Title: CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation
Abstract: Article15 January 2002free access CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation T. Arnould Corresponding Author T. Arnould Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author S. Vankoningsloo S. Vankoningsloo Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author P. Renard P. Renard Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author A. Houbion A. Houbion Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author N. Ninane N. Ninane Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author C. Demazy C. Demazy Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author J. Remacle J. Remacle Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author M. Raes M. Raes Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author T. Arnould Corresponding Author T. Arnould Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author S. Vankoningsloo S. Vankoningsloo Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author P. Renard P. Renard Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author A. Houbion A. Houbion Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author N. Ninane N. Ninane Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author C. Demazy C. Demazy Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author J. Remacle J. Remacle Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author M. Raes M. Raes Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium Search for more papers by this author Author Information T. Arnould 1, S. Vankoningsloo1, P. Renard1, A. Houbion1, N. Ninane1, C. Demazy1, J. Remacle1 and M. Raes1 1Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:53-63https://doi.org/10.1093/emboj/21.1.53 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We characterized a new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a ρ0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNALys (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21Waf1/Cip1, a p53-regulated cyclin-dependent kinase inhibitor. Introduction Cells can modulate the expression of nuclear genes in response to changes in the activity level of their organelles. For example, mitochondria dysfunction induces the activation of transcription factors in a response called ‘retrograde communication’ (Parikh et al., 1987; Liao and Butow, 1993). Several lines of evidence previously established that alterations in the mitochondrial activity result in changes in nuclear gene expression (Wang and Morais, 1997). In yeast, numerous studies have shown that a retrograde signaling pathway can act as a homeostatic or stress response mechanism to adjust metabolic activities to the alterations of mitochondria activity. It has been particularly well characterized for the CIT2 gene, encoding a peroxisomal isoform of citrate synthase that catalyzes the first step in the glyoxylate cycle. In cells lacking mitochondrial DNA (mtDNA), increased expression of CIT2 is dependent on the activation of RTG1 and RTG3 encoding basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors (Liao and Butow, 1993). In yeast, detailed mechanisms on the regulation of the mitochondria to nucleus signaling and metabolic control through Rtg proteins in response to mitochondria impairment have been described recently (Komeili et al., 2000; Sekito et al., 2000). The interorganelle ‘retrograde communication’ pathways have been described not only in yeast (Liao and Butow, 1993; Komeili et al., 2000; Sekito et al., 2000) but also in plant (Mayfield, 1990) and mammalian cells (Biswas et al., 1999). However, the signaling pathway(s) used by mitochondria to communicate the state of activity to the nucleus is still poorly understood in mammalian cells. Mitochondrial activity plays a crucial role in cellular physiology, as emphasized recently by the discovery of a wide variety of human diseases caused by or associated with mitochondrial alterations such as genetic defects in mtDNA or nuclear DNA that affect the respiratory chain and ADP phosphorylation (Wallace, 1999). Among these diseases, myoclonus epilepsy with ragged-red fibers (MERRF) is a maternally inherited disorder characterized by point mutations in the mtDNA-encoded tRNALys gene which impairs respiration and mitochondrial protein synthesis (Lombes et al., 1989). Cells cultured with ethidium bromide (EtBr) partly or completely lose their mtDNA (mtDNA-depleted or ρ0 cells) and consequently cannot carry out oxidative phosphorylation (OXPHOS) and thus glycolysis is their only source of ATP (King and Attardi, 1989). Recently, these cells have been used to study mitochondrial disorders due to genetic defects such as deletions in mtDNA, depletion of mtDNA or reduction of mtDNA copy number (King and Attardi, 1989; Moraes et al., 1991). These mtDNA-depleted cells usually have a proliferation defect characterized by a longer doubling time, as observed for mtDNA-depleted HeLa S3 and 143B cells (Vaillant and Nagley, 1995; Buchet and Godinot, 1998), but no molecular control of the cell cycle after inhibition of mitochondria has been evidenced so far. Mitochondrial dysfunction also greatly modifies nuclear gene expression (Epstein et al., 2001). Indeed, increased expression of ANT-1 and ATP synthase β-subunit mRNAs was described in skeletal muscle of patients with respiratory defects resulting from mtDNA point mutations (Heddi et al., 1993), and increased mRNAs encoding cytochrome oxidase (COX IV and COX VIaL) were also observed in ρ0 cells (Li et al., 1995). Biswas et al. (1999) recently described that in mouse C2C12 cells, the decrease of mtDNA content initiates a signaling pathway leading to the activation of JNK and increased transcription of ryanodine receptor-1 Ca2+ release channel and COX Vb genes. While eukaryotic cells can respond to mitochondrial dysfunction by a modulation of nuclear gene expression, evidence for signaling pathways and transcriptional mechanisms are lacking in most instances. Interestingly, several genes encoding mitochondrial proteins contain CRE (cAMP response element) sequences such as some respiratory chain subunits (Scacco et al., 2000), cytochrome c (Gopalakrishnan and Scarpulla, 1994), manganese superoxide dismutase (Kim et al., 1999) or carnitine palmitoyltransferase (Brady et al., 1992). In this study, we analyzed the effect of mitochondrial activity inhibition on the signaling and transcriptional machinery of mammalian cells. We identified CRE-binding protein (CREB) as a new transcription factor involved in the ‘retrograde communication’ pathways between impaired mitochondria and the nucleus. We used several cellular models: a mtDNA-depleted murine fibrosarcoma cell population (L929), a true ρ0 human osteosarcoma cell line (143B), a cybrid cell line with a nucleotide mutation (A8344G) in the mitochondria-encoded tRNALys gene and an acute inhibition of mitochondria in 293 cells treated with metabolic mitochondrial inhibitors. We found that mitochondrial activity impairment is sufficient to trigger CREB phosphorylation on Ser133 by calcium/calmodulin kinase IV (CaMKIV). In turn, phosphorylated CREB mediates a proliferation defect in mtDNA-depleted L929 cells by increasing p53 transcriptional activity that leads to the up-regulation of p21Waf1/Cip1, a cyclin-dependent kinase inhibitor gene regulated by p53. Our results suggest that the activation of a ‘retrograde communication’ signaling pathway initiated by a mitochondrial dysfunction could be an important modulator of the cell cycle in cells submitted to energetic stress conditions. Results Functional and morphological characterization of the mtDNA-depleted L929 cell line PCR analysis revealed that EtBr-treated L929 cells are dramatically depleted in but not completely devoid of mtDNA. As shown in Figure 1A, using specific primers for the D-loop fragment in mouse mtDNA, we amplified a fragment of the expected size (823 bp) in L929 cells, but only a very weak band was obtained for EtBr-treated cells. No amplification was obtained with nuclear DNA, confirming the specificity for mtDNA. Consistently, the mtDNA-encoded COX I protein was weakly stained in mtDNA-depleted L929 compared with parental cells (Figure 1B). The functional effect of EtBr treatment on oxidative phosphorylation was assessed further on in situ respiration as measured by the respiratory control ratio (RCR). EtBr treatment induces a complete inhibition of the oxidative respiration (RCR = 1) in mtDNA-depleted cells (see Supplementary data available at The EMBO Journal Online). The total cellular ATP measured in mtDNA-depleted L929 cells was reduced by 60% and was no longer sensitive to the uncoupler FCCP, suggesting that the residual ATP content in these cells is supplied by glycolysis (data not shown). Figure 1.Effects of EtBr-treatment on mitochondria in L929 cells. (A) PCR amplification of a specific mtDNA fragment in the mitochondrial D-loop performed on purified mtDNA (lanes 1 and 2) and nuclear DNA (lanes 4 and 5). (B) Immunofluorescence patterns of cells stained with a monoclonal antibody against the COX I subunit: (a) mtDNA-depleted cells; (b) L929 cells. Asterisks indicate the localization of nuclei. Scale bars = 10 μm. Download figure Download PowerPoint CREB is constitutively activated by phosphorylation on Ser133 in mtDNA-depleted L929 cells To address the question of whether or not the inhibition of mitochondrial activity modulates the transcriptional activity, L929 or mtDNA-depleted L929 cells were transiently co-transfected with a plasmid encoding β-galactosidase and different luciferase reporter constructs transactivated by various transcription factors. As illustrated in Figure 2A, the luciferase activity was increased specifically for the constructs driven either by the authentic α-inhibin promoter (Pei et al., 1991) or by the authentic c-fos promoter, both containing CRE sites. CREB transactivation is also observed in a ρ0 143B cell line or clonal cybrids obtained from ρ0 143B cells that have been repopulated with mtDNA from a MERRF patient harboring a tRNALys mutation (A8344G) (King and Attardi, 1989). Luciferase activity was compared in L929, mtDNA-depleted L929 cells, cybrids containing either 100% mutated or wild-type mtDNA, and 143B and ρ0 143B cells after transient transfection with the luciferase reporter construct driven by the α-inhibin promoter (Figure 2B). The different cell lines associated with severe defects in respiratory chain activity show a significant increase in CREB-dependent luciferase activity. This effect seems to be specific for mitochondrial inhibition, as glycolysis inhibition with 2-deoxy-D-glucose is without any effect on CREB activation (data not shown). In mtDNA-depleted L929 cells, the overexpression of K-CREB and M1-CREB, two dominant-negative mutants for CREB (Bonni et al., 1999), completely inhibited the luciferase activity driven by the α-inhibin promoter (Figure 2C). Western blotting analysis revealed that CREB phosphorylation on Ser133 is induced in mtDNA-depleted L929 cells, mutated MERRF cybrids and ρ0 143B cells (Figure 2D). Confocal microscopy and subcellular fractionation–western blot analyses show that phosphorylated CREB is found mainly in nuclei of the different cell lines associated with a mitochondrial dysfunction (data not shown). To make sure that phosphorylated CREB is able to modify gene expression, we checked the amount of c-fos expression, a CREB-regulated immediate-early gene (Hall et al., 2001). As can be seen in Figure 2E, c-fos protein is more abundant in mtDNA-depleted cells. Figure 2.(A) Luciferase activity in transiently transfected L929 control and mtDNA-depleted cells with luciferase constructs driven by c-myc, c-Jun, authentic MMCP-6, c-fos and α-inhibin promoters. L929 (black) and mtDNA-depleted L929 (hatched) cells, were transiently co-transfected with β-galactosidase and the luciferase constructs. (B) CREB-dependent luciferase activity in L929, mtDNA-depleted L929, wild-type (wt) MERRF cybrid, mutated (mut) MERRF cybrid, 143B and ρ0 143B cells transiently transfected with the luciferase reporter construct driven by the α-inhibin promoter and an expression plasmid encoding β-galactosidase. (C) CREB-dependent luciferase activity in L929 (black) and mtDNA-depleted L929 (hatched) cells transiently transfected with K-CREB or M1-CREB, together with the CREB-sensitive luciferase reporter construct and an expression plasmid encoding β-galactosidase. Luciferase activity was determined 24 h post-transfection. Results are expressed in relative light units (RLU) after normalization for β-galactosidase activity (A) or in fold increase of control cells (B and D) as means ± 1 SD (*** and ###, significantly different from L929 and mtDNA-depleted L929 cells with P <0.001). (D) Western blot analysis of phospho-CREB (p-CREB) and CREB assessed for the different cell lines used in (B). (E) Western blot analysis for c-fos and α-tubulin assessed for L929 (lane 2) and mtDNA-depleted L929 (lane 1). Download figure Download PowerPoint Identification of the kinase responsible for the phosphorylation of CREB We next examined the activity of several kinase cascades leading to CREB activation in mtDNA-depleted L929 cells. We first measured the in vitro cAMP-dependent protein kinase (PKA) activity in cell homogenates, but no difference was observed between L929 and mtDNA-depleted cells, while PKA is activated in a time-dependent manner in cells treated with 100 μM dibutyryl-cAMP (see Supplementary data). We then tested whether CREB phosphorylation was mediated by a Ca2+-dependent mechanism. Cell treatment with BAPTA, an intracellular Ca2+ chelator, inhibits luciferase activity observed in mtDNA-depleted L929 cells on both CREB reporter constructs (Figure 3A), while BCECF, an unrelated probe tested to rule out the potentially toxic effect, is without any effects (data not shown). We also confirmed that intracellular calcium concentration in cells loaded with Fluo-3, a fluorescent calcium indicator, is higher in mtDNA-depleted L929 cells (Figure 3B). Figure 3.(A) Effect of a 6 h treatment with 20 μM BAPTA-AM on the CREB-sensitive luciferase activity after transfection of L929 (black) or mtDNA-depleted L929 (hatched) cells with luciferase reporter genes driven by either CREB-regulated c-fos or α-inhibin promoters. Luciferase activity was determined and expressed as RLU after normalization for β-galactosidase activity (n = 6; ***, significantly different from mtDNA-depleted cells with P <0.001). (B) Intracellular calcium concentration ratio in L929 and mtDNA-depleted cells loaded with 10 μM Fluo-3-AM. Results are expressed in fold increase of control cells, and represent the mean ± SD for n = 3 (***, significantly different from L929 with P <0.001). Download figure Download PowerPoint As calcium is able to activate several kinase pathways leading to CREB phosphorylation on Ser133 such as CaMKI, II or IV, p70S6K, rsk2, MSK, PKCs, Ras/Raf/MAPK and MAPKAP-K2, allowing the interaction with the CREB-binding protein (CBP/p300) (De Cesare et al., 1999), we used different inhibitors to modulate the CREB-dependent luciferase activity. As shown in Figure 4A, only KN-93, a CaMK inhibitor, and W-7, a calmodulin antagonist, were able to inhibit CREB-dependent transcription significantly, while inhibitors of the PKA (H-89), PKC (calphostin C), mitogen-activated protein kinase (PD 95089) or phosphatidylinositol 3-kinase (wortmannin) pathway have no effect. We thus investigated the possible role of CaMKI, II, and IV in CREB activation. Expression of a dominant-negative form of CaMKIV (CaMKIV T200A) (Chatila et al., 1996) completely suppressed the activation of the CREB reporter construct in transfected mtDNA-depleted L929 cells, while overexpression of the wild-type enzyme enhanced the luciferase activity only in mtDNA-depleted cells (Figure 4B). When these cells are transiently transfected with plasmids encoding the wild type of different isoforms (CaMKI, II and IV), only CaMKIV overexpression is able to enhance the CREB-dependent luciferase activity by an additional 2-fold increase (Figure 4C). Figure 4.(A) Effect of kinase pathway inhibitors on CREB-dependent luciferase activity in L929 (black) and mtDNA-depleted (hatched) cells. After transfection, cells were incubated for 16 h before the assay with 50 μM KN-93, 50 μM W-7, 10 μM H-89, 20 μM PD 95089, 1 μM calphostin C or 1 μM wortmannin. (B) L929 (black) and mtDNA-depleted L929 (hatched) cells were transiently co-transfected with a plasmid encoding β-galactosidase, the CREB luciferase reporter construct driven by the α-inhibin promoter and an expression vector encoding wild-type CaMKIV or the dominant-negative form CaMKIV T200A. (C) Effect of overexpression of CaMKI, CaMKII and CaMKIV on the activation of the CREB luciferase reporter construct driven by the α-inhibin promoter. L929 (black) and mtDNA-depleted L929 (hatched) cells were transiently co-transfected as described in (B) with an expression vector encoding wild-type CaMKI, II or IV. Results are normalized for β-galactosidase activity, are expressed in fold increase of luciferase activity of the L929 control cells and represent the mean ± SD for n = 3 (***, significantly different from L929 with P <0.001; ###, significantly different from mtDNA-depleted L929 cells with P <0.001). Download figure Download PowerPoint The crucial role of CaMKIV in the phosphorylation of CREB in mtDNA-depleted L929 cells was evidenced further by the fact that overexpression of dominant-negative CaMKIV T200A or KN-93 treatment dramatically inhibited CREB phosphorylation, as observed by western blot analysis (Figure 5A). The overexpression of the dominant-negative M1-CREB also inhibited phosphorylation of endogenous CREB, probably by competing for binding and subsequent phosphorylation (Figure 5A). Finally, endogenous CaMKII and CaMKIV were immunoprecipitated and their activity was determined in vitro. The results shown in Figure 5B and C clearly indicate that the CaMKIV activity is increased by 3- to 4-fold in mtDNA-depleted L929 cells while CaMKII activity seems to be unaffected. The slight increase observed in the CaMKII activity is most likely to be the result of the higher amount of the immunoprecipitated kinase used for the assay (Figure 5C). In conclusion, these results clearly support the hypothesis that CREB phosphorylation in mtDNA-depleted L929 cells is mediated by CaMKIV. Figure 5.(A) Effect of dominant-negative CaMKIV T200A or M1-CREB overexpression and the calmodulin kinase inhibitor KN-93 on the CREB phosphorylation pattern in mtDNA-depleted L929 cells. mtDNA-depleted L929 cells were either transfected with a plasmid encoding CaMKIV T200A or M1-CREB or treated with 50 μM KN-93 for 24 h before phospho-CREB and CREB expression were analyzed by western blotting. (B) Endogenous CaMKIV or CaMKII activity in L929 and mtDNA-depleted cells was measured after immuno precipitation. Representative results of one experiment in duplicate are shown and are expressed as c.p.m. (C) The amounts of immunoprecipitated kinases were controlled by western blotting. Download figure Download PowerPoint CREB phosphorylation is induced by inhibitors of mitochondrial activity In order to determine whether CREB activation is induced directly by a mitochondrial activity impairment, we tested the effect of metabolic mitochondrial inhibitors on CREB activation. Phospho-CREB localization in cell lines treated with FCCP, oligomycin or antimycin A is almost exclusively nuclear, as shown for L929 cells in Figure 6A. Phosphorylated CREB is also transcriptionally active in cells treated with metabolic inhibitors, as demonstrated by the activation of the CREB reporter gene construct (Figure 6B). Cell incubation with BAPTA and overexpression of dominant-negative form M1-CREB or CaMKIV T200A almost completely inhibited the luciferase activity triggered by the different mitochondrial inhibitors (Figure 6B). Using the Ca2+-sensitive photoprotein apoaequorin as a calcium probe, we confirmed that free cytosolic [Ca2+]cyt is increased in 293 cells treated with mitochondrial inhibitors while mitochondrial [Ca2+]mt is decreased (see Supplementary data). Endogenous CaMKIV activity is also increased (5–7 times) in 293 cells after treatment with oligomycin, antimycin A or FCCP (see Supplementary data). Altogether, these results suggest that mitochondrial inhibitors are also able to trigger CREB phosphorylation through a CaMKIV-dependent mechanism and they demonstrate that CREB activation induced by mitochondria dysfunction is not cell specific as it is observed in two different cell lines. Figure 6.(A) Immunolocalization of phospho-CREB on Ser133 in L929 cells treated or not (a) for 6 h with 10 μM FCCP (b), 8 μM oligomycin (c) and 1 μM antimycin A (d) and analyzed the next day by immunofluorescence and confocal microscopy. Bars = 10 μm. Arrows indicate a nuclear localization. (B) CREB-dependent luciferase activity in 293 cells triggered by treatment with 1 μM antimycin A (hatched bars), 8 μM oligomycin (white bars) or 10 μM FCCP (black bars) is inhibited by 20 μM BAPTA for 6 h, or by expression of a dominant-negative form of CREB (M1-CREB) or a dominant-negative mutant of CaMKIV (CaMKIV T200A). Results are expressed as fold increase of control cells (vector) after normalization. The results are representative of two experiments in triplicate and are expressed as means ± 1 SD. Download figure Download PowerPoint CaMKIV activity is differentially regulated by PP2A in cells affected by mitochondria dysfunction Once activated by phosphorylation on a single threonine residue by the calmodulin-dependent kinase kinase (CaMKK), CaMKIV activity becomes independent of both calcium and calmodulin (Park and Soderling, 1995). However, CaMKIV is known to be inactivated by protein phosphatase 2A (PP2A) (Westphal et al., 1998). To determine whether these enzymes exist as a complex in L929 and mtDNA-depleted L929 cells, we immunoprecipitated endogenous CaMKIV from cell lysates and analyzed the immune complexes by immunoblotting with an antibody specific for the PP2A catalytic C subunit. As shown in Figure 7A, PP2A catalytic C subunit co-immunoprecipitated with CaMKIV in L929 cells (lane 1), but the interaction is dramatically weaker in mtDNA-depleted L929 cells (lane 2). To make sure the differential interaction is caused by mitochondrial inhibition, we compared the co-immunoprecipitated PP2A in L929 cells treated with the different inhibitors. As shown in Figure 7A, antimycin A, FCCP and oligomycin are all able to impair the interaction between both enzymes. These results suggest that PP2A interaction with CaMKIV is weaker in mtDNA-depleted L929 cells and in cells submitted to the metabolic inhibitors. A very important question to answer is whether or not the weaker association of CaMKIV with PP2A observed in mtDNA-depleted cells is caused by CaMKIV's increased activity as a result of the higher calcium concentration or if the impaired association with PP2A is reponsible for causing the increase in CaMKIV. To address this question, we performed co-immunoprecipitation experiments between CaMKIV and PP2A in L929 cells treated with ionomycin, a calcium ionophore, or in mtDNA-depleted cells treated with KN-93 to inhibit CaMKIV activity. The results presented in Figure 7B clearly show that ionomycin treatment inhibits the interaction while the association is increased in mtDNA-depleted L929 cells treated with KN-93. These results support the first hypothesis that the interaction can be modulated by the CaMKIV activity status. Figure 7.(A) Co-immunoprecipitation of PP2A catalytic C subunit with CaMKIV. CaMKIV was immunoprecipitated (IP) from L929 cells (lanes 1 and 3) treated or not with mitochondrial inhibitors (8 μM oligomycin, 1 μM antimycin A and 10 μM FCCP) (lanes 4–6) or from mtDNA-depleted L929 cells (lane 2). (B) Co-immunoprecipitation of PP2A catalytic C subunit with CaMKIV. CaMKIV was immuno precipitated from L929 cells (lane 3) treated or not with ionomycin at 10, 100 or 500 nM for 16 h (lanes 4–6) or from mtDNA-depleted L929 cells treated or not with 50 μM KN-93 for 16 h (lanes 1 and 2). Immune complexes were analyzed by immunoblotting with an anti-PP2A C subunit antibody (anti-PP2A) or an anti-CaMKIV antibody (anti-CaMKIV). Expression levels of the PP2A C subunit in the lysates are also shown. Download figure Download PowerPoint Involvement of phosphorylated CREB in the cell proliferation defect A slower proliferation of mtDNA-depleted cells than parental cells has been observed in several cell lines and is usually attributed to the lower ATP content in these cells (King and Attardi, 1996; Morais, 1996), but the molecular pathway is unknown. As stimulation of the cAMP pathway inhibits proliferation of some tumoral cell lines (Yokozaki et al., 1992), we hypothesized that phosphorylated CREB contributes to the lower proliferation of the mtDNA-depleted L929 or L929 cells treated with an inhibitor of mitochondrial activity. As shown in Figure 8A, the [3H]thymidine incorporation into mtDNA-depleted L929 cells and in oligomycin-treated L929 cells was strongly reduced as compared with L929. Therefore, we assessed the role of phosphorylated CREB on cell entry into S phase. L929 cells were transiently transfected with a control vector or with constructs encoding CREB dominant-negative mutants K-CREB and M1-CREB, 24 h before the addition of 8 μM oligomycin for 6 h. Oligomycin decreases [3H]thymidine incorporation by almost 40–50% in both untransfected control cells and transfected control cells. However, this effect is largely reduced when cells overexpressed either K-CREB or M1-CREB (Figure 8A). Similarly, mtDNA-depleted L929 cells incorporated only 50% of [3H]thymidine compared with parental cells, and the decrease can be significantly reduced by K-CREB or M1-CREB overexpression (Figure 8A). A reduced decrease in the [3H]thymidine incorporation was also observed when mtDNA-depleted L929 cells were transiently transfected with dominant-negative CaMKIV or treated with KN-93 (Figure 8B), two conditions that reduce the phosphorylation of CREB on Ser133 (Figure 5A). As a positive control, we showed that M1-CREB is also able to reduce the decrease of [3H]thymidine incorporation in forskolin-treated L929 cells (Figure 8B). In conclusion, mtDNA depletion or mitochondrial inhibitors such as oligomycin appear to inhibit cell cycle entry into S phase through the activation of CREB transcription factor. Figure 8.(A) Incorporation of [3H]thymidine into DNA was analyzed in L929 cells (treated or not with oligomycin) and compared with the incorporation for mtDNA-depleted L929 cells. In some conditions, these cells were transiently transfected before treatment with an empty