Mitochondrial pyruvate carrier (MPC), which is essential for mitochondrial pyruvate usage, mediates the transport of cytosolic pyruvate into mitochondria. Low MPC expression is associated with various cancers, and functionally associated with glycolytic metabolism and stemness. However, the mechanism by which MPC expression is regulated is largely unknown. In this study, we showed that MPC1 is down-regulated in human renal cell carcinoma (RCC) due to strong suppression of peroxisome proliferator-activated receptor-gamma co-activator (PGC)-1 alpha (PGC-1α). We also demonstrated that overexpression of PGC-1α stimulates MPC1 transcription, while depletion of PGC-1α by siRNA suppresses MPC expression. We found that PGC-1α interacts with estrogen-related receptor-alpha (ERR-α) and recruits it to the ERR-α response element motif located in the proximal MPC1 promoter, resulting in efficient activation of MPC1 expression. Furthermore, the MPC inhibitor, UK5099, blocked PGC-1α-induced pyruvate-dependent mitochondrial oxygen consumption. Taken together, our results suggest that MPC1 is a novel target gene of PGC-1α. In addition, low expression of PGC-1α in human RCC might contribute to the reduced expression of MPC, resulting in impaired mitochondrial respiratory capacity in RCC by limiting the transport of pyruvate into the mitochondrial matrix.

Introduction

Cancer cells reprogram their metabolism through various mechanisms to survive deleterious conditions in tumor microenvironment, such as energy or oxygen depletion [1]. Pyruvate has multiple roles in cells as it links cytoplasmic and mitochondrial energy metabolism. Most mammalian cells oxidize pyruvate for efficient energy production, whereas cancer cells prefer to convert pyruvate to lactate, which is accomplished via several mechanisms. For example, up-regulation of glycolytic enzymes, including lactate dehydrogenase A (LDHA), leads to transition of pyruvate kinase muscle isozyme M1 (PKM1) to less active pyruvate kinase muscle isozyme M2 (PKM2), and down-regulation of pyruvate dehydrogenase (PDH) activity leads to aerobic glycolysis in cancer cells [2]. In addition, tumor cells are known to exhibit reduced mitochondrial pyruvate uptake results in inefficient mitochondrial oxidation [3,4].

It has been well known that pyruvate is taken up into mitochondria through a specific carrier, according to studies using specific inhibitors such as UK5099 and α-cyano-4-hydroxycinnamate [5,6]. However, recent studies have elucidated the molecular identity of mitochondrial pyruvate carrier (MPC) [7,8]. MPC is localized to the mitochondrial inner membrane as a 150-kDa multimeric complex composed of MPC1 and MPC2. The absence of either MPC1 or MPC2 leads to degradation of the other, and results in defective mitochondrial pyruvate uptake and utilization [7,8]. Furthermore, mutations or altered expressions of MPC1/MPC2 are also associated with defects in pyruvate utilization in mitochondria, and result in various diseases including lactic acidosis, developmental defects, and cancer [7,9,10]. Recently, low MPC expression has been reported in colon cancer, prostate cancer, and esophageal squamous cell carcinoma. Blocking of MPC activity, by either knockout of MPC1 with Cas9/sgRNA or pharmacological inhibition, has been associated with poor survival as well as enhanced aerobic glycolysis and stemness [1114].

The functional significance of MPC is not limited to cancer biology. Metabolic implications of mitochondrial pyruvate transport have been unveiled by studies using inhibitors of mitochondrial pyruvate uptake. Inhibition of mitochondrial pyruvate uptake leads to a decrease in fatty acid synthesis from glucose and fructose in epididymal fat tissue and pyruvate-derived gluconeogenesis in the kidney [15]. In addition, inhibition of MPC expression disturbs glucose homeostasis via impaired insulin secretion [16]; hepatic MPC1 expression is involved in pyruvate-derived gluconeogenesis [17], and MPC inhibitors have been suggested as candidates for the prevention and treatment of Parkinson's disease [18]. Although altered expression of MPC has been closely associated with various pathologic conditions, little is known about the precise mechanism of MPC1 gene expression.

Peroxisome proliferator-activated receptor-γ co-activator (PGC) family, consisting of PGC-1α, PGC-1β, and PPRC, plays important roles in regulating metabolic pathways, including mitochondrial biogenesis, oxidative phosphorylation, and energy homeostasis, in various tissues and pathologic conditions including cancer [19,20]. Among PGC family members, PGC-1α is most studied, and its expression is stimulated by various factors such as cold exposure, fasting, and exercise. However, the roles of PGC-1α as a regulator of multiple metabolic pathways in cancer still remain controversial.

Based on previous reports of reduced expression of MPC1 in several types of cancer and the expected role of PGC-1α in cancer regarding mitochondrial metabolism, we investigated the link between PGC-1α and MPC1 in human renal cell carcinoma (RCC). In this report, we have shown that MPC1 expression is down-regulated in RCC through the marked suppression of PGC-1α expression. We have demonstrated that overexpression of PGC-1α up-regulates MPC1 mRNA and protein expression, which is dependent on estrogen-related receptor-alpha (ERR-α); knockdown of PGC-1α down-regulates MPC1 expression concomitant with decreased ERR-α expression. Finally, we found that MPC is required for the functioning of PGC-1α as a master regulator of mitochondrial biogenesis and respiration.

Materials and methods

Cell culture

Human cancer cell lines were obtained from A.T.C.C. (Manassas, VA, U.S.A.). All reagents related to animal cell culture were purchased from Life Technologies (Big Cabin, OK, U.S.A.). Caki1 and Caki2 cells were cultured in modified McCoy's 5A medium, A498 cells were cultured in Eagle's Minimum Essential medium, 786-0 cells were maintained in RPMI 1640, and MDA435S and SK-Hep1 cells were cultured in the Dulbecco's modified Eagle's medium. All media contained 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Cells were cultured at 37°C in a 5%-CO2 humidified atmosphere.

RCC tissues

All human kidney tissues, including cancer and corresponding adjacent normal types, were from surgically resected tissues and were undertaken with approval from the Institutional Review Board of Yonsei University (College of Medicine, Yonsei University).

Lentivirus-mediated overexpression and knockout

For stable overexpression of human PGC-1α and ERR-α, a fragment encoding the full-length cDNA of hPGC-1α was cloned into pLL-CMV-puro lentiviral vector. Lentiviral CRISPR/Cas9 system was designed for knockout of MPC1. Two oligomers, 5′-CACCGGGCTACTTCATTTGTTGCG-3′ and 5′-AAACCGCAACAAATGAAGTAGCCC-3′, were cloned to lenti-CRISPR-V2 vectors [21]. For the production of lentivirus, plasmid DNAs were transfected into human embryonic kidney (HEK293T) cells, along with a lentiviral packaging mix consisting of an envelope and packaging vector to produce lentiviruses. Puromycin (Sigma–Aldrich) was utilized for stable cell selection after infection.

Transient transfection of plasmid and siRNA

Cells were plated on the day before transfection. When the cells reached ∼70% confluence, they were transfected with the indicated plasmid utilizing Lipofectamine 2000® Reagent (Life Technologies, Carlsbad, CA, U.S.A.) according to the supplier's protocol. In the case of siRNA transfection, 20 nM of siRNA was transfected in cell suspension utilizing RNAiMax reagent (Life Technologies, Carlsbad, CA, U.S.A.) 48 h before cell harvesting. The siRNA sequences were 5′-GCAATAAAGCGAAGAGTATTTGTCA-3′ (#1) and 5′-GCACTACAGATATCATATTGAGGAT-3′ (#2) for PGC-1α, and 5′-GAGCATCCCAGGCTTCTCATT-3′ for ERR-α.

Cloning of MPC1 promoter and luciferase assays

For cloning of human MPC1 promoter, genomic DNA was prepared from HEK293T cells by the phenol/chloroform/isopropyl alcohol extraction method, followed by PCR to amplify ∼2.5 kbp with primers; sense 5′-CCACCAATCTTGCAGAGCAGTTTGG-3′ and antisense 5′-CTGCCTCTGCTGCCGCTTC-3′ and cloned into pGL3-basic vector (Promega). Luciferase activity in whole-cell lysates was measured using the Dual-Luciferase® reporter assay system (Promega). The 786-O and SK-Hep1 cells were harvested two days after transfection. Luciferase activity was obtained by normalizing the reporter luciferase activity to the value of Renilla luciferase from pRL-SV40 construct. All data shown are from three independent experiments performed in triplicate.

Total RNA isolation and quantitative real-time PCR

Total RNA was isolated from human tissues or cultured cells by using TRIzol (Invitrogen) according to the manufacturer's instructions. Human tissues were homogenized by TissueLyser II (Qiagen, Hilden, Germany). For quantitative real-time PCR (qPCR), cDNAs were synthesized from 4 µg of total RNA using random hexamer primers and SuperScript reverse transcriptase II (Invitrogen) following the manufacturer's instructions. Diluted cDNAs were analyzed for qPCR using SYBR Green PCR Master Mix (Applied Biosystems) and gene-specific primers, and then subjected to RT-PCR quantification using the ABI PRISM 7300 RT-PCR System (Applied Biosystems).

Western blotting

Cultured cells were washed twice with ice-cold phosphate-buffered saline, and harvested in whole-cell lysis buffer (1% sodium dodecyl sulfate, 60 mM Tris–HCl, pH 6.8). Protein concentrations were measured by the bicinchoninic acid assay. Equal amounts of protein extracts were subjected to SDS–PAGE, and transferred onto nitrocellulose transfer membranes (Whatman). Membranes were blocked in 5% (w/v) non-fat DifcoTM skimmed milk (BD Biosciences), followed by incubation with primary antibodies in 1% bovine serum albumin. The following antibodies were used: anti-TFAM (mitochondrial transcription factor A), MPC1, MPC2 (Cell Signaling), heat shock protein 60 (HSP 60) (Santa Cruz Biotechnology), and α-tubulin (Merck Millipore). Quantification of immunoblot was determined by densitometric analysis using ImageJ software.

Chromatin immunoprecipitation assay

To confirm PGC-1α recruitment into the MPC1 promoter region, a ChIP (chromatin immunoprecipitation) assay was performed using a PGC-1α antibody and ChIP assay kit (Millipore) by following the supplier's protocol. The 786-O cells that stably expressed PGC-1α were harvested in PBS, cross-linked with 1% formaldehyde, and sonicated. Anti-PGC-1α antibody (Santa Cruz) or normal rabbit IgG (Jackson ImmunoResearch) were used for immunoprecipitation. Primers for real-time qPCR analysis were designed to amplify the proximal region of ERR-binding site region (−910 to −760); sense 5′-TGATCCGAAAGTCGTGCTGC-3′, antisense 5′-TTAGGCTCGAGGAAGCGTGA-3′. Primer sequences for distal 3′UTR region for negative controls were as follows: sense 5′-TGAAGGGACAGCATTGCCAG-3′ and antisense 5′-CTTGTAAGGCAGCAGAGAGTTGG-3′.

Metabolic activity measurements

Oxygen consumption rates (OCR, pmol/min) were measured using Seahorse XF 24 Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA, U.S.A.) according to the manufacturer's instructions. Cells were in 1× MAS medium (2-mM HEPES, 10-mM KH2PO4, 1-mM EGTA, 5-mM MgCl2, 70-mM Sucrose, 220 mM Mannitol, 0.2% fatty acid free-BSA) supplemented with 1-nM Seahorse XF Plasma Membrane Permeabilizer, 4-mM ADP. Two mM of pyruvate, glutamate, or succinate and 0.2 mM of malate were used as substrates for analysis. Inhibitors were used for the tests at final concentrations of 3-µg/ml oligomycin, 5-µM FCCP, 0.5-µM rotenone, and 1-µM UK5099.

Mitochondrial DNA quantification

Total DNA was isolated utilizing G-spin TotalTM DNA extraction kit (Intron biotechnology, Seoul, Korea). Level of mitochondrial DNA genome (16S rRNA) was amplified with primers for sense: 5′-GCCTTCCCCCGTAAATGATA-3′ and antisense: 5′-TTATGCGATTACCGGGCTCT-3′, and normalized to nuclear gene (β2-microglobulin) amplified with sense: 5′-TGCTGTCTCCATGTTTGATGTATCT-3′ and antisense: 5′- TCTCTGCTCCCCACCTCTAAGT-3′.

Statistical analysis

Statistical analyses were performed by either Exel or SPSS. All data underwent normality test and were analyzed by two-tailed t-test.

Results

Expression of MPC1, MPC2, and PGC-1α in human RCC tissues

Implications of MPC1 in human cancer were documented in several reports. MPC1 protein expression has been reported to be decreased in human esophageal squamous cell carcinoma tissues compared with normal tissues [13], and low levels of MPC1 mRNA expression in various cancers have been associated with poor survival and maintenance of cancer cell stemness [1113]. However, MPC expression level in RCC and regulation of MPC expression in human cancer cells are largely unknown.

To determine the expression pattern of MPC complexes in the matched human normal kidney and advanced RCC, protein and mRNA levels of MPC1 and MPC2 were measured by western blot and qPCR analyses (Figure 1A,B, respectively). Both protein and mRNA levels of MPC1 and MPC2 showed a marked decrease in RCC tissues (C) compared with matched normal adjacent kidney tissues (N), although the MPC2 mRNA level in RCC decreased to a much less extent than MPC1 (Figure 1B). Notably, the level of PGC-1α, which is known to regulate mitochondrial biogenesis, showed a similar pattern of decrease that was observed in MPC1 and MPC2 expression.

Expressions of MPC1, MPC2, and PGC-1α in human RCC tissues.

Figure 1.
Expressions of MPC1, MPC2, and PGC-1α in human RCC tissues.

(A) Left panel: Western blots of protein extracts from four patients (P1–P4). Normal adjacent tissues (N) and RCC tissues (C). GAPDH expression was used as a loading control. Right panel: Band density of each target protein was normalized to corresponding GAPDH expression. Data are presented as mean ± SE. ***P < 0.001, **P < 0.01, *P < 0.05 by Student's two-tailed t-test. (B) qPCR analysis of the expressions of PGC-1α, MPC1, and MPC2 mRNA in human normal and cancer tissues. All bar graphs are plotted as mean ± SE.

Figure 1.
Expressions of MPC1, MPC2, and PGC-1α in human RCC tissues.

(A) Left panel: Western blots of protein extracts from four patients (P1–P4). Normal adjacent tissues (N) and RCC tissues (C). GAPDH expression was used as a loading control. Right panel: Band density of each target protein was normalized to corresponding GAPDH expression. Data are presented as mean ± SE. ***P < 0.001, **P < 0.01, *P < 0.05 by Student's two-tailed t-test. (B) qPCR analysis of the expressions of PGC-1α, MPC1, and MPC2 mRNA in human normal and cancer tissues. All bar graphs are plotted as mean ± SE.

It has been known that RCC reveals decreased mitochondrial functions due to reduced mitochondrial DNA, respiratory chain activities, and oxidative phosphorylation-related proteins [2224]. Therefore, it is intriguing to see whether drastic decrease in MPC in RCC might result from diminished mitochondrial contents. As shown in Figure 1A, decreased expression of mitochondrial marker proteins, such as HSP60 (heat shock protein 60), COX4 (cytochrome c oxidase subunit 4), and TFAM, showed statistically significant differences between cancer tissues and normal adjacent tissues. However, fold decreases in MPC1, MPC2, and PGC-1α (8-, 5-, and 5-fold, respectively) were much greater than those of HSP60, COX4, and TFAM (1.5-, 2.5-, and 1.8-fold, respectively). These data suggest there might be other mechanisms that lead to decreased expression of MPC1 in addition to diminished amount of mitochondria, in RCC.

PGC-1α stimulates the transcription of MPC1

In consistence with an essential role of PGC-1α in mitochondrial biogenesis in various tissues, exogenous PGC-1α expression stimulates mitochondrial oxidative phosphorylation in RCC cell lines [25]. Furthermore, our previous report showed that RCC cell lines exhibit suppression of pyruvate-dependent mitochondrial oxidation, and that re-expression of MPC1 and MPC2 recovered mitochondrial respiration [26]. Therefore, we hypothesized that PGC-1α regulates MPC1 gene expression. First, we stably overexpressed PGC-1α in RCC cell lines (786-O and Caki1) and a hepatocellular carcinoma cell line (SK-Hep1), which had low and no endogenous expression of PGC-1α, respectively, to evaluate the effect of PGC-1α on MPC1 expression. As shown in Figure 2A, PGC-1α-overexpressing cell lines exhibited a marked increase in protein levels of both MPC1 and MPC2. ERR-α, one of the most well-characterized transcription factors that interacts with PGC-1α, was also markedly up-regulated. However, no significant increase in mitochondrial TFAM and HSP60 was observed, although COX4 expression was increased in PGC-1α-overexpressing cells to less extent than MPC1 and MPC2 (Figure 2A, right panel). In addition, qPCR analysis showed that MPC1 mRNA was up-regulated (13-fold increase) in the presence of PGC-1α, whereas MPC2 mRNA expression increased to a much less extent (3-fold increase) as shown in Figure 2B.

PGC-1α stimulates MPC1 transcription.

Figure 2.
PGC-1α stimulates MPC1 transcription.

(A) Left panel: Western blot analysis of cancer cells that were stably transduced with mock- or PGC-1α-expressing lentiviruses. Right panel: Band density of each target protein was normalized to corresponding α-tubulin. Data are presented as mean ± SE. Statistical analysis performed as described in Materials and Methods. ***P < 0.001; *P < 0.05 by Student's two-tailed test. (B) qPCR analysis of MPC1 and MPC2 mRNA levels in PGC-1α overexpressing cells. ***P < 0.001 by Student's two-tailed t-test. (C) Left panel: Protein levels of indicated genes from MDA-MB435S cells that were transfected with 20 nM of short inferring RNAs (siRNAs) targeting PGC-1α (#1 and #2). Right panel: Band density of target proteins. *P < 0.05 by Student's two-tailed t-test. (DF) mRNA levels of indicated genes from MDA-MB435S cells that were knockdown PGC-1α. ***P < 0.001; **P < 0.01 compared with mock transfection group Student's two-tailed t-test. Data are representative of three experiments.

Figure 2.
PGC-1α stimulates MPC1 transcription.

(A) Left panel: Western blot analysis of cancer cells that were stably transduced with mock- or PGC-1α-expressing lentiviruses. Right panel: Band density of each target protein was normalized to corresponding α-tubulin. Data are presented as mean ± SE. Statistical analysis performed as described in Materials and Methods. ***P < 0.001; *P < 0.05 by Student's two-tailed test. (B) qPCR analysis of MPC1 and MPC2 mRNA levels in PGC-1α overexpressing cells. ***P < 0.001 by Student's two-tailed t-test. (C) Left panel: Protein levels of indicated genes from MDA-MB435S cells that were transfected with 20 nM of short inferring RNAs (siRNAs) targeting PGC-1α (#1 and #2). Right panel: Band density of target proteins. *P < 0.05 by Student's two-tailed t-test. (DF) mRNA levels of indicated genes from MDA-MB435S cells that were knockdown PGC-1α. ***P < 0.001; **P < 0.01 compared with mock transfection group Student's two-tailed t-test. Data are representative of three experiments.

To further investigate whether PGC-1α regulates MPC1 expression, we transfected small interfering RNA (siRNA) to knock down endogenous PGC-1α expression. We tested the endogenous mRNA expression level of PGC-1α in various human cancer cell lines (kidney cancer cell lines, breast cancer cell lines, and prostate cancer cell lines) using real-time qPCR analysis. MDA-MB435S cell line was selected, since it showed the highest expression of PGC-1α among the cell lines we tested. Therefore, we measured the changes in protein and mRNA levels of MPC1 in MDA-MD435S, a breast cancer cell line that strongly expresses endogenous PGC-1α. Knockdown of PGC-1α with two different siRNAs (#1 and #2, Figure 2C–E) significantly down-regulated both protein and mRNA expressions of MPC1 and MPC2 along with PGC-1α, whereas expression levels of mitochondrial proteins, including COX4, HSP60, and TFAM, were unchanged except ERR-α, which was down-regulated upon knockdown of PGC-1α (Figure 2C).

PGC-1 family co-activators share structural features, and they show functional redundancy in metabolic pathways and mitochondrial biogenesis [27,28]. Our data showed that knockdown of PGC-1α induced compensatory expression of other PGC-1 family members, PGC-1β and PPRC (Figure 2F). However, the elevation of these factors shown to be insufficient to recover MPC expression. Collectively, these results indicate that PGC-1α regulates the MPC expression that was not compensated by other family members.

PGC-1α regulates MPC1 expression through binding to MPC1 promoter region

Given the fact that PGC-1α strongly activates MPC1 gene expression (Figure 2B), we next assessed the role of PGC-1α in MPC1 regulation at the transcriptional level in more detail. We cloned the MPC1 promoter region contained within a 2.5-kb fragment (−2070 to −30 bp) into luciferase reporter vectors, as described in Materials and methods. As shown in Figure 3A, exogenous expression of PGC-1α elicited ∼3.5-fold increase in the MPC1 promoter activation. It has been reported that one of the core functions of PGC-1α, which is the elevation of oxidative metabolism, was conserved in two other PGC-1 families [2830]. Therefore, it was intriguing to test whether PGC-1β, which has the most functional similarity to PGC-1α, was also able to stimulate MPC1 promoter activity. As shown in Figure 3A, exogenous expression of PGC-1β alone stimulated MPC1 promoter activity to a similar extent than PGC-1α.

PGC-1α activates MPC1 gene expression and MPC1 promoter activity.

Figure 3.
PGC-1α activates MPC1 gene expression and MPC1 promoter activity.

(A) Effects of PGC-1α, PGC-1β, and ERR-α on MPC1 promoter activity in SK-Hep1 cells. ***P < 0.001 compared with mock transfection group, ###P < 0.001; #P < 0.05 compared with PGC-1α transfection group, by Student's two-tailed t-test. (B) PGC-1α-mediated MPC1 promoter activity with or without ERR-α siRNA transfection. **P < 0.01 by Student's two-tailed t-test. (C) Luciferase activity of deletion constructs of MPC1 promoter in the presence of PGC-1α. **P < 0.01 by Student's two-tailed t-test. (D) Luciferase activity of MPC1-promoter which has mutations on ERRE2 (CCAAggtgcccTTG to CCAAtttgtaaTTG), and ERRE3 (CCTGTgggtCAGA to CCTGTtttaCAGA) in the presence or absence of exogenous PGC-1α expression. *P < 0.05 by Student's two-tailed t-test. (E) ChiP assay was performed with anti-PGC-1α antibody. Normal rabbit IgG was used as control. 786-O cells, which express exogenous PGC-1α, were used for analysis. Real-time PCR analysis of MPC1 promoter region (−910/−770) with DNA samples from IgG or PGC-1α antibody immunoprecipitation. Primers for the distal region from MPC1 promoter, 3′-UTR (exon 5), were used as negative control. ***P < 0.001 by Student's two-tailed t-test. Experiments were performed three times, and all bar graphs are plotted as mean ± SE.

Figure 3.
PGC-1α activates MPC1 gene expression and MPC1 promoter activity.

(A) Effects of PGC-1α, PGC-1β, and ERR-α on MPC1 promoter activity in SK-Hep1 cells. ***P < 0.001 compared with mock transfection group, ###P < 0.001; #P < 0.05 compared with PGC-1α transfection group, by Student's two-tailed t-test. (B) PGC-1α-mediated MPC1 promoter activity with or without ERR-α siRNA transfection. **P < 0.01 by Student's two-tailed t-test. (C) Luciferase activity of deletion constructs of MPC1 promoter in the presence of PGC-1α. **P < 0.01 by Student's two-tailed t-test. (D) Luciferase activity of MPC1-promoter which has mutations on ERRE2 (CCAAggtgcccTTG to CCAAtttgtaaTTG), and ERRE3 (CCTGTgggtCAGA to CCTGTtttaCAGA) in the presence or absence of exogenous PGC-1α expression. *P < 0.05 by Student's two-tailed t-test. (E) ChiP assay was performed with anti-PGC-1α antibody. Normal rabbit IgG was used as control. 786-O cells, which express exogenous PGC-1α, were used for analysis. Real-time PCR analysis of MPC1 promoter region (−910/−770) with DNA samples from IgG or PGC-1α antibody immunoprecipitation. Primers for the distal region from MPC1 promoter, 3′-UTR (exon 5), were used as negative control. ***P < 0.001 by Student's two-tailed t-test. Experiments were performed three times, and all bar graphs are plotted as mean ± SE.

Since PGC-1 family co-activators transactivate target gene expression by binding to various nuclear receptors and transcription factors, we next searched for binding partners that could be responsible for PGC-1-dependent MPC1 promoter activation. We found that exogenous PGC-1α overexpression enhanced expression of ERR-α, one of the binding partners of PGC-1α (Figure 2A), while knockdown of PGC-1α down-regulated ERR-α expression (Figure 2C). Furthermore, our RNA sequencing analysis data revealed that PGC-1α-transduced 786-O and that SK-Hep1 cell lines notably increased ERR-α expression, while it did not affect the expression of other known binding partners such as NRFs (nuclear respiratory factors), PPARs, and FOXOs (Forkhead box transcription factors; Supplementary Materials, Table 1). Therefore, we attempted to establish the role of ERR-α in MPC1 promoter activity. As shown in Figure 3A, exogenous expression of ERR-α alone elicited weak activation of the MPC1 promoter; however, ERR-α stimulated MPC1 promoter activity in the presence of PGC-1α, whereas PGC-1β-mediated MPC1 promoter activity was marginally affected by ERR-α.

To determine whether the stimulatory effects of PGC-1α on MPC1 promoter activity were mediated through ERR-α, we depleted ERR-α via siRNA treatment and measured PGC-1α-induced MPC1 promoter activity. Notably, knockdown of ERR-α completely blocked the stimulatory effects of PGC-1α on MPC1 promoter activation (Figure 3B), which indicated that ERR-α is essential for PGC-1α-mediated MPC1 promoter activation.

To further assess the role of ERR-α in MPC1 activation, we next searched for the ERR-α binding motif responsible for PGC-1α-mediated MPC1 promoter activation. Since there were three putative ERR-binding elements within the 2.5-kb MPC1 promoter region, we cloned truncated mutants of MPC1 promoter. As shown in Figure 3C, MPC1 promoter activity was not affected by the deletion of ERR-α response element 1 (ERRE1), while the absence of ERRE2 completely abrogated the stimulatory action of PGC-1α on MPC1 promoter activity. Further deletion of ERRE3 showed no change in ERR-α responsiveness. In addition, mutagenesis of ERRE2 sites (CCAAggtgcccTTG to CCAAtttgtaaTTG) significantly impaired MPC1 promoter activity (Figure 3D).

Next, we performed a ChIP-qPCR assay to confirm that PGC-1α was recruited to the ERRE2 region in MPC1 promoter. Since the region compassing ERRE2 had excessively high levels of GC contents (∼80%) to be used for qPCR analysis, slight upstream region (−910/−770) was amplified for ChIP assay. As shown in Figure 3E, PGC-1α was enriched at the human MPC1 promoter region, whereas no recruitment of PGC-1α was detected in the 3′-UTR region, which was used as a control. These data suggest that PGC-1α stimulate transcription of MPC1 through binding MPC1 promoter region and ERRE2 is essential for PGC-1α-mediated MPC1 transcriptional activation.

ERR-α is necessary for PGC-1α–mediated MPC1 expression

Given that ERR-α itself increased MPC1 promoter activity (Figure 3A), it was intriguing to test whether ERR-α itself could be a transcriptional activator of MPC1 expression. Therefore, we overexpressed ERR-α in 786-O, an RCC cell line, and measured MPC1 expression. As shown in Figure 4A,B, exogenous expression of ERR-α itself showed 2-fold activation of MPC1 expression at both protein and mRNA levels, in contrast with 13-fold activation by PGC-1α itself (Figure 2B). These results were in accordance with previous reports which showed that ERR-α is a very week transcription activator without protein ligands [31]. To investigate the role of ERR-α in PGC-1α-mediated stimulation of MPC1 expression, we depleted ERR-1α utilizing siRNA transfection in both mock- and PGC-1α-overexpressing 786-O cells. As shown in Figure 4C, knockdown of ERR-α completely abrogated PGC-1α-induced elevation of MPC1 and MPC2 expression. Taken together, these data indicate that expression of ERR-α is critical for PGC-1α-mediated stimulation of MPC1 transcription.

ERR-α expression is essential for PGC-1α-mediated MPC1 expression.

Figure 4.
ERR-α expression is essential for PGC-1α-mediated MPC1 expression.

(A) Upper panel: Western blot analysis of protein of indicated targets. Lower panel: Statistical analysis of bands as described in Materials and Methods. *P < 0.05 by Student's two-tailed t-test. (B) mRNA level of indicated targets utilizing 786-O cells stably overexpressing ERR-α. (C) Effects of siRNA-induced ERR-α knockdown on the level of protein expression of indicated targets in 786-O cells in the presence or absence of PGC-1α overexpression.

Figure 4.
ERR-α expression is essential for PGC-1α-mediated MPC1 expression.

(A) Upper panel: Western blot analysis of protein of indicated targets. Lower panel: Statistical analysis of bands as described in Materials and Methods. *P < 0.05 by Student's two-tailed t-test. (B) mRNA level of indicated targets utilizing 786-O cells stably overexpressing ERR-α. (C) Effects of siRNA-induced ERR-α knockdown on the level of protein expression of indicated targets in 786-O cells in the presence or absence of PGC-1α overexpression.

MPC is required for stimulatory effects of PGC-1α on mitochondrial respiration capacity

PGC-1α is known to be responsible for mitochondria biogenesis, in addition to regulating mitochondrial functions in various tissues and cell lines [25,32]. To investigate whether PGC-1α overexpression is associated with oxidative phosphorylation and glycolysis, we measured OCR (pmol/min) using the Seahorse XF analyzer with permeabilized cells, as described in Materials and Methods. As shown in Figure 5A, overexpression of PGC-1α strongly elevated pyruvate-driven basal and maximal OCR. We previously demonstrated that impaired glucose oxidation in RCC cell lines was recovered by MPC1 and MPC2 overexpression [26]. Since MPC expression increased in association with PGC-1α expression, we assessed whether there was a functional relationship between MPC and PGC-1α regarding mitochondrial respiration. Notably, blocking of MPC activity with an MPC inhibitor, UK5099, completely abrogated the pyruvate-derived OCR in PGC-1α-overexpressing 786-O cells, whereas glutamate- or succinate-derived OCR was not affected by UK5099 treatment (Figure 5B). Taken together, these data indicate that increased pyruvate oxidation due to PGC-1α overexpression, occurred mostly through MPC expression.

MPC is required for PGC-1α-mediated mitochondrial oxidative phosphorylation and mitochondrial biogenesis.

Figure 5.
MPC is required for PGC-1α-mediated mitochondrial oxidative phosphorylation and mitochondrial biogenesis.

(A) Pyruvate-derived basal and maximal OCR of mock- and PGC-1α-overexpressing 786-O cells which were permeabilized as described in Materials and Methods. ***P < 0.001 by Student's two-tailed t-test. (B) Maximal OCR of permeabilized mock- and PGC-1α-overexpressing 786-O cells in the presence or absence of 1 μM UK5099 in the indicated 2 mM substrates with 0.2 mM malate. ***P < 0.001; **P < 0.01; *P < 0.05 compared with DMSO-treated group by Student's two-tailed t-test. (C) Quantification of mitochondrial DNA of mock- and PGC-1α-overexpressing 786-O cells in the presence or absence of CRISPR/Cas9-mediated knockout of MPC1. *P < 0.05 compared with PGC-1α transfection group by Student's two-tailed t-test. (D) Western blot analysis of whole-cell lysates from PGC-1a overexpressing 786-O cells, which stably express either non-targeting sgRNA or MPC1-targeting sgRNA.

Figure 5.
MPC is required for PGC-1α-mediated mitochondrial oxidative phosphorylation and mitochondrial biogenesis.

(A) Pyruvate-derived basal and maximal OCR of mock- and PGC-1α-overexpressing 786-O cells which were permeabilized as described in Materials and Methods. ***P < 0.001 by Student's two-tailed t-test. (B) Maximal OCR of permeabilized mock- and PGC-1α-overexpressing 786-O cells in the presence or absence of 1 μM UK5099 in the indicated 2 mM substrates with 0.2 mM malate. ***P < 0.001; **P < 0.01; *P < 0.05 compared with DMSO-treated group by Student's two-tailed t-test. (C) Quantification of mitochondrial DNA of mock- and PGC-1α-overexpressing 786-O cells in the presence or absence of CRISPR/Cas9-mediated knockout of MPC1. *P < 0.05 compared with PGC-1α transfection group by Student's two-tailed t-test. (D) Western blot analysis of whole-cell lysates from PGC-1a overexpressing 786-O cells, which stably express either non-targeting sgRNA or MPC1-targeting sgRNA.

These results were extended to explore the possibility that efficient carbon supply to mitochondria via MPC might have a role in mitochondrial biogenesis induced by PGC-1α. We utilized CRISPR/Cas9 system to knock out MPC1 gene in mock- or PGC-1α-overexpressing 786-O cells, and measured mitochondrial DNA (mtDNA) in 786-O cells as indicated (Figure 5C,D). In line with previous reports, PGC-1α up-regulated mitochondrial DNA contents by ∼2-fold. Notably, PGC-1α-induced mtDNA contents were mostly impaired in Crispr/Cas9-mediated MPC1- knockout cells without notable change of PGC-1α and ERR-α expressions, while basal mtDNA contents were not affected by the absence of MPC. These results indicate that MPC plays a critical role in PGC-1α-mediated pyruvate-dependent mitochondrial oxidative phosphorylation and mitochondrial biogenesis.

Discussion

MPC mediates the transport of cytosolic pyruvate into the mitochondria. There is growing evidence that MPC is important in pyruvate metabolism not only in normal physiological processes such as glucose homeostasis, but also in a variety of pathological conditions including cancer. Although altered expression of MPC gene is associated with diet-induced obesity and poor survival in various cancers, the precise mechanism by which MPC genes are regulated is largely unknown. In this study, we demonstrated that MPC1 and MPC2 are down-regulated in RCC, while providing evidence that PGC-1α, in concert with ERR-α, mediates transcriptional activation of human MPC1. Furthermore, we also proposed that MPC is essential for PGC-1α-dependent mitochondrial respiration.

First, we showed that expressions of MPC1 and MPC2 decreased at both protein and mRNA levels in human RCC tissues compared with matched normal tissues (Figure 1). These results were in line with our recent report, which showed that the expressions of MPC1 and MPC2 were lower in RCC cell lines compared with those in HEK239 and hepatocellular carcinoma cell lines [26]. The advantageous effects of loss of MPC in human cancers are supported by findings that MPC inhibition induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis, which is associated with resistance to chemotherapy [12,13]. It also shows that suppression of MPC expression leads to increased stemness via up-regulated expression of stem cell markers [11,14]. Contrary to these reports, one study demonstrated that increased uptake of pyruvate and high OCR were important in invasive ovarian cancer cells [33]. The role of PGC-1α in cancer seems diverse, and also dependent on the cell lines analyzed [20]. Recent study on human RCC showed that suppression of PGC-1α is associated with poor overall survival through metabolic reprogramming toward a decrease in oxidative phosphorylation [25]. However, the precise mechanisms of reduced respiration by PGC-1α suppression are largely unknown.

The loss of either MPC1 or MPC2 results in the degradation of the other [7,8,11,17]. In accordance with these reports, our data showed that MPC2 protein level is completely abrogated upon Crispr/Cas9-mediated MPC1 knockout (Figure 5E). Notably, we consistently observed that PGC-1α strongly elevates MPC complex (Figures 2A, 4C and 5E), although stimulatory effects of PGC-1α on MPC1 transcription are much more prominent compared with those on MPC2 transcription. Therefore, transcriptional regulation of MPC1 by PGC-1α might be critical to whole MPC complex level.

In this study, we first demonstrated the new regulatory mechanism of MPC1 expression at transcriptional level. Depletion of ERR-α totally compromised PGC-1α-induced elevation of MPC1 expression, indicating that ERR-α is a major partner of PGC-1α regarding MPC1 expression. We also demonstrated that the effects of ERR-α by itself on transcription of MPC1 as well as MPC1 promoter activity were marginal without PGC-1α co-expression. These results were consistent with a previous report demonstrating that ERR-α is a poor transcriptional activator by itself, and protein ligand such as PGC-1α is required for the full activation of ERR-α [31]. Importantly, ERR-α showed marked elevation of MPC1 promoter activity when co-ordinated with PGC-1α compared with PGC-1β.

There have been several mechanisms that were proposed to explain the low expression of MPC1. Schell et al. proposed that low expression of MPC1 in colon cancer might be caused by deletion of 6q27 where MPC1 is located. They also proposed that decreased MPC1 expression was not due to the genomic location of MPC1, as expression of an adjacent gene in 6q27, RPS6KA2, increased in colorectal cancer [11]. In human prostate cancer, up-regulation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) negatively regulated MPC1 expression, as knockdown and overexpression of COUP-TFII increased MPC1 expression and suppressed MPC1 promoter activity, respectively [34]. However, we conclude that the mechanism by which MPC1 expression is suppressed in prostate cancer might differ from that in human RCC, since COUP-TFII expression was rather down-regulated in human RCC tissues compared with matched normal tissues according to our microarray data.

Mitochondrial biogenesis and activity must be closely associated with cellular nutrient availability and energy status [3537]; however, nutrient-specific regulation of mitochondria still largely unknown. In this study, we investigated functions of MPC in a PGC-1α-overexpressing cell line regarding mitochondrial function and biogenesis. We found that the inhibition of MPC completely blocked PGC-1α-mediated pyruvate oxidation, as well as an increase in mitochondrial DNA, all of which imply that MPC could act as a limiting factor for mitochondrial function and biogenesis by controlling mitochondrial pyruvate utilization. Since there was no decrease in PGC-1α and ERR-α in CRISPR/Cas9-mediated MPC knockout cells, the precise mechanisms by which impaired pyruvate oxidation by MPC inhibition affects mitochondrial function and mtDNA contents need to be investigated. Taken together, our results suggest that low level of PGC-1α expression leads to decreased level of MPC expression, which results in reduced oxidation of pyruvate in human RCC.

The role of pyruvate metabolism in cancer depends on types of tumors. Reduced pyruvate oxidation is advantageous to prostate and colon cancer cells [1114], whereas increased pyruvate uptake is important for highly invasive ovarian cancer cells [33]. In addition, therapeutic applications based on modulating MPC activity have been reported for various diseases such as nonalcoholic steatohepatitis (NASH), diabetes, and Parkinson's disease [18,38,39]. Therefore, a deeper understanding of mechanisms regarding mitochondrial pyruvate uptake is essential for the development of therapeutic interventions for diseases related to mitochondrial dysfunction, such as cancer.

Abbreviations

     
  • COX4

    cytochrome c oxidase subunit 4

  •  
  • ERRE

    ERR-α response element

  •  
  • ERR-α

    estrogen-related receptor-alpha

  •  
  • FOXO

    Forkhead box transcription factors

  •  
  • HSP60

    heat shock protein 60

  •  
  • MPC

    mitochondrial pyruvate carrier

  •  
  • NRF

    nuclear respiratory factor

  •  
  • OCR

    oxygen consumption rate

  •  
  • PGC

    peroxisome proliferator-activated receptor-gamma co-activator

  •  
  • PGC-1α

    (PGC)-1 alpha

  •  
  • PPAR

    peroxisome proliferator-activated receptor

  •  
  • RCC

    Renal cell carcinoma

  •  
  • TFAM

    mitochondrial transcription factor A

Author Contribution

K.-S.K. and E.K. designed the study and wrote the paper. E.K., D.S. and Y.K.K. performed experiments and analyzed the data. All authors read and approved the final manuscript.

Funding

This work was supported by National Research Foundation of Korea (NRF) grants from the Korean government (MSIP) [NRF-2017R1A2B4007462, NRF-2017R1A2B4009674, and NRF-2011-0030086].

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Supplementary data