To explain why mitochondrial DNA (mtDNA)-depleted or rho0 cells still keep a mitochondrial membrane potential (Delta(psi)m) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1-ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase-polymerase chain reaction on L929 mtDNA-depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor alpha. Interestingly, up- or down-regulation of mtCLIC protein expression changes Delta(psi)m whereas the chloride channel inhibitor NPPB reduces the Delta(psi)m in mtDNA-depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA-depleted cells incorporate, in a NPPB-sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mitochondria.

To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energydemanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-1) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1⌬ in myoblasts to phosphorylated CREB-1␣ protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-1␣ in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis.In mammals, mitochondria are composed of at least 1,000 proteins, including components of the inner membrane electron transport and oxidative phosphorylation system (OXPHOS), metabolite carriers, matrix enzymes, subunits of the protein import machineries, factors necessary for replication and expression of the small mitochondrial DNA (mtDNA) genome, and components of the mitochondrial protein biosynthesis machinery (5). To synthesize these proteins in a reasonably economical way, it is essential to orchestrate the expression of their genes, which are predominantly located on nuclear chromosomes, and coordinate it with the expression of mtDNA. As both ATP demand and mitochondrial content are very different in the various cell types of the body and can change even in terminally differentiated cells, these regulatory mechanisms must operate during developmental programs as well as in adaptation processes in the adult. Indeed, cells are able to adjust energy metabolism by altering the architecture and dynamics of the mitochondrial reticulum (10), by modifying its enzyme equipment and/or the level of proton leak, or by adjusting total mitochondrial respiratory capacity when changes in energy demand persist for long periods (23). Among the factors known to strongly stimulate mitochondrial biogenesis in vivo, the most prominent examples are high levels of thyroid (67) and glucocorticoid (55, 66) hormones and also conditions like endurance exercise of muscle (1) and cold adaptation in brown fat tissue (31).Wh...

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‐1α) 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.

The up-regulation of cathepsin B by NF-kappaB, followed by its secretion into the extracellular environment, might be partly responsible for the previously reported invasiveness of the mtDNA-depleted 143B osteosarcoma cells.

We show that mitochondrial DNA (mtDNA)-depleted 143B cells are hypersensitive to staurosporine-induced cell death as evidenced by a more pronounced DNA fragmentation, a stronger activation of caspase-3, an enhanced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and a more dramatic cytosolic release of cytochrome c. We also show that B-cell CLL/lymphoma-2 (Bcl-2), B-cell lymphoma extra large (Bcl-X(L)), and myeloid cell leukemia-1 (Mcl-1) are constitutively less abundant in mtDNA-depleted cells, that the inhibition of Bcl-2 and Bcl-X(L) can sensitize the parental cell line to staurosporine-induced apoptosis, and that overexpression of Bcl-2 or Bcl-X(L) can prevent the activation of caspase-3 in ρ(0)143B cells treated with staurosporine. Moreover, the inactivation of cathepsin B with CA074-Me significantly reduced cytochrome c release, caspase-3 activation, PARP-1 cleavage, and DNA fragmentation in mtDNA-depleted cells, whereas the pan-caspase inhibitor failed to completely prevent PARP-1 cleavage and DNA fragmentation in these cells, suggesting that caspase-independent mechanisms are responsible for cell death even if caspases are activated. Finally, we show that cathepsin B is released in the cytosol of ρ(0) cells in response to staurosporine, suggesting that the absence of mitochondrial activity leads to a facilitated permeabilization of lysosomal membranes in response to staurosporine.