TGF-ß/NF1/Smad4-mediated suppression of ANT2 contributes to oxidative stress in cellular senescence.

Oxidative stress and persistent activation of DNA damage response (DDR) are causally involved in the development of cellular senescence, a phenomenon implicated in fundamental (patho)physiological processes such as aging, fetal development and tumorigenesis. Here, we report that adenine nucleotide translocase-2 (ANT2) is consistently down-regulated in all three major forms of cellular senescence: replicative, oncogene-induced and drug-induced, in both normal and cancerous human cells. We previously reported formation of novel NF1/Smad transcription repressor complexes in growth-arrested fibroblasts. Here we show that such complexes form in senescent cells. Mechanistically, binding of the NF1/Smad complexes to the NF1-dependent repressor elements in the ANT2 gene promoter repressed ANT2 expression. Etoposide-induced formation of these complexes and repression of ANT2 were relatively late events co-incident with production and secretion of, and dependent on, TGF-ß. siRNA-mediated knock-down of ANT2 in proliferating cells resulted in increased levels of reactive oxygen species (ROS) and activation of the DDR. Knock-down of ANT2, together with etoposide treatment, further intensified ROS production and DNA damage signaling, leading to enhanced apoptosis. Together, our data show that TGF-ß-mediated suppression of ANT2 through NF1/Smad4 complexes contributes to oxidative stress and DNA damage during induction of cellular senescence.

TGF-ß signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts.

We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G. Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-ß, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-ß is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-ß are prevented by inhibitors of TGF-ß RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-ß and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-ß signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.

Mutation in the beta subunit of F ATPase allows Kluyveromyces lactis to survive the disruption of the KlPGS1 gene.

The petite-negative yeast Kluyveromyces lactis does not tolerate the loss of phosphatidylglycerol (PG). We demonstrate that the lethality of PG loss is suppressed in strains carrying a mutation in the beta subunit of F(1) ATPase (mgi1-1). Phenotypic characterization shows that the strain lacking the phosphatidylglycerolphosphate synthase gene (KlPGS1) is able to grow only on glucose, but significantly more slowly and to substantially lower densities than the parental mgi1-1 strain. In addition, oxygen consumption in the DeltaKlpgs1 strain is <1% of the parental strain. Western blot analysis of mitochondrial membrane proteins shows that the amounts of some proteins are substantially decreased or even not detectable in this mutant. However, overexpression of the KlPGS1 gene under the inducible GAL1 promoter does not restore the ability of DeltaKlpgs1 cells to grow on galactose, indicating the presence of some other mutations and/or deletions in genes involved in oxidative phosphorylation. We also demonstrate that DeltaKlpgs1 cells do not spontaneously lose mtDNA, but are able to survive its loss after ethidium bromide mutagenesis. Deletion of the cardiolipin synthase gene (KlCLS1) in mgi1-1 has only a minimal effect on mitochondrial physiology, and additional experiments show that this deletion is also viable in wild-type K. lactis.

Lactate utilization in mitochondria prevents Bax cytotoxicity in yeast Kluyveromyces lactis.

In a search for the physiological conditions able to suppress the disruption of electron transport through the inner mitochondrial membrane induced by Bax, we found that respiratory substrate - lactate completely abolished Bax toxicity in yeast Kluyveromyces lactis. The effect of lactate was dependent on the presence of cytochrome c, as no effect was observed in the cytochrome c null strain. The investigation of lactate effect on markers of Bax toxicity showed that: (i) oxidation of lactate did not affect the decrease in oxygen consumption, but (ii) lactate was able to diminish the generation of reactive oxygen species and simultaneously to suppress Bax-induced cell death. We show that suppression of Bax lethality in K. lactis can be, in addition to anti-apoptotic proteins, achieved also by the utilization of lactate in the mitochondria.

The delivery of ADP/ATP carrier protein to mitochondria probed by fusions with green fluorescent protein and beta-galactosidase.

The import of proteins into mitochondria is an essential process, largely investigated in vitro with isolated mitochondria and radioactively labeled precursors. In this study, we used intact cells and fusions with genes encoding two reporter proteins, green fluorescent protein (GFP) and beta-galactosidase (lacZ), to probe the import of the ADP/ATP carrier (AAC). Typical mitochondrial fluorescence was observed with AAC-GFP fusions containing at least one complete transmembrane loop. This confirms the results of in vitro analysis demonstrating that an internal targeting signal was present in each one of the three transmembrane loops of the carrier. The fusions of AAC fragments to beta-galactosidase demonstrated that the targeting signal was capable of delivering the reporter molecule to the mitochondrial surface, but not to internalize it to a protease-inaccessible location. The delivery to a protease-inaccessible location required the presence of more distal sequences present within the third (C-terminal) transmembrane loop of the carrier molecule. The results of our study provide an alternative for investigation in a natural context of mitochondrial protein import in cells when the isolation of intact, functional mitochondria is not achievable.

The antiapoptotic protein Bcl-x(L) prevents the cytotoxic effect of Bax, but not Bax-induced formation of reactive oxygen species, in Kluyveromyces lactis.

The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effect on cell survival and production of reactive oxygen species (ROS). Bax expression decreased the number of cells capable of growing and forming colonies, and it increased the number of cells producing ROS, as detected by both dihydrorhodamine 123 fluorescence and the intracellular content of SH groups. Mutation in the beta-subunit of F(1)-ATPase, or mitochondrial deficiency resulting from deletion of mtDNA (rho(0) mutant), increased the sensitivity to Bax, indicating that Bax cytotoxicity does not require mitochondrial respiratory-chain functions. The antiapoptotic protein Bcl-x(L), when co-expressed with Bax, localized to the mitochondria and prevented Bax cytotoxicity. However, this co-expression did not prevent the production of ROS. These data suggest that in K. lactis cells expressing Bax, ROS are not the sine qua non of cell death and that the antiapoptotic function of Bcl-x(L) is not limited to its antioxidant property.