-3 in H₂O₂ treated cells

It has been previously established that induction of apoptosis by H₂O₂[30,48] and other oxidative stress agents like high glucose [36,43] proceeds through the activation of the mitochondrial apoptotic cascade, which involves the sequential activation of caspase-9 and caspase-3. The protective effect of PGC-1α on apoptosis prompted us to investigate whether PGC-1α activity prevents the activation of the mitochondrial apoptosis cascade. HUVEC were exposed to 250 μM H₂O₂, and activation of caspase-9 and caspase-3 was monitored at different times in WCE incubated with fluorescent caspase substrates (Fig. 7A). To directly monitor the activation of the caspases within living cells, HUVEC were labeled with fluorescent caspase-9 and caspase-3 substrates, and examined by confocal microscopy (Fig. 7B). Control cells showed the expected sequential activation of caspase-9, that peaked 4.5 h after H₂O₂ addition, and caspase-3, that peaked 9 h after H₂O₂ addition. PGC-1α expression both delayed and reduced the activation of caspase-9 and caspase-3. Caspase-9 activity was lost after 6 h, and caspase-3 activity could not be detected after 12 h. We concluded that PGC-1α can protect HUVEC from apoptosis induced by H₂O₂, preventing the activation of the mitochondrial apoptotic cascade.

Fig. 7

PGC-1α delays and reduces the sequential activation of caspase 9 and caspase 3 in HUVEC cells treated with H₂O₂. (A) Caspase 3 and Caspase 9 activity were analyzed in WCE. Values are relative to the signal for the non-treated control cells at time 0, that is given the arbitrary value of 1. (*) P ≥ 0.05. (B) Live cells were labeled with fluorescent substrates for Caspase 3 (green) and Caspase 9 (red) and visualized on a confocal microscope. Control samples were pre-incubated with the caspase inhibitor Z-VAD. (*) P ≥ 0.05.

3.9. Suppression of endogenous PGC-1α expression results in the down-regulation of the mitochondrial detoxification machinery

To determine whether the PGC-1α protein present in resting primary endothelial cells was required to sustain the basal levels of mitochondrial detoxification enzymes we decided to suppress PGC-1α expression, transfecting PGC-1α siRNA or GAPDH siRNA as a negative control in BAEC cells.

As shown in Fig. 8 we were able to suppress PGC-1α expression at the mRNA (Fig. 8A) and protein (Fig. 8B) levels, and this downregulation resulted in reduced levels of MnSOD, Prx3, Prx5, TRX2 and TRXR2, supporting the notion that endogenous PGC-1α does indeed regulate their expression.

Fig. 8

Suppression of PGC-1α expression in BAEC results in reduced levels of mitochondrial antioxidant proteins. (A) mRNA levels of PGC-1α, CytC, GAPDH and mitochondrial oxidative stress genes were monitored by rRT-PCR. Values are relative to those obtained for the 18S rRNA. For each gene (except GAPDH itself), values are given as the relative % of levels obtained for the control siRNA GAPDH samples. (*) P ≥ 0.05. (B) Levels of PGC-1α, CytC, GAPDH and mitochondrial oxidative stress proteins were monitored by Western blot.

4. Discussion

Oxidative stress is the leading cause of various pathological conditions that affect systems as diverse as the central nervous system, heart, lung, liver or the vascular wall. However, our understanding of the mechanisms that allow cells to protect themselves from oxidative stress is still very poor. Our results allow us to propose that PGC-1α is a major transcriptional regulator of the mitochondrial detoxification system.

Our results demonstrate that PGC-1α, a positive regulator of oxidative metabolism, co-regulates the induction of a set of proteins that participate in the cellular response to mitochondrial oxidative stress. This positive regulation increases the cellular capacity to detoxify mitochondrial ROS, preventing endothelial dysfunction and apoptotic cell death in response to oxidative stress conditions, such as high glucose.

We interpret these results as evidence that reduced ROS levels allow a better function of mitochondrial ETC. Cells that expressed PGC-1α showed a higher survival rate, supporting the hypothesis that PGC-1α-mediated increase in the cell scavenging activity enable the mitochondria to respond better to metabolic overload. These results have important implications since they point to PGC-1α as a potential target for drug development.

Furthermore, we showed that suppression of PGC-1α expression in BAEC cells by siRNA results in a strong reduction in the levels of the mitochondrial detoxification proteins MnSOD, Prx3, Prx5, TRX2, TRXR2, and UCP-2. These results suggest that the endogenous endothelial PGC-1α protein plays a crucial protective role in the endothelium.

To our knowledge this is the first report of a mechanism that coordinately regulates the mitochondrial antioxidant defense system at the transcriptional level. In order to support the notion that PGC-1α could be directly involved in the transcriptional regulation of antioxidant genes, we tested PGC-1α interaction with the promoter regions of MnSOD [2,17,19,38,40] UCP-2 [15,47], and Prx5. Our data showed that PGC-1α could be found associated with the regulatory promoter sequences of MnSOD, UCP-2 and Prx5. Importantly, UCP-2 has already been proposed to be a direct target of PGC-1α transcriptional regulation [45]. We believe that our results indicate that PGC-1α is likely to be directly involved in the transcriptional regulation of the complete set of oxidative stress genes tested here, although alternative indirect mechanisms are also possible.

One interesting observation that came along with the characterization of PGC-1α activity in the endothelium was the effect of high glucose on the expression of oxidative stress protective proteins. We observed that high glucose had a positive effect on the mRNA levels of the detoxification genes, both in the absence and presence of PGC-1α. However, this positive regulation was not paralleled by a corresponding increase in protein levels. We interpret this result as evidence of a dual regulatory mechanism that might render ineffective the initial increase in mRNA levels. Previous studies on the topic do not shed any light on the possible mechanisms involved, although a decrease in the expression levels of protein synthesis genes has been described in streptozotocin-induced diabetic mice [4]. However, future studies will be necessary to elucidate this point.

Acknowledgements

This work was supported by an institutional grant from CNIC, by the Cardiovascular National Network “RECAVA”, by European Union grant FEDER 2FD97-1432, Plan Nacional de I+D+I grant SAF2003-01039 and, grant-in-aid from the Spanish Society of Nephrology to S.L. and, by grants Plan Nacional de I+D+I SAF2003-04901 and 04.4/0027.1/2003 from the Consejeria de Educación de la Comunidad de Madrid to M.M. Inmaculada Valle is from October 2003 holder of a CNIC-Bancaja predoctoral fellowship and was previously supported by the “Fundación Renal Iñigo Alvarez de Toledo”. María Monsalve is holder of a Ramon y Cajal contract from the Ministerio de Ciencia y Tecnología.

We thank Dr. Luisa Botella and Antonio Quesada for their help with endothelial cell culture, Estrella Soria, Dr. Mariano Redondo, Raquel Nieto, and Pilar Torralbo for their technical assistance. Dr. Ana Aranda, Dr. Juan Bolaños, Dr. Antonio Miranda and Sara Borniquel for careful reading of the manuscript.

Author notes

Time for primary review 28 days