HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/14/2145 03-0075fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ARNOULD, T. Articles by RAES, M. Search for Related Content PubMed PubMed Citation Articles by ARNOULD, T. Articles by RAES, M.

mtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells¹

Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (F.U.N.D.P), 5000 Namur, Belgium

²Correspondence: Laboratoire de Biochimie et Biologie Cellulaire, University of Namur (F.U.N.D.P), 61 rue de Bruxelles, 5000 Namur, Belgium. E-mail: thierry.arnould{at}fundp.ac.be

SPECIFIC AIMS

The aim was to identify differentially expressed genes in mtDNA-depleted cells that could play a role in two interesting features of these cells such as mitobiogenesis and the generation of a mitochondrial potential. The functional characterization led us to identify mtCLIC, a mitochondrial chloride channel, as a protein that participates in generating a mitochondrial membrane potential in these cells.

PRINCIPAL FINDINGS

1. Mitochondria are present in mtDNA-depleted L929 cells
We first confirmed that mitochondria are present in mtDNA-depleted L929 cells. When rhodamine 123 (R123) accumulation is used to measure the mitochondrial membrane potential (m), mitochondria from mtDNA-depleted L929 cells seem to incorporate less probe than the parental cells. These results suggest that mitobiogenesis still occurs in mtDNA-depleted L929 cells even in the absence of functional respiration and mtDNA, but also that mitochondria of these cells retain an electrochemical gradient that generates a potential while the respiration is absent. It is accepted that the ANT mediates an exchange of ATP^4- for ADP^3- to maintain the mitochondrial potential in mtDNA-depleted cells. According to this hypothesis, ATP produced by glycolysis in the cytosol is imported into mitochondria and used by the F1-ATPase to generate the mitochondrial membrane potential. However, when we next compared ADP and ATP incorporation in mitochondria of mtDNA-depleted and L929 cells, we found no reverse translocase activity leading to ATP incorporation in mitochondria of mtDNA-depleted L929 cells.

2. mtCLIC is a mitochondria-responsive gene in mtDNA-depleted L929 cells
In an attempt to study differentially expressed genes in response to chronic mitochondrial dysfunction, we used mRNA differential display reverse transcriptase-PCR to identify mitochondria-responsive genes in mtDNA-depleted L929 cells. We found that mtCLIC is up-regulated in response to mitochondrial impairment and is involved in maintaining a m in these cells. Mouse mtCLIC belongs to a superfamily of putative intracellular anion channels collectively called chloride intracellular channel (CLIC) proteins. Northern blot, reverse Northern dot-blot analysis, confocal microscopy, and Western blot analyses were used to confirm that the differentially expressed cDNA fragment represents a mRNA whose steady-state level changes in mtDNA-depleted L929 cells (Fig. 1 ).

View larger version (30K): [in this window] [in a new window]   Figure 1. mtCLIC is an overexpressed gene in mtDNA-depleted cells. A) Total RNA was extracted from L929 or mtDNA-depleted cells and analyzed by Northern blot using a 32P-labeled oligonucleotide probe specific for mtCLIC mRNA. After autoradiography, the membrane was reprobed with a 32P-labeled oligonucleotide specific for GAPDH mRNA. B) Reverse Northern dot-blot of differentially expressed mtCLIC. Cloned fragment was amplified and ± 200 ng of the amplified product was immobilized on membranes, next hybridized with 32P-labeled cDNA synthesized from poly(A+) RNA prepared from either L929 or mtDNA-depleted L929. C) Visualization of COXI (green) and mtCLIC (red) expression levels in L929 and mtDNA-depleted L929 cells by immunostaining and confocal microscopy.

3. mtCLIC is also overexpressed in L929 cells treated with mitochondria metabolic inhibitors
We also found that mtCLIC is overexpressed in L929 cells treated with mitochondria metabolic inhibitors such as FCCP, antimycin A, or oligomycin. Furthermore, CLIC4, the human mtCLIC ortholog, was found to be overexpressed in rho0 143B and mutated MERRF (A8344G) cybrid cells. Finally, Western blot analysis performed on mitochondrial fractions confirmed that mtCLIC is more abundant in mitochondria of mtDNA-depleted cells.

4. mtCLIC is a target gene regulated by calcium and CREB
We also bring some evidence that mtCLIC is a target gene regulated by calcium and the activated cyclic AMP-responsive element binding protein (CREB) operating through its physical interaction with p53. Indeed the activity of a p53-dependent luciferase reporter gene is strongly and constitutively increased in mtDNA-depleted L929 cells while antimycin A, oligomycin, and TNF- enhance p53-dependent luciferase activity in L929 cells. Thus, molecules that induce mtCLIC expression also increase p53 transcriptional activity. We next tried to understand how p53 transcriptional activity is increased in mtDNA-depleted cells. As we recently showed that phosphorylated CREB physically interacts with p53 in mtDNA-depleted L929 cells. mtDNA-depleted L929 cells were transiently transfected with dominant negative mutants for CREB (K-CREB) or p53 (p53R175H), separately or in combination, before mtCLIC protein expression was analyzed by Western blot. K-CREB and p53R175H overexpression both reduce the expression level of the putative intracellular chloride channel. Furthermore overexpression of K-CREB and p53R175H completely inhibits the increased p53 activity in mtDNA-depleted L929 cells. The inhibitory effect of K-CREB on p53-dependent luciferase activity could be explained by the fact that p53 activity is controlled by the interaction with CREB and that overexpression of K-CREB disrupts the interaction between the two proteins. Indeed, when endogenous p53 protein was immunoprecipitated from L929 and mtDNA-depleted L929 cells transiently transfected with a plasmid encoding K-CREB, the amount of coimmunoprecipitated CREB was dramatically decreased in mtDNA-depleted cells that overexpress K-CREB. This explains why K-CREB overexpression inhibits p53 transcriptional activity in mtDNA-depleted L929 cells. Altogether, these results suggest that p53 is a key transcription factor involved in mtCLIC expression in the L929 cell line and support the hypothesis that its role in the regulation of mtCLIC expression is mainly controlled by its interaction with CREB.

5. mtCLIC biological function in mtDNA-depleted L929 cells
Finally, we addressed the question of mtCLIC biological function in mtDNA-depleted L929 cells by showing that chloride influx is higher in mitochondria of mtDNA-depleted cells (in which mtCLIC is more abundant), and we saw that the abundance and/or activity of this intracellular chloride channel contribute(s) to maintain a m in mtDNA-depleted L929 cells. Using ³⁶chloride, we showed that purified mitochondria from mtDNA-depleted cells incorporate more chloride and in a faster way than mitochondria from the parental cell line. Chloride uptake is also sensitive to NPPB, a nonspecific chloride channel inhibitor. We next showed that NPPB decreased the R123 fluorescent signal in mtDNA-depleted L929 cells (Fig. 2 ). These results agree with the effects of the inhibitor on the chloride uptake by mitochondria from mtDNA-depleted cells. Finally, to further address the role of mtCLIC in this m, mtDNA-depleted L929 cells were transiently transfected with either the dominant negative K-CREB or p53 R175H mutants in order to decrease mtCLIC expression or with different constructs encoding 5' or 3'-GFP tagged and nontagged-mtCLIC protein. We found a perfect correlation between the abundance of the protein and the R123 fluorescent signal (Fig. 2) . We also confirmed that mtCLIC expression plays an important role in mitochondrial membrane potential by showing that siRNA for mtCLIC specifically down-regulates the expression of the protein and decreases the m in mtDNA-depleted L929 cells. These data suggest that the mitochondrial membrane potential detected in mtDNA-depleted cells could be directly or indirectly modulated by the level of mtCLIC expression.

View larger version (40K): [in this window] [in a new window]   Figure 2. mtCLIC is involved in the mitochondrial membrane potential measured in mtDNA-depleted cells. A) Effect of NPPB, a nonspecific chloride channel inhibitor, on m of L929 (black columns) or mtDNA-depleted L929 cells (hatched columns). Cells were treated with different concentrations of NPPB for 16 h before the assay. B) Effects of mtCLIC expression modulation on m determined by accumulated R123 fluorescence. L929 mtDNA-depleted cells seeded at 80,000 cells/well were transiently transfected with 1 µg of pGL2 vector (control) or 1 µg of a plasmid encoding the dominant negative mutants K-CREB, p53(R175H), GFP-tagged mtCLIC at the 5' or 3' end or full-length untagged mtCLIC. After incubation or 24 h post-transfection, cells were loaded for 30 min with 10 µM R123 and fluorescence was measured in a spectrofluorimeter. Representative results of 2 independent experiments are expressed in arbitrary fluorescence units and normalized for control values as means ± SD (n=4).

CONCLUSIONS AND SIGNIFICANCE

We identified mtCLIC as a differentially overexpressed gene in cells deprived from mitochondrial ATP production. MtCLIC, a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We demonstrated that its expression is dependent on CREB and p53 and that it is sensitive to calcium and TNF-. We also noted that chloride influx is higher in mitochondria of mtDNA-depleted cells and can be inhibited in a NPPB-dependent manner. The role of chloride influx is demonstrated by up- or down-regulation of mtCLIC protein expression using dominant negative mutants for CREB and p53, changes m measured with the fluorescent probe rhodamine 123. This is supported by the fact that chloride channel inhibitor NPPB reduces the m in mtDNA-depleted L929 cells. We thus described a new mechanism that could help to explain how mitochondria from mtDNA-depleted cells maintain a mitochondrial membrane potential (m) in the absence of an active respiration. We identified a molecular actor to explain charges that partitioning adds a new hypothesis to the well-accepted one that describes the generation of a mitochondrial potential by the reverse action of ANT. These data should be confirmed by electrophysiology; questions that remain to be addressed are why these mtDNA-depleted cells maintain a mitochondrial potential and what is the role of it in apoptosis prevention and mitochondrial protein import as part of the mitobiogenesis process.

View larger version (28K): [in this window] [in a new window]   Figure 3. A new hypothesis to explain how mtDNA-depleted cells or rho0 cells maintain a mitochondrial membrane potential. Mitochondrial deficiency triggers the overexpression of a CREB and p53-regulated intracellular chloride channel (mtCLIC). The inner mitochondrial membrane localization leads to chloride uptake and resulting H+/Cl- separation might be responsible for m. The other hypothesis related to the exchange between ATP4- and ADP3-, followed by hydrolysis by the F1-ATPase is also represented.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0075fje; doi: 10.1096/fj.03-0075fje

This article has been cited by other articles:

				E. Ferraro, A. Pulicati, M. T. Cencioni, M. Cozzolino, F. Navoni, S. di Martino, R. Nardacci, M. T. Carri, and F. Cecconi Apoptosome-deficient Cells Lose Cytochrome c through Proteasomal Degradation but Survive by Autophagy-dependent Glycolysis Mol. Biol. Cell, August 1, 2008; 19(8): 3576 - 3588. [Abstract] [Full Text] [PDF]

				O. Bachmann, A. Heinzmann, A. Mack, M. P. Manns, and U. Seidler Mechanisms of secretion-associated shrinkage and volume recovery in cultured rabbit parietal cells Am J Physiol Gastrointest Liver Physiol, March 1, 2007; 292(3): G711 - G717. [Abstract] [Full Text] [PDF]

				L. Huc, X. Tekpli, J. A. Holme, M. Rissel, A. Solhaug, C. Gardyn, G. Le Moigne, M. Gorria, M.-T. Dimanche-Boitrel, and D. Lagadic-Gossmann c-Jun NH2-Terminal Kinase-Related Na+/H+ Exchanger Isoform 1 Activation Controls Hexokinase II Expression in Benzo(a)Pyrene-Induced Apoptosis Cancer Res., February 15, 2007; 67(4): 1696 - 1705. [Abstract] [Full Text] [PDF]

				J. Satrustegui, B. Pardo, and A. del Arco Mitochondrial Transporters as Novel Targets for Intracellular Calcium Signaling Physiol Rev, January 1, 2007; 87(1): 29 - 67. [Abstract] [Full Text] [PDF]

				Y. Shiio, K. S. Suh, H. Lee, S. H. Yuspa, R. N. Eisenman, and R. Aebersold Quantitative Proteomic Analysis of Myc-induced Apoptosis: A DIRECT ROLE FOR Myc INDUCTION OF THE MITOCHONDRIAL CHLORIDE ION CHANNEL, mtCLIC/CLIC4 J. Biol. Chem., February 3, 2006; 281(5): 2750 - 2756. [Abstract] [Full Text] [PDF]

				S. Vankoningsloo, M. Piens, C. Lecocq, A. Gilson, A. De Pauw, P. Renard, C. Demazy, A. Houbion, M. Raes, and T. Arnould Mitochondrial dysfunction induces triglyceride accumulation in 3T3-L1 cells: role of fatty acid {beta}-oxidation and glucose J. Lipid Res., June 1, 2005; 46(6): 1133 - 1149. [Abstract] [Full Text] [PDF]

				K. S. Suh, M. Mutoh, K. Nagashima, E. Fernandez-Salas, L. E. Edwards, D. D. Hayes, J. M. Crutchley, K. G. Marin, R. A. Dumont, J. M. Levy, et al. The Organellular Chloride Channel Protein CLIC4/mtCLIC Translocates to the Nucleus in Response to Cellular Stress and Accelerates Apoptosis J. Biol. Chem., February 6, 2004; 279(6): 4632 - 4641. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/14/2145 03-0075fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ARNOULD, T. Articles by RAES, M. Search for Related Content PubMed PubMed Citation Articles by ARNOULD, T. Articles by RAES, M.