Mitochondrial biogenesis in mtDNA-depleted cells involves a Ca2+-dependent pathway and a reduced mitochondrial protein import ´ ´ Ludovic Mercy, Aurelia de Pauw, Laetitia Payen, Silvia Tejerina, Andree Houbion, Catherine Demazy, Martine Raes, Patricia Renard and Thierry Arnould Laboratory of Biochemistry and Cellular Biology, University of Namur (FUNDP), Namur, Belgium Keywords biogenesis; calcium ⁄ CaMKIV; gene expression; mitochondrial dysfunction; protein import Correspondence T Arnould, Laboratory of Biochemistry and Cellular Biology, University of Namur (F.U.N.D.P.), 61 rue de Bruxelles, 5000 Namur, Belgium Fax: +32 81 724135 Tel: +32 81 724321 E-mail: thierry.arnould@fundp.ac.be (Received 31 May 2005, revised August 2005, accepted 11 August 2005) doi:10.1111/j.1742-4658.2005.04913.x Alterations in mitochondrial activity resulting from defects in mitochondrial DNA (mtDNA) can modulate the biogenesis of mitochondria by mechanisms that are still poorly understood In order to study mitochondrial biogenesis in cells with impaired mitochondrial activity, we used rho-L929 and rho0143 B cells (partially and totally depleted of mtDNA, respectively), that maintain and even up-regulate mitochondrial population, to characterize the activity of major transcriptional regulators (Sp1, YY1, MEF2, PPARgamma, NRF-1, NRF-2, CREB and PGC-1a) known to control the expression of numerous nuclear genes encoding mitochondrial proteins Among these regulators, cyclic AMP-responsive element binding protein (CREB) activity was the only one to be increased in mtDNA-depleted cells CREB activation mediated by a calcium-dependent pathway in these cells also regulates the expression of cytochrome c and the abundance of mitochondrial population as both are decreased in mtDNA-depleted cells that over-express CREB dominant negative mutants Mitochondrial biogenesis in mtDNA-depleted cells is also dependent on intracellular calcium as its chelation reduces mitochondrial mass Despite a slight increase in mitochondrial mass in mtDNA-depleted cells, the mitochondrial protein import activity was reduced as shown by a decrease in the import of radiolabeled matrix-targeted recombinant proteins into isolated mitochondria and by the reduced mitochondrial localization of ectopically expressed HA-apoaequorin targeted to the mitochondria Decrease in ATP content, in mitochondrial membrane potential as well as reduction in mitochondrial Tim44 abundance could explain the lower mitochondrial protein import in mtDNA-depleted cells Taken together, these results suggest that mitochondrial biogenesis is stimulated in mtDNA-depleted cells and involves a calcium-CREB signalling pathway but is associated with a reduced mitochondrial import for matrix proteins Abbreviations ANT2, adenine nucleotide translocase isoform 2; ATF2, activating transcription factor 2; b-ATPase, beta subunit of Fo-F1-ATPase; CaMKIV, calmodulin-dependent kinase IV; COX I, II, IV and VIII, cytochrome c oxidase subunit I, II, IV and VIII; CPT-1, carnitine palmitoyl transferase-1; CREB, cAMP-responsive element binding protein; cyt c, cytochrome c; DHFR, dihydrofolate reductase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HA, hemaglutinin; mtDNA, mitochondrial DNA; MEF2, myocyte enhancer factor 2; mtTFA ⁄ Tfam, mitochondrial transcription factor A; NAO, nonyl acridine orange; NFAT, nuclear factor of activated T cells; NFjB, nuclear factor kappaB; NRF-1 and 2, nuclear respiratory factor-1 and 2; OXPHOS, oxidative phosphorylation; PGC-1a and b, PPARc coactivator-1 a and b; PPARc, peroxisome proliferator-activated TATA-box receptor c; PRC, PGC-1a-related coactivator; R123, rhodamine 123; ROS, reactive oxygen species; Sp1, specificity protein 1; TBP, TATA-binding protein; TNFa, tumor necrosis factor a; TIM, translocase of inner membrane; TOM, translocase of outer membrane; USF-2, upstream stimulatory factor-2; YY1, ying-yang 1; Dwm, mitochondrial membrane protential FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS 5031 Mitochondrial biogenesis in mtDNA-depleted cells Mitochondria play crucial functions in health and diseases and many mitochondrial disorders, that mainly affect tissues with high energy demands, result from mutations or deletions in the mitochondrial genome that impair the synthesis of one or more of the mitochondrial encoded respiratory protein leading to a decrease in oxidative phosphorylation (OXPHOS) capacity [1–3] Mitochondrial proliferation and increase in the expression of respiratory proteins are a common manifestations found in patients with mitochondrial myopathies or mtDNA depletion that is responsible for the so-called ‘raggedred fibers’ phenotype of skeletal muscle [4] In addition, several studies have now shown that mitochondrial dysfunction leads to the stimulation of mitochondrial biogenesis For example, muscle from mouse with myopathy and hypertrophic cardiomyopathy resulting from the targeted inactivation of the gene encoding the heart muscle isoform of the adenine nucleotide translocator (ANT 1) display abnormal proliferation of mitochondria [5] In a conditional knockout mice for mitochondrial transcription factor A (Tfam), a transcription factor involved in the regulation of the mitochondrial genome replication and transcription [6], leading to mtDNA-depletion and prolonged respiratory chain deficiency, Hansson et al recently reported that the mitochondrial mass increases in respiratory chain deficient embryos and differentiated mouse tissues [7] If each mammalian cell contains several hundreds to more than a thousand mitochondria, it is thus now clear that the size, shape, and abundance of mitochondria vary dramatically in different cell types and may change under different energy demand [8] The abundance of mitochondria in a cell is determined by division and ⁄ or biogenesis of the organelle [9] that can be defined as a complex biological process requiring the synthesis of phospholipids and cooperative interactions between proteins encoded by both the nuclear and mitochondrial genes [10,11], the mitochondrial protein import and their assembly [3] However, mechanisms leading to mitochondrial biogenesis in cells deficient for mitochondrial activity are still poorly understood As the protein-coding capacity of mammalian mtDNA is limited to 13 respiratory subunits that are necessary for mitochondrial function and integrity, more than 95% of the genes necessary for mitochondrial biogenesis are encoded in the nucleus and their expression is regulated by the activation of a small set of specific transcription factors and signalling pathways [9,12,13] The first class of nuclear transcriptional regulators involved in the biogenesis 5032 L Mercy et al of the organelle includes specific DNA-binding transcription factors such as nuclear respiratory factors and (NRF-1 and NRF-2) that act on the genes coding for constituent subunits of the OXPHOS system and mtDNA replication [14–17] Other factors such as CREB (cyclic-AMP responsive element-binding protein) [18], PPARc (peroxisome proliferator activated receptor gamma) [19,20],or the muscle-specific transcription factor MEF2 (myocyte enhancer factor 2) [21,22] and general factors such as YY-1 (ying yang 1) [23], USF-2 (upstream stimulatory factor-2) [24], and Sp1 (specificity protein 1) [25] have been described to act as activators or repressors of nuclear genes encoding mitochondrial proteins and more particularly proteins involved in the OXPHOS complexes A second class of regulators contains coactivators that are unable to bind DNA such as PGC-1a (peroxisome proliferator activated receptor gamma coactivator-1alpha) and related family members (PRC and PGC-1b) [26] These proteins can interact with DNA-bound transcription factors in order to coordinate their action in the expression of genes essential for cellular energetics and mitochondrial biogenesis [27] as recently shown in exerciseinduced skeletal muscle adaptation [28] Numerous signalling pathways have been reported to act upstream of these transcriptional regulators involved in mitochondrial biogenesis by stimulating the expression of nuclear genes encoding respiratory proteins Firstly, reactive oxygen species (ROS) have been described to promote expression of cytochromes c1 and b through a H2O2-dependent signalling in human cells that respond to defective respiratory function [29] Moreover, a treatment of human MRC-5 lung cells with antimycin A that elevated the intracellular ROS production induced an increase in the mitochondrial mass in the cells [30] ROS have also been reported to enhance the expression of nuclear genes involved in mitochondrial biogenesis such as NRF-1 and Tfam in rho0 HeLa S3 cells [31] Secondly, a nitric oxide (NO)-cGMPdependent pathway has been reported to control mitochondrial biogenesis in several mammalian cell types [32] On the other hand, many links exist between a high cytosolic calcium concentration and the increase in mitochondrial biogenesis as a treatment of muscle cells with A23187 (a calcium ionophore) triggers the expression of cyt c in a PKC-dependent manner [33] Ojuka et al also demonstrated that intermittent increases in cytosolic calcium stimulate mitochondrial biogenesis in muscle cells and suggested that calcium is the mediator responsible for the increase in mitochondrial population in response to exercise [34,35] Furthermore, mitochondrial dysfunction and calcium homeostasis are closely FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al interdependent in cell signalling and cell death [36] Indeed, it was observed that depletion of mtDNA below a certain level as well as treatment of mammalian cells with respiratory inhibitors increased steady-state levels of cytosolic calcium that may change activities of several Ca2+-dependent transcription factors such as CREB [37], nuclear factor of activated T-cells (NFAT), activating transcription factor (ATF2) and nuclear factor kappa B (NFjB) that increase OXPHOS gene expression including subunit Vb of cytochrome c oxidase (COXVb) and thus stimulate mitochondrial biogenesis [38] A role of calcium and calmodulin-dependent kinases (CaMKs) in the control of mitochondrial biogenesis has also been demonstrated in skeletal muscle of transgenic mice that over-express a muscle-specific constitutively active form of CaMKIV [39], a kinase we previously found to be activated in L929 and 143B mtDNA-depleted cells and responsible for CREB activation [37] During the mitochondrial biogenesis process, the majority of the thousand or more mitochondrial proteins are required to be imported from nuclear-encoded cytosolically synthesized precursors The import of these proteins is achieved by different mechanisms known to operate during the import of the two major classes of mitochondrial proteins such as the hydrophilic proteins with cleavable presequences and hydrophobic proteins with multiple internal signals [40] The mitochondrial protein import involves an important group of proteins including translocase of the outer membrane (TOM) such as Tom40, Tom20, Tom70 and translocase of the inner membrane (TIM) such as Tim22 and Tim23 family members [40] as well as numerous chaperones such as Hsp70 [41] forming effectors, adaptors and receptors of the mitochondrial protein import machinery Several reports mentioned the significance of the protein import in the rate of mitochondrial protein import as several stimuli, including contractile activity of skeletal muscle, thyroid hormone treatment, and muscle differentiation can alter the expression of the import proteins that ultimately lead to a change in protein import rate and mitochondrial phenotype [42–46] Numerous mtDNA-depleted cell lines have been generated by long-term treatment with ethidium bromide [47,48] or DNA polymerase-c inactivation [49] to study important mitochondrial defects in OXPHOS, calcium homeostasis alteration, ROS production and more recently resistance to apoptosis [37,38,50] It is also interesting to emphasize that mtDNA-depleted cells maintain their ability to generate mitochondria-like structure and a mitochondrial membrane potential (Dwm) [51–53] Thus, even if the FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS Mitochondrial biogenesis in mtDNA-depleted cells mechanisms involved in the mitochondrial biogenesis of mtDNA-depleted cells are poorly understood, it is now more evident that mtDNA is not essential for the biogenesis of mitochondrial-like structure in proliferating cells In this study, to address the question of mitochondrial biogenesis in cells depleted of mtDNA, we used rho-L929 and rho0143 B cells (partially and totally depleted of mtDNA, respectively) to evaluate the retrograde signalling that controls the expression of nuclear genes encoding mitochondrial proteins and the activity of mitochondrial matrix-tageted protein import We first showed that cells depleted of mtDNA not only maintain but up-regulate the biogenesis of mitochondria, as the mitochondrial staining with specific fluroescent dyes and the expression of cyt c are both increased in these cells We next studied the activity status of several key transcriptional regulators known to control the biogenesis of mitochondria such as NRF-1 ⁄ 2, PPARc, MEF2, CREB, Sp1 and YY-1, as well as the abundance of the coactivator PGC1a and found that CREB is the only overactivated factor in mtDNA-depleted cells We also showed that CREB regulates cyt c expression and could play a role in mitochondrial biogenesis in mtDNA-depleted cells as the over-expression of dominant negative mutants (K1-CREB and M1-CREB) decreases both cyt c expression and nonyl acridine orange (NAO) accumulation used to monitor mitochondrial mass in cells The dependence of mitochondrial biogenesis on intracellular calcium in mtDNA-depleted cells was also evidenced as chelation of intracellular calcium reduces the abundance of mitochondrial population However, despite a slight increase in mitochondrial population and cyt c abundance in mtDNA-depleted cells, the mitochondrial import activity for matrix proteins is reduced in these cells as we observed a decrease in the import of radiolabeled matrix-targeted recombinant proteins into isolated mitochondria and a lower mitochondrial localization of ectopically expressed HA-apoaequorin addressed to the mitochondria We also clearly showed that lower mitochondrial import for matrix proteins in mtDNA-depleted cells is associated with a decrease in ATP content, in mitochondrial membrane potential as well as with a reduction in mitochondrial Tim44 abundance, an important effector of mitochondrial import apparatus Taken together, these results suggest that mitochondrial biogenesis leading to the accumulation of ‘abnormal’ mitochondria in mtDNA-depleted cells could be mediated, at least partly, by a calcium-CREB signalling pathway but is associated with a reduced mitochondrial import for matrix proteins 5033 Mitochondrial biogenesis in mtDNA-depleted cells Results L Mercy et al A Maintenance of mitochondrial structure in mtDNA-depleted cell lines Rho-L929 cells [54] as well as rho0143B cells were previously characterized by our group for mtDNA-depletion and impaired mitochondrial function [37,51] To compare mtDNA-depletion in rho-L929 and rho0143B cells used in this study, COXI expression was determined by western blot analysis in parental and mtDNA-depleted cells (Fig 1A) As expected, this mtDNA-encoded subunit of cytochrome c oxidase is not expressed in rho0143B and its expression is barely detectable in rho-L929 cells To investigate whether or not mtDNA depletion leads to modifications in mitochondrial content, the abundance and the morphology of mitochondria were compared in rho-L929 and L929 cells using transmission electron microscopy (TEM) (Fig 1B) In rho-929 cells, the morphology of mitochondria is clearly different as they appear rounder, swollen and less dense to electrons as already reported for several other mtDNA-depleted cell lines [53,55] When mitochondrial population abundance was assessed with Mitotracker Red, a specific mitochondrial fluorescent probe used for mitochondrial mass detection [56,57], we found a punctuated pattern of staining that is compatible with a mitochondrial retention and localization of the fluorescent probe in both mtDNA-depleted and parental cells (Fig 1C) Quantitative analysis of Mitotracker Red accumulation using spectrofluorimetry also revealed that staining is dependent on loading time and suggests a slight increase in rho-L929 cells when assessed after 30 in the presence of the dye (supplementary Fig S1) Similar results were also found for rho0143B cells stained with Mitotracker Red or NAO, a lipophilic cation that has a high affinity for mitochondrial cardiolipin rich membranes [58] (Figure 7A) We are aware that in several cell types NAO staining has been described recently to be also dependent on the mitochondrial membrane potential [59] As the NAO staining is not reduced, and is even slightly increased in mtDNAdepleted cells while the mitochondrial membrane potential is lower in these cells [51,52], these data suggest a higher mitochondrial mass in mtDNA-depleted cells This statement is also supported by the analysis of mitochondrial population abundance perfomed by the quantification of the cell area occupied by mitochondria on transmission electron microscopy (TEM) micrographs from L929 and rho-L929 cells Indeed, using the nih image software free online (http://rsb info.nih.gov/nih-image/Default.html) we analysed three 5034 B C Fig Mitochondrial structures are still observed in mtDNA-depleted cells that not express mitochondrial-encoded markers (A) Western blotting analysis of COXI subunit expression in 143B, rho0143B, L929 and rho-L929 cells Equal loading was determined by the immunodetection of TBP (B) Electron micrographs of rhoL929 and parental L929 cells showing the presence of rounder shaped mitochondria (arrows) (magnification: 25 200 X) (C) Staining for mitochondrial population with Mitotracker Red in L929, rhoL929, 143B and rho0143B incubated with 250 nM of the cationic dye for 30 and processed for confocal microscopy observation Scale bars ¼ 10 lm and arrows indicate punctuated mitochondrial staining section images (10.7 square inches; magnification 15 600·) taken from random observations and found that the surface corresponding to mitochondria represents 11.2 ± 2.3% and 18.8* ± 2.5% (*P < 0.05) for L929 and rho-L929 cells, respectively FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al These data show that the abundance of mitochondrial population in cells without mtDNA is maintained and even slightly increased when compared to parental cells Effect of mtDNA depletion on some mitochondrial markers As mitochondrial biogenesis is dependent on the expression of numerous nuclear-encoded genes, we next determined the expression level of several key mitochondrial markers that cover energetic pathway or mitochondrial protein import machinery such as the b-subunit of the Fo-F1-ATPase (b-ATPase), the adenine nucleotide translocator isoform (ANT2), COXVb or Tom40 and Tim44 The relative mRNA abundance of b-ATPase, COXVb and Tim44, determined by realtime PCR, is significantly up-regulated (> twofold increase) in rho0143B cells compared to parental cells (Fig 2A) However, we found that some of these genes might be variously expressed at the protein level when assessed on cleared cell lysates Indeed, the Fo-F1-ATPase b-subunit is similarly expressed in both 143B and rho0143B cells (Fig 2B), a data in agreement with a previous report showing that the expression of b-ATPase is unchanged in rho0 HeLa S3 and rho0143B [52] The reason for this discrepancy between mRNA and protein abundance is unknown but could involve a post-transcriptional regulation as it has been proposed before for the over-expression of Tfam and NRF-1 at the transcriptional level that was not reflected at the protein level in mtDNA-depleted cells [31,60] However, this regulation might also be relatively specific as Tim44 was found to be over-expressed at the protein level in rho0143 B cells, a data in accordance with the increase in the messenger RNA for this marker (Fig 2A) To discriminate between a transcriptional regulation and mRNA stabilization in the accumulation of these transcripts, we next transfected cells with plasmids encoding chloramphenicol acetyl transferase (CAT) reporter gene driven by the authentic promoter of the cyt c or the b-ATPase gene (Fig 2C) CAT activity was significantly up-regulated (respectively three and sixfold increase) in rho0143B, a result that is consistent with a positive transactivation of these genes In order to make sure that the activation of the cyt c promoter is really the result of a mitochondrial inhibition and not a consequence of an indirect long-term cell adjustment to mtDNA depletion, we tested the effect of mitochondrial metabolic inhibitors on the promoter activity 143B cells were first transiently transfected with the cyt c-CAT plasmid and then incubated for 24 h with lm FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS Mitochondrial biogenesis in mtDNA-depleted cells antimycin A (a complex III inhibitor) or 10 lm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler that both impair the OXPHOS We found that cyt c promoter was also activated in response to both treatments (Fig 2D) These results show that mitochondrial activity impairment per se is responsible for the up-regulation of cyt c gene expression and several other mitochondrial markers while ANT2 does not seem to be overexpressed in mtDNA-depleted cells (Fig 2C) As cyt c is a common marker used to characterize mitochondrial biogenesis [32] and in order to directly address both the expression and the distribution of the protein, the endogenous expression of cyt c was first analysed by western blotting performed on proteins extracted from enriched-mitochondrial fractions of mtDNA-depleted cell lines (Fig 3A) The protein is more abundant (two- to threefold increase) in the mitochondria of both mtDNA-depleted cell lines suggesting that not only the protein is over-expressed but is also imported into mitochondria These data have been confirmed by immunostaining of cyt c in the different cell lines (Fig 3B) Quantification of fluorescence signals in cell sections indicates both over-expression and a wider distribution of the protein in mtDNA-depleted cells (Fig 3C) Taken together, these data strongly suggest an over-expression of cyt c that might be associated with a more abundant mitochondrial population in mtDNA-depleted cells The role and the functional significance of cyt c over-expression in mitochondria of mtDNA-depleted cells is an intriguing observation that should be addressed in the future The regulation of mitochondrial marker expression in mtDNA-depleted cells is a process that might involve the activation of transcription factors described to control the biogenesis of mitochondria Reduced expression and activity of NRF-1, NRF-2 and Tfam in mtDNA-depleted cells It has been reported previously that mtDNA-depleted HeLa cells display increased mRNA levels of NRF-1 and Tfam genes [61] We thus evaluated NRF-1 and NRF-2, two major transcription factors that control the expression of several nuclear genes encoding mitochondrial proteins [6,14,62] and Tfam expression [60,63,64] Interestingly, while NRF-1 expression is only slightly reduced in both mtDNA-depleted cells (10–20%), NRF-2 expression is strongly decreased in rho0143B and rho-L929 cells by 60 and 80%, respectively (Fig 4A) As both factors have been implicated in the control of Tfam expression [6] which is known to regulate mtDNA transcription and replication [65], 5035 Mitochondrial biogenesis in mtDNA-depleted cells L Mercy et al A B C D Fig Expression of mitochondrial markers in mtDNA-depleted cells (A) Total RNA was isolated from 143B (white) and rho0143B cells (black) before the relative amount of transcript encoding the b-subunit of the Fo-F1-ATPase, Tim44 and COXVb was determined by real-time PCR using SYBR green staining and normalized for TBP (TATA-box binding protein) used as a reference gene Results are expressed as relative mRNA abundance compared to control 143B cells and represent means ± SD for independent extractions (B) Western blot analyses of clear lysate proteins (35 lg) prepared from 143B and rho0143B cells using specific antibody to Tim44 and to the F1-ATPase b subunit Equal protein loading between lanes was determined by the immunodetection of a-tubulin (C) Promoter activity of ANT2, cyt c and b subunit of F1-ATPase determined by CAT activity in transiently cotransfected 143B (white) and rho0143B (black) cells with CAT reporter constructs driven by the authentic promoter of these genes and a plasmid encoding b-galactosidase CAT activity (cpm: count per minute) was determined 48 h post-transfection and normalized for b-galactosidase activity Results are expressed in percentages of control cells (n ¼ 4) (*,***): significantly different from control cells with, respectively, P < 0.05, and P < 0.001 (D) Effect of antimycin A and FCCP on the promoter activity of cyt c determined by CAT activity 143 B cells were transiently transfected with a CAT reporter construct driven by the cyt c promoter and were incubated or not (control, CTL) for h with lM antimycin A or 10 lM FCCP CAT activity (cpm) was determined 48 h post-transfection and normalized for b-galactosidase activity Results are expressed in percentages of control cells as means for n ¼ we thus monitored the expression of Tfam in the murine cell line [66] and show that this factor is downregulated in rho-L929 cells (Fig 4B) These results are 5036 in agreement with data obtained in rho-C2C12 cells [60] and suggest that the activity of NRFs is decreased in mtDNA-depleted cells To test this hypothesis, cells FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al Mitochondrial biogenesis in mtDNA-depleted cells A B Fig Expression of cytochrome c protein is enhanced in mtDNA-depleted cells (A) Western blot analysis of mitochondrial cyt c (mtcyt c) abundance performed on proteins extracted from mitochondrial-enriched fractions of 143B, rho0143B, L929, and rhoL929 cells Equal protein loading between mtDNA-depleted and corresponding parental cell lines was determined by the immunodetection of the nuclear-encoded COXIV (B) Immunostaining of cyt c and confocal microscopy analysis performed on paraformaldehyde-fixed and Triton permeabilized 143B, rho0143B, L929 and rho-L929 cells (C) Analysis of fluorescence intensity perfomed on cell sections presented in B using the QUANTIFY software from Leica Fluorescence intensity profiles are plotted from A to B direction for the different cell lines C were transiently transfected with luciferase reporter constructs driven by a minimal TK promoter linked to either four copies of the binding site for NRF-1 (4X NRF-1) or the Tfam authentic promoter responsive to NRF-1 [57] Under these conditions, a dramatic and highly significant decrease in luciferase activity was obtained for both constructs in rho0143 B (Fig 4C) These results suggest that the transactivation mediated by NRF-1 is reduced in mtDNA-depleted cells As NRF-1 DNA-binding and activity have been shown to be positively regulated after phosphorylation by casein kinase II [67], the activity of this enzyme was assessed in vitro after immunoprecipitation of the kinase from FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L929 and rho-L929 Results show an important decrease in the activity of casein kinase II in mtDNAdepleted cells (Fig 4D) Furthermore, in human fibroblasts, ROS production has also been reported to mediate a retrograde signalling pathway that can enhance the expression of NRF-1 and Tfam mRNA in rho0HeLa or antimycin A-treated cells [29,31] ROS production was thus determined in L929 and rho-L929 cells using the dichlorofluorescein (DCF) probe In these conditions, while antimycin A, used as a positive control, triggers a significant increase in ROS production in L929, we found that ROS generation was reduced by almost 40% in rho-L929 cells (Fig 4E) as 5037 Mitochondrial biogenesis in mtDNA-depleted cells already reported for other cell lines depleted of mtDNA [68,69] These cells are also less-responsive to an antimycin A treatment Taken together, these results support a lower expression and activity of NRF-1 and Tfam in mtDNA-depleted cells A B C D E 5038 L Mercy et al Activity of YY1, Sp1, PPARc, MyoD, MEF2 and CREB in mtDNA-depleted cells Beside the crucial role of NRF-1 and NRF-2 in the regulation of OXPHOS genes [15], the transcriptional control of numerous nuclear genes encoding mitochondrial proteins also involves other transcription factors such as YY1, Sp1, PPARc, MyoD, MEF2 and CREB [13,70–74] Using a sensitive colorimetric assay system as previously described for NF-jB [75], we thus measured the DNA-binding activity of these transcription factors to specific synthetic DNA consensus sequence in nuclear protein extracts prepared from 143B L (L929) and rho0143B cells (Fig 5A) or from L929 and rho-929 cells (Fig 5B) The amount of Sp1, PPARc and MyoD that binds to DNA is reduced in both mtDNA-depleted cell lines while MEF2 DNAbinding activity is unchanged in these cells These results suggest that a chronic inhibition of mitochondrial activity impairs the DNA-binding activity of Fig Decrease in NRF-1, NRF-2 and Tfam expression in mtDNAdepleted cells is associated with a reduction in casein kinase II activity and a lower ROS production (A) Western blot analysis of NRF-1 and NRF-2 expression performed on 20 lg of proteins from clear lysates of 143B, rho0143B, L929, and rho-L929 cells Equal loading between mtDNA-depleted and corresponding parental cell line was determined by the immunodetection of a-tubulin (B) Western blot analysis of Tfam expression performed on 20 lg of proteins from clear lysates of L929 and rho-L929 cells Equal loading between lanes was determined by the immunodetection of a-tubulin (C) mtDNA-depletion decreases the activity of a NRF-1responsive synthetic promoter as well as the activity of the authentic Tfam promoter 143B and rho0143 B cells were transiently cotransfected with 0.25 lg of a CMV ⁄ b-gal expression plasmid and 0.5 lg of the 4X NRF-1-Luc construct or 0.5 lg of the Tfam promoter-Luc construct Luciferase activity was determined 24 h posttransfection and normalized for b-galactosidase activity Results are expressed in percentages of control 143B cells as means ± SD for n ¼ (**: significantly different from control cells with P < 0.01) (D) Casein kinase II activity is reduced in rho-L929 cells The enzyme was immunoprecipitated from cleared lysates of L929 and rho–L929 cells In vitro activity was then determined in the presence of a synthetic peptide and [c-32P]ATP as described in the ‘experimental procedures’ Results represent the radioactivity associated with the substrate and are expressed in cpm as means for two samples The amount of immunoprecipitated kinase in the different conditions is shown on the western blot below (E) ROS production is reduced in rho-L929 cells Cells were incubated for 30 at 37 °C with lM DCF and then incubated or not with lM antimycin A for 60 Cells were then lysed before cellassociated fluorescence was measured with a spectrofluorimeter Results are expressed in arbitrary fluorescence units as means ± S.D for n ¼ **, ***: significantly different from L929 cells as ´ determined by an ANOVA I and Sheffe’s contrasts with, respectively, P < 0.01 and P < 0.001 FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al Mitochondrial biogenesis in mtDNA-depleted cells structs encoding a luciferase reporter gene driven by promoters responsive to these factors (Fig 5C) The transcriptional activity of these factors was either unchanged (YY1) or slightly decreased (PPARc) in rho0143B cells Using luciferase constructs that are specific ally activated by Sp1 or NRF-2, we also observed a decrease in the transcriptional activity of these factors in both mtDNA-depleted cells (data not shown) On the contrary, CREB, a transcription factor we previously identified as specifically activated in cells with impaired mitochondrial activity [37], is activated in rho0143B cells as shown by a 2.5-fold increase in the luciferase activity encoded by a reporter construct driven by the a-inhibin promoter that contains several CRE sites [76] Several of the nuclear transcription factors that control the expression of genes encoding mitochondrial proteins are coordinated by PGC-1a, which induces NRF-1 and NRF-2 expression and coactivates several mitochondrial regulatory factors such as NRF-1, MEF2 or PPARc [76a] In skeletal muscle, it has been shown that a p38 MAPK signalling stimulates PGC1a expression and promotes mitochondrial biogenesis [28] Indeed, consequently to its activation, PGC1a causes an increase in mRNA for several genes encoding mitochondrial proteins such as cyt c, COXII and COXIV, the b-ATPase, CPT-1 and uncoupling proteins (UCPs) in a cell type-selective manner [19,57,77] In both rhoL929 and rho0143B cell lines, PGC1a expression is decreased, as shown by a strong reduction of PGC-1a promoter activity and protein abundance analysed by western blotting and immunostaining (supplementary Fig S2) Fig Effect of mtDNA-depletion on Sp1, PPARc, MyoD and MEF2 DNA-binding activity and transactivation Microwells containing the DNA probes were incubated with 10 lg of nuclear proteins prepared either (A) from 143B (white) and rho0143B (black) or (B) from L929 (white) and rho-L929 (black) After the colorimetric reaction, absorbance was measured at 490 nm and the results were expressed in percentages of corresponding controls as means ± SD for n ¼ (C) Effect of mtDNA-depletion on the transcriptional activity of YY1, PPARc and CREB 143B and rho0143B cells were transiently transfected with 0.25 lg of a CMV ⁄ b-gal plasmid and 0.5 lg of responsive luciferase constructs responsive to either YY1 (Msx2SSLuc), PPARc (3X-PPRE-TK-Luc) or CREB Luciferase activity was determined 24 h post-transfection and normalized for b-galactosidase activity Results are expressed as percentages of controls as means ± SD for n ¼ *, **, ***: significantly different from ´ corresponding controls as determined by an ANOVA I and Sheffe’s contrasts with, respectively, P < 0.05, P < 0.01, and P < 0.001 several key transcription factors involved in the control of genes encoding mitochondrial proteins In order to study the activity of YY1 and PPARc further, cells were transiently transfected with conFEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS Role of a CREB ⁄ CaMKIV pathway in the mitochondrial biogenesis of mtDNA-depleted cells We showed previously that a CaMKIV-CREB signalling pathway is specifically activated in cells with impaired mitochondrial activity [37] and more recently several studies reported the importance of this pathway in the regulation of mitochondrial biogenesis in skeletal muscle [39,78] To test the potential role of CREB in the biogenesis of mitochondria in mtDNA-depleted cells, we over-expressed K-CREB and M1-CREB, two dominant negative mutants of CREB [79] and measured their effect on the CAT reporter gene driven by the cyt c promoter, containing two functional CRE sites [18] While inhibition efficiency is rather different for both dominant negative forms that could be explained by either their different mechanism of action or their respective level of expression, the over-expression of both dominant mutants significantly reduces 5039 Mitochondrial biogenesis in mtDNA-depleted cells A B C Fig Cytochrome c up-regulation is dependent on CREB in mtDNA-depleted cells (A) cyt c promoter activity in transiently cotransfected 143B (white) and rho0143B (black) cells with plasmids encoding K-CREB, M1-CREB, a CREB-sensitive CAT reporter construct driven by the cyt c promoter and an expression plasmid encoding the b-galactosidase CAT activity was determined in cell lysates 48 h post-transfection Substrate-associated radioactivity (cpm) was normalized for b-galactosidase activity and results are expressed in percentages of 143B control cells as means ± SD for n ¼ (***: significantly different from 143B control cells with P < 0001; + and + + + : significantly different from rho0 cells with, respectively, P < 0.05 and P < 0.001) (B) Representative western blot image of cyt c expression assessed in L929 and rho-L929 cells transiently transfected with either plasmids encoding K-CREB, M1-CREB, CaMKIV(T200A) or a pGL2 empty vector (L929 and rho-L929 control cells) Equal loading was determined by the immunodetection of a-tubulin (C) Quantification of cyt c expression after optical density determination of the different signals and normalization by the abundance of a-tubulin The mean value in L929 cells was set as a reference for comparison and results are expressed in fold-increase as means ± SD (n ¼ 3) *: significantly different from L929 cells with P < 0.05 the activity of cyt c promoter in rho0143B cells (Fig 6A) The over-expression of K-CREB and M1-CREB also decreases the expression of endogenous cyt c in rho-929 cells (Fig 6B,C) Furthermore, the over-expression of a CaMKIV dominant mutant 5040 L Mercy et al (CaMKIVT200A) [80] is also able to repress cyt c expression in rho–L929 cells (Fig 6B,C), suggesting that a calcium ⁄ CaMKIV-CREB pathway might be involved in the induction of cyt c expression in mtDNA-depleted cells As a single mitochondrial protein marker is not enough to characterize mitochondrial biogenesis, we next used NAO dye to monitor total abundance of the mitochondrial population in rho0143 B cells that over-express either K-CREB or M1-CREB After 48 h, we consistently found a reduction of about 15–20% in the NAO signal in these cells (Fig 7A), suggesting that global mitochondrial abundance can be reduced by inhibiting CREB activity Taken together, these data suggest that the presence of mitochondria in mtDNA-depleted cells could be dependent on an active CaMKIV-CREB pathway As mtDNA-depletion causes a sustained increase in cytosolic calcium that activates cell signalling such as CaMKIV-CREB or JNK pathway [37,38], and because intermittent or sustained increase in cytosolic calcium of skeletal muscle during exercise results in an increase Fig Mitochondrial mass is dependent on CREB and calcium in mtDNA-depleted cells (A) Spectrofluorimetric determination of mitochondrial abundance measured by NAO accumulation in 143B (white) and rho0143B (black) cells transiently transfected with a plasmid encoding K-CREB or M1-CREB for 48 h or (B) in cells incubated in the presence of BAPTA (10 lM) for 72 h Results are expressed in arbitrary fluorescence unit normalized for protein content as means ± SD for n ¼ (A) or means for n ¼ (B) FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al in mitochondria [34,35], we next incubated mtDNAdepleted cells for 72 h with 10 lm BAPTA, an intracellular calcium chelator, before the mitochondrial abundance was assessed with NAO staining (Fig 7B) In these conditions, NAO fluorescence was reduced by almost 40% in both mtDNA-depleted cell lines, suggesting a decrease in mitochondrial population Mitochondrial protein import for matrix-targeted proteins is reduced in mtDNA-depleted cells As mitochondrial biogenesis in mtDNA-depleted cells most likely requires mitochondrial protein import, we next assessed this process by two different experimental approaches We first adapted an in vitro assay, that has been used mainly in yeast [81], to quantitatively determine the import of radiolabeled mitochondrial proteins targeted to the matrix in purified mitochondria as visualized on the electron micrograph of enriched mitochondrial fractions (Fig 8A) Two precursor fusion proteins targeted to the mitochondrial matrix, the subunit-9 of the ATPsynthase– dihidrofolate reductase (DHFR) and a truncated form of the cytochrome b2 (b2(167)D-DHFR), have been translated and radiolabeled with [35S]methionine in vitro (Fig 8B) The mitochondria-associated radioactivity was then measured on mitochondrial fractions of 143B and rho0143B cells treated with proteinase K after Fig The mitochondrial protein import is reduced in mitochondria isolated from mtDNA-depleted cells (A) Electron micrograph of an enriched mitochondrial fraction prepared for the import assay and illustrated for mitochondria purified from 143B cells analysed by transmission electron microscopy Scale bar: 100 nm (B) Autoradiography of the cytochrome b2 and the ATPase subunit-9 chimeric proteins translated and radiolabeled in vitro (10% of the output) b2(167D)-DHFR consists of the first 167 amino acids of the cytochrome b2 precursor fused to the full-length mouse DHFR by a linker of two amino acids The cytochrome b2 presequence consists of an amino-terminal matrix-targeting sequence (residues 1–31) and a sorting sequence (residues 32–80) Su9-DHFR contains the first 66 amino acids of the subunit-9 of Neurospora crassa ATPase fused to DHFR (C,D) For the mitochondrial protein import assay, isolated mitochondria (30 lg) from 143B and rho0143B cells were incubated for 10 at 25 °C with reticulocyte lysate containing 35 S-labelled Su9-DHFR (C) or b2(167D)-DHFR (D) The import assay was then stopped by the addition of lM valinomycin To remove nonimported preproteins, all samples were treated with proteinase K (40 lgỈmL)1) for 15 on ice After mitochondria isolation, associated radioactivity was counted Results are presented as representative data for three independent experiments and expressed in c.p.m as means ± SD (E) Western blotting analysis of b-ATPase, Tom40 and Tim44 abundance performed on mitochondrial purified fractions of 143B and rho0143B cells FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS Mitochondrial biogenesis in mtDNA-depleted cells the import assay The global mitochondrial import was reduced by 66% and 85%, respectively, for Su9-ATPase-DHFR and b2(167)D-DHFR proteins (Fig 8C,D) We next wondered if the reduced global mitochondrial import of matrix proteins in mtDNAdepleted cells could be due to a decrease in the b-barrel Tom40 core of the TOM complex, through which the precursor proteins are passing before being transferred to other mitochondrial compartments [82] This is not likely the case as the amount of Tom40 in purified mitochondria is comparable in rho0 and parental 143B (Fig 8E) despite a strong increase in the expression of Tom40 in both mtDNA-depleted cell B A C D E 5041 Mitochondrial biogenesis in mtDNA-depleted cells lines as shown by western blot analysis of clear cell lysates and immunostaining (supplementary Fig S3) In addition, western blot analysis of mitochondrial fractions prepared from 143B and rho0143B cells revealed that the abundance of b-ATPase and Tom40 is similar in both cell lines while Tim44, an important effector of mitochondrial protein import that interacts with mtHsp70 (mitochondrial heat-shock protein 70) [41], could only be detected in parental cells (Fig 8E) While we cannot rule out that the abundance of the various markers in the mitochondria of mtDNA-depleted cells may result from a different degradation of the different proteins in the mitochondria of cells depleted of mtDNA, these results indicate that endogenous mitochondrial proteins might be differentially imported in the mitochondria of mtDNA-depleted cells L Mercy et al In order to extend our data on the mitochondrial protein import in mtDNA-depleted cells in situ, the import activity was also determined in rho-L929 cells transfected with a cDNA encoding a chimeric protein containing HA-tagged apoaequorin and the mitochondrial presequence of the COXVIII subunit that specifically targets the fusion protein to the mitochondrial matrix [83] Confocal microscopy observations (Fig 9A) and quantitative analysis of fluorescence signals on sections of cells immunostained for HA-tagged apoaequorin and cyt c, used as a mitochondrial marker, revealed a decrease in the colocalization between both proteins in the mitochondria of rho-L929 as evidenced by the reduced match of fluorescence signals found in the overlapping fluorescence profiles for these cells (Fig 9B) A B 21.37 µm 16.87 µm 5042 Fig The mitochondrial protein import is reduced in mtDNA-depleted cells (A) L929 and rho-L929 cells were transiently transfected with an expression plasmid encoding HA-tagged apoaequorin targeted to the mitochondria (mtAEQ ⁄ pcDNA1) Expression of HA-apoaequorin in a transfected cell (green), abundance of endogenous cyt c (red), and colocalization (overlay) were then visualized by confocal microscopy after the immunostaining of both proteins (B) Analysis of fluorescence intensity perfomed for HA-apoaequorin (green) and cyt c (red) on cell sections using the QUANTIFY software from Leica Fluorescence intensity profiles showing expression level and colocalization are plotted from A to B direction for L929 and rho-L929 cells (representative of about 30 analyses) FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al Effect of mtDNA depletion on membrane potential and ATP content It is well known that the protein import into mitochondria is driven by the mitochondrial membrane potential (Dwm) and the ATPase activity of mtHsp70 that requires ATP [84,85] As expected, and as already reported for other mtDNA-depleted cell lines such as L6 myocytes [86], the ATP content in the mtDNAdepleted cells was also decreased by 60–70% in both rho0143B and rho-L929 cells (Fig 10A) We thus qualitatively assessed the mitochondrial membrane potential (Dwm) with the cationic fluorescent dye Rhodamine 123 (R123) at 500 nm (the lowest concentration that gave a significant fluorescence signal above background in our experimental conditions) and found that the mitochondrial membrane potential was reduced in both mtDNA-depleted cell lines when compared with their related parental cell lines showing a normal oxidative capacity (Fig 10B,C) An argument in favor that relative mitochondrial membrane potential (Dwm) measurement can be assessed in these conditions is brought by the fact that FCCP induces a significant decrease in the R123 fluorescence These results clearly show that both driving forces required for matrix mitochondrial protein import are reduced in cells with impaired mitochondrial activity and could explain why mitochondrial matrix-targeted proteins import is reduced in mtDNA-depleted cells Mitochondrial biogenesis in mtDNA-depleted cells A B C Discussion In this study, we have used two different cell lines that differ mainly by their origin and the severity of mtDNA-depletion to investigate the nature of some mechanisms involved in the mitochondrial biogenesis of cells with a chronic mitochondrial dysfunction Indeed, stimulation of mitochondrial biogenesis and increase in the expression of nuclear genes encoding mitochondrial proteins such as respiratory enzymes seem to be a common cell response to mitochondrial dysfunction or high energy demand observed in many pathophysiological conditions [86a,70,86b] and experimental models [5,7] While mitochondrial biogenesis has been thought to be dependent on the expression and replication of mitochondrial genome [87], several studies have reported later on significantly higher steady-state levels of nuclear-encoded mRNAs for mitochondrial proteins in rho0 cells [88,89] Moreover, mitochondria-like structures can still be observed in the cytoplasm of mtDNA-depleted cells suggesting an active biogenesis of the organelle in these cells [52,53] FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS Fig 10 Mitochondrial protein import driving forces are decreased in mtDNA-depleted cells (A) ATP content was measured in the various cell lines using a luciferin-luciferase assay and results calculated in RLU (relative light unit) were normalized for protein content and expressed in percentages of control cells as means ± SD for n ¼ 3.: Significant differences from L929 or 143B control cells with *P < 0.05, ***P < 0.001 Effect of mtDNA-depletion on mitochondrial membrane potential as determined by the accumulation of R123 Rho-L929 and L929 (B) or 143B and rho0143B (C) seeded at 50 000 cells per well in a 12-well plate were preincubated or not with 10 lM FCCP for h and then loaded 30 with Rhodamine 123 (500 nM) before fluorescence was measured in a spectrofluorimeter Results are expressed in fluorescence intensity unit normalized for protein content as means ± SD for n ¼ + + , + + +: significantly different from parental cell lines with, respectively, P < 0.01 and P < 0.001 *, ** and ***: significantly different from corresponding untreated cells with P < 0.05, P < 0.01 and P < 0.001 respectively 5043 Mitochondrial biogenesis in mtDNA-depleted cells Using electron and confocal microscopy as well as quantitative determination assays to monitor total mitochondrial population based on the analysis of the surface occupied by mitochondria in rho-L929 cells and on the accumulation of fluorescent dyes (Mitotracker Red and NAO) that stain mitochondria by two different mechanisms, we found that not only mitochondrial population is maintained in mtDNA-depleted cells but there is even a slight increase in the abundance of mitochondria in these cells A previous study reporting the fragmentation of the mitochondrial network leading to a distribution of small individual organelles in rho0 MRC5 fibroblasts and rho0143B emphasized the fact that while the structure ⁄ morphology of mitochondria is modified in mtDNA-depleted cells, the total amount of mitochondrial volume did not appear modified between normal and rho0 cells [90] However, no quantitative analysis was performed by these authors As many mitochondrial proteins are encoded by the nuclear genome, it is likely that rho0 cells are able to express many components of mitochondria-like structures despite a depletion of mtDNA [91] and to import mitochondrial proteins through TOM and TIM complexes Indeed, mtDNA-depleted cells maintain a reduced mitochondrial membrane potential by mechanisms reported elsewhere [51,52,91] Mitochondrial membrane potential and ATP content are known to be crucial for matrix protein import as they both act as driving forces [92] While mitochondrial biogenesis has been intensively studied in yeast [93] and in the context of muscle exercice [46,70], the signalling pathway linking bioenergetic stress and mitochondrial biogenesis [94] is still poorly understood, particularly in mtDNA-depleted cells The goal of the present study was thus to analyse the impact of a chronic bioenergetic defect induced by mtDNA-depletion on the activity status of key mechanisms involved in mitochondrial biogenesis (key transcriptional regulators and mitochondrial protein import activity) in cell lines that are partially (rhoL929) or totally (rho0143B) depleted of mtDNA First, our data show that several genes encoding mitochondrial proteins such as bATPase, Tim44, COXVb and cyt c are up-regulated in mtDNA-depleted cells while ANT2 is not affected by a chronic metabolic stress Interestingly, the over-expression of COXVb was already reported previously in rho-C2C12 cells [38] Tom40 is also clearly over-expressed in both mtDNA-depleted cells while its mitochondrial abundance is unchanged in these cells As demonstrated for several mitochondrial markers, the up-regulation involves an active transcription of these genes but additional regulation might be specifically involved in 5044 L Mercy et al the control of the protein synthesis as only Tim44 and cyt c but not b-ATPase subunit were found to be overexpressed at the protein level (Fig 11) In conclusion, even if mtDNA-depleted cells are unable to generate ATP by the OXPHOS, it seems that some proteins involved in the respiratory chain are over-expressed and accumulate in the mitochondrial structure as demonstrated for cyt c We are aware that only few proteins were analysed to assess mitochondrial biogenesis but as several mitochondrial markers are up-regulated at the transcriptional level in mtDNA-depleted cells, we thus next investigated the activity status of several transcriptional regulators known to control the expression of nuclear genes encoding mitochondrial proteins and thus the mitochondrial biogenesis [13,16] A key observation of this study is that the expression and ⁄ or the activity of major factors described to control mitochondrial biogenesis in various cell types such as NRF-1, NRF-2, Tfam and other regulators such as MEF2, MyoD, PPARc or the coactivator PGC-1a [13,16,17,27,57] are either down-regulated or not modified in both mtDNA-depleted cell lines (Fig 11) One can also emphasize that NRF-1 and Tfam down-regulation is correlated with a decrease in ROS production and casein kinase II activity in rho-L929 cells supporting the positive role of these molecules in the expression and the activity of this transcription factor [29,31,67] Tfam, a NRF-1 regulated gene [6], was already described to be down-regulated in other rho0 cell lines [95,96] In addition, as PGC-1a affects the expression as well as the transcriptional activity of NRF-1 [57], it is thus likely that lower abundance of the protein observed in rho-L929 cells results from the decrease in NRF-1 and PGC-1a expression in these cells Among the various regulators that control the expression of genes known to encode mitochondrial proteins analysed in this study, we clearly confirmed that CREB is the only transcription factor found to be activated in mtDNA-depleted cells [37] CREB has been described as a key regulator of several nuclear genes encoding mitochondrial proteins such as cyt c [18,97], CPT-1 [98] and MnSOD [99] Furthermore, the promoter of murine and human Tom40 (NM_016871 and NM_006114), a mitochondrial marker we found to be over-expressed in mtDNAdepleted cells, also contains putative CRE sites (TGACGT) within a fragment of 1000 bp upstream the transcription start site (in silico analysis performed with dbtss: http://dbtss.hgc.jp/ and tfsearch: http:// molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html softwares), suggesting that Tom40 could also be a CREB-target gene Furthermore, promoter analysis for FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al Mitochondrial biogenesis in mtDNA-depleted cells Fig 11 Schematic representation of a molecular pathway that potentially contributes to mitochondrial biogenesis in mtDNA-depleted cells leading most likely to ‘abnormal’ mitochondria Chronic mitochondrial dysfunction induces a calcium-CaMKIV-CREB-dependent pathway leading to the over-expression of several nuclear genes encoding mitochondrial proteins and the adaptative ⁄ compensatory mitochondrial biogenesis The role of this pathway is probably important in these conditions as major regulators of mitochondrial biogenesis are either downregulated (Sp1, PPARc, MyoD, PGC-1a) or unchanged (MEF2, YY1) in mtDNA-depleted cells However, some up-regulated transcripts (+) like b-ATPase not lead to protein accumulation (¼) Furthermore, some up-regulated proteins are not found in the mitochondria (– : Tim44), some not accumulate more in the organelle (¼ : Tom40, b-ATPase) while others (+ : cyt c), suggesting a differential import and ⁄ or degradation in the mitochondria of mtDNA-depleted cells The thickness of arrows that symbolize the mitochondrial import is related to the importance of the recovered protein in the mitochondria Discontinued arrows represent potential control not analysed in this study b-ATPase, beta subunit of ATPsynthase; CaMKIV, calmodulin-dependent kinase IV; CREB, cAMP-responsive element binding protein; cyt c, cytochrome c; DHFR, dihydrofolate reductase; mtDNA, mitochondrial DNA; NRF-1 and 2, nuclear respiratory factor-1 and 2; MEF2, myocyte enhancer factor 2; PGC-1a, PPARc coactivator-1 a; PPARc, peroxisome proliferator-activated receptor c; Sp1, specificity protein 1; TIM, translocase of inner membrane; TOM, translocase of outer membrane; YY1, Ying-Yang Tim44 (NM_006351) and for beta subunit of F1-ATPase (NM_001686) using the new CREB Target Gene Database (http://natural.salk.edu/CREB/) [100] revealed that both genes are also potentially regulated by CREB as, using chromatin immunoprecipitation assay (ChIP), the factor was found to bind their promoter in hepatocytes and other cell types Taken FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS together, these data suggest that several of the up-regulated nuclear genes encoding mitochondrial proteins in response to mitochondrial dysfunction could be controlled by activated CREB It might be apparently surprising to find an up-regulation of mitochondrial biogenesis in mtDNA-depleted cells that is accompanied by a decrease in the abundance of 5045 Mitochondrial biogenesis in mtDNA-depleted cells PGC1a However, Wang et al have recently identified PPARd as a major and direct effector in the adaptative muscle response to endurance exercise characterized by an increase of mitochondrial biogenesis, up-regulation of mtDNA and over-expression of mitochondrial markers without any modification in PGC1a protein [101] The activation of a calcium ⁄ CaMKIV ⁄ CREB signalling pathway initiated by a chronic mitochondrial dysfunction (Fig 11) has been previously characterized in the mtDNA-depleted cells used in this study [37,51] Here we found and extend our data by showing that this pathway is also crucial for mitochondrial biogenesis in mtDNA-depleted cells as the over-expression of CREB dominant negative mutants (K-CREB and M1-CREB) or of a negative form of CaMKIV (CaMKIVT200A) decreases both the activation of the cyt c promoter and the expression of the endogenous protein Furthermore, the mitochondrial abundance is lower in mtDNA-depleted cells that over-express K-CREB or M1-CREB or in cells incubated with BAPTA, a calcium chelator In a recent study using rho-C2C12 cells and MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) fibroblasts dealing with the protein import machinery and transcription factors involved in mitochondrial biogenesis, it was shown that differential behavior and gene expression can be observed depending on mtDNA defects Indeed, in rho-cells, Tom20 and Tim23 protein levels were reduced whereas mtHSP70 was induced, leading to a small increase in enhanced yellow fluorescent protein (EYFP) import into mitochondria in these cells, while EYFP import was not altered in MELAS cells [60] In both mtDNA-depleted cell lines used in the present study, the expression of mtHSP70 is unchanged (data not shown) Furthermore, these authors have shown that NRF-1 and transcription factor A (Tfam) expression declined in rho-cells whereas no change was observed for PGC-1alpha [60] Finally, we addressed the mitochondrial protein import activity in rho-L929 and rho0143B cells by two different approaches that both support a reduction of mitochondrial matrix protein import in mtDNAdepleted cells First, using chimeric recombinant proteins (Su9ATPsynthase–DHFR and cytochrome b2 [b2(167)D-DHFR] translated and radiolabeled in vitro, we found that mitochondrial import by purified mitochondria from rho0143B is dramatically reduced Second, the quantitative analysis of fluorescence signal profiles showing a reduction in the colocalization between the mitochondrial-targeted HA-apoaequorin ectopically over-expressed and the endogenous cyt c in L929 mtDNA-depleted cells suggests a decrease in the import of the protein in situ Finally, the abundance of 5046 L Mercy et al Tim44, an essential component of the machinery that mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane [102,103], is also dramatically reduced in the mitochondria of rho0143B while the gene is over-expressed both at the transcript and the protein levels The reduction of Tim44 abundance in the mitochondria of mtDNAdepleted cells could thus result from a lower mitochondrial protein import and contribute to explain, in addition with a reduced ATP content and a decrease in the Dwm (Fig 10), how matrix protein import is reduced in the mitochondria of mtDNA-depleted cells Furthermore, these results showing a deficit in the protein import for Tim44 not observed for b-ATPase subunit, Tom40 or cyt c that even accumulates in the mitochondria, suggest that mitochondrial dysfunction might impair mitochondrial protein import differently according to the protein of interest (Fig 11) It is noteworthy that while the mechanisms of cyt C import are still obscure [104] mitochondrial import of apocytochrome c is apparently independent of the major receptors Tom20, Tom70 and even Tom40 [104] Thus, a reduction in matrix protein import can probably not be extended to all mitochondrial proteins Taken together, these results show that mitochondria of mtDNA-depleted cells are qualitatively different than the ones found in parental cells In conclusion, our results show that nuclear factors usually described as key effectors in the control of mammalian mitochondrial biogenesis are down-regulated in mtDNA-depleted cells while CREB is activated Furthermore, several CREB-responsive nuclear genes encoding mitochondrial proteins are up-regulated in mtDNA-depleted cells For example, we previously showed that CaMKIV-dependent CREB activation in mtDNA-depleted cells is a pathway activated by mitochondrial dysfunction [37] Here, we show that this pathway could be involved in the mitochondrial biogenesis of mtDNA-depleted cells While the function of mitochondria-like structures in cells depleted of mitochondrial genome is unclear and should be addressed in the future, other mitochondrial activities than ATP production as well as prevention of apoptosis have been proposed Experimental procedures Cell cultures The characterization of L929, rho-L929, 143B and rho0143B cells was described previously [37,54,105] Cells were grown in Dulbecco’s modified Eagle’s medium containing 4.5 mgỈmL)1 glucose and 10% fetal bovine FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al serum (Gibco, BRL, Paisley, UK) and maintained at 37 °C in a humidified incubator (Heraeus, Hassau, Germany) under 5% (v ⁄ v) CO2 As a polyclonal cell population, rhoL929 were kept in media containing ethidium bromide (400 ngỈmL)1), uridine (50 lgỈmL)1) and pyruvate (1 mm) to compensate for the respiratory metabolism impairment and support cell growth Transmission electron microscopic (TEM) L929, rho-L929 cells or mitochondria-enriched fractions prepared from 143B cells were processed for electron microscopy as described previously [106] Briefly, samples were fixed overnight with 2.5% (v ⁄ v) glutaraldehyde (Ladd, Williston, USA) in 0.1 m sodium cacodylate buffer (pH 7.4) then rinsed three times with 0.1 m sodium cacodylate buffer (pH 7.4) and postfixed with 1% (w ⁄ v) OsO4 (Sigma, St Louis, MO, USA) in the same buffer After dehydration with a graded series of ethanol (Merck, Rahway, NJ, USA) [3 · 10 in 50, 70, 90 and 100% (v ⁄ v) ethanol], they were immersed in acetone and embedded in EPON resin Section of 40 nm were cut (Ultramicrotome NOVA, LKB, Bromma, Sweden), counterstained with uranyl acetate and examined with an electron microscope (Phillips EM 301, Eindhoven, the Netherlands) To quantify mitochondrial population abundance on transmission electron microscopy micrographs, we analysed the area occupied by mitochondria on the micrographs showing the ultrastructure of rho-L929 and parental L929 cells by using the NIH image software (http://rsb.info.nih.gov/nih-image/Default.html) We analysed three section images (magnification 15 600·) taken from random observations and calculated the total area occupied by mitochondria (cumulative data) out of a global surface of 10.7 square inches Determination of intracellular reactive oxygen species Intracellular H2O2 was detected using 2¢-7¢ dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR, USA) as described previously [50] with a slight modification Cells were seeded at a density of 100 000 cells per well in a 24-well plate format (Corning, NY, USA) 16 h before being incubated (30 at 37 °C) with lm DCFH-DA in HBSS (8 gỈL)1 NaCl, 0.4 gỈL)1 KCl, 60 mgỈL)1 Na2HPO4Ỉ2 H2O, 60 mgỈL)1 KH2PO4, 100 mgỈL)1 MgSO4Ỉ7 H2O, 0.147 gỈL)1 CaCl2, gỈL)1 glucose and 100 mgỈL)1 MgCl2) Cells were then rinsed once with HBSS, lysed using Passive Lysis Buffer (Promega, Madison, WI, USA) and ROS production was assessed on 100 lL aliquotes by fluorescence intensity determination using a spectrofluorimeter (FluoStar, BMG Lab Technologies, Offenburg, Germany) (excitation wavelength, 485 nm; emission wavelength, 530 nm) Fluorescence intensity values are reported as arbitrary units after subtracting background Where indicated, FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS Mitochondrial biogenesis in mtDNA-depleted cells cells were incubated with lm antimycin A for 60 after cell loading with the probe Transient transfection and luciferase reporter gene assay To determine the transcriptional activity of NRF-1, YY1, PPARc and CREB, or to assess the activity of the authentic Tfam promoter, 143B and rho0143B cells were seeded in 12-well plates (50 000 cells per well) and transiently cotransfected by the Superfect reagent (Qiagen, Valencia, CA, USA) for h in a : ratio, with a luciferase reporter construct responsive to one of these factors (0.5 lg per well) or with the mtTFA-RC4 ⁄ pGL3 reporter construct that contains the authentic promoter of Tfam driving the expression of the luciferase gene and an expression plasmid encoding b-galactosidase (0.25 lg per well) The activity of PGC-1a promoter was assessed by transient cotransfection of L929, rho-L929, 143B and rho0143B cells with 0.5 lg per well of a pGL3-mPGC-luc plasmid that contains a truncated 230 bp fragment of the murine PGC-1a promoter and 0.25 lg per well of a plasmid encoding b-galactosidase Cells were lysed 24 h post-transfection and the luciferase activity was determined using the Luciferase Reporter Assay (Promega) and then normalized for b-galactosidase activity Transient transfection and CAT reporter gene assay To analyse the expression of mitochondrial markers such as cyt c, ANT2 and b-ATPase genes, 143B and rho0143B cells were seeded in 60 mm Ø culture dishes (Corning) at 500 000 cells per dish The next day, cells were transiently cotransfected for h by the Superfect reagent with 0.75 lg of a plasmid encoding b-galactosidase and 2.5 lg of a CAT reporter gene expressed under the control of either the authentic promoter of cyt c, ANT2 and b-ATPase genes For cyt c expression, when indicated, cells were transiently cotransfected with 1.75 lg of a pGL2 empty vector or expression plasmids encoding K-CREB (Arg287Leu) or M1-CREB (Ser133Ala) Cells were lysed 48 h after transfection in cell culture lysis reagent (Promega) and CAT activity was measured as described previously, after a simple phase-extraction step [107] Briefly, cell lysates were incubated for 10 at 60°C to inactivate endogenous deacetylase activity and then aliquots corresponding to 500 lg protein were incubated for 18 h at 37°C with ll of n-butyryl-CoA (Promega) and ll of [14C]chloramphenicol (PerkinElmer, Boston, MA, USA) in a total reaction volume of 125 ll The n-butyryl chloramphenicol was then extracted with 300 ll of xylene (Sigma) and the associated radioactivity was counted on a 200 lL aliquot of the xylene phase in a scintillation counter (Hewlett Packard, Palo Alto, USA) Results were then normalized for b-galactosidase activity 5047 Mitochondrial biogenesis in mtDNA-depleted cells Casein kinase II (CKII) assay Confluent rho-L929 cells were rinsed with NaCl ⁄ Pi and lysed in mL of cold lysis buffer containing 1% (v ⁄ v) Triton X-100, 150 mm NaCl, 10 mm Tris ⁄ HCl pH 7.5, mm EDTA, mm EGTA, mm Na3VO4, and a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) CKII was then immunoprecipitated from cleared lysates with 2.5 lg of a monoclonal antibody, anti-CKII (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for h at °C Immune complexes were immobilized by adding 60 lL of Protein G Plus ⁄ Protein A-Agarose beads (Oncogene, Boston, MA, USA) and washed twice with 800 lL of lysis buffer For the determination of immunoprecipitated kinase, aliquotes of resuspended beads were resolved by 10% SDS ⁄ PAGE and western blotting analysis Immunoprecipitates were washed with 500 lL of kinase reaction buffer (20 mm Tris ⁄ HCl; pH 7.5, 10 mm MgCl2, mm dithiothreitol) and resuspended in 50 lL of kinase reaction buffer containing 10 lm of a synthetic peptide (RRRDDDSDDD) used as the substrate for CKII (Cell Signalling, Beverly, CA, USA) The assay was carried out in the presence of 20 lm unlabeled ATP (Sigma) and 10 lCi [c-32P]ATP (PerkinElmer) for 30 at 30 °C A 25 lL aliquote was applied to a phosphocellulose membrane spin column (Pierce, Rockford, IL, USA), washed with 500 lL of 75 mm H3PO4, and membrane-associated radioactivity was counted Immunofluorescence confocal laser scanner microscopy (CLSM) Immunolocalization of cyt c, PGC-1a and Tom40 proteins was performed on L929, rho-L929 and ⁄ or on 143B and rho0143B cells Cells were fixed for 10 with NaCl ⁄ Pi containing 4% (v ⁄ v) paraformaldehyde, washed three times with NaCl ⁄ Pi, permeabilized with NaCl ⁄ Pi ⁄ 1% (v ⁄ v) Triton-X 100 for and unspecific sites were blocked with NaCl ⁄ Pi ⁄ 1% (w ⁄ v) BSA (Sigma) Cells were then incubated at °C for 16 h with either a rabbit antibody raised against cyt c (Santa Cruz Biotechnology), PGC-1a or Tom40 at ⁄ 100 dilution Cells were then washed three times with NaCl ⁄ Pi ⁄ 1% (w ⁄ v) BSA and incubated for h at 37 °C with an AlexaFluor (568 nm) goat polyclonal anti-rabbit IgG (Molecular Probes) at a ⁄ 500 dilution Cells were then processed for confocal microscopy with Mowiol (Aldrich, Bornem, Belgium) solution and observed with a confocal microscope TCS (Leica, Solms, Germany) using a constant photomultiplicator To determine cyt c abundance and distribution, quantitative analysis of the fluorescence signals was carried out on cell sections using quantify software (Leica) and fluorescence profiles were plotted on charts To assess mitochondrial protein import activity in situ, L929 and rho-L929 cells were seeded on glass cover slips in 24 well-culture plates (25 000 cells per well) and transiently transfected with lg of a plasmid encoding mitochondrial 5048 L Mercy et al apoaequorin that contains a HA-tag (mtHA-apoaequorin) (Molecular Probes) and the pre-sequence of COXVIII that targets the protein to mitochondria After 24 h, cells were fixed for 10 with NaCl ⁄ Pi containing 4% (v ⁄ v) paraformaldehyde, washed three times with NaCl ⁄ Pi, permeabilized with NaCl ⁄ Pi 1% Triton-X 100 for and unspecific sites were blocked with NaCl ⁄ Pi and % (w ⁄ v) BSA (Sigma) For double staining of mtHA-apoaequorin and endogenous cyt c, cells were incubated at 4°C for 16 h with an anti-HA mouse monoclonal antibody (Roche) and with a rabbit antibody raised against cyt c (Santa Cruz) both at a ⁄ 100 dilution Cells were then washed three times with NaCl ⁄ Pi ⁄ % (w ⁄ v) BSA and incubated for h at 37°C with an Alexa Fluor (488 nm) goat polyclonal antimouse IgG (1 ⁄ 500 dilution) and with an Alexa Fluor (568 nm) goat polyclonal anti-rabbit IgG (1 ⁄ 500 dilution; both Molecular Probes) After several washes with NaCl ⁄ Pi ⁄ 1% (w ⁄ v) BSA and NaCl ⁄ Pi, cells were processed for confocal microscopy To assess the import of HA-apoequorin into mitochondria and its colocalization with cyt c, quantitative analysis of the fluorescence signals was carried out on cell sections for both proteins using quantify (Leica) and fluorescence profiles were compared To assess mitochondrial abundance, cells were seeded in 24-well dishes (50 000 cells per well) on glass cover slides (Merck) Cells were incubated with 250 nm MitoTracker Red (Molecular Probes) for 30 in Dulbecco’s modified Eagles’ medium containing 4.5 mgỈmL)1 glucose and 10% (v ⁄ v) fetal bovine serum, before being fixed for 10 with a sodium cacodylate buffer (0.1 m, pH 7.4) containing 0.5 % (v ⁄ v) glutaraldehyde and processed for confocal microscopy Spectrofluorimetric quantitation of mitochondria L929, rho-L929, 143B and rho0143B cells were seeded in 24-well plates (50 000 cells per well) When indicated, 143B and rho0143B cells were incubated with 10 lm BAPTA-AM or transiently transfected with 0.75 lg of a pGL2 empty vector or expression plasmids encoding K-CREB or M1-CREB After 72 h, they were rinsed once with HBSS buffer: gỈL)1 NaCl, 0.4 g KCl, 60 mgỈL)1 KH2PO4, 60 mgỈL)1 NaH2PO4, pH 7.4, 100 mgỈL)1 MgSO4, gỈL)1 glucose, 100 mgỈL)1 MgCl2, 350 mgỈL)1 NaHCO3, 140 mgỈL)1 CaCl2) and incubated with 250 nm Mitotracker Red or 10 lm NAO for the indicated times Cells were washed twice with HBSS and lysed in 150 lL of Passive Lysis Buffer (Promega) for 15 The lysates were then centrifuged 15 at 15 000 g and the fluorescence was quantitated on supernatants with a spectrofluorimeter (Fluostar, BMG lab technologies: Mitotracker Red: excitation wavelength, 585 nm; emission wavelength, 612 nm; NAO: excitation wavelength, 485 nm; emission wavelength, 520 nm) Fluorescence intensities were normalized for protein content determined by a Bio-Rad Protein assay [108] FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al Reverse transcription and real-time PCR Total RNA was extracted from 143B and rho0143B cells with the RNAgents total RNA isolation system (Promega) according to the manufacturer’s instructions Reverse transcription was performed with lg of total RNA and U of reverse transcriptase enzyme (SuperscriptRII, Invitrogen, Carlsbad, CA, USA) in a final volume of 20 lL Quantitative PCR amplification of Tim44, COXVb and F1-ATPase b-subunit (b-ATPase) was performed with specific reverse and forward primers at 300 nm with an Applied Biosystem 7100 To monitor amplification, SYBR Green QPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was used according to manufacturer’s instructions Primers for the amplification of Tim44, COXVb, b-ATPase and the housekeeping gene TATA-box binding protein (TBP) were as follows: Tim44 sense 5¢-TC CATTCTCGCATCCTAGACATT-3¢, antisense 5¢-GTGC CTGGAAGGTGATGATCA-3¢; COXVb sense 5¢-TGCG CTCCATGGCATCT-3¢, antisense 5¢-CTTCTTTGCAGC CAGCATGAT-3¢; b-ATPase sense 5¢-CCATCCTGGGTA TGGATGAACT-3¢, antisense 5¢-GGCTGAGACAAGAA ACGCTGTAT-3¢, TBP sense 5¢-CCTCACAGGTCAAAG GTTTACAGTAC-3¢, antisense 5¢-GCTGAGGTTGCAG GAATTGAA-3¢) PCR amplifications were denaturation at 95°C for 15 s, annealing at 60°C for and polymerization at 72°C for 1.5 (40 cycles) Samples were then subjected to melting curve analysis All reactions were performed on RNA extracted from three independent cell cultures and values were normalized for TBP used as reference Mitochondrial protein import assay in situ or into isolated mitochondria The procedure for standard isolation of cell mitochondria and in vitro import of radiolabeled precursors was previously described [109,110] The chimeric proteins encoded by plasmids was either Neurospora crassa ATPase subunit fused to the dihydrofolate reductase (pSu9-DHFR) or a fusion protein consisting of the 167 NH2-terminal amino acids of the cytochrome b2 precursor with a 19-residue deletion in the sorting sequence and entire DHFR [pb2(167)D19-DHFR] Mitochondria were isolated as described previously [11] Briefly, 143B and rho0143B cells were washed twice with cold NaCl ⁄ Pi before being scraped in sucrose ⁄ imidazole (SI) buffer (3 mm imidazol, pH 7.4, 0.23 gỈL)1 sucrose) The cell suspension was then passed 40 times through a Dounce homogenizer, centrifuged for 10 at 260 g (4°C), and the supernatant collected and centrifuged for at 14 000 g (4°C) The pellet was finally washed once with SI buffer and mitochondria were resuspended in SEM buffer [250 mm sucrose, mM EDTA, 10 mm morpholinopropanesulfonic acid (MOPS ⁄ KOH) FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS Mitochondrial biogenesis in mtDNA-depleted cells pH 7.2] Radiolabelled proteins were synthesized by in vitro translation in the presence of [35S]methionine after in vitro transcription using SP6 polymerase and the TNT Coupled Reticulocyte Lysate System (Promega) according to the manufacturer’s instructions Import of precursor proteins used 30 lg of mitochondrial proteins diluted in import buffer [3% (w ⁄ v) fatty acid-free BSA, 10 mm Mops; pH 7.2, 80 mm KCl, mm MgCl2, mm KH2PO4 and mm methionine) Prior to the import assay, samples were supplemented with mm ATP, 10 mm succinate, 10 mm malate (all Sigma) and mm methionine The import reaction of Su9-DHFR and b2(167)D-DHFR precursors was performed for 10 at 25°C, stopped by the addition of lm valinomycin for and placed on ice Each sample was divided into two aliquots that were either treated or not with 40 lgỈmL)1 proteinase K for 15 at 4°C Proteinase K was inhibited by a protease inhibitor cocktail (Roche) Mitochondria were subsequently reisolated by centrifugation for at 14 000 g), washed twice with SEM buffer, then lysed in 0.5 m NaOH for 16 h The import was determined by the radioactivity counted in proteinase K-treated samples (Hewlett Packard) To control that the proteins were specifically imported into mitochondria, membrane potential was eliminated by treatment with lm valinomycin before the import assay and the values subtracted from each test Colorimetric DNA binding assay To assess the DNA binding activity of Sp1, PPARc, MyoD and MEF-2 transcription factors, TransAM colorimetric DNA binding assay (Active Motif, Carlsbad, CA, USA) were performed based on the protocol described previously [75] Briefly, lg of nuclear proteins were incubated for h in a 96 well-plate coated with either a double strand oligonucleotide containing the consensus sequence for Sp1, PPARc, MyoD or MEF-2 DNA binding activity was detected with respectively an anti-Sp1, anti-PPARc, anti-MyoD and anti-MEF-2 Igs (all from Santa Cruz Biotechnology) and revealed by a colorimetric reaction with a HRP-conjugated secondary antibody The enzymatic reaction was stopped and A450 measured with a spectrophotometer (BioRad, Hercules, CA, USA) High salt nuclear protein extractions were prepared by incubating cells (in 75 cm2 flasks) on ice for with 10 mL cold hypotonic buffer (HB): 20 mm Hepes, mm NaF, mm Na2MoO4, 0.1 mm EDTA Cells were then harvested in 500 lL HB containing 0.2% (v ⁄ v) NP-40 (Sigma), protease inhibitors (Roche) and phosphatase inhibitors (1 mm Na3VO4, mm NaF, 10 mm p-nitrophenylphosphate, 10 mm b-glycerophosphate) Cell lysates were centrifuged 30 s at 15 000 g and the sedimented nuclei were resuspended in 50 lL HB containing 20% (v ⁄ v) glycerol, protease ⁄ phosphatase inhibitors Nuclear extracted proteins 5049 Mitochondrial biogenesis in mtDNA-depleted cells were obtained by the addition of 100 lL HB containing 20% (v ⁄ v) glycerol, 0.8 m NaCl and protease ⁄ phosphatase inhibitors, and the protein concentration was determined [108] Western blotting analysis Cells were washed with cold NaCl ⁄ Pi and lysed in a buffer containing 40 mm Tris, pH 7.5, 150 mm KCl, mm EDTA, 1% (v ⁄ v) Triton X-100, a protease inhibitor cocktail (Roche) and phosphatase inhibitors (250 mm NaVO3, 10 mm p-nitrophenylphosphate, 10 mm b-glycerophosphate and mm NaF) The clear lysates were collected and protein concentration was determined using BioRad protein assay [108] After adding sample buffer containing 50 mm Tris ⁄ HCl, pH 6.8, 2% (v ⁄ v) SDS, 8% (v ⁄ v) glycerol and 0.4% (v ⁄ v) 2-mercaptoethanol, proteins were size-separated by 4–12% Nu-PAGE gel (Invitrogen) and transferred to PVDF membranes (Amersham Pharmacia Biotech) Blots were blocked for 18 h at °C with TBS [Tris 200 mm; pH 7.4, 140 mm NaCl, 0.1% (v ⁄ v) Tween] containing 5% (w ⁄ v) fat milk (Gloria, Vevey, Switzerland) After incubations with appropriate primary and secondary antibodies, target proteins were visualized using ECL kit (Amersham Biosciences) For the detection of the various proteins, antibodies against NRF-1 (dilution : 2000), NRF-2 (dilution : 2000), cyt c and Tom40 (both Santa Cruz Biotechnology; dilutions : 2000 and : 1000, respectively), PGC1a (dilution : 1000), Tfam (dilution : 5000) and COXI (Molecular Probes, dilution : 1000) were used and blots were probed with appropriate horseradish peroxydaseconjugated secondary antibodies (Amersham Biosciences, dilution : 100 000) Blots were then washed (three times for 20 each) and proteins visualized using enhanced chemiluminescence (Amersham Biosciences) Equal loading was assessed using antibody raised against either TBP (Santa Cruz; dilution : 200), a-tubulin (Sigma, dilution : 5000) or COXIV (Molecular Probes, dilution : 1000) For cytochrome c abundance analysis, L929 and rho0L929 cells seeded in 25 cm2 flasks (Costar) were transiently transfected with either lg of expression plasmids encoding K-CREB, M1-CREB, a dominant negative form of CaMKIV (CaMKIVT200A), or an empty vector (pGL2) After 48 h cells were harvested and processed for western blot analysis Protein signals were quantified on films with TotalLab image master software (Amersham Biosciences) and data were normalized for a-tubulin signals used as a loading control Cellular ATP content ATP contents were measured using a somatic cell ATP assay kit (Sigma) based on the assay of ATP-driven luciferin luciferase activity [111] Briefly, cells were seeded at 5050 L Mercy et al 50 000 cells per well in 12-multiwell plates, harvested at °C, lysed with 400 lL of ATP releasing reagent and the lysates were assayed for luciferase activity according the manufacturer’s procedure in a Luminometer (Lumac, Landgraaf, the Netherlands) Proteins were determined by the Bradford assay [108] and results were calculated in relative light units (RLU) per lg of protein and expressed arbitrarily in percentages of controls Mitochondrial membrane potential measurements Mitochondrial membrane potential was assesed using the Rhodamine 123 fluorescent probe [52,112] L929, rho-L929, 143B and rho0143B cells, seeded in 12 well-plates (50 000 cells per well), were preincubated or not with FCCP (10 lm) for h before being loaded 30 with Rhodamine 123 (500 nm) Cells were then washed with HBSS buffer (8 gỈL)1 NaCl, 0.4 g KCl, 60 mgỈL)1 KH2PO4, 60 mgỈL)1 NaH2PO4, pH 7.4, 100 mgỈL)1 MgSO4, gỈL)1 glucose, 100 mgỈL)1 MgCl2, 350 mgỈL)1 NaHCO3, 140 mgỈL)1 CaCl2) and lysed with passive lysis buffer (Promega) for 15 Lysates were centrifuged at 15 000 g and Rhodamine 123 fluorescence (excitatory wavelength, 485 nm; emission wavelength, 520 nm) was measured on the supernatant with a spectrofluorimeter (FluoStar) Statistical analysis Data from at least three separate experiments were presented as means ± SD and analysed with anova I and Sche´ ffe’s contrasts Differences were considered statistically significant if P < 0.05 Acknowledgements T Arnould and A De Pauw are Research Associate and Research Assistant, respectively, of the Fonds National de la Recherche Scientifique, Belgium (FNRS) L Mercy has a doctoral fellowships from the Fonds pour la Recherche dans l’Industrie et l’Agriculture (FRIA), Brussels, Belgium and S Tejerina has a ´ doctoral fellowship from the Cooperation Universitaire ´ au Developpement (CUD) This paper presents results of the Belgian Programme on Interuniversity Attraction Poles (IAP) initiated by the Belgian State, Prime Minister’s Office Science Policy Programming and the ´ Action de Recherche Concertee (ARC) funded by the French-speaking community of Belgium The scientific responsibility is assumed by the authors We thank Prof J Grooten (University of Ghent, Belgium) for the rho-L929 and L929 cell lines, Dr G Janssen (Leiden University Medical Center, the Netherlands) and Prof Attardi (California Institute of Technology, USA) for FEBS Journal 272 (2005) 5031–5055 ª 2005 FEBS L Mercy et al the generous gifts of 143B and rho0143B cell lines The 4xNRF-1-luciferase reporter, anti-NRF-1, mtTFARC4 ⁄ pGL3 and PGC-1a expression plasmids were a generous gift from Dr Scarpulla (North-Western University Medical School, Chicago, USA) The PGC1a antibody was kindly provided by Dr Kelly (Washington University School, USA) We also thank Dr Wiesner (University of Kholn, Germany) for antiă Tfam All the CAT reporter plasmids were kindly provided by Dr Zaid (Department of Biochemistry, Stockholm, Sweden) The plasmids K-CREB and M1 CREB were a generous gift from Prof Greenberg (Children’s Hospital, Department of Neurobiology, Harvard Medical School, Boston, USA) and the construct encoding the dominant negative mutant CaMKIV (T200A) was from Prof K.A Anderson and Prof A.R Means (Duke University, USA) The plasmid pMsx2SS-luciferase that contains three copies of YY1 sites upstream of the firefly luciferase was a gift from Dr Shum (National Institute of Arthitis and Muscoskeletal and Skin Diseases, National Institute of Health, Bethesda, USA) 3xPPRE-luciferase was a gift from Prof Evans (The Salk Institute for Biological Studies, San Diego, USA) and the plasmid containing the authentic a-inhibine promoter with four CRE sites driving the expression of the luciferase gene was from Dr Jameson (North-Western University, Evanston, USA) pGL3-mPGC-luc was a generous gift from Dr Herzig (Peptide Biology Laboratories, Salk Institute for Biological Studies, La Jolla, USA) We also thank Pr Voos (Albert-ludwigUniversitat, Freiburg, Germany) for the plasmids ă pSu9-DHFR and pb2(167)D19-DHFR References Wallace DC (1999) Mitochondrial diseases in man and mouse Science 283, 1482–1488 Duchen MR (2004) Mitochondria in health and disease: perspectives on a new 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Mitochondrial protein import driving forces are decreased in mtDNA-depleted cells (A) ATP content was measured in the various cell lines using a luciferin-luciferase assay and results calculated... by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation EMBO J 21, 53–63 38 Biswas G, Adebanjo OA, Freedman BD, Anandatheerthavarada HK, Vijayasarathy C, Zaidi