Mitochondrion 14 (2014) 18–25

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Loss of MED1 triggers mitochondrial biogenesis in C2C12 cells Jialing Yu 1, Yun Xiao 1, Junxia Liu, Yanchun Ji, Hao Liu, Jing Xu, Xiaofen Jin, Li Liu, Min-Xin Guan, Pingping Jiang ⁎ Department of Genetics, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Article history: Received 21 July 2013 Received in revised form 31 October 2013 Accepted 12 December 2013 Available online 22 December 2013 Keywords: MED1 Mitochondrial biogenesis Nrf1 Oxygen consumption

a b s t r a c t Under stress conditions transcription factors, including their coactivators, play major roles in mitochondrial biogenesis and oxidative phosphorylation. MED1 (Mediator complex subunit 1) functions as a coactivator of several transcription factors and is implicated in adipogenesis of the lipid and glucose metabolism. This suggests that MED1 may play a role in mitochondrial function. In this study, we found that both the mtDNA content and mitochondrial mass were markedly increased and cell proliferation markedly suppressed in MED1-deficient cells. Upon MED1 loss, Nrf1 and its downstream target genes involved in mitochondrial biogenesis (Tfam, Plormt, Tfb1m), were up-regulated as were those genes in the OXPHOS pathway. Moreover, the knockdown of MED1 resulted in significant changes in the profile of mitochondrial respiration, accompanied by a prominent decrease in the generation of ATP. Collectively, these observations strongly suggest that MED1 has an important affect on mitochondrial function. This further elucidates the role of MED1, particularly its role in the energy metabolism. © 2013 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction Mitochondrion is a multi-functional cellular organelle. Their normal and pathological activities are dependent on the expression of the mitochondrial genome which is organized as circular, double-stranded DNA which codes for 13 proteins, 22 transfer RNAs and 2 ribosomal RNAs (rRNAs) (Anderson et al., 1981; Scheffler, 1999). Their function is also dependent upon the expression of numerous nuclear genes, the products of which (~ 1500 proteins) are transported into the mitochondria (Calvo and Mootha, 2010; Schmidt et al., 2010). The limited coding capacity of mitochondrial DNA (mtDNA) necessitates a big contribution from nuclear genes towards mitochondrial biogenesis, redox regulation and molecular architecture (Garesse and Vallejo, 2001). Recently, evidences have pointed towards the nucleus-encoded transcription factors such as NFRs, and STAT3, and nuclear receptors such as the thyroid receptors (TRs), estrogen receptor (ERs), estrogenrelated receptor (ERRs), peroxisome proliferator-activated receptor (PPARs)) and their co-regulators (PGC-1, RIP140), all being major regulators of mitochondrial biogenesis. When cells have a high metabolic demand for mitochondrial energy or are responding to other stress conditions these regulators induce the gene transcription of oxidative phosphorylation (OXPHOS) and its biosynthesis (Arany et al., 2007; Chen et al., 2009a; Gianotti et al., 2011; Leigh-Brown et al., 2010; Scarpulla et al., 2012; Weitzel and Iwen, 2011).

⁎ Corresponding author at: Department of Genetics, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China. 310058. Tel.: +86 571 8898 2356; fax: +86 571 8898 2377. E-mail address: [email protected] (P. Jiang). 1 These authors contributed equally to this work.

MED1 (also known as TRAP220/DRIP205/PPARBP) is a component of the TRAP/Mediator complex in the nucleus that plays an essential role as a co-regulator in RNA polymerase II-dependent transcription (Zhang et al., 2005). It also regulates signaling transduction by interacting with several nuclear receptors, such as PPARs, ERs, TRs, GR, and RXR (Chen and Roeder, 2011; Ge et al., 2002; Malik et al., 2004). Previously, it has also been suggested that MED1 plays a major role in the transcriptional control of specific genes associated with the energy metabolism. An earlier study has shown that MED1 is essential for the PPAR-stimulated adipogenesis in mouse embryonic fibroblasts (MEFs) (Ge et al., 2002). A liver-specific Med1 knockout mouse showed a defect in PPARα-mediated oxidation of fatty acids (Jia et al., 2004). Chen et al. (2009b, 2010) demonstrated that MED1 was not only involved in the metabolism of brown adipose tissue by increasing the specific gene expression of the mitochondrial uncoupling protein UCP-1, but also had a potential role in the metabolism of glucose in the muscle tissue as the Med1 knockdown mouse exhibited enhanced insulin sensitivity and improved glucose tolerance. Recently, a microarray analysis has also suggested that MED1 is involved in the control of energy homeostasis by regulating mitochondrial metabolic pathways (Becerril et al., 2012). Nevertheless, there is no direct evidence supporting the idea that MED1's ability to regulate the energy metabolism is closely linked to its appropriate function on mitochondria. As the function of MED1 relates to the energy metabolism, we hypothesize that MED1 plays an important role in mitochondrial function under an as yet unknown mechanism. Here, we knock down the expression of MED1 in C2C12 cells, and find that both the mtDNA content and mitochondrial mass are markedly increased and cell proliferation markedly suppressed. The gene expression level of Nrf1 and its downstream target genes: Tfam, Plormt, and Tfb1m, were all up-regulated and exhibited increased transcription levels in the

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J. Yu et al. / Mitochondrion 14 (2014) 18–25

OXPHOS pathway. Interestingly, the lack of MED1 also changed the mitochondrial respiratory profile with a prominent enhancement of glycolysis, and a sharply decreased intracellular ATP level. 2. Materials and methods 2.1. Cell proliferation and western blotting The C2C12 myoblasts were maintained in DMEM in the presence of 10% FBS. The myoblasts were transfected with MED1 shRNA and scramble shRNA (gifts from Dr. Xiaoting Zhang, University of Cincinati) using Lipofectamine 2000 (Invitrogen) and selected with 4 μg/ml puromycin (Invitrogen). Cells were collected at various numbers of days after transfection for analysis of mRNA and protein levels. Cell proliferation was measured using a Click-iT EdU Alexa Fluor® 488 Flow Cytometry assay kit (Invitrogen) by directly determining the level of DNA synthesis. 2 × 105 cells were plated in 6-well tissue culture plates. 24 h later the cells were incubated with EdU (10 μM) for 2 h. Cells were then harvested from tissue culture plates by incubation in 0.25% trypsin, and subsequently washed with ice-cold 1× PBS. Fixation was then subjected to the Click-iT reaction as described in the manufacturer's manual. Cells were then treated with RNase A followed by DNA staining with propidium iodide (PI) for 30 min at 37 °C. Data was collected and analyzed using flow cytometry analysis (Beckman FC500MCL) with a 530/30 nm and 610/20 nm emission for detection of EdU and PI, respectively. 3 days after puromycin selection, the levels of the MED1 protein in the control and in MED1−/− cells were analyzed via western blot as describe elsewhere (Zhang et al., 2005). We also analyzed the level of the protein Nrf1 (abcam) at 2, 3 and 4 days after selection as a time course analysis. Anti-β-actin (abcam) was used as a loading control. 2.2. Quantitative RT-PCR, mtDNA copy number analysis For quantitative RT-PCR, the total cellular RNA was extracted using an RNeasy Plus Mini kit (Qiagen) and first-strand cDNA was synthesized by reverse transcription of mRNA using an oligo(dT)20 primer and SuperScriptTM III Reverse Transcriptase (Invitrogen). Gene expression was analyzed on a 7900 HT Fast Real-time PCR System (Applied Biosystems) using a SYBR Green Master Mix (Roche Applied Science). Experiments were repeated in triplicate, and the relative gene expression was analyzed using the 2−ΔΔCT method (Livak and Schmittgen, 2001) by normalization to the 18S rRNA levels. All primers used in this study are listed in Supplemental Table S1. The copy number of mtDNA was determined by comparing the ratio of mtDNA to nDNA (18S rRNA) by real-time quantitative PCR as described previously (Schneider et al., 2011).

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were seeded in 96-well FluxPaks (Seahorse Bioscience Inc.) at the density of 10,000 cells/well and cultured overnight at 37 °C. Before running the Seahorse assay, cells were incubated for half hour without CO2 in un-buffered DMEM. The OCR and ECAR measurements were taken simultaneously under basal conditions and after the addition of oligomycin (1 μM), fluoro-carbonyl cyanide phenylhydrazone (FCCP) (0.5 μM), rotenone (1 μM) and antimycin A (5 μM) sequentially. The steady state OCR was defined as the baseline OCR before any reagents had been added. The OCR from non-mitochondrial and from mitochondrial respiration was identified separately. Results from these experiments were used to determine the basal respiration OCR (defined as the difference between the OCR of the basal conditions and nonmitochondrial OCR after R&A treatment); proton leakage (defined as the difference between the OCR following oligomycin exposure and non-mitochondrial OCR after rotenone treatment); maximal respiration (defined as the maximum OCR after FCCP uncoupling); and reserve capacity (defined as the difference between the maximum respiration OCR and the basal respiration OCR) (Chacko et al., 2013; Dranka et al., 2011). As the ATP-linked OCR may serve as a good indicator of the capacity of the mitochondria, we defined the coupling efficiency as the ratio of the ATP-linked OCR to that of the steady state OCR (Martin and David, 2011). After measurement of OCR and ECAR, data was normalized to the cell number in each well. All the chemicals were purchased from Sigma. 2.5. Measurement of intracellular ATP levels The relative intracellular ATP levels were measured using the CellTiter-Glo® Luminescent kit (Promega) according to the manufacturer's instructions. In brief, samples of 10,000 cells were seeded in a white opaque tissue culture plate, then lysed and incubated at room temperature for 10 min to stabilize the luminescent signal. Samples were measured by a Syneregy H1 (Bio-Tek) under the luminescence module. 2.6. Measurement of cellular ROS ROS measurements were performed using the DCFDA Cellular ROS Detection Assay Kit (Abcam/Mitosciences) in a 96-well dark microplate. Briefly, 10,000 cells were seeded into each well and cultured for at least 4 h before measurements were taken. Then, cells were washed once with PBS supplemented with 25 uM of 2′, 7′-dichlorofluorescein diacetate (DCFDA). After incubation at 37 °C for 40 min, cells were washed again and resuspended in PBS in the presence of freshly prepared 1 mM H2O2 and 10% FBS, followed by another 30 min culture at 37 °C. Samples with or without H2O2 stimulation were analyzed using a Syneregy H1 (Bio-Tek) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

2.3. Assessment of mitochondrial mass 2.7. Statistical analysis Cells were incubated with 80 nM Mitotracker® Red CMXRos (Invitrogen) for 30 min, and then fixed with 3.7% (w/v) formaldehyde plus 0.2% (v/v) Triton-X 100 in PBS for 15 min at room temperature. After washing with PBS, cells were further colabelled with the nuclear marker-DAPI. Coverslips were mounted onto slides and then sealed with regular nail polish. Confocal images were acquired using an Olympus Fluoview FV1000 with a 60 × objective. The mitochondrial mass was analyzed by measuring the relative pixel intensity of the CMXRosstained red signal.

All numerical values in the text and figures were presented as mean ± STDEV. Statistical significance was performed by the unpaired, two-tailed Student's t-test contained in Microsoft Office Excel (version 2007). Differences were considered significant at a P b0.05. 3. Results 3.1. Both mtDNA copy number and mitochondrial mass increase with the knockdown of MED1

2.4. Measurement of OCR and ECAR The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a XF96 Analyzer (Seahorse Bioscience) which allows real-time determination of oxygen (O2) and proton (H+) concentrations (Ferrick et al., 2008; Wu et al., 2007). Cells

In order to understand the role of MED1 as associated with mitochondria, we reduced the gene expression of MED1 by shRNA transfection in C2C12. MED1 expression was decreased distinctly by shMED1 transfection as compared with the control cells transfected by scramble shRNA (Fig. 1A). To determine the population of cells that were in the S-

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Fig. 1. Loss of MED1 increases the mtDNA content with suppression of cell proliferation. (A) Cells were transfected with shRNAs against MED1, with scramble as a control. Western blotting was performed 3 days after selection. (B) Cell proliferation assay after transfection. The Click-iT® EdU Alexa Fluor® 488 Flow Cytometry assay and PI staining were used for analysis of DNA replication in proliferating cells. (C) mtDNA copy numbers were quantified using real-time PCR. Mitochondrial DNA was normalized to 18S nuclear DNA. n = 3. Data is represented as the mean ± STDEV of duplicates. *p b 0.05, Student's t test, as compared to control.

phase and undergoing active DNA synthesis, we labeled cells with the thymidine analog EdU. As shown in the upper F panel of Fig. 1B, the Sphase (EdU-positive) population of the MED1−/− cells had been reduced to 22.2%, as compared to that of 53.2% in the control cells. More than 70% of the population of the MED1−/− cells was at rest at the G0/G1-phase. This indicated that the cell proliferation of C2C12 had been significantly decreased with the knockdown of MED1. This was consistent with previous reports that the suppression of MED1 causes a decrease in the proliferation of prostate and breast cancer cells (Vijayvargia et al., 2007; Zhu et al., 1999). We supposed that the suppression of MED1−/− cell proliferation may be a survival strategy elicited under such stress circumstances. Therefore, we examined the mitochondrial DNA copy number in both control and in the MED1−/− cells. At days 2 and day 3 after selection, a dramatic increase (105 fold (P = 0.022) and 12.10-fold (P = 0.048 respectively) in the mtDNA copy number was found in the MED1−/− cells, as compared to that of the control cells (Fig. 1C). It seems that an increase of mtDNA content was an early event in response to the loss of MED1. A dramatic increase of mitochondrial mass in MED1−/− cells was also detected by using a Mitotracker® Red CMXRos. As shown in Fig. 2A, C2C12 cells were stained by the Mitotracker at 2 days after selection. Interestingly, the mitochondrial mass in the MED1−/− cells (Fig. 2B) had increased by approximately 80% (P b0.0001) over that in the controls'. Most likely, this indicated that the knockdown of

MED1in C2C12 cells triggered mitochondrial biogenesis, accompanied by the suppression of cell proliferation. 3.2. Nrf1 and OXPHOS genes were up-regulated in MED1-deficient cells Transcriptional coactivators are major regulators of mitochondrial biogenesis by inducing gene transcription of oxidative phosphorylation. (Campbell et al., 2012; Van-Tienen et al., 2010). Was the increased mitochondrial transcription part of a secondary response to increase mitochondrial biogenesis under knockdown of MED1? The nuclear respiratory factors (NRFs: Nrf1 and Nrf2) are the key transcription factors required for mitochondrial respiration, mtDNA transcription and replication. It has been shown that over-expression of Nrf1 results in an increased mtDNA content (Bonawitz et al., 2006; Li et al., 2012; Piantadosi et al., 2008; Scarpulla et al., 2012; Suliman et al., 2010). Thus, we firstly evaluated the mRNA level of Nrf1 and Nrf2 before and after suppression of MED1 expression in the C2C12 cells. As shown in Fig. 3A, the mRNA level of Nrf1 had increased by 119% on day 2 and by 198% on day 3 after selection. In contrast, no significant change was found in Nrf2 transcription. The Nrf1 protein level was analyzed by western blot at 2, 3 and 4 days after selection. As shown in Fig. 3E, the Nrf1 protein had increased significantly in MED1−/− cells at 2, 3 and 4 days compared to that of the controls'. The loss of MED1 indeed up-regulated the level of Nrf1.

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Fig. 2. Mitochondrial mass is increased in MED1-deficient cells. (A) Cells transfected by scramble (“-”) and MED1shRNA (“+”) were labeled using a Mitotracker® Red CMXRos (Invitrogen) for mitochondrion and a DAPI for the nucleus. Images were captured with an Olympus Fluoview FV1000 using a 60× objective. Bar equals 10 μm. (B) The pixel intensity was measured by an FV10-ASW3.1 viewer from (A) and was plotted as the relative density of the mitochondrion. Data is represented as the mean ± STDEV of duplicates. *p b 0.05, Student's t test, compared to control.

Several genes, identified as Nrf1 downstream target genes, have also been reported to be involved in mtDNA maintenance. These include Tfam, Plormt and Tfb1m (Campbell et al., 2012; Falkenberg et al., 2002;

Larsson et al., 1998; Takamatsu et al., 2002). Transcription of Tfam, Plormt and Tfb1m was all significantly up-regulated by 73%, 240% and 429% respectively, compared to controls, in MED1−/− cells (Fig. 3B).

Fig. 3. Knockdown of MED1 increased the expression of mitochondrial biogenesis related genes. (A) mRNA levels of nuclear respiratory factors (NRFs); (B) mRNA levels of Tfam, Plormt and Tfb1m, regulators for expression and replication of the mitochondrial genome; (C)) mRNA levels of OXPHOS genes encoded by the nucleus; (D) mRNA levels of OXPHOS genes and 12S rRNA encoded by mitochondrial genome per se. (E) The protein level of Nrf1 by Western blotting at 2, 3 and 4 days after selection. Data is represented as the mean ± STDEV of duplicates.

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As gene transcription of oxidative phosphorylation is another major contributor to mitochondrial mass, we extended our analysis to the transcription of genes involved in the OXPHOS pathway in both the nucleus (Ndufs3, Sdhb, Sdhc, Cox5b) (Dhar et al., 2008; Kelly and Scarpulla, 2004; Scarpulla, 2008) and mitochondria (ND4L, CYTB, ND1, COII, 12SrRNA). As shown in Fig. 3C and D, all these genes, which encode for respiratory complexes from nucleus and mitochondrion, were upregulated to cope with the increase of mitochondrial mass. These results suggest that the loss of MED1 triggered an increase in mitochondrial mass through inducing both the gene transcription of Nrf1 and oxidative phosphorylation. 3.3. Mitochondrial respiration altered upon MED1 knockdown with increased glycolysis Increased mitochondrial biogenesis usually serves as a cellular attempt to compensate for a severe respiratory chain deficiency. As MED1 was reported to have an influence on energy balance (Chen et al., 2010), could it be that the mitochondrial respiratory function has changed in MED1−/− cells? We therefore assessed parameters in OCR and ECAR under different respiratory conditions induced by sequential injections of oligomycin, FCCP, and rotenone plus antimycin A (Fig. 4). The progress curve seemed higher in MED1−/− cells than in the control's. This implied that the respiratory function had changed upon MED1 loss. The steady state OCR in MED1−/− cells was similar to that of control cells (Fig. 4A). The average OCR in the steady state, detected from 4 different time points, was 101.1 pMoles/min and 107 pMoles/min in

control cells and MED1−/− cells, respectively. There was a significant decrease in the basal respiration OCR in the MED1−/− cells as compared to that of the control's (P b0.01, B-OCRC = 62.63, B-OCRM = 32.65 pMoles/min). This was in accordance with the suppression of cell proliferation in MED1−/− cells. The OCR decreased in response to oligomycin (which inhibits the ATP synthase, complex V) to the extent that the cells were using mitochondria to generate ATP. In this study, beyond injection of oligomycin, the control samples exhibited a deeper fall in OCR (ATP-linked A-OCR: P b 0.01, A-OCRC = 52.13, OCRM = 22.65 pMoles/ min) than did the MED1-deficient cells. This indicates that the mitochondrial oxygen consumption used for ATP synthesis in control cells was greater than that in the MED1-deficient cells. No difference was observed in the OCR ascribed to proton leakage across the mitochondrial membrane. A lower basal respiration and ATP-linked OCR seemed to lead to a significant decrease in the maximal OCR after FCCP uncoupling in MED1−/− cells (Maximal OCR: P b0.05, M-OCRC = 90.61, M-OCRM = 81.03 pMoles/min). In contrast, the reserve capacity in MED1−/− cells was stimulated to a significantly higher degree (reserve capacity OCR: P b 0.01, R-OCRC = 28.11, R-OCRM = 48.03 pMoles/min) suggesting that there was a high demand upon the mitochondrial energy metabolism in response to MED1 loss. Lastly, rotenone and antimycin A were injected to inhibit whole electron transfer in both complex I and complex III. This caused a dramatic suppression of the OCR. What remained was an OCR that was attributable to O2 consumption from non-mitochondrial sources. There was a significant increase in non-mitochondrial OCR upon MED1 loss (Non-mito OCR: P b 0.01, N-OCRC = 39.47, N-OCRM = 75.31 pMoles/min). It is well known that non-mitochondrial respiration is dependent on glycolysis (Herst

Fig. 4. Loss of MED1 alters mitochondrial respiration. Cells were seeded into 96-well FluxPaks (10,000 cells/well) and allowed to grow for 24 h before the measurement of OCR and ECAR. (A) OCR measurement under the basal condition followed by the sequential addition of oligomycin (O) 1 μM, FCCP (F) 0.5 μM, Rotenone (R) 1 μM and antimycin A (A) 5 μM. (B) Parameters of the cellular oxygen consumption from the progress curve are plotted as basal OCR, APT-linked OCR, proton-leak OCR, maximal OCR, the reserve capacity of cells and the contribution of non-respiratory-chain oxygen consumption. (C) Coupling efficiency, calculated as the ratio between the ATP-linked OCR and the steady state OCR. (D) ECAR was recorded synchronously. (E) The increase in ECAR was compared between the control and MED1shRNA cells and was plotted as the function of enhanced aerobic glycolysis. Data is the mean ± STDEV of duplicates.*p b 0.05, **p b 0.01 Student's t test, compared to controls.

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et al., 2004). This increase in non-mitochondrial OCR indicated that the O2 consumption from glycolysis had increased significantly in MED1−/− cells. The coupling efficiency was calculated here using the ratio of the ATP-linked OCR to that of the steady state OCR. The coupling efficiency in MED1−/− cells (22.2%) was clearly lower than that of the controls cells (51.1%). To confirm the occurrence of an increase of O2 consumption in aerobic glycolysis, the ECAR level, as a surrogate for glycolysis (Watanabe et al., 2006), was monitored simultaneously. In MED1-deficient cells, we noticed that a significant increase of ECAR appeared after injection of oligomycin and after FCCP. The maximal increase of ECAR in the presence of FCCP was up to 138% (P b 0.0001), of that of the control's (Fig. 4C. This implies an increased requirement for glycolysis. This was correlated with the results of increased non-mitochondrial OCR above. These results support our notion that the loss of MED1 had altered the mitochondrial respiratory profile towards increasing aerobic glycolysis.

3.4. Marked change in ATP generation but not in ROS production Fig. 4B shows the loss of MED1 suppressed ATP-linked OCR. This implies that ATP generation by the mitochondria may be decreased in MED1−/− cells. Thus, we confirmed whether the loss of MED1 decreases ATP generation. Relative intracellular ATP levels were measured using the CellTiter-Glo® Luminescent kit. As expected, a significant decrease of ATP generation was observed in MED1−/− cells (Fig. 5A), accounting for an average of 85% ±3% (P b 0.0001) relative to the mean of the control cells. This finding further supports the notion that MED1 plays a key role in the energy metabolism (Becerril et al., 2012; Ge et al., 2002). The reduced generation of ATP in MED1−/− cells directly reflected a deficiency in the respiratory chain. This led to an increase in mitochondrial biogenesis as a cellular attempt to compensate for such an ATP generation reduction. The levels of ROS generation as cellular indicators under oxidative stress were measured via a microplate assay under normal conditions and H2O2 stimulation. The mean fluorescence intensity was recorded to measure the level of ROS for each sample. The ratio of mean intensity between unstimulated samples and samples stimulated with H2O2 in each of the cell lines was calculated to delineate the reaction when levels of ROS increase under oxidative stress. As shown in Fig. 5B, there was no significant difference observed in the ROS level between MED1-deficient cells and the control cells.

Fig. 5. ATP & ROS generation in MED1-deficient cells. (A) Measurement of cellular ATP levels using a bioluminescence assay (10,000 cell/well) by Synergy H1 under the luminescence module. (B) The ROS ratio of mean intensity between with or without H2O2 stimulation. The rates of ROS generation from MED1shRNA cells and the controls s were analyzed by Synergy H1 with or without H2O2 stimulation. The relative ratio of intensity (stimulated vs unstimulated with H2O2) was calculated. Data is the mean ± STDEV of duplicates.*p b 0.05, **p b 0.01 Student's t test, compared to controls.

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4. Discussion In the present in vitro study, we have endeavored to elucidate a novel aspect of MED1 and its association with mitochondria. We have concluded that MED1 does have effect on mitochondrial function. The loss of MED1 decreases ATP generation which elicits mitochondrial biogenesis by the increased transcription of genes related to mitochondrial genome maintenance and OXPHOS as a futile compensatory response. With the knockdown of MED1, in the progression of mitochondrial biogenesis, respiration is deficient, glycolysis enhanced and the coupling efficiency decreased. This phenomenon is highly similar to that of the response of increased mitochondrial biogenesis in cases of human and mouse mitochondrial disease (Wenz et al., 2008; Wredenberg et al., 2002). In previous studies, MED1 has been shown to participate in the regulation of energy homeostasis. It has been reported that Med1−/− cells exhibit a defect in PPARγ-stimulated adipogenesis in cultured mouse embryonic fibroblasts (Ge et al., 2002) and in the PPARα-mediated oxidation of fatty acids in liver parenchymal cells (Jia et al., 2004). These studies strongly imply that the mitochondrion may be the target organelle of MED1 due to the essential role that the mitochondrion has in energy homeostasis. The Med1 knockdown mice had enriched mitochondria in white muscle, relative to the control mice (Chen et al., 2010). During the life of mice in vivo, increased mitochondrial biogenesis has usually been considered to be an adaptive response to muscle exercises (Nisoli and Carruba, 2006; Rowe et al., 2012) or caloric restriction (Civitarese et al., 2007). Our results here indicate that the loss of MED1 prominently enhanced both mtDNA levels and mitochondrial mass in C2C12 cells cultured in vitro. Corresponding to its function in the energy metabolism, the loss of MED1 causes a significant decrease of ATP production. This, in turn, triggers mitochondrial biogenesis as a compensation pathway for this respiratory chain deficiency in a similar manner to that of many other mitochondrial diseases (Wenz et al., 2008). As there is no change of proton leak OCR in MED1-deficient cells, the decreased generation of ATP is consistent with the finding of reduced ATP-linked OCR — may contribute to the lower basal OCR because of the inhibition of cell proliferation. A decreased coupling efficiency may be another reason for the lesser generation of ATP. However, as suggested by the Warburg hypothesis, rapidly increasing mtDNA and mitochondrial mass has a great dependence on glycolysis to provide both metabolic intermediates and rapid ATP support. Upon the knockdown of MED1, decreased ATP generation induces a compensatory up-regulation in the processes of cytoplasmic glycolysis as was noted by Kang et al. (2013). Thus, we suggest that the major reason for a lesser generation of ATP was that the increased contribution of glycolysis to the total cellular ATP generation was not sufficient to compensate for the decrease in ATP generation by OXPHOS. Interestingly, the MED1-deficient cells had a higher reserve capacity OCR, which was available for the cells to call upon when the bioenergetic demand was changed (Dranka et al., 2010; Hill et al., 2009). This renders the cells more flexible to energy and growth demands (Yoshida et al., 2013). We suggest that the increase of aerobic glycolysis may account, at least in part, for the enhanced reserve capacity to protect cells from death or apoptosis as a response to MED1 loss. Thus, as loss of MED1 decreases the generation of ATP, it leads to a higher demand for more mtDNA content and mitochondrial mass to compensate for this deficient respiratory function. Mitochondrial biogenesis is a multifactorial coordinated processes of the nuclear and mitochondrial genomes (Garesse and Vallejo, 2001; Scarpulla, 2008; Scarpulla et al., 2012). It is significant that the nuclear respiratory factor-1 (NRF1) has been identified as a transcription factor that activates the expressions of OXPHOS components, mitochondrial transporters, mitochondrial ribosomal proteins and the expression of Tfam, Tfb1m and other genes in mtDNA replication and expression (Hock and Kralli, 2009; Li et al., 2012; Scarpulla, 2008). NRF2 (also known as GABP) has also been reported to facilitate the increase of mitochondrial biogenesis by increased expression of the estrogen

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related receptor α (ERRα), under a different regulation pathway to that of NRF1 (Ohtsuji et al., 2008; Wenz, 2013). Importantly, gene expression analysis in MED1-deficient and control cells revealed that the loss of MED1 led to induction of biogenesis via an increased expression of Nrf1 rather than Nrf2. The over-expression of biogenesis and OXPHOS genes was considered as a part of mitochondrial biogenesis (Falkenberg et al., 2002; Larsson et al., 1998; Scarpulla, 2008). PGC-1α was widely thought to be an indispensable transcriptional cofactor for mitochondrial density, playing a direct role in the transcription of the nuclear OXPHOS genes or an indirect role in the transcription of nuclear and mitochondrial OXPHOS genes via the Nrf1 pathway (Psarra and Sekeris, 2008; Scarpulla, 2011; Ventura et al., 2008; Wenz, 2013). On the other hand, Rowe et al. (2013) suggested that the lack of PGC-1 s leads to a deficiency in mitochondrial respiration, whilst preserving mitochondrial content. Nevertheless, our result showed that the higher mitochondrial mass was disconnected from the up-regulation of PGC-1α in MED1deficient cells (Fig. 1S). This is consistent with a previous report by Chen et al. (2010) who noted that the expression of PGC-1α was not altered in the white muscle of MED1 knockout mice relative to that of wildtype mice. This finding suggests that an increase in PGC-1α expression is not required for the enhanced mitochondrial biogenesis by knockdown of MED1 in C2C12 cells. Although MED1 has also been shown to directly interact with PGC1α via multiple domains (Viswakarma et al., 2010; Wallberg et al., 2003), the underlying mechanism requires further investigation. Additionally, as there was no significant difference in ROS generation as compared by the control and MED1−/− cells. ROS, acting as signaling molecules to induce mitochondrial biogenesis (Acin-Perez et al., 2009), should be inattentive. Thus, both increased mtDNA content and mitochondrial mass in this study were clearly induced by the upregulation of Nrf1 and OXPHOS gene transcription. Given together, our study establishes that suppression of MED1 increases both mtDNA content and mitochondrial mass via upregulation of Nrf1 and OXPHOS genes, accompanied by an increase in glycolysis. This seems to be a compensatory response to cope with a decrease of ATP production in MED1−/− cells. These observations highlight that MED1 does play a role in mitochondrion function and mitochondrial control of energy homeostasis. Acknowledgments We thank Dr. Xiaoting Zhang for his plasmids and MED1 antibody. We also thank Chris Wood and Yongmei Xi for their advice and critical reading of the manuscript. This work was supported by the National Key Technologies R&D Program Grant (2012BAI09B03) from the Ministry of Science and Technology of China (MXG and PJ) and a grant from the Ministry of Science and Technology of Zhejiang Province (2013R10042) to PJ. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mito.2013.12.004. References Acin-Perez, R., Salazar, E., Brosel, S., Yang, H., Schon, E.A., Manfredi, G., 2009. Modulation of mitochondrial protein phosphorylation by soluble adenylyl cyclase ameliorates cytochrome oxidase defects. EMBO Mol. Med. 1, 392–406. Anderson, S., Bankier, A., Barrell, B.G., De Bruijn, M., Coulson, A., Drouin, J., Eperon, I., Nierlich, D., Roe, B., Sanger, F., 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465. Arany, Z., Lebrasseur, N., Morris, C., Smith, E., Yang, W., Ma, Y., Chin, S., Spiegelman, B.M., 2007. The transcriptional coactivator PGC-1beta drives the formation of oxidative type IIX fibers in skeletal muscle. Cell Metab. 5 (1), 35–46. Becerril, S., Rodríguez, A., Catalán, V., Sáinz, N., Ramírez, B., Gómez-Ambrosi, J., Frühbeck, G., 2012. Transcriptional analysis of brown adipose tissue in leptin-deficient mice lacking inducible nitric oxide synthase: evidence of the role of Med1 in energy balance. Physiol. Genomics 44 (13), 678–688.

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